Chemical Modification of Membrane Proteins in Relation to Inhibition of Anion Exchange in Human Red Blood Cells L. ZAKI, H. FASOLD, B. SCHUHMANN AND H. PASSOW Max-Planck-Institut fiir Biophysik and Institut fiir Biochemie der Universitat, Frankfurt/Main

ABSTRACT Mono-, di-, and trisulfonic acids, including 4,4’-diacetamido stdbene-2,2’-disulfonic acid (DAS) and 2-( 4’-amino phenyl)-6-methylbenzene thiazol-3’,7-disulfonic acid ( APMB ) produce a reversible inhibition of sulfate equilibrium exchange in human red cells. A study of the sidedness of the action of a number of these sulfonic acids in red cell ghosts revealed that some, like DAS, inhibit only a t the outer membrane surface while others, like APMB, inhibit at either surface. This finding suggests that at least two different types of membrane sites are involved in the control of anion permeability. The nature of the anion permeability controlling sites in the outer cell surface was investigated by studying the effects of DAS on the inhibition by dinitrofluorobenzene (DNFB) of anion equilibrium exchange and on the binding of DNFB to the proteins of the red blood cell membrane. After exposure to DNFB in the presence of DAS for a certain period of time, there was a reduction of both the inhibitory effect of DNFB on sulfate exchange and the binding of DNFB to the protein in band 3 of SDS polyacrylamide gel electropherograms (nomenclature of Steck, J. Cell. Biol., 62: 1, ’74). Since binding to other membrane proteins was not affected, this observation supports the assumption that the protein in band 3 plays some role in anion transport. In accordance with the absence of an inhibitory effect at the inner membrane surface, internal DAS does not affect DNFB binding to the protein in band 3. DAS protected the anion exchange system not only against inhibition by DNFB but also by m-isothiocyanato benzene sulfonic acid. In contrast to DAS, the equally inhibitory phlorizin does not reduce the rate of dinitrophenylation of the protein in band 3. This suggests that either not all inhibitors of anion exchange exert their action by a combination with sites on the protein in band 3 or that in spite of the described evidence this protein is not involved in the control of anion movements. The effect of the irreversibly binding inhibitor 4-acetamido-4’-isothioc~anatostilbene-2,2’-disulfonicacid (SITS) on DNFB binding to the protein in band 3 was studied in an attempt to differentiate DNFB binding related to inhibition of anion permeability from DNFB binding which is not involved. At least three distinguishable populations of DNFB binding sites were found: ( 1 ) binding sites common for DNFB and SITS which are probably related to inhibition, ( 2 ) other common sites which are not related to inhibition and ( 3 ) different sites whose dinitrophenylation is not affected by SITS. The number of sites in population ( 1 ) was estimated to be 0.8-1.2 . 106/cell. A study of the concentration dependence of the inhibition of anion equilibrium exchange with 4,4’-isothiocyanato2,2’-stilbene disulfonic acid (DIDS) and APMB further suggests that among the sites in population (1) a major fraction is susceptible to modification by APMB and DIDS while the rest is only susceptible to DIDS. It remains undecided whether these differences of susceptibility reflect differences of accessibility or reactivity. Received Oct. 15, ’74. Accepted Mar. 21, ’75.

J. CELL. PHYSIOL.,86: 471-494.

471

472

L. ZAKI, H . FASOLD, B. SCHUHMANN AND H . PASSOW

The obvious procedure for the identification of sites which participate in the control of anion exchange across the red blood cell membrane would consist of labeling with an irreversibly acting, site specific modifier of anion exchange and isolating the labeled product. The effects of most modifiers of anion exchange such as dipyridamole, dinitrophenol, salicylate, phlorizin, and its aglycone phloretin (Deuticke, '67, Schnell and Passow, '69, Gunn and Tosteson, '71, Wieth, '71, Schnell, '72) are completely reversible. Hence they are not suited for labeling of anion permeability controlling sites. However, it was found that many amino-reactive reagents, such as dinitrofluorobenzene (DNFB, Passow, '69a,b; Poensgen and Passow, '71) methoxynitrotropone (MNT, Schnell and Passow, '69), and trinitrobenzene sulfonate (TNBS, Zaki et al., 1970) are powerful and irreversibly acting inhibitors. Knauf and Rothstein ('71 ) confirmed these findings and added to the list of amino reactive inhibitors the monoisothiocyanate of a stilbene disulfonic acid (SITS)'. This compound had first been used by Maddy ('64) as a non-penetrating surface label of the red blood cell membrane. The diisothiocyanate (DIDS, Cabantchik and Rothstein, '74a,b) proved to be most effective among the irreversibly binding inhibitors of anion transfer known so f a r . Amino reactive agents are capable of interacting with amino groups in both membrane proteins and lipids. However, the action on anion permeability does not seem to be related to a modification of the lipids. This was inferred from the observation that anion permeability could be inhibited by the proteolytic enzyme pronase without measurable esterolytic effects on the principal membrane lipids (Passow, '71), and that maleylation of the red blood cell membrane inhibits anion permeability without significant labeling of the amino lipids (Obaid et al., '72). The search for the anion controlling sites in the red blood cell membrane focussed, therefore, on membrane proteins. Cabantchik and Rothstein synthesized radioactive derivatives of stilbene disulfonic acids and studied their distribution over the various membrane protein fractions by SDS polyacryl-

amide gel electrophoresis. The initially used derivative labeled a large variety of different membrane constituents (Cabantchik and Rothstein, '72). However, using a hydrogenated diisothiocyanato derivative (DIDS) they observed that binding was confined to the protein in the 95,000 Dalton range. There was no binding to membrane lipids. (Cabantchik and Rothstein, 74a,b). In our laboratory we also made use of Rothstein's and his associates' discovery of the high inhibitory power of stilbene disulfonic acids. We followed up the finding of Knauf and Rothstein ('71) that pretreatment with DNFB reduces SITS binding to whole red cells. However, we first exposed the cells to SITS and subsequently to DNFB and then showed that the reduction of DNFB binding by SITS was confined to DNFB binding sites on the protein in the 95,000 Dalton range of SDS gel electropherograms (band 3 of the nomenclature of Steck, '74). Since the 95,000 Dalton proteins were the only membrane proteins at which two different inhibitors of anion permeability affected each others binding, we concluded that these proteins are possibly involved in the control of anion permeation. This work, which had only been presented at the International Biochemistry Congress in Stockholm (Zaki and Passow, '73) and subsequent meetings (Passow et al., '75), forms the basis of the experiments reported below. We first describe the properties of the inhibitors which we used as tools in this study, then we deal with the modification of the binding sites on the protein in band 3 and the relation of these sites to anion permeability. Finally, we describe experiments on the sidedness of the action of a number of reversibly 1 Abbreviations used in the paper. APMB, 2-(4'-aminophenyl)-6-methylbenzenethiazol3',7-disulfonic acid IPMB, 2-(4'-isothiocyanopheny1)-6-methylbenzenethiazol-3',7-disulfonicacid IPT, 8-(isothiocyano)pyrene-1,3,6-trisulfonicacid DIDS, 4,4'-isothiocyano stilbene-2,2'-disulfonicacid SITS, 4-acetamido 4'-isothiocyano stilbene-2,2'disulfonic acid DAS, 4,4'-diacetamido stilbene-2,2'-disulfonicacid 12DAS, 4,4'-diacetamidodiiodo stilbene-2,2'-disulfonic acid 12DIDS, 4,4'-diisothiocyanodiiodo stilbene-2,2'disulfonic acid IBS, m-isothiocyano benzene sulfonic acid DNFB, l-fluoro-2,4-dinitrobenzene TCA, trichloro acetic acid PAS, periodic acid-Schiff's technique

CHEMICAL MODIFICATION OF MEMBRANE PROTEINS

acting inhibitors. These experiments suggest that more than one type of binding sites is involved in the control of anion transfer. MATERIALS AND METHODS

oRh+ blood from healthy donors was obtained from the blood bank of the Red Cross in Frankfurt and stored at 4°C for no longer than 2-5 days. Before use, the cells were washed thrice in isotonic saline. Chemicals : 14C DNFB (specific activity 17-18 Ci/M) was purchased from Amersham/Buchler, SITS was obtained from British Drug House. On thin layer chromatograms developed i n CHC13/CH30H/ H,O 65:25:4 at least six different spots could be observed. The commercial SITS was purified on a silica column ( 4 X 50 cm, Kieselgel Woelm 0.063-0.1 m m ) by elution with the same mixture of CHCl,/ CH30H/H20 described above and monitoring at 345 nm. The two isomers of SITS were identified by their fluorescence and then combined. IPMB and IPT were gifts from Professor Braunitzer (MPI Miinchen) and APMB from Professor Petersen (Bayer Leverkusen).

