Biochimica et Biophysica Acta, 1040 (1990) 267-275
267
Elsevier BBAPRO 33727
A monoclonal antibody against a native conformation of the porcine renal Na+/K+-ATPase a-subunit protein Osamu
Urayama
1,2, H i d e a k i N a g a m u n e
1.., Makoto
Nakao I and Yukichi Hara 1
t Department of Biochemistry, Tokyo Medical and Dental University School of Medicine, Bunkyo, Tokyo (Japan) and : Department of Chemistry, University of California at San Diego, La Jolla, CA (U.S.A.)
(Received 20 November 1989) (Revised manuscript received 7 March 1990)
Key words: Enzymeconformation; Monoclonal antibody; ATPase, Na+/K +-
A monoclonal antibody (mAb50c) against the native porcine renal Na+/K+-transporting adenosinetriphosphatase (EC 3.6.1.37, ATP phosphohydrolase) (Na+/K+-ATPase) was characterized. The antibody could be classified as a conformation-dependent antibody, since it did not bind to N a + / K +-ATPase denatured by detergent and its binding was affected by the normal conformational changes of the enzyme induced by ligands. The binding was the greatest in the presence of Na +, ATP or Mg 2+ (El form), slightly less in the presence of K ÷ (E2K form) and the least when the enzyme was phosphorylated, especially in the actively hydrolyzing form in the presence of Na +, Mg 2+ and ATP. The antibody inhibited both the Na÷,K ÷-ATPase activity and the K ÷-dependent p-nitrophenylphosphatase activity by 25%, but it had no effect on Na÷-dependent ATPase activity. The antibody partially inhibited the fluorescence changes of the enzyme labeled with 5'-isothiocyanatofluorescein after the addition of orthophosphate and Mg 2+, and after the addition of ouabain. Proteolytic studies suggest that a part of the epitope is located on the cytoplasmic surface of the N-terminal half of the a-subunit.
Introduction Na+/K+-transporting adenosinetriphosphatase ( N a + / K + - A T P a s e ) , which is a membrane-embedded protein, is primarily responsible for the active transport of N a + and K + in animal cells. It consists of two folded polypeptides, a catalytic subunit ( a ) and a glycosylated subunit (/3). The et subunit contains the A T P binding, ouabain binding and phosphorylation sites. The enzyme is phosphorylated on the a subunit by Mg2+-ATP in the presence of N a + or by orthophos-
* Present address: Department of Oral Microbiology and Immunology, University of Tokushima, Tokushima, Japan. Abbreviations: Na+/K+-ATPase, Na+,K+-transporting ATPase (EC 3.6.1.37, ATP phosphohydrolase); ATPase, adenosinetriphosphatase; K+-p-NPPase, K+-dependent p-nitrophenylphosphatase; TBS, 154 mM NaCI/20 mM Tris-HC1 (pH 7.5); FITC, 5'-isothiocyanatofluorescein; SDS, sodium dodecyl sulfate; SET, 0.32 M sucrose/1 mM EDTA/20 mM Tris-HCl (pH 7.5); SDS-PAGE, electrophoresis on polyacrylamidegels poured in a solution of SDS. Correspondence: O. Urayama, Department of Biochemistry, Tokyo Medical and Dental University School of Medicine, Yushima 1-5-45, Bunkyo-ku, Tokyo 113, Japan.
phate in the presence of Mg 2+, and the phosphoenzyme is dephosphorylated in the presence of K +. The phosphorylation promotes the binding of ouabain. The structure responsible for these catalytic functions is the a subunit. There are at least four conformations of the enzyme, two conformations of the unphosphorylated enzyme and two of the phosphorylated enzyme. The conformation designated E1 is the unphosphorylated form binding N a + as a substrate of transport and E1P is the phosphorylated form that has occluded N a +. The conformation designated E2P is the phosphorylated form binding K + as a substrate and E2 is the unphosphorylated form that has occluded K +. These occluded ions are transported to the opposite side of the membrane. The normal sequence of events is for the enzyme to pass from E1 to E1P to E2P and to E2 during turnover and these transitions are believed to be coupled to cation transfer across the membrane (reviewed in Refs. 1-3). These four conformations differ in their affinity for nucleotides, binding of cardiotonic steroids, susceptibility to proteolytic enzymes, intrinsic fluorescence and response to N a + and K +. Models of the transmembrane organization of the a subunit have recently been proposed, based on its amino acid sequence [4-6]. The importance of the N-terminal
0167-4838/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
268 half of the a subunit for the transition between E2 and E1 has been demonstrated [7-9]. The first 90 amino acids from the N-terminus, which are located on the cytoplasmic side of the membrane [10-12], have a high frequency of hydrophilic, especially basic, amino acids [4,13]. Protonation of the enzyme facilitates the transition from the E1 to the E2 conformation [14], and the removal of 30 residues from the N-terminus by tryptic cleavage causes an increase in the affinity of the enzyme for Na ÷ at pH 6 [9]. Chymotrypsin cleaves a cytoplasmic segment of the polypeptide between residues 150 and 280 in the sequence of the a subunit, and this also shifts the equilibrium toward the E1 form [8]. Conformational changes of Na+/K+-ATPase can be induced by specific antibodies [15-18]. Some monoclonal antibodies have been shown to affect the transition between the E1 and the E2 forms of the enzyme. Antibody 9-A5 [15], which can bind to a denatured chymotryptic fragment containing residues 200 to 430 and including the phosphorylation site [19], stabilizes the E1 conformation [20]. On the other hand, antibody M45-80 [18] stabilizes the E2K conformation, probably by binding to the sequences in the N-terminal half of the a subunit on the extracellular surface of the enzyme. In this paper, we characterize one of the murine monoclonal antibodies mAb50c that was obtained by immunization and screening with a native porcine renal Na÷/K+-ATPase [21]. The antibody can detect ligandinduced conformational changes of the enzyme, and its binding affects the transition between enzyme conformations. A part of the epitope whose structure is altered during these conformational changes may be located in the N-terminal half of the amino acid sequence of the a subunit. A portion of this work was presented at the Tonomura Memorial Synposium of the Yamada Conference XI [50]. Materials and Methods
Materials All reagents were of reagent grade. Bio-Gel A5m was purchased from Bio-Rad. Protein A-Sepharose CL-4B was purchased from Pharmacia. Trypsin-TPCK was from Worthington. Soybean trypsin inhibitor, a-chymotrypsin, phosphoenolpyruvate, /3-NADH, pyruvate kinase, lactic dehydrogenase, Tris salt of p-nitrophenyl phosphate, fluorescein 5'-isothiocyanate, prestained SDS-PAGE molecular weight marker proteins and anti-rabbit IgG alkaline phosphatase conjugate were obtained from Sigma. Mouse myeloma protein MOPC21 IgG1 was purchased from Litton Bionetics. Clear Blot Membrane-P hydrophobic membrane was purchased from Atto.
Preparation of Na +/ K +-A TPase and its polypeptides Na+/K+-ATPase was purified from porcine renal outer medulla essentially according to Jergensen [22].
The a and fl polypeptides were separated chromatographically on a column (0.3 × 86 cm) of Bio-Gel A5m equilibrated with a solution of sodium dodecyl sulfate (SDS) [23,24]. The SDS was removed from the isolated polypeptides by the method described by Nicholas [24]. This produces fully soluble proteins dissolved in aqueous buffer [23].
Determination of Na +/ K +-A TPase activity and protein The enzymatic activity of Na+/K+-ATPase was measured by using one of two assay methods. One was an assay in which orthophosphate liberated by the hydrolysis of ATP was determined [21,25] by the method of Fiske and SubbaRow [26]. In the other method, the oxidation of NADH was coupled to the hydrolysis of ATP using pyruvate kinase and lactic dehydrogenase and the loss of NADH was monitored continuously by measuring the absorbance at 340 nm [27] in a spectrophotometer equipped with a thermally controlled cell holder maintained at 37°C. The standard mixture for the Na+/K+-ATPase assay contained 140 mM NaC1, 10 mM KC1, 5 mM MgC12, 0.5 mM EDTA, 3 mM ATP and 50 mM Tris-HC1 (pH 7.5) with or without 0.2 mM ouabain. The activity of Na+-dependent ATPase was assayed by using the above assay mixture except that KC1 was omitted. The activity of K+-dependent pnitrophenylphosphatase (K+-p-NPPase) was measured by our method [28]. The assay mixture contained 10 mM Tris salt of p-nitrophenyl phosphate, 10 mM KC1, 5 mM MgC12, 0.5 mM EDTA and 20 mM imidazolium (pH 7.5). Protein was determined by the method of Lowry et al. [29] using BSA as a standard.
Effect of antibody on Na + / K +-A TPase activity Porcine renal Na+/K+-ATPase (10 ~g) was incubated with the purified antibody (30/~g) in 100/~1 of 154 mM NaC1 and 20 mM Tris-HC1, pH 7.5 (TBS) for 45 min at room temperature. In a competition experiment, the antibody (30 #g in 80/~1) was preincubated at 4°C overnight with Na÷/K+-ATPase that had been inactivated (0 /~mol Pi/mg per h) with N-ethylmaleimide [30], and thereafter it was mixed with unalkylated Na+/K+-ATPase (10/xg in 20/~l). A portion (20 #1) of the mixture was transferred to 1 ml of the coupled assay medium [27], which had been warmed to 37°C, for Na+/K+-ATPase assay. The reaction reached a steadystate rate within 1 min of its initiation and proceeded at a constant rate for at least 4 rain either in the presence or in the absence of the antibody.
Na +/ K +-A TPase labeled with 5'-isothiocyanatofluorescein Renal enzyme was labeled with 5'-isothiocyanatofluorescein (FITC) according to Hegyvary and Jorgensen [31]. The ratio of incorporated FITC for each a subunit was estimated to be 1.1-1.6, as described before [28].
269 Fluorescence of the enzyme (excitation at 496 nm and emission at 520 nm) was measured with a Hitachi 850 fluorescence spectrophotometer.
Hybridoma production and antibody purification Porcine renal Na+/K÷-ATPase, with a specific activity of 1600 ttmol Pi/mg per h at 37°C, was injected into mice, and sera were screened for specific antibodies by assessing binding to the holoenzyme followed by an assay for Na÷/K÷-ATPase as described before [21]. The monoclonal antibody mAb50c was an IgG 1 immunoglobulin. The monoclonal IgG 1 immunoglobulin mAb38 also raised against the native porcine renal Na+/K+-ATPase was used as a control in some experiments. Monoclonal immunoglobulins were purified from the ascites fluid of pristine-primed mice inoculated with cells of the hybridoma 50c by affinity chromatography on a column of Protein A-Sepharose [32].
Binding of antibody Ligand-dependent binding of antibody to renal Na÷/K÷-ATPase was assessed as described before [21,33] with some modifications. A sample of the enzyme (60 gg in 300 gl) was incubated with mAb50c (100 ttg) in 10 mM Tris-HC1 (pH 7.5) containing either 140 mM NaC1; 140 mM KC1; 140 mM choline chloride; 3 mM ATP; 3 mM MgCI2; 3 mM MgC12 and 3 mM orthophosphate; 140 mM NaC1, 3 mM MgC12, and 3 mM ATP; or 140 mM NaC1, 3 mM MgC12, 3 mM ATP, and 1 mM ouabain for 60 min at 23°C. The amount of antibody chosen was a saturating concentration in the presence of Na ÷. The enzyme-antibody complex was pelleted by centrifugation (100000 rpm, 5 min) in a Beckman TL-100 centrifuge, washed twice with the respective solution of buffer and ligands, and dissolved in SDS. A portion of the solution of polypeptides (20 /tg of Na+/K÷-ATPase) was heated at 70°C for 5 rain and applied to a discontinuous polyacrylamide gel system [34] with 8% acrylamide separating gel. After electrophoresis, the gel was stained with Coomassie brilliant blue and scanned. The stoichiometry of binding (mol of IgG per mol of aft protomer) was calculated from the ratio between the area of the peak of the light chain (M r = 23 000) of the immunoglobulin G and the area of the peak of the a polypeptide ( M r = l l 0 0 0 0 ) of Na+/K÷-ATPase. Calibration curves for the extinction coefficients of complexes between protein and dye were prepared before estimation. The total absorbance of the a polypeptide and that of the light chain complexed to the dye were proportional to the amount of protein added to the gel over the range of 5-20 /~g for Na+/K+-ATPase and the range of 2-10 #g for the immunoglobulin.
Proteolytic digestion Selective tryptic cleavage of the a subunit of renal Na÷/K÷-ATPase was carried out as described by Far-
ley et al. [19]. The epitope of mAb50c on the a subunit was investigated by submitting the antigen-antibody complex to proteolysis [35,36]. Porcine renal N a + / K ÷ATPase (99/xg) was preincubated alone or with mAb50c (200/xg) or with mAb38 (200 ttg) in 100/~1 of TBS at 4°C overnight. The mixture was centrifuged (Beckman TL-100) at 100000 rpm for 5 min. The pellet was washed and then suspended in a minimum vol. of 0.32 M sucrose, 1 mM EDTA and 20 mM Tris-HC1 (pH 7.5) (SET). The suspension was divided into three portions. The first portion was subjected to tryptic digestion, the second was subjected to chymotryptic digestion, and the third was subjected to electrophoresis to quantify formation of the complex. Tryptic digestion was carried out as follows. A complex between Na+/K+-ATPase (30 ttg) and antibody, suspended in SET containing 15 mM KC1, was incubated with 0.3 ttg of trypsin at 37 o C. Aliquots were removed and quenched with soybean trypsin inhibitor at the indicated times and dissolved for electrophoresis as described [34] but with the addition of saturated phenylmethylsulfonyl fluoride and 2-mercaptoethanol to 1%. The samples were heated to 70°C for 5 min and applied to the discontinuous gel system with a 10% separating gel. After electrophoresis, proteins were transferred [37] to hydrolqhobic membranes, and the membranes were immunostained with anti-porcine renal Na÷/K+-ATPase rabbit serum (Antibody PK) as described before [38]. For chymotryptic cleavage, the complex between Na+/K÷-ATPase (30 /~g in 125#1) and antibody was digested with chymotrypsin (1.5 ttg) in the presence of 15 mM Tris-HC1 (pH 7.5) and 10 mM NaC1 at 37°C. Aliquots were quenched in 100 mM NaC1 [8] at the indicated times and subjected to SDS-PAGE followed by the above immunoblotting. Results
Binding of antibody The monoclonal antibody was shown to recognize native Na+/K+-ATPase by an enzyme-linked immunoadsorbance assay. In this procedure, wheat germ agglutinin associated with native Na÷/K÷-ATPase was chemically immobilized on microtiter plates as the antigen [39]. The half-value of the maximum binding of mAb50c to a microtiter well coated in this way was 130 # g / m l (0.8 /tM using M r = 162000). In solution, mAb50c and mAb38 with higher affinity [21,39] showed similar titration for native Na÷/K+-ATPase (not shown). In contrast, the antibody did not bind to any polypeptide of the Na÷/K÷-ATPase in Western blot analysis. Even a concentrated solution of mAb50c (40 ttl of the ascites fluid in 8 ml of TBS) did not stain either a or B polypeptide after their separation from the native
270 TABLE I Ligand-dependent binding of mA b50c to Na +/K +-A TPase a
Antibodies Ligands mAb50c
mAb38
Na + K+ ATP Mg1+ Mgz+ + orthophosphate Na ÷ +Mg 2+ +ATP Na + +Mg 2+ +ATP + ouabain Na +
IgG/afl (mol/mol) Expt. b: 1 2 3 0.80 0.74 0.65 0.72 0.25 0.25
0.78 0.63 0.80 0.52 0.33
0.49 0.37 -
0.85 c
a Enzyme (final concentration 0.2 mg/ml) was mixed with appropriate solutions of the ligands. Monoclonal antibody (final concentration 0.34 mg/ml) was then added. After 1 h at 23°C, binding was assessed by electrophoresis. b Different preparations of Na+/K+-ATPase were used in Expts. 1, 2 and 3, respectively. c This has been shown to be ligand-independent binding [21].
enzyme (16 ttg) by SDS-PAGE followed by electrophoretic transfer to nitrocellulose paper. Moreover, neither the isolated a polypeptide nor the isolated fl polypeptide, after being stripped of their SDS, competed with the native renal enzyme for the antibody binding. The isolated a polypeptide (42 /~g) or the isolated fl polypeptide (30 #g) was mixed with mAb50c (30 /zg) and the mixture was incubated with native enzyme (10 ~tg in 100/~1) for a competition assay (see the legend to Fig. 1), but neither of the isolated polypeptides had any effect on the inhibition of the N a + / K + - A T P a s e activity by the antibody. These resuits suggest that the binding of mAb50c requires the antigen to be in its native conformation. The binding of mAb50c to native N a + / K + - A T P a s e was measured by centrifugation of antigen-antibody complexes followed by analysis of their composition by SDS-PAGE (Table I). The level of binding (IgG per ctfl-protomer) was calculated to be 0.80 in the presence of Na ÷ (E1Na), 0.80 in the presence of Mg 2÷ (E1Mg), 0.72 in the presence of ATP (E1ATP), 0.65 in the presence of K ÷ (E2K), 0.52 in the presence of Mg 2÷ and orthophosphate, 0.25 in the presence of N a ÷, Mg 2÷ and ATP (E2P), and 0.49 in the presence of N a ÷, Mg 2÷, ATP and ouabain (complex between ouabain and E2P). Choline chloride as a control of ionic strength gave the value of 0.68. The highest binding was obtained with E1 and the least binding with E2P, especially the actively hydrolyzing enzyme form. Binding numbers were slightly different with different enzyme preparations. In addition, the stoichiometry never reached unity. This may be due to an overestimation of the enzyme protein that can react with the antibody. It is known that N a + / K + - A T P a s e purified by extraction with SDS con-
tains a small amount of denatured enzyme [40]. If we could calculate accurately the amount of the active enzyme by subtraction of the amount of the inactive enzyme in the preparation of N a + / K + - A T P a s e , higher binding numbers should be obtained. Effect on N a + / K +-A TPase functions A partial inhibition (25%) of the activity of N a + / K + - A T P a s e by the antibody was observed when activity was assayed with a timed test tube assay. Because the binding of antibody depended on the ligands present (Table I), the time-course of the inhibition was examined by means of the coupled assay (Fig. 1). Both in the presence and in the absence of the antibody, the enzymatic reaction proceeded linearly after a short lag. The maximum inhibition (25.9 + 2.4 (mean + S.D.) % for three experiments) was observed with 3-fold weight excess of antibody over enzyme. Mouse myeloma IgG1 (another control as well as the no-antibody control) had no effect on the N a + / K + - A T P a s e activity. N-Ethylmaleimide-inactivated N a + / K + - A T P a s e [30] partially reversed the inhibition caused by the antibody by competition (Fig. 1). Ouabain inhibited the renal N a + / K ÷ATPase when it was in a complex with the antibody. The half-value for the maximum inhibition by ouabain was estimated to be 30 nM, which was identical to that for the uncomplexed native enzyme.
-
-
v
i
i
0.2
o
':~,,
o.~
~
,>.,, "%x. x
0.6
:,,
I
',>\ \
'~' ',
0.8 1
0
__
i
- - ~
1 2 Reaction tirne (rnin)
\
'l ~,
3
Fig. 1. Effect of mAb50c on Na+/K+-ATPase activity. Renal Na+/K+-ATPase (10 /~g in 100 /d) was incubated with 30 /~g of
mAb50c ( - - - - - ) or with no antibody ( ). In a competition experiment, fully active enzyme(10/~g in 100/~1) was incubated with the antibody (30/ill) that had been preincubated with N-ethylmaleimide-inactivated enzyme (15 /zg, •. . . . or both 30 and 60 /~g, . . . . . . . . ). Portions of the incubation mixtures were transferred to the coupled assay medium for Na+/K+-ATPase activity to initiate the reaction. The oxidation of NADH coupled to the hydrolysis of ATP was monitored by measuring the absorbance at 340 nm.