Synthetic procedures ( a ) Preparation of m-isothiocyanatobenzenesulfonic acid: 2 g of m-aminobenzenesulfonic acid were dissolved in 250 ml of water at pH 6 by addition of 4 N NaOH, and 4 ml of freshly distilled thiophosgene were added. The mixture was stirred for at least one hour, or until all of the excess thiophosgene had evaporated. After cooling, the compound was precipiated and purified as described below for diacetamido stilbene disulfonic acid. Analysis: CrH5NS2 Calculated: C = 39.03%; H = 2.32%; N = 6.52%; S = 29.76%; Found: C = 39.30%; H = 2.56% ; N = 6.65% ; S = 29.32%;

( b ) Preparation of the diiodo derivative of stilbenes : 4,4'-diamino-2,2'-stilbene disulfonic acid or the acetylated compound were iodinated by the addition in several portions of the calculated volume of a 10% iodine solution in ethanol, to a solution of 1 g of stilbene in 350 ml of water

473

at pH 6-7. The diiodo-4,4'-diacetamido2,2'-stilbene disulfonic acid was precipitated as described for the unmodified diacetamido derivative. The diamino compound was immediately converted into the diisothiocyanate derivative by adjusting the pH to 5, and adding a 100-fold molar excess of thiophosgene. After stirring for one hour, the product was again precipitated by the addition of concentrated sulfuric acid to the cooled solution. Fractionated precipitation controlled by paper electrophoresis at pH 1.9 was used to obtain the pure product. The suggested formula is presented in figure 2. Analysis: C I ~ H I O ~ ~ N Z S ~ I ~ Calculated: C = 27.12%; H = 1.41%; N = 3.95%; S = 18.15%; I = 35.87% ; Found: C = 27.72% ; H = 1.72%; N = 4.32% ; S = 18.29%; I = 35.21%;

4,4'-diamino stilbene8,2'-disulfonic acid was purified by silica gel column chromatography (Kotaki et al., '71), and its diisothiocyanate derivative was prepared as described in the literature (Cabantchik and Rothstein, '74a). ( c ) Preparation of 4,4'-diacetamido stilbene-2,2'-disulfonic acid: 2 g of the diamine were dissolved at pH 6-8 in 350 ml of water by addition of NaOH; a fourfold molar excess of acetic anhydride was added in small portions while the sample was cooled and stirred. The pH was maintained constant by the addition of 4 N NaOH. The mixture was then acidified to pH 1.5, and a small amount of precipitate was removed by filtration. The compound was then precipitated by addition of concentrated sulfuric acid ( 7 5 m l ) . During precipitation, the sample was cooled to temperatures below 10°C. For purification, the compound was dissolved in appraximately 70 ml of water, and reprecipitated as described above. Finally, the precipitate was washed on a glass filter with cold concentrated HC1 and ethanol. Paper electrophoresis at pH 6.5 and pH 1.9 showed a single, fluorescent band. The absence of contaminations of the original diamino derivative or of the monoacetyl derivative was demonstrated by exposing in a glass tank the paper sheets to nitrous fumes for a few minutes and spraying with a solution of N = naphthyl-ethenediamine-l,2 hydrochloride.

474 Analysis:

L. ZAKI, H. FASOLD, B. SCHUHMANN AND H. PASSOW CI~HI~O~N~SZ C = 47.63%; H = 3.97%; N =

Calculated:

6.17%; S = 14.11%; F o u n d : C = 47.82% ; H S = 14.01%;

= 3.76% ; N = 6.08% ;

result does not depend on the choice of the sampling times. Before determining the rate constants by the initial slope method, we always plot the full curve with six data points to assure ourselves that there are no obvious deviations from a single exponential (fig. 1 ) . Table 1 represents the rate constants pertaining to the experiment depicted in figure 8c as estimated by a least square fit, a fit by eye on a semilog plot (fig. I), and the initial slope method. As one would expect, the initial slope method systematically yields somewhat lower values (about 10% ) than the other two methods. Estimates of the degree of inhibition are not significantly affected by this difference. I n experiments with type I1 ghosts, the radioactive sulfate was incorporated during the resealing period. Otherwise the experimental procedure was identical to that explained above. Labeling with I4C DNFB: the washed red cells or type I1 ghosts were incubated in isotonic media containing 151 mM NaC1, 1 0 mM Na-phosphate, and 20 mM sucrose, pH 7.4-7.6. In experiments in which flux measurements were also made, the media contained sulfate and had the composition given above. Added to these media were those agents whose effects on DNFB binding or protection of anion permeability controlling sites were to be studied. Usually, the incubation time was 10-15 minutes for reversibly acting disulfonic acids and 20-30 minutes for SITS or LDIDS. The incubation temperature

Experimental procedures Resealed type I1 ghosts (Bodemann and Passow, '72) were prepared as described by Schwoch and Passow ('73). Measurement of "SO0, equilibrium exchange: intact red blood cells were washed, equilibrated, and subsequently loaded with 3 5 S 0 ,in an incubation medium containing 5 mM NaSO,, 20 mM Na-phosphate, 130 mM NaC1, 20 mM sucrose, pH 7.2 as described by Schwoch et al. (('74), and exposed to isothiocyanate derivatives of disulfonic acids and DNFB as described below. After the removal of the unreacted modifiers by three washes at 0°C in the described incubation medium, the cells were resuspended in the same medium at a hematocrit of 2.5% at 37°C and the outflow of "SO0, was followed. During the flux measurements, the medium contained neither DNFB nor other irreversibly binding inhibitors, except in the experiment shown in figure 4. When the effects of reversibly acting inhibitors on sulfate movements were studied, the inhibitors were present during the flux measurements. Rate constants, Oks, were calculated by dividing the initial slope of the curve relating "SO0,concentration in the supernatant, y, to time t by the concentration of radioactivity in the supernatant at infinite time, y,. This unorthodox method of calculating rate constants is based on a series TABLE 1 expansion of the exponential describing the time course of the appearance of the Method radioactivity in the supernatant: y/y- = 1 DNFB (1) (2,) (3) Initial Semilog conc. Least square - e-Okst which, for small values of t, slope plot IlM fit leads to y/ym =_"kst. This method has the 1.09 1.18 0 1.25 disadvantage that it uses only the data 1.03 0 1.18 1.13 points at the first two or three sampling 0.99 8.75 1.05 1.04 17.5 0.99 0.99 0.82 times (when the curve is still nearly lin0.87 0.68 35 0.88 ear) and at infiniate time. However, it has 0.45 70 0.48 0.48 the advantage that in contrast to the con140 0.30 0.32 0.27 0.24 280 0.23 0.25 ventional techniques of calculating "kS ___ ___ (fitting by eye on a semilog scale, or by Rate constants derived by three different methods from the data in figure 1 and from additional data the method of least squares on a computer) which are not shown in this figure. Rate constants in 102 min-I. In figure 8c the rate constants calculated the contribution of the experimental error by method (3) are. plotted against the DNFB concenof the individual data points to the final tration in the medium.

CHEMICAL MODIFICATION OF MEMBRANE PROTEINS

pM DNFB

- 0.4-

- 0.2-

30

60

90

, 120 rmnutes

Fig. 1 Semilogarithmic representation of the time course of release of 35s04from 35S0,loaded red cells. Prior to the initiation of the effiux of the labeled sulfate, the cells had been dinitrophenylated at the concentrations indicated i n the figure. The curves pertain to the experiment represented in figure 8c. For details of the experimental conditions see the legend to that figure. y, yo, y. represent, respectively, the radioactivity i n the supernatant at times t, t = 0, and t = m . To facilitate the representation of the data, two curves (at 8.75 and 140 pM DNFB) have been omitted. The corresponding rate constants are represented i n table 1.