271 i
100
t
i
o
'~-'0~
_._>,
A w
0.6
Q.
i
/
50
o
I
I
5 10 Antibody (IJg)
I
I
20
Fig. 2. Effect of mAb50c on K÷-p-NPPase as a function of antibody concentration. Renal Na+/K+-ATPase (1 /~g in 500 btl) was preincubated with mAb50c(t) or mAb38 (o) as a control in the amounts indicated on the abscissa and then assayed by adding the reaction medium for the K+-p-NPPase assay. The remaining activity is expressed as a percentage of that without antibodies. The fact that mAb50c was poorly bound to the enzyme in the presence of N a ÷, Mg 2+ and A T P (Table I), but partially inhibited N a + / K + - A T P a s e activity (Fig. 1), suggested that the antibody might inhibit the enzyme by affecting not an Na÷-dependent step but a K÷-de pendent step during its turnover. Na+,K÷-transporting ATPase has a p-NPPase activity in the presence of K÷; the K÷-bound unphosphorylated enzyme is probably responsible for the hydrolysis [41,42]. The effect of the antibody on the K+-p-NPPase was examined as a function of its concentration (Fig. 2). The antibody inhibited the activity by 22-25% at 10-fold weight excess by decreasing both the K m value for p-NPP from 1.3 to 1.1 mM and the Vm~, from 3.57 #mol p - N P / m g per min to 2.78 /xmol p - N P / m g per min (Fig. 3). The inhibition was of an intermediate type between noncompetitive and uncompetitive. The antibody increased the K m for K ÷ from 0.8 mM with a Hill coefficient of 1.7 to 1.2 mM with a Hill coefficient of 1.9 (Fig. 4). The K m values for K ÷ were identical in another experiment. In contrast, the antibody had no effect on the N a ÷dependent ATPase either with 140 mM N a ÷ or with 700 mM N a ÷ (5 mg mAb50c per mg N a ÷ / K + - A T P a s e ) , which suggests poor binding of the antibody to the actively hydrolyzing enzyme form. Two N a ÷ concentrations were tested, since the level and decay of the phosphoenzymes of the Na÷-dependent ATPase [41] depend on N a + concentration [43]. In order to observe the effect of the antibody on the ligand-induced conformational changes of the enzyme directly, N a + / K ÷ - A T P a s e was labeled with FITC. In the presence of Mg 2÷, orthophosphate quenches F I T C fluorescence while phosphorylating the enzyme [31]. When the fluoresceinyl enzyme was complexed with
0.5 1.o 1.5 1/[p-NPP](mM) Fig. 3. Effect of the concentrationof p-NPP on K+-p-NPPaseactivity in the presenceof mAb50c. Renal Na+/K+-ATPase (1/~g in 500/~1) was preincubated with 10/tg of mAb50c (t) or without any antibody (o) in the medium for the K+-p-NPPase assay at 10 mM KC1. The reaction was initiated by adding p-NPP (0.66-10 mM) to the mixture. The initial velocities as a function of concentration are presented in the form of a Lineweaver-Burkplot.
mAb50c, the fluorescence change after the addition of orthophosphate and Mg 2÷ was inhibited compared to that of the enzyme complexed with mAb38 (Fig. 5A). The quenching with mAb38 was almost the same as that with the enzyme unbound by antibodies. The fluorescence change after adding these ligands was 35% less than that of the control at higher concentrations of mAb50c (Fig. 5B). Addition of ouabain to the phosphoenzyme caused a large drop in fluorescence resulting from the formation of the phosphoenzyme-ouabain complex. The quenching of the enzyme bound to mAb50c by binding of ouabain was slightly slower than
0.4
'
0
X
E
o
.J
- 0.4
I
-0.4
I
___
I
t
0 Log[KCI] (raM)
i
0.4
Fig. 4. Effect of the concentration of KC! on K+-p-NPPase in the presence of mAb50c. Renal Na+/K+-ATPase (1 rig in 500 btl) was preincubated with 10/tg of mAb50c(IP)or without any antibodies (o) in the reaction medium for the K+-p-NPPase assay at various concentrations of KCI (0.67-6 mM). The reaction was initiated by adding p-NPP to the mixture. The data are presented as a Hill plot.
272 A
Mg
Pi o
Ou
. ~11
mAb38 7
§
~ so
mAb50c
";" 100,
1 ,"""'
01, 1rain
i
i
1
10
J
100 1 00
Antibody(ug )
Fig. 5. Effect of mAb50c on the change in fluorescence caused by orthophosphate in the presence of MgC12. Renal Na+/K+-ATPase labeled with FITC (30/~g in 100/~1) was preincubated with the particular antibody or without any antibody in 50 mM Tris-HC1 (pH 7.5) and 0.1 mM EDTA. A portion of the mixture was transferred to 1 ml of the Tris buffer and the observation was initiated by adding MgC12 (Mg) and then orthophosphate (Pi) to final concentrations of 2 mM. Ouabain (Ou) was then added to a final concentration of 100 /~M. (A) Changes of the intensity of the fluorescence (from left to fight). (B) The fluorescence change after the addition of orthophosphate as a percentage of that in the absence of antibodies is plotted against the concentration of mAb50c (O) or mAb38 (o).
that of enzyme bound to mAb38 (Fig. 5A). When ouabain binding was examined in the presence of Mg 2÷, it was found that mAb50c reduced the rate of quenching caused by ouabain binding by 50% (Fig. 6). The maximum guenching after the further addition of orthophosphate, however, resulting from the formation of the phosphoenzyme-ouabain complex in the presence of mAb50c was similar to that in the presence of mAb38 or in the absence of antibodies (Fig. 5A). By shifting the enzyme into the E2 form, K ÷ quenched the FITC fluorescence on the enzyme in the presence of mAb50c i
100 80
-~ 60
!,o
2o tL
20
0
I
I
2
I
I
i
4
i
6
Time after addition of ouabain (rain) Fig. 6. Time course of the quenching of fluorescence by ouabain. Renal Na+/K+-ATPase labeled with FITC (30 /~g in 100 #1) was preincubated either with mAb50c (3/~g, × ; 30 #g, zx; 300/~g, &) or mAb38 (300 #g, D) or without antibodies (o). An equal portion of each mixture was transferred to 1 ml of the medium used to follow change in fluorescence: 2 mM MgC12, 0.1 mM EDTA and 50 mM Tris-HC1 (pH 7.5). The reaction was initiated by adding ouabain to 100 /xM arid the quenching was monitored with time. The fluorescence change is expressed as a percentage of the difference in fluorescence between the initial level (conformation EIMg 2+ ) and the level that was obtained by the further addition of orthophosphate to give ouabain-bound E2P.
or in the presence of mAb38 (the concentration giving half-maximal change was 0.07 mM in each case).
Localization of epitope The effects of mAb50c on the catalytic rates and equilibria of Na+/K+-ATPase suggested that the antibody recognized the a subunit. We have previously shown that mAb50c was raised against a cytoplasmic site of Na÷/K+-ATPase [21]. Segments of the folded a polypeptide that are on the cytoplasmic surface of the subunit were investigated as candidates for the epitope by using ligand-dependent proteolysis. In the presence of K ÷, trypsin initially cleaves a peptide bond at the center of the a polypeptide in the cytoplasmic portion of the ct subunit followed by a secondary cleavage in the N-terminal region [9,44]. Chymotrypsin cleaves the a polypeptide between transmembrane segments M2 and M3 in the cytoplasmic portion of the a subunit in the presence of low Na ÷ [8,9]. The binding of mAb50c to various types of digested enzyme was analyzed by centrifugation and SDS-PAGE (Fig. 7). The binding of mAb50c decreased with the decrease of the intact a polypeptide after tryptic digestion (Fig. 7A). During the digestion the fl polypeptide was not lost, because its electrophoretic mobility was unchanged when it was identified by immunostaining. Selective chymotryptic cleavage of the a subunit also reduced the binding of antibody (Fig. 7B). These results confirm that the epitope is located in the a subunit. Epitopes have also been studied by examining the ability of antibodies to protect one region of the antigen from proteolysis [35,36]. To characterize further the epitope of mAb50c on the a subunit, the complex between Na+/K+-ATPase and mAb50c was treated with trypsin or chymotrypsin followed by immunoblotting with anti-porcine-renal Na÷/K+-ATPase rab-
273 bit serum (Fig. 8). Trypsin produced the N-terminal fragment of M r = 42 000 early in the digestion (2.5 min in both Fig. 8A and B) in the presence of mAb50c as well as in the presence of mAb38 as a control. This N-terminal fragment in the control complex was further digested progressively to several subfragments with M r = 41000-37000 (highlighted in Fig. 8B at 10 rain), in agreement with previous observations [12,19,44], but mAb50c clearly decreased the rate of the further fragmentation of the N-terminal fragment (compare 10 rain between A and B, and 40 rnin between A and B, respectively, in Fig. 8). Larger subfragments of the N-terminal fragment persisted in the presence of mAb50c than in the presence of mAb38. The behavior of the complex between Na+/K÷-ATPase and mAb38 was the same as that of uncomplexed Na+/K÷-ATPase, another control. On the other hand, there was no difference between the complex of mAb50c and the enzyme and the complex of mAb38 and the enzyme or between the complex of mAb50c and the enzyme and
A
I
2
B
I
2
-Fc
-F m
A 0
2.5
B I0
40
Mr
0
2.5
I0
40
Fig. 8. Tryptic digestion of the complex between Na*/K*-ATPase and monocional antibodies. The complex between the enzyme and mAb50c (A) or the complex between the enzyme and mAb38 (B) was treated with trypsin for the indicated number of minutes. Samples of the digested enzyme were submitted to electrophoresis in SDS and transferred to a sheet of hydrophobic membrane. The blot was incubated with Antibody PK and the bound antibodies were detected with goat anti-rabbit immunoglobulin conjugated to alkaline phosphatase. The polypeptides were labeled as follows: a, a subunit; fl, fl subunit; N, N-terminal fragment; Ns, subfragments from the Nterminal fragment. The lane designated M r contained prestained molecular weight markers of 180000, 116000, 84000, 58000, 48500, 36500 and 26600.
the enzyme alone in the rate of production of the primary product (M r = 78000) [8] from the a subunit during chymotryptic cleavage. The protection of the N-terminal fragment from later digestion by mAb50c could be due to a direct steric effect of the bound antibody or to a structural change of the region of the a subunit surrounding the site of cleavage after the binding of the antibody.
Discussion
Fig. 7. Binding of mAb50c to Na+/K÷-ATPase cleaved with proteolytic enzymes. Renal Na÷/K+-ATPase (30 /~g) was treated with 0.3 /~g of trypsin in 20 #1 containing 15 mM KC1 (A) or with 1.5 #g of chymotrypsin in 150/xl containing 10 mM NaC! (B), respectively, for 30 min at 37°C. After quenching of each reaction, the cleaved enzyme was washed with TBS and incubated with 2-fold weight excess of mAb50c at 4°C overnight. The complex of antigen and antibody was collected by centrifugation and then analyzed by SDS-PAGE (7.5% gel for A and 10% gel for B). Lane 1: binding to the uncleaved enzyme. Lane 2: binding to the cleaved enzyme. The polypeptides are labeled as follows: a, a polypeptide; fl, fl polypeptide; H, heavy chain (a doublet) of mAb50c; L, light chain; Ft, tryptic fragments from a subunit; Fc, a chymotryptic fragment from a subunit.
As specific probes for Na+/K+-ATPase, we prepared mouse monoclonal antibodies by using porcine renal Na+/K+-ATPase with high specific activity as an antigen. Most of the monoclonal antibodies obtained bound native Na+/K÷-ATPase, but not its constituent polypeptides, denatured with SDS. On this basis, these antibodies might be classified as conformation-dependent antibodies [45]. In conformation-dependent epitopes, discontinuous determinants [reviewed in Refs. 46, 47], which may be composed of residues far apart in the primary structure, are brought together on the surface of the protein by the folding of the native structure. The identification of such separate determinants has sometimes been made from binding studies between the
274 antibody and either peptide fragments of the antigen or evolutionarily related proteins. Recently, both proteolysis [35,361 and cross-linking [48] of complexes between antigen and antibody have been applied to identify these types of epitopes. In these experiments, we have used ligand-dependent proteolysis to identify the epitope of mAb50c in the subunit of porcine renal Na+/K+-ATPase. The ability of the antigen to bind the monoclonal antibody was lost by selective chymotryptic cleavage between Leu-266 and Ala-267 [9] of the a polypeptide in the native enzyme (Fig. 7) as well as by tryptic digestion. The results suggest that the epitope is near this point of cleavage or is dependent on the integrity of the polypeptide in this region. During trypsinolysis of the complex between antibody and enzyme, mAb50c had the ability to protect the N-terminal fragment (Gly-1Arg-438) produced by tryptic cleavage between Arg-438 and Ala-439 [9] from further digestion (Fig. 8). In the unprotected enzyme some of this further digestion occurs at sites in this N-terminal fragment close to its N-terminus containing the site T2 between Lys-30 and Glu-31 [9,44]. It cannot be ruled out, however, that some of this further digestion also occurs at a site near the C-terminus of this N-terminal fragment in addition to those sites near its N-terminus [12,19]. Therefore, a portion of the epitope for mAb50c responsible for this protection may be located at either the N-terminus of the a polypeptide or in the sequence of the a polypeptide surrounding Arginine-438. The N-terminal regions of the a subunit are involved in the conformational changes that accompany cation binding and occlusion [4]. Tryptic cleavage at Lys-30 increases the affinity of the enzyme for Na + and chymotryptic cleavage at Leu-266 stabilizes the E1 form of the enzyme [9]. Monoclonal antibody 9-A5, the epitope of which was identified as being on the cytoplasmic surface of the enzyme in a region containing the portion of the a polypeptide between Ala-267 and Arg-438 [19], also stabilizes the E1 form of the enzyme [20]. The antibody '50c inhibited the formation of E2P from E1 in the presence of Mg 2+ (Fig. 5). The controlled proteolysis, the binding of antibody 9-A5, and the binding of mAb50c all show a similar effect on the conformational change of native Na+/K+-ATPase. On the other hand, another monoclonal antibody, M45-80, inhibited the enzymatic reactions by shifting the enzyme to the E2 form [18]. The epitope of M45-80 is thought to be located on the extracellular surface in the Nterminal half of the a subunit. It has been proposed that the formation and dissociation of hydrogen bonds [49] between the N-terminal segment of the folded a polypeptide which contains excess positive charges [4,13] and other cytoplasmic portions of the a subunit may be involved in cation translocation [9], but the spatial organization of the a polypeptide within the N-terminal
half of the a subunit remains to be elucidated. Further study of the conformationally dependent epitopes of mAb50c or monoclonal antibody M10-P5-Cll [17] might give us some information on the three-dimensional structure of the N-terminal half. The binding activity of mAb50c is ligand-dependent (Table I) and the site is exposed in the E1 form but protected in the E2P form. This property may be useful for the detection of the intactness of Na+/K÷-ATPase and for further purification of the active enzyme.
Acknowledgments We thank Dr. Jack Kyte (University of California at San Diego), in whose laboratory a part of this research was performed, for his advice and helpful comments. We also thank Dr. Hiroyuki Sugiyama (National Institute for Physiological Science, Japan) for assisting us in the production of monoclonal antibodies, and Toshiyuki Kojima (Tokyo Medical and Dental University) and Katherine Charles (University of California at San Diego) for preparation of porcine renal enzymes. This work was supported by Grants-in-Aid for Special Project Research on Bioenergetics from the Ministry of Education, Science and Culture of Japan and by Grant GM-33962 from the National Institutes of Health.
References 1 Kyte, J. (1981) Nature 292, 201-204. 2 Jorgensen, P.L. (1982) Biochim. Biophys. Acta 694, 27-68. 3 Glynn, I.M. (1985) in The Enzymes of Biological Membranes (Martonosi, A.N., ed.), Vol. 3, pp. 35-114. Plenum, New York. 4 Shull, G.E., Schwartz, A. and Lingrel, J.B. (1985) Nature 316, 691-695. 5 Kawakami, K., Noguchi, S., Noda, M., Takahashi, H., Ohta, T., Kawamura, M., Nojima, H., Nagano, K., Hirose, T., Inayama, S., Hayashida, H., Miyata, T. and Numa, S. (1985) Nature 316, 733-736. 60vchinnikov, Y.A., Modyanov, N.N., Broude, N.E., Petrukhin, K.E., Grishin, A.V., Arzamazova, N.M., Aldanova, N.A., Monastyrskaya, G.S. and Sverdlov, E.D. (1986) FEBS Lett. 201,237-245. 7 Jorgensen, P.L. and Karlish, S.J.D. (1980) Biochim. Biophys. Acta 597, 305-317. 8 Jorgensen, P.L. and Petersen, J. (1985) Biochim. Biophys. Acta 821, 319-333. 9 Jorgensen, P.L. and Collins, J.H. (1986) Biochim. Biophys. Acta 860, 570-576. 10 Jorgensen, P.L., Karfish, S.J.D. and Gitler, C. (1982) J. Biol. Chem. 257, 7435-7442. 11 Ball, W.J., Jr. and Loftice, C.D. (1987) Biochim. Biophys. Acta 916, 100-111. 12 Felsenfeld, D.P. and Sweadner, K.J. (1988) J. Biol. Chem. 263, 10932-10942. 13 Shull, G.E., Greeb, J. and Lingrel, J.B. (1986) Biochemistry 25, 8125-8132. 14 Skou, J.C. and Esmann, M. (1980) Biochim. Biophys. Acta 601, 386-402. 15 Schenk, D.B., Hubert, J.J. and Leffert, H.L. (1984) J. Biol. Chem. 259, 14941-14951. 16 Ball, W.J., Jr. (1984) Biochemistry 23, 2275-2281.
275 17 Ball, W.J., Jr. (1986) Biochemistry 25, 7155-7162. 18 Satoh, K., Nakao, T., Nagai, F., Kano, I., Nakagawa, A., Ushiyama, K., Urayama, O., Hara, Y. and Nakao, M. (1989) Biochim. Biophys. Acta 994, 104-113. 19 Farley, R.A., Ochoa, G.T. and Kudrow, A. (1986) Am. J. Physiol. 250, 896-906. 20 Friedman, M.L. and Ball, W.J., Jr. (1987) Biophys. J. 51, 384a. 21 Urayama, O., Nagamune, H., Nakao, M., Hara, Y., Sugiyama, H., Sato, K. and Nakao, T. (1985) J. Biochem. 98, 209-217. 22 Jorgensen, P.L. (1974) Methods Enzymol. 32, 277-290. 23 Kyte, J. (1972) J. Biol. Chem. 247, 7642-7649. 24 Nicholas, R.N. (1984) Biochemistry 23, 888-898. 25 Kyte, J. (1971) J. Biol. Chem. 246, 4157-4165. 26 Fiske, C. and SubbaRow, Y.H. (1935) J. Biol. Chem. 66, 375-400. 27 Schwartz, A., Nagano, K., Nakao, M., Lindenmayer, G.E., Allen, J.C. and Matsui, H. (1971) Methods Pharmacol. 1, 361-388. 28 Nagamune, H., Urayama, O., Hara, Y. and Nakao, M. (1986) J. Biochem. 99, 1613-1624. 29 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 30 Winslow, J.W. (1981) J. Biol. Chem. 256, 9522-9531. 31 Hegyvary, C. and Jorgensen, P.L. (1981) J. Biol. Chem. 256, 6296-6303. 32 Ey, P.L., Prowse, S.J. and Jenkin, C.R. (1978) Immunochemistry 15,429-436. 33 Kyte, J. (1974) J. Biol. Chem. 249, 3652-3660. 34 Laemmli, U.K. (1970) Nature 227, 680-685. 35 Jemmerson, R. and Paterson, Y. (1986) Science 232, 1001-1004. 36 Sheshberadaran, H. and Payne, L.G. (1988) Proc. Natl. Acad. Sci. USA 85, 1-5.
37 Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354. 38 Urayama, O., Shutt, H. and Sweadner, K.J. (1989) J. Biol. Chem. 264, 8271-8280. 39 Nagamune, H., Urayama, O., Hara, Y., Ohta, F., Hirota, K., Satomi, Y., Seo, K., Fukui, K. and Nakao, M. (1989) Anal. Biochem. 180, 362-367. 40 Hayashi, Y. and Post, R.L (1980) Fed. Proc. 39, 1704. 41 Post, R.L., Hegyvary, C. and Kume, S. (1972) J. Biol. Chem. 247, 6530-6540. 42 Robinson, J.D., Levine, G.M. and Robinson, L.J. (1983) Biochim. Biophys. Acta 731, 406-414. 43 Hara, Y. and Nakao, M. (1981) J. Biochem. 90, 923-931. 44 Castro, J. and Farley, R.A. (1979) J. Biol. Chem. 254, 2221-2228. 45 Tzartos, S.J. and Lindstrom, J.M. (1980) Proc. Natl. Acad. Sci. USA 77, 755-759. 46 Atassi, M.Z. (1978) Immunochemistry 15, 909-936. 47 Benjamin, D.C., Berzofsky, J.A., East, I.J., Gurd, F.R.N., Harthum, C., Leach, S.J., MargoUash, E., Michael, J.G., Miller, A., Prager, E.M., Reichlin, M., Sercal"z, E.E., Smith-Gill, S.J., Todd, P.E. and Wilson, A.C. (1984) Annu. Rev. Immunol. 2, 67-101. 48 Hamada, H. and Tsuruo, T. (1987) Anal. Biochem. 160, 483-488. 49 Karlish, S.J.D., Rephaeli, A. and Stein, W.D. (1985) in The Sodium Pump (Glynn, I.M. and Ellory, J.C., eds.), pp. 487-499, Company of Biologists, Cambridge. 50 Urayama, O., Nagamune, H. and Nakao, M. (1988) in Proc. of Yamada Conference XI on Energy Transduction in ATPases (Mukohata, Y., Morales, M.F. and Fleischer, S. eds.), pp. 441-442 (abstr.), Yamada Science Foundation, Osaka.