475

ghosts either in a solution containing 0.1 % saponin, or in a phosphate EDTA buffer without saponin ( 1 mM EDTA, 9.6 mM NaC1, 3.6 Na2HP04, 1.2 mM KH2P04,pH 7.2). Subsequently, the hemoglobin was removed by washing in the saponin medium or in TRIS buffer, respectively. Hemolysis by saponin did neither affect the protein recovery nor the distribution pattern on the gel electropherograms. The final suspension of w h t e ghosts was stored at - 16°C until further processing. SDS polyacrylamide gel electrophoresis The packed white ghosts were solubilized in 5% or 1 % SDS in 0.1 M phosphate buffer, pH 7.2 and heated to 100°C for 4 minutes. The solubilized membranes were diluted 1: 10 with 0.01 m phosphate buffer pH 7.2 to give final SDS concentrations of 0.5 or 0.1%. 30-50 yg membrane protein as determinted by the method of Lowry et al. ('51) was used for electrophoresis. The membranes dissolved in 0.5% SDS were layered on top of 5% polyacrylamide gels (ratio acrylamide :bisacrylamide 33: l ) , those dissolved in 0.1% SDS on top of 7.5% gels (ratio acry1amide:bisacrylamide 20 : 1). Both types of gels contained 0.1 M Naphosphate pH 7.1, 6 M urea and either 0.1 or 0.5% SDS. The electrode buffers contained the same SDS-phosphate medium that was incorporated into the gels except that there was no urea present. Electrophoresis was performed at 5 mA/ gel for four hours (5% gels) or for 16-18 hours (7.5% gels). The proteins were fixed in 20% sulfosalicylic acid for 16 hours. Staining with 0.2% Coomassie's

was 5"C, 20"C, 25"C, or 37"C, the hematocrit 10%. After incubation, the excess of the irreversibly acting inhibitors was removed by washing2 in the media described above, and cells and ghosts were incubated in the dark at 37°C for 30 _ _ _ ~ minutes in these media containing 14C 2 Cabantchik and Rothstein noted that within the DNFB at the desired concentration. The 20 minutes of exposure only part of the binding of SITS or DIDS is irreversible. The reversibly bound isohematocrit was usually either 10 or 25% thiocyanate derivatives of the stilbenes could be removed by washing i n albumine containing media but and is indicated in tables and figures. If not by washing i n protein-free solutions. In expenthe effects of reversibly acting disulfonic ments with 12DIDS we could confirm these results. However, we found that depending on the method of acids were studied, the agents were also iodination of the compound, the ratio between reversibly and irreversibly bound IzDIDS vaned considerably present during washing and subsequent from one experiment to another and from one prepdinitrophenylation. Dinitrophenylation was aration of IzDIDS to another. For this reason we preferred to wash the cells after exposure to SITS or terminated by three washings at 0°C in IzDIDS i n the absence of albumin and to analyze the the media described above. The final sedi- effect on the isolated 95,000 Dalton proteins rather on the red cell a s a whole a s did Cabantchik ment was used for the preparation of than and Rothstein ('74a) in their work on the relationship between of sulfate exchange and 3H white ghosts and subsequent measurement DIDS binding.inhibition Moreover, we showed that i n the conof DNFB binding. The white ghosts were centration range used in our experiments the protection against dinitrophenylation by SITS was indeobtained by hemolysis of cells or type I1 pendent of the SITS concentration (table 3).

476

L. ZAKI, H . FASOLD, B. SCHUHMANN AND H. PASSOW

blue for one hour at 37°C. Destaining in 10% acetic acid. Carbohydrate staining by the periodic acid Schiffs technique of Zacharius et al. ('69). The gels were run in duplicates or triplicates for staining and for the determination of the distribution of the radioactive label. Densitometric tracings were obtained with the Gilford 240 spectrophotometer at 540 n m for proteins or at 560 n m for carbohydrates. Determination of radioactivity in gels Immediately after the end of electrophoresis the gels were subdivided into about 40 slices. The slices were incubated for 6-12 hours at 55°C in soluene (Packard). After addition of 10 ml acidified Insta Gel (Packard) the radioactivity was determined in a liquid scintillation counter. The recovery of the radioactivity in the 5% gels was greater than 9 5 % . The distribution pattern of the radioactivity in unstained gels was the same as in stained gels. Using the known specific activity of 14C DNFB, the number of 14C DNP residues was calculated from each band on the electropherogram and expressed i n sites/ mg membrane protein as determined by the Lowry method. Assuming that 1 mg of membrane protein corresponds to the membranes of 1.43 . lo9 cells (Dodge et al. ('63), these figures were converted into binding sites/cell.

TABLE 2

Inhibition of sulfate equilibrium exchange b y disulfonic acids ( A P M B and I P M B ) and a trisulfonic acid (IPT) Compound

APMB

Concentration Inhibition /LM percent

Temperature

37°C

500 2500

11.7 35.2 63.8

APMB

2500 5000

67.5 70.7

30°C

IPMB

500

76.7

30°C

IPT

500

90.5

30°C

100

For inhibition with the isothiocyano derivatives (IPMB and IPT), the cells were reacted with the agents at the concentrations indicated i n the table for 60' at 37'C. Hematocrit: 20%. Subsequently, the unreacted modifier was removed by washing and 35S04 movements were measured at Donnan equilibrium, pH 6.9, 10 vol. %. The reversibly acting inhibitor APMB was added to the final medium in which the sulfate exchange was measured.

discussion of the effects of stilbene disulfonic acids such as SITS or DIDS. SITS and its derivative DIDS contain two types of functional groups which are capable of reacting with amino groups : sulfonic acid groups and isothiocyanate groups. According to Cabantchik and Rothstein, the specificity of binding of the isothiocyanate derivatives of the stilbene disulfonic acids to anion permeability controlling sites is related to the high affiity of the two sulfonic acid groups to the control sites while irreversible fixation is due to a reaction of the NCS groups with RESULTS other amino groups in their vicinity. The experiments in figure 2 would be in accord 1. Sulfonic acids as inhibitors of with the view that the spatial arrangement anion exchange of the sulfonic acid groups is of decisive The chemical modifiers used in the influence on inhibition. They show that present work as tools for elucidating the stilbene disulfonic acids with no isothiorelationship between modification of mem- cyanate groups (4,4'-diacetamido stilbenebrane proteins and inhibition of anion ex- 2,2'-disulfonic acid, DAS; and 4,4'-diacetchange comprise DNFB and a variety of amido diiodo stilbene disulfonic acid, IZ sulfonic acids. The inhibitory action of DAS) are effective, reversibly acting inDNFB has been extensively dealt with in hibitors. However, the stilbene backbone two previous publications (Poensgen and of the disulfonic acids is not a necessary Passow, '71, Schwoch et al., '74) and thus prerequisite for a n inhibitory action since does not need to be discussed again. The other disulfonic acids with similar disinhibitory effects on anion exchange of tances between the sulfonic acid groups the sulfonic acids are illustrated partly in also inhibit. For instance disulfonic acids figures 2, 3 and table 2, and partly in the such as 2-( 4'-amino phenyl)-6-methyl bentext below. It seems best to consider these zene thiazole-3',7-disulfonic acid ( APMB, data within a conceptual frame provided fig. 2) and anthraquinone-l,5-disulfonic by Cabantchik and Rothstein ('72) for a acid produced 50% inhibition at 0.9 and

477

CHEMICAL MODIFICATION OF MEMBRANE PROTEINS

sog

so; AC

H N-@-CHI

- CHI

Q-NHAC

AC

HN

-@-CH= C

/

so;

Of0

100-0

0

HN HAC

~

/

so;

OIO

0

0

loo-&

50/L 5op\:-:

O-0

0

- 0

0

0.7 5 1.5 mM 12 DAS

0

0.7 5 mM DAS

1.5

100

50

0 PM 1, DlDS

0

2

4

mM APMB

Fig. 2 Effects of various disulfonic acids on sulfate equilibrium exchange i n intact red blood cells. Hematocrit 2.5%. Temperature 30"C, pH 7.1-7.2, except in the experiment with LDIDS, where the pH was 6.9. In the experiment with IsDIDS, the cells (10 vol. % ) were exposed to the various concentrations of the agent at 5°C for 30 minutes. Subsequently, the unreacted agent was removed by washing at 4°C and the cells were resuspended i n a medium free of IzDIDS at a hematocrit of 2 . 5 % for measuring =SO4 exit at 30°C. In the experiments with the reversibly acting inhibitors, the lower curves represent flux measurements i n the presence of the inhibitor concentrations indicated on the abscissa, the upper curves refer to flux measurements after removal of the inhibitor by washing. Ordinate: penetration rate i n percent of control value i n the absence of inhibitor. Abscissa: concentration of inhibitor.