267
Elsevier BBAPRO 33727
A monoclonal antibody against a native conformation of the porcine renal Na+/K+-ATPase a-subunit protein Osamu
Urayama
1,2, H i d e a k i N a g a m u n e
1.., Makoto
Nakao I and Yukichi Hara 1
t Department of Biochemistry, Tokyo Medical and Dental University School of Medicine, Bunkyo, Tokyo (Japan) and : Department of Chemistry, University of California at San Diego, La Jolla, CA (U.S.A.)
(Received 20 November 1989) (Revised manuscript received 7 March 1990)
Key words: Enzymeconformation; Monoclonal antibody; ATPase, Na+/K +-
A monoclonal antibody (mAb50c) against the native porcine renal Na+/K+-transporting adenosinetriphosphatase (EC 3.6.1.37, ATP phosphohydrolase) (Na+/K+-ATPase) was characterized. The antibody could be classified as a conformation-dependent antibody, since it did not bind to N a + / K +-ATPase denatured by detergent and its binding was affected by the normal conformational changes of the enzyme induced by ligands. The binding was the greatest in the presence of Na +, ATP or Mg 2+ (El form), slightly less in the presence of K ÷ (E2K form) and the least when the enzyme was phosphorylated, especially in the actively hydrolyzing form in the presence of Na +, Mg 2+ and ATP. The antibody inhibited both the Na÷,K ÷-ATPase activity and the K ÷-dependent p-nitrophenylphosphatase activity by 25%, but it had no effect on Na÷-dependent ATPase activity. The antibody partially inhibited the fluorescence changes of the enzyme labeled with 5'-isothiocyanatofluorescein after the addition of orthophosphate and Mg 2+, and after the addition of ouabain. Proteolytic studies suggest that a part of the epitope is located on the cytoplasmic surface of the N-terminal half of the a-subunit.
Introduction Na+/K+-transporting adenosinetriphosphatase ( N a + / K + - A T P a s e ) , which is a membrane-embedded protein, is primarily responsible for the active transport of N a + and K + in animal cells. It consists of two folded polypeptides, a catalytic subunit ( a ) and a glycosylated subunit (/3). The et subunit contains the A T P binding, ouabain binding and phosphorylation sites. The enzyme is phosphorylated on the a subunit by Mg2+-ATP in the presence of N a + or by orthophos-
* Present address: Department of Oral Microbiology and Immunology, University of Tokushima, Tokushima, Japan. Abbreviations: Na+/K+-ATPase, Na+,K+-transporting ATPase (EC 3.6.1.37, ATP phosphohydrolase); ATPase, adenosinetriphosphatase; K+-p-NPPase, K+-dependent p-nitrophenylphosphatase; TBS, 154 mM NaCI/20 mM Tris-HC1 (pH 7.5); FITC, 5'-isothiocyanatofluorescein; SDS, sodium dodecyl sulfate; SET, 0.32 M sucrose/1 mM EDTA/20 mM Tris-HCl (pH 7.5); SDS-PAGE, electrophoresis on polyacrylamidegels poured in a solution of SDS. Correspondence: O. Urayama, Department of Biochemistry, Tokyo Medical and Dental University School of Medicine, Yushima 1-5-45, Bunkyo-ku, Tokyo 113, Japan.
phate in the presence of Mg 2+, and the phosphoenzyme is dephosphorylated in the presence of K +. The phosphorylation promotes the binding of ouabain. The structure responsible for these catalytic functions is the a subunit. There are at least four conformations of the enzyme, two conformations of the unphosphorylated enzyme and two of the phosphorylated enzyme. The conformation designated E1 is the unphosphorylated form binding N a + as a substrate of transport and E1P is the phosphorylated form that has occluded N a +. The conformation designated E2P is the phosphorylated form binding K + as a substrate and E2 is the unphosphorylated form that has occluded K +. These occluded ions are transported to the opposite side of the membrane. The normal sequence of events is for the enzyme to pass from E1 to E1P to E2P and to E2 during turnover and these transitions are believed to be coupled to cation transfer across the membrane (reviewed in Refs. 1-3). These four conformations differ in their affinity for nucleotides, binding of cardiotonic steroids, susceptibility to proteolytic enzymes, intrinsic fluorescence and response to N a + and K +. Models of the transmembrane organization of the a subunit have recently been proposed, based on its amino acid sequence [4-6]. The importance of the N-terminal
0167-4838/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
268 half of the a subunit for the transition between E2 and E1 has been demonstrated [7-9]. The first 90 amino acids from the N-terminus, which are located on the cytoplasmic side of the membrane [10-12], have a high frequency of hydrophilic, especially basic, amino acids [4,13]. Protonation of the enzyme facilitates the transition from the E1 to the E2 conformation [14], and the removal of 30 residues from the N-terminus by tryptic cleavage causes an increase in the affinity of the enzyme for Na ÷ at pH 6 [9]. Chymotrypsin cleaves a cytoplasmic segment of the polypeptide between residues 150 and 280 in the sequence of the a subunit, and this also shifts the equilibrium toward the E1 form [8]. Conformational changes of Na+/K+-ATPase can be induced by specific antibodies [15-18]. Some monoclonal antibodies have been shown to affect the transition between the E1 and the E2 forms of the enzyme. Antibody 9-A5 [15], which can bind to a denatured chymotryptic fragment containing residues 200 to 430 and including the phosphorylation site [19], stabilizes the E1 conformation [20]. On the other hand, antibody M45-80 [18] stabilizes the E2K conformation, probably by binding to the sequences in the N-terminal half of the a subunit on the extracellular surface of the enzyme. In this paper, we characterize one of the murine monoclonal antibodies mAb50c that was obtained by immunization and screening with a native porcine renal Na÷/K+-ATPase [21]. The antibody can detect ligandinduced conformational changes of the enzyme, and its binding affects the transition between enzyme conformations. A part of the epitope whose structure is altered during these conformational changes may be located in the N-terminal half of the amino acid sequence of the a subunit. A portion of this work was presented at the Tonomura Memorial Synposium of the Yamada Conference XI [50]. Materials and Methods
Materials All reagents were of reagent grade. Bio-Gel A5m was purchased from Bio-Rad. Protein A-Sepharose CL-4B was purchased from Pharmacia. Trypsin-TPCK was from Worthington. Soybean trypsin inhibitor, a-chymotrypsin, phosphoenolpyruvate, /3-NADH, pyruvate kinase, lactic dehydrogenase, Tris salt of p-nitrophenyl phosphate, fluorescein 5'-isothiocyanate, prestained SDS-PAGE molecular weight marker proteins and anti-rabbit IgG alkaline phosphatase conjugate were obtained from Sigma. Mouse myeloma protein MOPC21 IgG1 was purchased from Litton Bionetics. Clear Blot Membrane-P hydrophobic membrane was purchased from Atto.
Preparation of Na +/ K +-A TPase and its polypeptides Na+/K+-ATPase was purified from porcine renal outer medulla essentially according to Jergensen [22].
The a and fl polypeptides were separated chromatographically on a column (0.3 × 86 cm) of Bio-Gel A5m equilibrated with a solution of sodium dodecyl sulfate (SDS) [23,24]. The SDS was removed from the isolated polypeptides by the method described by Nicholas [24]. This produces fully soluble proteins dissolved in aqueous buffer [23].
Determination of Na +/ K +-A TPase activity and protein The enzymatic activity of Na+/K+-ATPase was measured by using one of two assay methods. One was an assay in which orthophosphate liberated by the hydrolysis of ATP was determined [21,25] by the method of Fiske and SubbaRow [26]. In the other method, the oxidation of NADH was coupled to the hydrolysis of ATP using pyruvate kinase and lactic dehydrogenase and the loss of NADH was monitored continuously by measuring the absorbance at 340 nm [27] in a spectrophotometer equipped with a thermally controlled cell holder maintained at 37°C. The standard mixture for the Na+/K+-ATPase assay contained 140 mM NaC1, 10 mM KC1, 5 mM MgC12, 0.5 mM EDTA, 3 mM ATP and 50 mM Tris-HC1 (pH 7.5) with or without 0.2 mM ouabain. The activity of Na+-dependent ATPase was assayed by using the above assay mixture except that KC1 was omitted. The activity of K+-dependent pnitrophenylphosphatase (K+-p-NPPase) was measured by our method [28]. The assay mixture contained 10 mM Tris salt of p-nitrophenyl phosphate, 10 mM KC1, 5 mM MgC12, 0.5 mM EDTA and 20 mM imidazolium (pH 7.5). Protein was determined by the method of Lowry et al. [29] using BSA as a standard.
Effect of antibody on Na + / K +-A TPase activity Porcine renal Na+/K+-ATPase (10 ~g) was incubated with the purified antibody (30/~g) in 100/~1 of 154 mM NaC1 and 20 mM Tris-HC1, pH 7.5 (TBS) for 45 min at room temperature. In a competition experiment, the antibody (30 #g in 80/~1) was preincubated at 4°C overnight with Na÷/K+-ATPase that had been inactivated (0 /~mol Pi/mg per h) with N-ethylmaleimide [30], and thereafter it was mixed with unalkylated Na+/K+-ATPase (10/xg in 20/~l). A portion (20 #1) of the mixture was transferred to 1 ml of the coupled assay medium [27], which had been warmed to 37°C, for Na+/K+-ATPase assay. The reaction reached a steadystate rate within 1 min of its initiation and proceeded at a constant rate for at least 4 rain either in the presence or in the absence of the antibody.
Na +/ K +-A TPase labeled with 5'-isothiocyanatofluorescein Renal enzyme was labeled with 5'-isothiocyanatofluorescein (FITC) according to Hegyvary and Jorgensen [31]. The ratio of incorporated FITC for each a subunit was estimated to be 1.1-1.6, as described before [28].
269 Fluorescence of the enzyme (excitation at 496 nm and emission at 520 nm) was measured with a Hitachi 850 fluorescence spectrophotometer.
Hybridoma production and antibody purification Porcine renal Na+/K÷-ATPase, with a specific activity of 1600 ttmol Pi/mg per h at 37°C, was injected into mice, and sera were screened for specific antibodies by assessing binding to the holoenzyme followed by an assay for Na÷/K÷-ATPase as described before [21]. The monoclonal antibody mAb50c was an IgG 1 immunoglobulin. The monoclonal IgG 1 immunoglobulin mAb38 also raised against the native porcine renal Na+/K+-ATPase was used as a control in some experiments. Monoclonal immunoglobulins were purified from the ascites fluid of pristine-primed mice inoculated with cells of the hybridoma 50c by affinity chromatography on a column of Protein A-Sepharose [32].