5.8 mM, respectively. The trisulfonic acid Coomassie's blue produced 50% inhibition at 60-70 pM, but this figure is not redly comparable to the others since the dye seems to bind irreversibly. Even a monosulfonic acid, 6-hydroxy naphthalene-2sulfonic acid (HNS) was found to inhibit,

with 50% inhibition at 1.9 mM. This raises doubts about the justification of the assumption that the action of stilbene disulfonic acids or other disulfonic acids crucially depends on the spatid arrangement of the sulfonic acid groups and thus is essentially different from that of the

4 78

L. ZAKI, H. FASOLD, B. SCHUHMANN AND H. PASSOW

tend to level out at an inhibition which is lower than that produced by a maximally effective concentration of LDIDS. This would suggest that only part of the binding sites are capable of reacting with both LDIDS and a reversibly binding stilbene disulfonic acid. Unfortunately, the low solubility of the stilbene disulfonic acids available for our work did not permit us to decide whether at high concentrations inhibition by DAS and DIDS tends to approach the same or different limiting values. APMB, however, is soluble enough to allow a n increase of the concentration to the range where a further increase produces little further inhibition. In this concentration range, sulfate exchange shows about 85% inhibition instead of about 98% as observed with DIDS (figure 4 ) .

so, I OIO

100

50

0

.-. 0

2

4

5

mM I B S Fig. 3 Effects of m-isothiocyanato benzene sulfonic acid on sulfate equilibrium exchange i n intact red blood cells. Ordinate: penetration rate i n percent of control value without modifier. Abscissa: concentration in mM. Prior to measuring the rate of 35S04 exchange at 37"C, pH 7.55 and at a hematocrit of 2.5%, the cells had been exposed to the modifier at that pH for 30 minutes at 37°C. The hematocrit was 10% .

many other inhibitors of anion exchange which do not carry pairs of negatively charged groups, e.g. dipyridamole, phlorizin, phenol etc. However, the experiments described below on the effects of disulfonic acids on the one hand and phlorizin on the other on dinitrophenylation of anion permeability controlling sites on the protein in band 3 demonstrate differences which tend to support the notion that differences of the modes of action do in fact exist. Figures 2,3 and table 2 show that the isothiocynate derivatives of mono-, di-, and trisulfonic acids inhibit anion exchange. The inhibition is irreversible. Figure 2 suggests, however, that there exists an import ant difference between the reactions of an isothiocyanate derivative like LDIDS and the reversibly binding disulfonic acids. The curves relating the concentration of the disulfonic acids to penetration rate

11. Dinitrophenylation in the presence and absence o f an irreversibly binding derivative o f a stilbene disulfonic acid (SITS) and correlation with inhibition of anion exchange In the work described below the effect of previous exposure to SITS on subsequent DNFB-binding to the protein in band 3 was studied and correlated with the inhibition of anion exchange. The procedure employed is illustrated in figures 5-7. Intact red cells are dinitrophenylated with I4C DNFB and ghosted as explained under materials and methods. The SDS polyacrylamide electropherogram (5% polyacrylamide, 0.5% SDS) of the membrane preparation shows that virtu100;

"/

I

,

,

1

2

0.25 mM ,

3

,

4

DIDS ~

5 6 mM APMB

,

,

7

,

8

,

9

0

1

0

Fig. 4 Effects of APMB and APMB plus a fixed concentration of DIDS on sulfate equilibrium exchange. The inhibitors were present during the flux measurements. Temperature 30°C, pH 7.4; hematocrit: 10% ; Ordinate: penetration rate i n percent of control value in the absence of inhibitor. Abscissa: concentration of APMB.

479

SLICE NUMBER Fig. 5 Distribution of I4C DNP residues over the various proteins of the red blood cell membrane. Tracing on top of the photograph of the gel: densitogram of distribution of Coomassie blue staining. Tracing below the photograph of the gel: distribution of radioactivity. Membranes from intact red blood cells which had been dinitrophenylated at a DNFB concentration of 0.14 mM at pH 7.6 for 30 minutes. Temperature 37"C, hematocrit: 10%.

ally all of the protein bands are labeded. If, however, prior to dinitrophenylation the cells are exposed to SITS for 30 minutes at 25"C, labeling in the region of the band 3 protein (Steck, '74) is greatly reduced while the labeling of all other protein species remains unaltered (fig. 6). Obviously, by occupying DNFB binding sites, SITS protects these sites against dinitrophenylation. In the region of the 95,000 Dalton band on the electropherogram two protein species are superimposed. They were sep-

arated by electrophoresis in a denser gel (7.5% ) at a lower concentration of SDS (0.1% , Hubard and Cohn, '72) into a PAS positive and a PAS negative component as shown in figure 7. It can be seen that most of the SITS protectable I4C DNFB binding sites reside in the PAS negative protein of band 3. The finding that two different inhibitors of anion permeability bind to only one common site suggests that this site may be involved in anion transfer. Hence we studied the relationship between the dini-

480

L. ZAKI, H. FASOLD, B. SCHUHMANN AND H. PASSOW

COUNTS 1200

1

C14

n

-

without SITS

c--. with SITS

SLICE NUMBER

Fig. 6 Protection of '4C-DNFB binding sites by 500 yM SITS. Upper curve: control without SITS, lower curve: cells treated prior to dinitrophenylation with 500 pM SITS for 30 minutes at 25°C. Otherwise same experimental conditions as in figure 5. The distribution pattern of the Coomassie blue stain is the same in the presence or absence of SITS. The peak of band 3 is located at slice No. 12. The molecular weight as determined at the front of this band is 95,000 Daltons.

trophenylation of SITS-protectable DNFB binding sites and inhibition of anion exchange as a function of the DNFB concentration i n the medium. The experiments were performed with resealed red blood cell ghosts to minimize a deviation of the penetrating DNFB to intracellular proteins. Figure 8c shows that inhibition of anion exchange is nearly complete at a DNFB concentration i n the medium at which a considerable fraction of the SITS protectable binding sites on the 95,000 Dalton proteins is not yet dinitrophenylated (figs. 8b,c). The evidence for the absence of saturation of SITS protectable DNFB binding sites at maximally inhibitory concentrations may appear doubtful since it is based on the formation of only inaccurately determinable differences between two large numbers of DNFB binding sites in the presence and absence of SITS. In view of this inaccuracy, a method analogous to an end point titration was used for exploring whether or not at a maximally inhibitory concentration of DNFB all of the SITS protectable binding sites are actually

dinitrophenylated. The method is as follows: resealed ghosts are exposed to increasing concentrations of non-radioactive DNFB under the same conditions as in the experiment on 14C DNFB binding shown in figure 8b. Subsequently, the unreacted DNFB is removed by washing and the ghosts which had been treated at the various DNFB concentrations are exposed to equal and very low concentrations of 14C DNFB. Under these conditions, the labeling of SITS protectable DNFB binding sites should decrease with increasing concentrations of the non-radioactive DNFB used for pretreating the cells. Our data show the expected effect quite clearly (fig. 8 a ) . However, they also show that a fraction of SITS protectable DNFB binding sites is not saturated with DNFB at concentrations which produce a nearly maximal inhibition of sulfate exchange. For example, after dinitrophenylation at 0.265 mM non radioactive DNFB, inhibition is virtually maximal (fig. 8 c ) ; yet, further dinitrophenylation with 14C DNFB reveals that there are at least another 50,000 SITS protectable DNFB binding sites avail-

CHEMICAL MODIFICATION OF MEMBRANE PROTEINS

-

COUNTS

m1600

-

481

CU 7.5 *I* Gd

-without SITS -with SITS

1200-

800-

400-

PA.S.

Fig. 7 Protection against dinitrophenylation by SITS of the 95,000 Dalton proteins in the membrane of intact red blood cells. Upper tracing: distribution of 14C DNP residues i n unprotected and SITS protected membranes. Tracing i n the middle: densitogram of PAS stained electropherogram; lower tracing: densitogram of Coomassie blue stained electropherogram. Pretreatment with SITS does not affect the location of the stained bands. Same experimental conditions as i n figures 5 and 6 . Peak of band 3 at slices Nos. 14 and 15.

482

L. ZAKI, H. FASOLD, B. SCHUHMANN AND H. PASSOW

X105

l L C - DNFB binding on the 95K protein

after prelooding with cold DNFB

a o-: \ ‘

0

.?o aJ cn

unprotected

0.2

0.L

0.6 mM

DNFB(toto1)

.-

Y

m

X105

DNFB binding sites on the 9 5 K Drotein

toctod

b

/ --

/ O

TSl1’

01 0

0.8-

I

I

I

protected

I

I

0.2

0.1

I

0.3

mM DNFB

\ C

‘0

01 0

I

I

0.1

I

I

0.2

1

I

0.3

mM

DNFB

Fig 8 The effect of varying concentrations of DNFB in the medium on sulfate equilibrium exchange and on DNFB binding to the 95,000 Dalton proteins (band 3 ) i n resealed ghosts. a The ordinate represents the number of labeled binding sites on the 95,000 Dalton proteins i n each ghost i n resealed ghosts which had been exposed to 0.035 mM I4C-DNFB after dinitrophenylation with increasing concentrations of non radioactive DNFB. Dinitrophenylation with hot and cold DNFB for 30 minutes at 37”C, pH 7.6, hematocrit 25%. The non radioactive DNFB was removed by washing before the subsequent labeling with I4C-DNFB, and the latter was removed before the preparation of the membranes for gel electrophoresis. Both dinitrophenylation steps were applied to ghosts which had first been exposed to SITS for 15 minutes at 25”C, hematocrit 10% (“SITS protected”), or to untreated ghosts (“unprotected”). Abscissa: Sum of the concentrations of labeled and cold DNFB in the incubation medium. Note the differences of the scales on the abscissa when comparing figure 8c with figures a and b. b Ordinate: number of occupied DNFB binding sites on the 95,000 Dalton proteins i n each ghost. DNFB binding was measured on gel electropherograms after dinitrophenylation for 30 minutes a t 37”C, pH 7.6, hematocrit 25% of untreated, resealed ghosts (“unprotected’) and of resealed ghosts which, prior to dinitrophenylation, h a d been exposed to 500 pM SITS for 15 minutes at 25”C, hematocrit 10% (“SITS protected”). The gel electropherograms were made from membranes which had been freed of excess DNFB by three washes at 0°C. Abscissa: DNFB concentration at which the ghosts were dinitrophenylated. c Ordinate: rate constant (“k, ) for sulfate equilibrium exchange. After dinitrophenylation of the resealed ghosts type 11) a t the DNFB concentrations indicated on the abscissa for 30 minutes at 37”C, pH 7.6, hematocrit 2 5 % , the unreacted DNFB was removed by three washes at 0°C. The penetration of 3 5 S 0 4 was subsequently followed after suspension of the modified ghosts i n a DNFB free medium at 37”C, pH 7.6, hematocrit 1 0 % .

CHEMICAL MODIFICATION OF MEMBRANE PROTEINS

able for further modification. At 0.53 mM, the number of these binding sites is still about 20,000 (fig. 8 a ) . These findings suggests that SITS protects at least two different populations of amino groups on the 95,000 Dalton proteins, one of which is possibly involved in the control of anion permeability, while the other is certainly not. The experiments described above were performed at a constant and maximally inhibitory SITS concentration of 500 pM. Table 3 shows that at a given DNFB concentration in the medium, the number of SITS protected binding sites remains constant when the SITS concentration is varied over the range 2.5-500 ,M. These findings would suggests that under our experimental conditions all SITS protectable DNFB binding sites on the 95,000 TABLE 3

Protection against dinitrophenylation b y increasing concentrations of S I T S Expt. no.

la

SITS fiM

2.5 5.0 10.0 20.0

50.0 500.0 2 a

5.0

10.0 100.0 500.0

Protected sites on 95 K protein per cell 105

0.12 0.12 0.06 0.11 0.11 0.10 7.0 7.0 6.4 3.8

8.18 81.8 409.0

11.8 11.8

3b

8.18 81.8 409.0

10.9 11.2 12.3

4 a

10.0 100.0 500.0

8.8 9.5 8.7

4b

10.0 100.0 500.0

11.6 11.4 13.0

3a

12.3

Intact cells (expt. 1 ) and resealed ghosts (type 11, expts. 2-4) had been dinitrophenylated for 30 minutes at 37°C in the presence of 0.14 mM DNFB except in experiment 2 where the DNFB concentration was 0.2 mM. During dinitrophenylation the density of the cell and ghost suspensions was 2 5 % , the pH 7.6. Prior to dinitrophenylation, the ghosts and cells had been exposed to SITS at the indicated concentrations for 20 minutes at 5°C (expts. designated a ) or 25°C (expts. designated b ) and a hematocrit of 10%.

483

Dalton proteins are actually occupied by SITS. On the basis of the described observations it is possible to estimate, from experiments of the type represented in figures 8a and b the number of SITS protectable DNFB binding sites which could maximally be involved in the control of anion permeability. We find a nearly maximal inhibition of sulfate exchange if about 0.8 . 106-1.2 . lo6 of these sites are dinitrophenylated. The previous discussion was based on experiments with resealed ghosts. For this reason one of the reviewers of this paper raised the question whether or not results obtained with these ghosts are comparable with those obtained with intact cells. This question is quite pertinent since the concentrations of DNFB required to produce the same degree of inhibition of sulfate exchange are about one order of magnitude higher in the experiment with intact cells represented in figure 9a, than in the experiment with ghosts i n figure 8c. The discrepancy is due to the fact that DNFB rapidly penetrates into the cells and reacts with hemoglobin. This leads to a reduction of the effective DNFB concentration at which dinitrophenylation of the binding sites in the membrane takes place. In control experiments, a comparison of DNFB binding to membranes in intact cells with binding to ghosts suspended in hemolysates showed that DNFB binding is governed by the ratio membrane protein/ hemoglobin. On the basis of this information it is possible to compare the effects of bound DNFB in intact cells and ghosts. For example, in the experiment with intact cells, represented in figure 9a, after dinitrophenylation at 0.5 mM DNFB, there is about 62% inhibition. Without taking the intracellular hemoglobin into account, this would correspond, according to table 4, to about 80,000 SITS protected DNFB binding sites/cell. In ghosts, dinitrophenylation of this number of sites should yield an inhibition of about 25% (figs. 8b,c). The large discrepancy between 62% and 25% is considerably reduced if one takes into account that in the experiments with intact cells binding was measured at a hematocrit of 3 3 % , inhibition at 1 0 % . To correct for this difference one

484

L. ZAKI, H. FASOLD, B. SCHUHMANN AND H. PASSOW

A k rnin-1 0.10 x 10-1

-I

0 1

0

1

0.5

I

I

1.0 1.5 mM DNFB

I

1

2.0

2.5

Ak rn1n-l 0.10 x lo-’

j

04 0

I

0.5

I

1

1.0 1.5 mM I B S

I

I

2.0

2.5

Fig. 9 Partial protection of anion permeability controlling sites by DAS against reaction with DNFB ( a ) or m-isothiocyanato benzene sulfonic acid (IBS) ( b ) . After incubation i n the absence (“without DAS”) or presence of 1.5 mM DAS (“with DAS”) for 5-10 minutes sufficient DNFB or IBS was added to establish i n the absence or presence of a constant concentration of 1.5 mM DAS the final concentrations indicated o n the abscissa. The cell density was 10 vol. percent. Temperature 37°C. The reaction with IBS and DNFB took place i n the presence or absence of DAS at pH 7.6 and 7.4, respectively. The time of reaction with DNFB was 30 minutes, with IBS 60 minutes. After removal by washing at 4°C of DAS and the other unreacted modifiers 35S04movements were measured at 2.5 vol. percent, 37”C, pH 7.6 and 7.4, respectively. The insets represent the protection afforded by DAS against reaction with the irreversibly acting inhibitors. Ordinate: difference of rate constants measured i n the presence and absence of DAS. Abscissa: concentration of irreversibly acting inhibitor.

may assume that 0.5 mM DNFB in a suspension of a hematocrit of 33% is equivalent to (10/33) x 0.5 = 0.15 mM in a suspension of a hematocrit of 1 0 % . At this concentration, inhibition in figure 9a

is about 23% and thus agrees with the value of about 25% inferred from the experiments with ghosts in figure 8. This agreement between three different experiments done under widely differing condi-