Binding of antibody Ligand-dependent binding of antibody to renal Na÷/K÷-ATPase was assessed as described before [21,33] with some modifications. A sample of the enzyme (60 gg in 300 gl) was incubated with mAb50c (100 ttg) in 10 mM Tris-HC1 (pH 7.5) containing either 140 mM NaC1; 140 mM KC1; 140 mM choline chloride; 3 mM ATP; 3 mM MgCI2; 3 mM MgC12 and 3 mM orthophosphate; 140 mM NaC1, 3 mM MgC12, and 3 mM ATP; or 140 mM NaC1, 3 mM MgC12, 3 mM ATP, and 1 mM ouabain for 60 min at 23°C. The amount of antibody chosen was a saturating concentration in the presence of Na ÷. The enzyme-antibody complex was pelleted by centrifugation (100000 rpm, 5 min) in a Beckman TL-100 centrifuge, washed twice with the respective solution of buffer and ligands, and dissolved in SDS. A portion of the solution of polypeptides (20 /tg of Na+/K÷-ATPase) was heated at 70°C for 5 rain and applied to a discontinuous polyacrylamide gel system [34] with 8% acrylamide separating gel. After electrophoresis, the gel was stained with Coomassie brilliant blue and scanned. The stoichiometry of binding (mol of IgG per mol of aft protomer) was calculated from the ratio between the area of the peak of the light chain (M r = 23 000) of the immunoglobulin G and the area of the peak of the a polypeptide ( M r = l l 0 0 0 0 ) of Na+/K÷-ATPase. Calibration curves for the extinction coefficients of complexes between protein and dye were prepared before estimation. The total absorbance of the a polypeptide and that of the light chain complexed to the dye were proportional to the amount of protein added to the gel over the range of 5-20 /~g for Na+/K+-ATPase and the range of 2-10 #g for the immunoglobulin.
Proteolytic digestion Selective tryptic cleavage of the a subunit of renal Na÷/K÷-ATPase was carried out as described by Far-
ley et al. [19]. The epitope of mAb50c on the a subunit was investigated by submitting the antigen-antibody complex to proteolysis [35,36]. Porcine renal N a + / K ÷ATPase (99/xg) was preincubated alone or with mAb50c (200/xg) or with mAb38 (200 ttg) in 100/~1 of TBS at 4°C overnight. The mixture was centrifuged (Beckman TL-100) at 100000 rpm for 5 min. The pellet was washed and then suspended in a minimum vol. of 0.32 M sucrose, 1 mM EDTA and 20 mM Tris-HC1 (pH 7.5) (SET). The suspension was divided into three portions. The first portion was subjected to tryptic digestion, the second was subjected to chymotryptic digestion, and the third was subjected to electrophoresis to quantify formation of the complex. Tryptic digestion was carried out as follows. A complex between Na+/K+-ATPase (30 ttg) and antibody, suspended in SET containing 15 mM KC1, was incubated with 0.3 ttg of trypsin at 37 o C. Aliquots were removed and quenched with soybean trypsin inhibitor at the indicated times and dissolved for electrophoresis as described [34] but with the addition of saturated phenylmethylsulfonyl fluoride and 2-mercaptoethanol to 1%. The samples were heated to 70°C for 5 min and applied to the discontinuous gel system with a 10% separating gel. After electrophoresis, proteins were transferred [37] to hydrolqhobic membranes, and the membranes were immunostained with anti-porcine renal Na÷/K+-ATPase rabbit serum (Antibody PK) as described before [38]. For chymotryptic cleavage, the complex between Na+/K÷-ATPase (30 /~g in 125#1) and antibody was digested with chymotrypsin (1.5 ttg) in the presence of 15 mM Tris-HC1 (pH 7.5) and 10 mM NaC1 at 37°C. Aliquots were quenched in 100 mM NaC1 [8] at the indicated times and subjected to SDS-PAGE followed by the above immunoblotting. Results
Binding of antibody The monoclonal antibody was shown to recognize native Na+/K+-ATPase by an enzyme-linked immunoadsorbance assay. In this procedure, wheat germ agglutinin associated with native Na÷/K÷-ATPase was chemically immobilized on microtiter plates as the antigen [39]. The half-value of the maximum binding of mAb50c to a microtiter well coated in this way was 130 # g / m l (0.8 /tM using M r = 162000). In solution, mAb50c and mAb38 with higher affinity [21,39] showed similar titration for native Na÷/K+-ATPase (not shown). In contrast, the antibody did not bind to any polypeptide of the Na÷/K÷-ATPase in Western blot analysis. Even a concentrated solution of mAb50c (40 ttl of the ascites fluid in 8 ml of TBS) did not stain either a or B polypeptide after their separation from the native
270 TABLE I Ligand-dependent binding of mA b50c to Na +/K +-A TPase a
Antibodies Ligands mAb50c
mAb38
Na + K+ ATP Mg1+ Mgz+ + orthophosphate Na ÷ +Mg 2+ +ATP Na + +Mg 2+ +ATP + ouabain Na +
IgG/afl (mol/mol) Expt. b: 1 2 3 0.80 0.74 0.65 0.72 0.25 0.25
0.78 0.63 0.80 0.52 0.33
0.49 0.37 -
0.85 c
a Enzyme (final concentration 0.2 mg/ml) was mixed with appropriate solutions of the ligands. Monoclonal antibody (final concentration 0.34 mg/ml) was then added. After 1 h at 23°C, binding was assessed by electrophoresis. b Different preparations of Na+/K+-ATPase were used in Expts. 1, 2 and 3, respectively. c This has been shown to be ligand-independent binding [21].
enzyme (16 ttg) by SDS-PAGE followed by electrophoretic transfer to nitrocellulose paper. Moreover, neither the isolated a polypeptide nor the isolated fl polypeptide, after being stripped of their SDS, competed with the native renal enzyme for the antibody binding. The isolated a polypeptide (42 /~g) or the isolated fl polypeptide (30 #g) was mixed with mAb50c (30 /zg) and the mixture was incubated with native enzyme (10 ~tg in 100/~1) for a competition assay (see the legend to Fig. 1), but neither of the isolated polypeptides had any effect on the inhibition of the N a + / K + - A T P a s e activity by the antibody. These resuits suggest that the binding of mAb50c requires the antigen to be in its native conformation. The binding of mAb50c to native N a + / K + - A T P a s e was measured by centrifugation of antigen-antibody complexes followed by analysis of their composition by SDS-PAGE (Table I). The level of binding (IgG per ctfl-protomer) was calculated to be 0.80 in the presence of Na ÷ (E1Na), 0.80 in the presence of Mg 2÷ (E1Mg), 0.72 in the presence of ATP (E1ATP), 0.65 in the presence of K ÷ (E2K), 0.52 in the presence of Mg 2÷ and orthophosphate, 0.25 in the presence of N a ÷, Mg 2÷ and ATP (E2P), and 0.49 in the presence of N a ÷, Mg 2÷, ATP and ouabain (complex between ouabain and E2P). Choline chloride as a control of ionic strength gave the value of 0.68. The highest binding was obtained with E1 and the least binding with E2P, especially the actively hydrolyzing enzyme form. Binding numbers were slightly different with different enzyme preparations. In addition, the stoichiometry never reached unity. This may be due to an overestimation of the enzyme protein that can react with the antibody. It is known that N a + / K + - A T P a s e purified by extraction with SDS con-
tains a small amount of denatured enzyme [40]. If we could calculate accurately the amount of the active enzyme by subtraction of the amount of the inactive enzyme in the preparation of N a + / K + - A T P a s e , higher binding numbers should be obtained. Effect on N a + / K +-A TPase functions A partial inhibition (25%) of the activity of N a + / K + - A T P a s e by the antibody was observed when activity was assayed with a timed test tube assay. Because the binding of antibody depended on the ligands present (Table I), the time-course of the inhibition was examined by means of the coupled assay (Fig. 1). Both in the presence and in the absence of the antibody, the enzymatic reaction proceeded linearly after a short lag. The maximum inhibition (25.9 + 2.4 (mean + S.D.) % for three experiments) was observed with 3-fold weight excess of antibody over enzyme. Mouse myeloma IgG1 (another control as well as the no-antibody control) had no effect on the N a + / K + - A T P a s e activity. N-Ethylmaleimide-inactivated N a + / K + - A T P a s e [30] partially reversed the inhibition caused by the antibody by competition (Fig. 1). Ouabain inhibited the renal N a + / K ÷ATPase when it was in a complex with the antibody. The half-value for the maximum inhibition by ouabain was estimated to be 30 nM, which was identical to that for the uncomplexed native enzyme.
-
-
v
i
i
0.2
o
':~,,
o.~
~
,>.,, "%x. x
0.6
:,,
I
',>\ \
'~' ',
0.8 1
0
__
i
- - ~
1 2 Reaction tirne (rnin)
\
'l ~,
3
Fig. 1. Effect of mAb50c on Na+/K+-ATPase activity. Renal Na+/K+-ATPase (10 /~g in 100 /d) was incubated with 30 /~g of
mAb50c ( - - - - - ) or with no antibody ( ). In a competition experiment, fully active enzyme(10/~g in 100/~1) was incubated with the antibody (30/ill) that had been preincubated with N-ethylmaleimide-inactivated enzyme (15 /zg, •. . . . or both 30 and 60 /~g, . . . . . . . . ). Portions of the incubation mixtures were transferred to the coupled assay medium for Na+/K+-ATPase activity to initiate the reaction. The oxidation of NADH coupled to the hydrolysis of ATP was monitored by measuring the absorbance at 340 nm.
271 i
100
t
i
o
'~-'0~
_._>,
A w
0.6
Q.
i
/
50
o
I
I
5 10 Antibody (IJg)
I
I
20
Fig. 2. Effect of mAb50c on K÷-p-NPPase as a function of antibody concentration. Renal Na+/K+-ATPase (1 /~g in 500 btl) was preincubated with mAb50c(t) or mAb38 (o) as a control in the amounts indicated on the abscissa and then assayed by adding the reaction medium for the K+-p-NPPase assay. The remaining activity is expressed as a percentage of that without antibodies. The fact that mAb50c was poorly bound to the enzyme in the presence of N a ÷, Mg 2+ and A T P (Table I), but partially inhibited N a + / K + - A T P a s e activity (Fig. 1), suggested that the antibody might inhibit the enzyme by affecting not an Na÷-dependent step but a K÷-de pendent step during its turnover. Na+,K÷-transporting ATPase has a p-NPPase activity in the presence of K÷; the K÷-bound unphosphorylated enzyme is probably responsible for the hydrolysis [41,42]. The effect of the antibody on the K+-p-NPPase was examined as a function of its concentration (Fig. 2). The antibody inhibited the activity by 22-25% at 10-fold weight excess by decreasing both the K m value for p-NPP from 1.3 to 1.1 mM and the Vm~, from 3.57 #mol p - N P / m g per min to 2.78 /xmol p - N P / m g per min (Fig. 3). The inhibition was of an intermediate type between noncompetitive and uncompetitive. The antibody increased the K m for K ÷ from 0.8 mM with a Hill coefficient of 1.7 to 1.2 mM with a Hill coefficient of 1.9 (Fig. 4). The K m values for K ÷ were identical in another experiment. In contrast, the antibody had no effect on the N a ÷dependent ATPase either with 140 mM N a ÷ or with 700 mM N a ÷ (5 mg mAb50c per mg N a ÷ / K + - A T P a s e ) , which suggests poor binding of the antibody to the actively hydrolyzing enzyme form. Two N a ÷ concentrations were tested, since the level and decay of the phosphoenzymes of the Na÷-dependent ATPase [41] depend on N a + concentration [43]. In order to observe the effect of the antibody on the ligand-induced conformational changes of the enzyme directly, N a + / K ÷ - A T P a s e was labeled with FITC. In the presence of Mg 2÷, orthophosphate quenches F I T C fluorescence while phosphorylating the enzyme [31]. When the fluoresceinyl enzyme was complexed with
0.5 1.o 1.5 1/[p-NPP](mM) Fig. 3. Effect of the concentrationof p-NPP on K+-p-NPPaseactivity in the presenceof mAb50c. Renal Na+/K+-ATPase (1/~g in 500/~1) was preincubated with 10/tg of mAb50c (t) or without any antibody (o) in the medium for the K+-p-NPPase assay at 10 mM KC1. The reaction was initiated by adding p-NPP (0.66-10 mM) to the mixture. The initial velocities as a function of concentration are presented in the form of a Lineweaver-Burkplot.
mAb50c, the fluorescence change after the addition of orthophosphate and Mg 2÷ was inhibited compared to that of the enzyme complexed with mAb38 (Fig. 5A). The quenching with mAb38 was almost the same as that with the enzyme unbound by antibodies. The fluorescence change after adding these ligands was 35% less than that of the control at higher concentrations of mAb50c (Fig. 5B). Addition of ouabain to the phosphoenzyme caused a large drop in fluorescence resulting from the formation of the phosphoenzyme-ouabain complex. The quenching of the enzyme bound to mAb50c by binding of ouabain was slightly slower than
0.4
'
0
X
E
o
.J
- 0.4
I
-0.4
I
___
I
t
0 Log[KCI] (raM)
i
0.4
Fig. 4. Effect of the concentration of KC! on K+-p-NPPase in the presence of mAb50c. Renal Na+/K+-ATPase (1 rig in 500 btl) was preincubated with 10/tg of mAb50c(IP)or without any antibodies (o) in the reaction medium for the K+-p-NPPase assay at various concentrations of KCI (0.67-6 mM). The reaction was initiated by adding p-NPP to the mixture. The data are presented as a Hill plot.
272 A
Mg
Pi o
Ou
. ~11
mAb38 7
§
~ so
mAb50c
";" 100,
1 ,"""'
01, 1rain
i
i
1
10
J
100 1 00
Antibody(ug )
Fig. 5. Effect of mAb50c on the change in fluorescence caused by orthophosphate in the presence of MgC12. Renal Na+/K+-ATPase labeled with FITC (30/~g in 100/~1) was preincubated with the particular antibody or without any antibody in 50 mM Tris-HC1 (pH 7.5) and 0.1 mM EDTA. A portion of the mixture was transferred to 1 ml of the Tris buffer and the observation was initiated by adding MgC12 (Mg) and then orthophosphate (Pi) to final concentrations of 2 mM. Ouabain (Ou) was then added to a final concentration of 100 /~M. (A) Changes of the intensity of the fluorescence (from left to fight). (B) The fluorescence change after the addition of orthophosphate as a percentage of that in the absence of antibodies is plotted against the concentration of mAb50c (O) or mAb38 (o).
that of enzyme bound to mAb38 (Fig. 5A). When ouabain binding was examined in the presence of Mg 2÷, it was found that mAb50c reduced the rate of quenching caused by ouabain binding by 50% (Fig. 6). The maximum guenching after the further addition of orthophosphate, however, resulting from the formation of the phosphoenzyme-ouabain complex in the presence of mAb50c was similar to that in the presence of mAb38 or in the absence of antibodies (Fig. 5A). By shifting the enzyme into the E2 form, K ÷ quenched the FITC fluorescence on the enzyme in the presence of mAb50c i
100 80
-~ 60
!,o
2o tL
20
0
I
I
2
I
I
i
4
i
6
Time after addition of ouabain (rain) Fig. 6. Time course of the quenching of fluorescence by ouabain. Renal Na+/K+-ATPase labeled with FITC (30 /~g in 100 #1) was preincubated either with mAb50c (3/~g, × ; 30 #g, zx; 300/~g, &) or mAb38 (300 #g, D) or without antibodies (o). An equal portion of each mixture was transferred to 1 ml of the medium used to follow change in fluorescence: 2 mM MgC12, 0.1 mM EDTA and 50 mM Tris-HC1 (pH 7.5). The reaction was initiated by adding ouabain to 100 /xM arid the quenching was monitored with time. The fluorescence change is expressed as a percentage of the difference in fluorescence between the initial level (conformation EIMg 2+ ) and the level that was obtained by the further addition of orthophosphate to give ouabain-bound E2P.
or in the presence of mAb38 (the concentration giving half-maximal change was 0.07 mM in each case).
Localization of epitope The effects of mAb50c on the catalytic rates and equilibria of Na+/K+-ATPase suggested that the antibody recognized the a subunit. We have previously shown that mAb50c was raised against a cytoplasmic site of Na÷/K+-ATPase [21]. Segments of the folded a polypeptide that are on the cytoplasmic surface of the subunit were investigated as candidates for the epitope by using ligand-dependent proteolysis. In the presence of K ÷, trypsin initially cleaves a peptide bond at the center of the a polypeptide in the cytoplasmic portion of the ct subunit followed by a secondary cleavage in the N-terminal region [9,44]. Chymotrypsin cleaves the a polypeptide between transmembrane segments M2 and M3 in the cytoplasmic portion of the a subunit in the presence of low Na ÷ [8,9]. The binding of mAb50c to various types of digested enzyme was analyzed by centrifugation and SDS-PAGE (Fig. 7). The binding of mAb50c decreased with the decrease of the intact a polypeptide after tryptic digestion (Fig. 7A). During the digestion the fl polypeptide was not lost, because its electrophoretic mobility was unchanged when it was identified by immunostaining. Selective chymotryptic cleavage of the a subunit also reduced the binding of antibody (Fig. 7B). These results confirm that the epitope is located in the a subunit. Epitopes have also been studied by examining the ability of antibodies to protect one region of the antigen from proteolysis [35,36]. To characterize further the epitope of mAb50c on the a subunit, the complex between Na+/K+-ATPase and mAb50c was treated with trypsin or chymotrypsin followed by immunoblotting with anti-porcine-renal Na÷/K+-ATPase rab-
273 bit serum (Fig. 8). Trypsin produced the N-terminal fragment of M r = 42 000 early in the digestion (2.5 min in both Fig. 8A and B) in the presence of mAb50c as well as in the presence of mAb38 as a control. This N-terminal fragment in the control complex was further digested progressively to several subfragments with M r = 41000-37000 (highlighted in Fig. 8B at 10 rain), in agreement with previous observations [12,19,44], but mAb50c clearly decreased the rate of the further fragmentation of the N-terminal fragment (compare 10 rain between A and B, and 40 rnin between A and B, respectively, in Fig. 8). Larger subfragments of the N-terminal fragment persisted in the presence of mAb50c than in the presence of mAb38. The behavior of the complex between Na+/K÷-ATPase and mAb38 was the same as that of uncomplexed Na+/K÷-ATPase, another control. On the other hand, there was no difference between the complex of mAb50c and the enzyme and the complex of mAb38 and the enzyme or between the complex of mAb50c and the enzyme and
A
I
2
B
I
2
-Fc
-F m
A 0
2.5
B I0
40
Mr
0
2.5
I0
40
Fig. 8. Tryptic digestion of the complex between Na*/K*-ATPase and monocional antibodies. The complex between the enzyme and mAb50c (A) or the complex between the enzyme and mAb38 (B) was treated with trypsin for the indicated number of minutes. Samples of the digested enzyme were submitted to electrophoresis in SDS and transferred to a sheet of hydrophobic membrane. The blot was incubated with Antibody PK and the bound antibodies were detected with goat anti-rabbit immunoglobulin conjugated to alkaline phosphatase. The polypeptides were labeled as follows: a, a subunit; fl, fl subunit; N, N-terminal fragment; Ns, subfragments from the Nterminal fragment. The lane designated M r contained prestained molecular weight markers of 180000, 116000, 84000, 58000, 48500, 36500 and 26600.
the enzyme alone in the rate of production of the primary product (M r = 78000) [8] from the a subunit during chymotryptic cleavage. The protection of the N-terminal fragment from later digestion by mAb50c could be due to a direct steric effect of the bound antibody or to a structural change of the region of the a subunit surrounding the site of cleavage after the binding of the antibody.
Discussion
Fig. 7. Binding of mAb50c to Na+/K÷-ATPase cleaved with proteolytic enzymes. Renal Na÷/K+-ATPase (30 /~g) was treated with 0.3 /~g of trypsin in 20 #1 containing 15 mM KC1 (A) or with 1.5 #g of chymotrypsin in 150/xl containing 10 mM NaC! (B), respectively, for 30 min at 37°C. After quenching of each reaction, the cleaved enzyme was washed with TBS and incubated with 2-fold weight excess of mAb50c at 4°C overnight. The complex of antigen and antibody was collected by centrifugation and then analyzed by SDS-PAGE (7.5% gel for A and 10% gel for B). Lane 1: binding to the uncleaved enzyme. Lane 2: binding to the cleaved enzyme. The polypeptides are labeled as follows: a, a polypeptide; fl, fl polypeptide; H, heavy chain (a doublet) of mAb50c; L, light chain; Ft, tryptic fragments from a subunit; Fc, a chymotryptic fragment from a subunit.