485

CHEMICAL MODIFICATION OF MEMBRANE PROTEINS OlO

tions is better than one is entitled to anticipate. Nevertheless, i t supports the assumption that there is no reason to believe that ghosts show a n essentially different behaviour from that of intact cells. 111. Dinitrophenylation in the presence and absence of a reversibly binding derivative of a stilbene disulfonic acid (DAS) and correlation with inhibition of anion exchange In the experiments described in the previous section, the treatment with the irreversibly acting modifier SITS lead to the conversion of a certain fraction of the DNFB binding sites in the membrane into products which are incapable of reacting with DNFB. This “protection” against dinitrophenylation is permanent. If dinitrophenylation is performed in the presence of a reversibly binding modifier like DAS, protection is only temporary. DAS reduces the concentration of free DNFB binding sites and hence the rate of reaction between DNFB and these sites. As the reaction goes on, those binding sites which reacted with DNFB will be partially replaced by others which had previously combined with the reversibly binding DAS. Since dinitrophenylation is irreversible, this process will continue until eventually either all of the DNFB binding sites will be dinitrophenylated or all of the DNFB in the medium is used up. Therefore, protection by a reversibly acting inhibitor is due to a change of a reaction rate. As a rough measure of this change we determined the protection afforded against dinitrophenyation for a given time under specified conditions. In the experiment shown in figure 9a red cells or ghosts had been exposed to DNFB in the presence or absence of DAS for 30 minutes under the conditions described in the figure legend. After exposure, the modifiers had been removed by washing and the rate constant for SO, movements was measured in the absence of added modifiers. The figure demonstrates that there is less inhibition of anion exchange when the membrane is modified with DNFB in the presence of DAS than in the absence of DAS. Similar results were obtained when m-isothiocyanato benzene sulfonic acid (IBS) was used in place

12

no DAS X

I 80 l \

I

I

I

1

1

, 2

rnMDNFB

Fig. 10 Effect of DNFB on DAS inhibited sulfate equilibrium exchange. Prior to the flux measurements, intact cells had been dinitrophenylated i n the absence of DAS at the concentrations indicated on the abscissa. During dinitrophenylation (30 min. 37°C) the cell density was 10 vol. percent, the pH 7.2. 3 5 S 0 4 movements were measured at 2.5 vol. percent, pH 7.2, 30°C i n the presence ( 0 ) or absence ( x ) of 2.5 mM DAS. Ordinate: rate constant for SO4 equilibrium exchange i n percent of the value i n the absence of inhibitors.

of DNFB as an irreversibly acting modifier (fig. 9b). Figure 10 shows that dinitrophenylation increases the inhibition beyond that produced by DAS alone and that DAS increases the inhibition beyond that produced by DNFB alone. This indicates that at least at the concentration used in the experiments on protection against dinitrophenylation, DAS does not protect all DNFB sensitive anion permeability controlling sites. As one would expect, at very high concentrations of either DNFB or IBS, DAS is somewhat less effective in protecting the binding sites than at lower concentrations (see insets in fig. 9). This emphasizes that, at high concentrations, the rate at which the irreversibly acting inhibitors DNFB or IBS replace DAS at the anion permeability controlling sites is faster than at lower concentrations. Dinitrophenylation in the presence of DAS not only reduces the modification of the anion transfer system by DNFB. It also leads to a decreased binding of DNFB to the protein in band 3 (table 4 ) . This suggests that the DAS protectable DNFB binding sites on the protein in band 3 are

486

L. ZAKI, H. FASOLD, B. SCHUHMANN AND H. PASSOW lLC

DNFE 2360 CPm

0 28mM DNFB

control

9LL

L7 2

23601 1888

DAS L - L ' diacelomido, 2 - 2 ' disulfonic stilbene

ilSmM)

L7 2

18 81

2360

I2 DIDS 4 - 4 ' diisothyocyano, 2 - 2 ' disulfonic - dtiodostilbene (25pMl

0

1 1

10

20

30

LO

slice no

7.5% acrylamid gels, 0 1 % SDS Fig. 11 SDS polyacrylamide gel electropherograms of membranes protected against dinitrophenylation by DAS or 12DIDS. Same experimental conditions as i n table 4. The peak at the position of slice 10 represents the 95,000 Dalton proteins. The total amount of membrane protein and the specific activity of the 14C-DNFBis the same in each gel. The quantitative evaluation of this and other similar experiments i s presented i n table 4. Electrophoresis was performed under the same conditions as i n figure 7. Resealed ghosts.

487

CHEMICAL MODIFICATION OF MEMBRANE PROTEINS TABLE 4

E f f e c t s of DAS a n d lzDIDS o n **CD N F B b i n d i n g DNFB conc. in the medium

70 pM resealed ghosts

280 pM

Experimental condition Control

1.5 mMDAS 25 pM IzDIDS 50 pM IzDIDS Control

1.5 mM DAS 25 pM LDIDS 50 pM IzDIDS intact

cells

500 pM

Control

2.5 mM DAS 50 p M IzDIDS

No. of DNFB binding sites on 95 K protein

7.0 x 3.3 x 3.5 x 2.7 x 23.4 x 13.4 x 12.7 x 10.2 x 2.0 x 1.2 x 1.3 X

105 105 105 105 105 105

105 105 105 105 10:

No. of protected DNFB binding sites

2.7 x 105 2.5 x 1 0 5 4.3 x 105

-

10.0 x 105 11.7 x 105 13.2x 105 -

x 105 0.7 x 105 0.8

Ten minutes after exposure at 25°C of the cells or resealed ghosts to the concentrations of DAS or IzDIDS indicated in the table sufficient 14C DNFB was added to establish the concentrations shown above. After 30 minutes of incubation at 37°C (hematocrit 25% in the ghost experiments, 33% in the experiments with intact cells) the unreacted inhibitors were removed by washing and hemoglobin-free membranes were prepared. SDS gel electrophoresis was performed on 7.5% gels in 0.1% SDS. See figure 11.

identical with the anion permeability controlling sites. The number of DAS protected DNFB binding sites as determined under the conditions specified in table 4 is of the same order of magniture as the number of sites protected against dinitrophenylation by the irreversibly binding diisothiocyanate of a stilbene disulfonic acid, LDIDS. With increasing DNFB concentration protection by a fixed concentration of DAS against DNFB binding passes through a maximum like protection against inhibition. This can be inferred from figure 12 where it is shown that the number of those DNFB binding sites on the proteins in band 3 which, for a given length of time, can be protected against dinitrophenylation passes through a maximum when the total number of dinitrophenylated sites (i.e. protectable plus unprotectable sites) on the protein in band 3 increases beyond a certain limit. The maximum in figure 12 corresponds to the maximum of inhibition in the inset of figure 9a. In a previous section the question had been raised whether or not the site of action for disulfonic acids such as DAS is different from that for the other inhibitors of sulfate exchange, such as dipyridamol, phenol, or phlorizin. It seemed interesting therefore to study the effect of one of these latter agents on dinitropheny-

lation of the protein in band 3. For our experiments we chose phlorizin which produces inhibition of sulfate equilibrium exchange in the same concentration range as DAS (compare table 1 of Schnell '72 with fig. 2 of this paper). The results presented in table 5 show that phlorizin, in contrast to DAS, does not protect against dinitrophenylation and that slight variations of the conditions under which dinitrophenylation takes place do not affect the result. If anything, there is a facilitation by phlorizin of DNFB binding. IV. Sidedness o f the effects o f reversibly acting disulfonic acids on anion exchange and D N F B binding to the proteins in band 3 For the study of the sidedness of action the same techniques were employed as in previous experiments on the sidedness of the actions of phlorizin on equilibrium exchange of anions and sugars in red cell ghosts (Lepke and Passow, '73, Schnell et al., '73). Table 6 shows that disulfonic acids can be incorporated into the ghosts, and remain trapped for the length of time required for measuring 35S0,movements across the membrane. The presence of hemoglobin at the same concentration which exists inside the resealed ghosts (about 5% of the original hemoglobin concentration) has no effect on the inhibition of anion exchange by external stilbene di-

488

L. ZAKI, H. FASOLD, B. SCHUHMANN AND H. PASSOW

/B

0

P .

sulfonic acids (table 7)3. The sidedness of the effect of the various disulfonic acids on anion exchange varied with the chemical nature of the disulfonic acids employed. The stilbene disulfonic acids only inhibited at the outer surface (table 7, fig. 1 3 ) and thus behaved like phlorizin. In contrast, APMB inhibited anion exchange at either surface to about the same extent (fig. 14). In addition to APMB both anthraquinone disulfonic acid and hydroxynaphthalene monosulfonic acid also inhibited at the outer and inner membrane surface. However, the concentrations required to achieve half maximal inhibition were different at the two membrane surfaces, amounting to 2.65 mM and 5.75 mM, respectively, €or external and internal anthraquinone disulfonic acid, and 2.3 mM and 15 mM, respectively, for the monosulfonic acid. In contrast to the disulfonic acids, hydroxynaphthalene monosulfonic acid leaks out of the ghosts until, at the end of the flux measurements, no more than about 10% of the original concentration can be found within the ghosts. For this reason the concentration for half maximal inhibition by this sulfonic acid only represents a crude estimate. Effects on DNFB binding to the proteins in band 3 were only studied with DAS but not yet with the other sulfonic acids. Corresponding to the asymmetry of inhibition of anion exchange, DAS reduced the dinitrophenylation of band 3 protein only if applied to the outer cell surface. There was no protection by internal DAS (table 8 ) . DISCUSSION

x 0 1

Our results are compatible with the hypothesis that the protein in band 3 of SDS polyacrylamide gel electropherograms is involved in the control of anion permeability. They show ( 1 ) that the only common binding sites for two different inhibitors of anion permeability, DNFB and SITS, are associated with this protein and --~ 3 The absence of inhibition was not only observed when hemolysis was performed at a ratio between cells and medium of 1:20 as under our standard conditions, but also if this ratio was changed to 1:60. Under the latter conditions a t the fixed concentration of the disulfonic acid in the hemolysing medium (1.5 mM) the ratio between the amounts of disulfonic acid and cell contents i n the final hemolysate is shifted in favour of DAS by a factor of 3.0 (table 7).

489

CHEMICAL MODIFICATION OF MEMBRANE PROTEINS

DAS protectable"C DNFB binding t o 9 5 K Dalton proteins sites lcell 111

in5

DNFB binding to 95K Dalton proteins sites lcell Fig. 12 DAS protectable DNFB binding sites on the band 3 proteins versus DNFB binding to these proteins in the absence of DAS. For details of experimental conditons see legend to table 4. total "C

( 2 ) that a reversibly acting inhibitor of

anion permeability, DAS, reduces both the development of the inhibitory effect of DNFB on anion exchange and the rate of dinitrophenylation of the protein in band 3. These findings are in accord with those of Cabantchik and Rothstein ('74a) who observed that inhibition by DIDS is accompanied by DIDS binding to the protein in this band but not by binding to other proteins or lipids in the red cell membrane.

I I external,removei' 111 internal

20 40 60 80 100 120min Fig. 13 Sidedness of the inhibition of sulfate equilibrium exchange by IzDAS. The disulfonic acid was present at a concentration of 1.5 mM during resealing (111, internal) o r during the last 5 minutes before the end of the resealing period at 37°C and then removed by washing (11, external, removed); or during the flux measurement (IV, external). Control ( I ) , no LDAS present. pH 7.2. Flux measurements at 30°C. Hematocrit 2.5%.

In spite of the described evidence it still remains open whether or not in place of the protein in band small numbers of other membrane constituents are involved in the control of anion permeability but remained undetected by the techniques employed SO far. One may conclude, therefore, that the results of Cabantchik and Rothstein ('74a) and those presented in this paper suggest a role of the protein in band 3 in anion transfer but provide by no means conclusive evidence.

TABLE 6

Incorporation and retention of disulfonic acids in red cell ghosts Added to the hemolysis medium 1, mM

Recovered i n the hemolysate 2 , mM

(2'1

(7%)

Recovered i n the resealed ghosts 2 . 3 . mM (4)

DAS

1.5 1.5 1.5 1.5

APMB

2.0

APMB

5.0 7.5 5.0 7.5 10.0

1.3 1.3 1.2 1.1 1.5 4.2 6.4 5.2 7.9 11.1

1.6 1.2 1.1 1.2 2.0 4.7 5.7 5.5 7.5 9.6

Disulfonic acid (1)

__

1 Final concentration after hemolysis. 2 Determined photometrically at 330 mfi ( D A S ) or 390 mp (APMB) i n the TCA filtrate (7.5% ). The readings were taken after storing the filtrate in the dark for 10 minutes. 3 The ghosts had been resealed in the hemolysate at the disulfonic acid concentrations indicated in column ( 3 ) . The internal disulfonic acids had been determined after removal of external disulfonic acids by three washes and incubation in their absence at 30°C for 110-120 minutes. The concentrations inside the ghosts were obtained by dividing the disulfonic acid concentrations i n the TCA filtrates by the independently determined ratio: ( K + conc. in the TCA filtrate of the cell suspension)/ ( K + conc. i n the TCA filtrate of the hemolysate).

490

L. ZAKI, H. FASOLD, B. SCHUHMANN AND H. PASSOW TABLE 7

Sidedness of action of 4,4’-diacetamido stilbene-2,2‘-disulfonic acid ( D A S ) ___ __ ~