As specific probes for Na+/K+-ATPase, we prepared mouse monoclonal antibodies by using porcine renal Na+/K+-ATPase with high specific activity as an antigen. Most of the monoclonal antibodies obtained bound native Na+/K÷-ATPase, but not its constituent polypeptides, denatured with SDS. On this basis, these antibodies might be classified as conformation-dependent antibodies [45]. In conformation-dependent epitopes, discontinuous determinants [reviewed in Refs. 46, 47], which may be composed of residues far apart in the primary structure, are brought together on the surface of the protein by the folding of the native structure. The identification of such separate determinants has sometimes been made from binding studies between the
274 antibody and either peptide fragments of the antigen or evolutionarily related proteins. Recently, both proteolysis [35,361 and cross-linking [48] of complexes between antigen and antibody have been applied to identify these types of epitopes. In these experiments, we have used ligand-dependent proteolysis to identify the epitope of mAb50c in the subunit of porcine renal Na+/K+-ATPase. The ability of the antigen to bind the monoclonal antibody was lost by selective chymotryptic cleavage between Leu-266 and Ala-267 [9] of the a polypeptide in the native enzyme (Fig. 7) as well as by tryptic digestion. The results suggest that the epitope is near this point of cleavage or is dependent on the integrity of the polypeptide in this region. During trypsinolysis of the complex between antibody and enzyme, mAb50c had the ability to protect the N-terminal fragment (Gly-1Arg-438) produced by tryptic cleavage between Arg-438 and Ala-439 [9] from further digestion (Fig. 8). In the unprotected enzyme some of this further digestion occurs at sites in this N-terminal fragment close to its N-terminus containing the site T2 between Lys-30 and Glu-31 [9,44]. It cannot be ruled out, however, that some of this further digestion also occurs at a site near the C-terminus of this N-terminal fragment in addition to those sites near its N-terminus [12,19]. Therefore, a portion of the epitope for mAb50c responsible for this protection may be located at either the N-terminus of the a polypeptide or in the sequence of the a polypeptide surrounding Arginine-438. The N-terminal regions of the a subunit are involved in the conformational changes that accompany cation binding and occlusion [4]. Tryptic cleavage at Lys-30 increases the affinity of the enzyme for Na + and chymotryptic cleavage at Leu-266 stabilizes the E1 form of the enzyme [9]. Monoclonal antibody 9-A5, the epitope of which was identified as being on the cytoplasmic surface of the enzyme in a region containing the portion of the a polypeptide between Ala-267 and Arg-438 [19], also stabilizes the E1 form of the enzyme [20]. The antibody '50c inhibited the formation of E2P from E1 in the presence of Mg 2+ (Fig. 5). The controlled proteolysis, the binding of antibody 9-A5, and the binding of mAb50c all show a similar effect on the conformational change of native Na+/K+-ATPase. On the other hand, another monoclonal antibody, M45-80, inhibited the enzymatic reactions by shifting the enzyme to the E2 form [18]. The epitope of M45-80 is thought to be located on the extracellular surface in the Nterminal half of the a subunit. It has been proposed that the formation and dissociation of hydrogen bonds [49] between the N-terminal segment of the folded a polypeptide which contains excess positive charges [4,13] and other cytoplasmic portions of the a subunit may be involved in cation translocation [9], but the spatial organization of the a polypeptide within the N-terminal
half of the a subunit remains to be elucidated. Further study of the conformationally dependent epitopes of mAb50c or monoclonal antibody M10-P5-Cll [17] might give us some information on the three-dimensional structure of the N-terminal half. The binding activity of mAb50c is ligand-dependent (Table I) and the site is exposed in the E1 form but protected in the E2P form. This property may be useful for the detection of the intactness of Na+/K÷-ATPase and for further purification of the active enzyme.
Acknowledgments We thank Dr. Jack Kyte (University of California at San Diego), in whose laboratory a part of this research was performed, for his advice and helpful comments. We also thank Dr. Hiroyuki Sugiyama (National Institute for Physiological Science, Japan) for assisting us in the production of monoclonal antibodies, and Toshiyuki Kojima (Tokyo Medical and Dental University) and Katherine Charles (University of California at San Diego) for preparation of porcine renal enzymes. This work was supported by Grants-in-Aid for Special Project Research on Bioenergetics from the Ministry of Education, Science and Culture of Japan and by Grant GM-33962 from the National Institutes of Health.
References 1 Kyte, J. (1981) Nature 292, 201-204. 2 Jorgensen, P.L. (1982) Biochim. Biophys. Acta 694, 27-68. 3 Glynn, I.M. (1985) in The Enzymes of Biological Membranes (Martonosi, A.N., ed.), Vol. 3, pp. 35-114. Plenum, New York. 4 Shull, G.E., Schwartz, A. and Lingrel, J.B. (1985) Nature 316, 691-695. 5 Kawakami, K., Noguchi, S., Noda, M., Takahashi, H., Ohta, T., Kawamura, M., Nojima, H., Nagano, K., Hirose, T., Inayama, S., Hayashida, H., Miyata, T. and Numa, S. (1985) Nature 316, 733-736. 60vchinnikov, Y.A., Modyanov, N.N., Broude, N.E., Petrukhin, K.E., Grishin, A.V., Arzamazova, N.M., Aldanova, N.A., Monastyrskaya, G.S. and Sverdlov, E.D. (1986) FEBS Lett. 201,237-245. 7 Jorgensen, P.L. and Karlish, S.J.D. (1980) Biochim. Biophys. Acta 597, 305-317. 8 Jorgensen, P.L. and Petersen, J. (1985) Biochim. Biophys. Acta 821, 319-333. 9 Jorgensen, P.L. and Collins, J.H. (1986) Biochim. Biophys. Acta 860, 570-576. 10 Jorgensen, P.L., Karfish, S.J.D. and Gitler, C. (1982) J. Biol. Chem. 257, 7435-7442. 11 Ball, W.J., Jr. and Loftice, C.D. (1987) Biochim. Biophys. Acta 916, 100-111. 12 Felsenfeld, D.P. and Sweadner, K.J. (1988) J. Biol. Chem. 263, 10932-10942. 13 Shull, G.E., Greeb, J. and Lingrel, J.B. (1986) Biochemistry 25, 8125-8132. 14 Skou, J.C. and Esmann, M. (1980) Biochim. Biophys. Acta 601, 386-402. 15 Schenk, D.B., Hubert, J.J. and Leffert, H.L. (1984) J. Biol. Chem. 259, 14941-14951. 16 Ball, W.J., Jr. (1984) Biochemistry 23, 2275-2281.
275 17 Ball, W.J., Jr. (1986) Biochemistry 25, 7155-7162. 18 Satoh, K., Nakao, T., Nagai, F., Kano, I., Nakagawa, A., Ushiyama, K., Urayama, O., Hara, Y. and Nakao, M. (1989) Biochim. Biophys. Acta 994, 104-113. 19 Farley, R.A., Ochoa, G.T. and Kudrow, A. (1986) Am. J. Physiol. 250, 896-906. 20 Friedman, M.L. and Ball, W.J., Jr. (1987) Biophys. J. 51, 384a. 21 Urayama, O., Nagamune, H., Nakao, M., Hara, Y., Sugiyama, H., Sato, K. and Nakao, T. (1985) J. Biochem. 98, 209-217. 22 Jorgensen, P.L. (1974) Methods Enzymol. 32, 277-290. 23 Kyte, J. (1972) J. Biol. Chem. 247, 7642-7649. 24 Nicholas, R.N. (1984) Biochemistry 23, 888-898. 25 Kyte, J. (1971) J. Biol. Chem. 246, 4157-4165. 26 Fiske, C. and SubbaRow, Y.H. (1935) J. Biol. Chem. 66, 375-400. 27 Schwartz, A., Nagano, K., Nakao, M., Lindenmayer, G.E., Allen, J.C. and Matsui, H. (1971) Methods Pharmacol. 1, 361-388. 28 Nagamune, H., Urayama, O., Hara, Y. and Nakao, M. (1986) J. Biochem. 99, 1613-1624. 29 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 30 Winslow, J.W. (1981) J. Biol. Chem. 256, 9522-9531. 31 Hegyvary, C. and Jorgensen, P.L. (1981) J. Biol. Chem. 256, 6296-6303. 32 Ey, P.L., Prowse, S.J. and Jenkin, C.R. (1978) Immunochemistry 15,429-436. 33 Kyte, J. (1974) J. Biol. Chem. 249, 3652-3660. 34 Laemmli, U.K. (1970) Nature 227, 680-685. 35 Jemmerson, R. and Paterson, Y. (1986) Science 232, 1001-1004. 36 Sheshberadaran, H. and Payne, L.G. (1988) Proc. Natl. Acad. Sci. USA 85, 1-5.
37 Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354. 38 Urayama, O., Shutt, H. and Sweadner, K.J. (1989) J. Biol. Chem. 264, 8271-8280. 39 Nagamune, H., Urayama, O., Hara, Y., Ohta, F., Hirota, K., Satomi, Y., Seo, K., Fukui, K. and Nakao, M. (1989) Anal. Biochem. 180, 362-367. 40 Hayashi, Y. and Post, R.L (1980) Fed. Proc. 39, 1704. 41 Post, R.L., Hegyvary, C. and Kume, S. (1972) J. Biol. Chem. 247, 6530-6540. 42 Robinson, J.D., Levine, G.M. and Robinson, L.J. (1983) Biochim. Biophys. Acta 731, 406-414. 43 Hara, Y. and Nakao, M. (1981) J. Biochem. 90, 923-931. 44 Castro, J. and Farley, R.A. (1979) J. Biol. Chem. 254, 2221-2228. 45 Tzartos, S.J. and Lindstrom, J.M. (1980) Proc. Natl. Acad. Sci. USA 77, 755-759. 46 Atassi, M.Z. (1978) Immunochemistry 15, 909-936. 47 Benjamin, D.C., Berzofsky, J.A., East, I.J., Gurd, F.R.N., Harthum, C., Leach, S.J., MargoUash, E., Michael, J.G., Miller, A., Prager, E.M., Reichlin, M., Sercal"z, E.E., Smith-Gill, S.J., Todd, P.E. and Wilson, A.C. (1984) Annu. Rev. Immunol. 2, 67-101. 48 Hamada, H. and Tsuruo, T. (1987) Anal. Biochem. 160, 483-488. 49 Karlish, S.J.D., Rephaeli, A. and Stein, W.D. (1985) in The Sodium Pump (Glynn, I.M. and Ellory, J.C., eds.), pp. 487-499, Company of Biologists, Cambridge. 50 Urayama, O., Nagamune, H. and Nakao, M. (1988) in Proc. of Yamada Conference XI on Energy Transduction in ATPases (Mukohata, Y., Morales, M.F. and Fleischer, S. eds.), pp. 441-442 (abstr.), Yamada Science Foundation, Osaka.