~

~~~

~

--

Rate constant for sulfate equilibrium exchange 10-3 min-1 Expt. no.

No disulfonic acid Disulfonic acid removed by washing External disulfonic acid Internal disulfonic acid

-. ~.

___.--

(1) 7.9

(2)

(3)

(4)

(5)

8.1

7.8

8.1

7.0

(6) 6.5

(7) 8.4

7.9

8.3

6.4

6.2

7.8

1.8

1.7

1.9

1.9

2.0

1.7

2.1

7.1

7.9

8.3

8.7

6.7

7.9

7.6

Hemolysis of one volume of cells in 20 (experiments ( 1 ) - ( 4 ) ) or 60 (experiments ( 5 ) - ( 7 ) ) volumes of medium. In experiments ( 3 ) and ( 4 ) the external medium contained a 5% hemolysate. Flux measurements at 30”C, pH 7.2, hematocrit 2 . 5 % .

k s ~ Lmm-‘ * removed by washing

o

O*l/ 06 OL-

inside.

\\:--2

02,

,

1

2

. 3

outside.

,

,

L

5

. 6

,

,

7

0mMAPMB

Fig. 14 Sidedness of action of APMB on sulfate equilibrium exchange in resealed ghosts. Ordinate: rate constant for sulfate exchange; abscissa: concentration of APMB in mM. “Inside” and “outside” refer to APMB inside or outside the ghosts. “Removed by washing” refers to ghosts to which the agent h a d been added at the end of the resealing period but subsequently been removed by washing to demonstrate the reversibility of the effect of the externally applied agent. pH 7.4 temperature 37”C, hematocrit 5 % .

A more detailed analysis of the effects of SITS on DNFB binding suggests that there exist at least three distinguishable populations of DNFB binding sites on the protein in band 3 : ( 1 ) Binding sites common to DNFB and SITS which are possibly related to inhibition of anion permeability ( 2 ) other common sites which are not related to inhibition and ( 3 ) different sites whose dinitrophenylation is not affected by SITS. We did not try to obtain quantitative estimates of the number of sites in each population except for the anion permeability controlling sites in population ( 1). In this latter population we found 0.8 . lO“l.2 . lo6 sites/cell. Population ( 2 ) is probably smaller than population ( 1 ) and population ( 3 ) is larger than the sum of the populations ( 1 ) and ( 2 ) . The finding that a certain fraction of

TABLE 8

Effects of internal a n d external DAS ( 1 . 5 m M ) on I4C D N F B binding to the 95,000 Dalton proteins in human red cell ghosts Expt. no.

1

2

3

14C binding to the

DAS

None Inside Inside and outside Outside None Inside Inside and outside Outside None Inside Inside and outside Outside

95 K proteins sites/cell 105

29.3 30.8 18.2 19.7 37.0 39.0 28.5 28.4 44.5 43.9 37.7 36.3

Change produced by DAS sites/cell lo5

+

-

1.5

- 11.1

- 9.6 -

+

2.0 8.5 - 8.6 -

-

- 0.6 - 6.8 - 8.2

Type I1 ghosts were dinitrophenylated in the presence or absence of DAS for 30’ at 37”C, pH 7.4, hematocrit 10%. DNFB.concentration: 0.28 mM.

CHEMICAL MODIFICATION OF MEMBRANE PROTEINS

the SITS bindings sites on the protein in band 3 cannot be related to the control of anion permeability is in qualitative accord with observations of Cabantchik and Rothstein ('74b) who showed that after pronase treatment of 3H DIDS labeled red cells, the radioactivity is distributed over three different bands. These bands represent a pronase resistant fraction and two peptides which result from the enzymatic cleavage of the rest of the protein in band 3. Only one of these peptides, which carries about 80% of the label, could be related to anion permeability. The sites in population (1 ) do not seem to be homogeneous, This may be inferred from the finding that APMB causes little further inhibition beyond 85% while DIDS produces 98% inhibition. Such difference cannot be accounted for by the fact that DIDS binds irreversibly and hence drives the reaction with the anion permeability controlling sites to completion. Instead the difference points to the existence of two types of anion permeability controlling sites in population ( I ) , one of which is capable of reacting with both APMB and DIDS, the other with DIDS only. An alternative explanation would consist of the assumption that bound APMB reduces either the accessibility or the affinity of still unoccupied binding sites to additional APMB. However, such allosteric effect should also be apparent with the isothiocyanate derivatives of the stilbenes and hence it seems more likely that the difference in the response to APMB and DIDS reflects the existence of two populations of anion permeability controlling sites which are distinguished by differences of accessibility or reactivity for the two inhibitors. Perhaps, some of the binding sites are only accessible to DIDS which is more lipophilic than APMB. The existence of the various populations of binding sites on the protein in band 3 raises the question whether the protein in this band consists of a single peptide or of a mixture of several different peptides which move at the same rate in SDS polyacrylamide gels. So far no unequivocal answer can be offered. Tanner and Boxer ('72) were unable to detect terminal amino groups in band 3 while in reports

491

from two other laboratories, it has been claimed that there are five different terminal amino groups in the protein in this band (Langdon, '74; Knufermann et al., '73). However, the end groups determined in these two laboratories do not agree. Moreover, it has been suggested that a single protein in band 3 may contain several cross linked peptide chains (Langdon, '74). A heterogeneity of the protein in band 3 is suggested by the work of Cabantchik and Rothstein ('74a), who found that maximal inhibition of anion exchange was obtained when about 300.000 binding sites on the protein in this band are occupied by DIDS. Since the total number of peptide chains in band 3 is about 940.000 (Steck, '74) this would indicate that maximally one third of the peptide chains in band 3 carries anion permeability controlling sites. Our own estimate of an involvement in the control of anion permeability of about 800.000-1.200.000 DNFB binding sites/cell agrees with the total number of peptides in band 3 per cell. Since the anion permeability controlling binding sites are capable of reacting with both stilbene disulfonic acids and isothiocyanates (fig. gb), it would be tempting to assume that each molecule of SITS prevents the binding of three molecules of DNFB. Hence our figure for DNFB binding would be equivalent to 267.000400.000 SITS binding sites/cell which is in the range estimated by Cabantchik and Rothstein. However, we hesitate to place much emphasis on this conclusion since the stoichiometrical relationship between the binding of disulfonic acids and the effect on dinitrophenylation is not yet really clear.4 Thus, the effects of DAS and of the diisothiocyanato derivative 1,DIDS on DNFB binding to the protein in band 3 are almost too similar to account for the fact that 1,DIDS carries two NCS groups while DAS carries none (table 4 ) . Moreover, any site which reacted with the irreversibly binding LDIDS is permanently protected against subsequent dinitrophenylation while dinitrophenylation in the 4 Recent work in our laboratory on the relationship between 3H-DIDS binding and the inhibition of sulfate equilibrium exchange suggests that there are about 1.7 . 108 3H-DIDS binding sites/cell on the prv tein in band 3 in place of 0.3 . 106 sites/cell as estlmated by Cabantchik and Rothstein ('74a).

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presence of the reversibly binding DAS is by reacting with the same binding sites unlikely to provide the full protection in the membrane. This question was not which one would expect to obtain on the systematically studied but it was shown basis of the stoichiometry of the reaction that at least phlorizin which carries an between DAS and the sites on the protein acidic phenol group instead of a sulfonic in band 3. Finally, it should be pointed acid group and which inhibits in the out that even if the number of anion per- same concentration range as DAS, does meability controlling sites should be equal not reduce DNFB binding to the protein to or larger than the number of peptide in band 3 . If anything, phlorizin slightly molecules in band 3 it remains unknown promotes dinitrophenylation. This would whether or not the anion permeability suggest that phlorizin and DAS do not controlling sites are equally distributed react at the same sites. Alternatively, if among all of the peptides or if a small one would postulate a common inhibitory number of specific peptide molecules site, one had to conclude that the protein in band 3 does not contain this site and carries many of them. What is the chemical nature of the anion that some other membrane constituent is permeability controlling sites on the pro- involved in the control of anion permetein in band 3? It would be conceivable ability. that the sulfonic acid groups of SITS The stilbene disulfonic acid DAS inhibmodify anion permeability by electrostatic its anion exchange and dinitrophenylation interactions with positive charges in the of the protein in band 3 at the outer, but membrane such as imidazole, amino, or not at the inner membrane surface. The guanidino groups while the NCS group same sidedness of the effect on anion exfixes the disulfonic acid to an adjacent change is observed with 1,DAS and phloriNH, group which may or may not be in- zin. The similarity between the actions of volved in anion transfer (Cabantchik and DAS and phlorizin is remarkable since the Rothstein, '72). The experiments with effects of the two inhibitors on DNFB reversibly binding disulfonic acids show binding to the externally located sites on that the isothiocyanate group is not essen- the protein in band 3 are different. tial for about 85% of that inhibition which There are several possible explanations can be maximally brought about by the for the observed asymmetry of action. For isothiocyanate derivatives of stilbene di- example, the anion permeability controlsulfonic acids (figs. 2,4). The finding ling sites to which the inhibitors bind that DAS is capable of reducing the dini- could be immobile and exclusively located trophenylation of anion permeability con- at the outer membrane surface; alternatrolling sites indicates that the DAS bind- tively, binding sites on a mobile carrier ing sites are susceptible to arylation with could change their affinities for the inhibiDNFB. This finding supports the previous tors when they traverse the membrane claim (e.g. Schnell and Passow, '69, Pas- such that binding and inhibition only sow, '69a,b) that the anion permeability occur at the outer but not at the inner controlling sites are amino groups. How- membrane surface. In contrast the symever, i t does not prove the participation of metrical inhibition by APMB would be comamino groups in anion transfer since in- patible with the presence of identical imhibition could be due to modification of mobile anion permeability controlling sites sites which are not normally involved in at both surfaces or with the existence of the transfer process. a mobile carrier which has the same affinOur experiments with a number of sul- ity to APMB at the two surfaces. It seems fonic acids, including a monosulfonic acid difficult to reconcile the different behaviour and a trisulfonic acid, show that neither of DAS and APMB with a mechanism inthe stilbene backbone nor the presence volving only one single type of common of two sulfonic acid groups is a prerequi- binding sites at either surface. Thus, our site for the inhibition of anion permea- findings would be easily reconcilable with bility. This raises the question whether the hypothesis that two types of sites are or not the various different inhibitors of involved, for example, sites on a mobile anion perme ability produce their effects carrier and immobile sites in the outer

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Konzentration, Anionen-Milieu und Zell-Volumen. Pflugers Arch. ges. Physiol. 296: 21. Dodge, J. T., C. Mitchell and D. Hanahan 1963 The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes. Arch. Biochem. Biophys., 100: 119. Gunn, R. B., and D. C. Tosteson 1971 The effect of 2, 4, 6 Trinitro-m-Cresol on cation and anion transport in sheep red blood cells. J. Gen. Physiol., 5 7 : 593. Hubard. A. N., and Z. A. Cohn 1972 The enzymatic iodination of the red cell membrane. J. Cell, Biol., 5 5 : 390. Knauf, P. A., and A. Rothstein 1971 Chemical modifications of membranes. I. Effects of Sulfhydyl and amino reactive reagents o n anion and cation permeability of the human red blood cell. J . Gen. Physiol., 5 8 : 190. Knufermann, H., S. Bhakdi, R. Schmidt-Ullrich and D. F. Hoelzl Wallach 1973 N-Terminal amino acid analysis reveal peptide heterogeneity i n major electrophoretic protein components of erythrocyte ghosts. Biochim. Biophys. Acta, 330: 356. Kotaki, A,, M. Naoi and K. Yagi 1971 A. Diaminostilbene dye as a hydrophobic probe for proteins. Biochim. Biophys. Acta, 229: 547. Langdon, R. G. 1974 Serum lipoprotein apoproteins as major protein constituents of the human erythrocyte membrane. Biochim. Biophys. Acta, 342: 213. Lepke, S., and H. Passow 1973 Asymmetric inhibition by phlorizin of sulfate movements across the red blood cell membrane. Biochim. Biophys. Acta, 298: 529. Lowry, 0.H., N. J. Rosebrough, A. L. Farr and R. J. Randall 1951 Protein measurement with the folin phenol reagent. J. Biol. Chem., ACKNOWLEDGMENT 193: 265. We thank Drs. J. Kaplan and P. K. Lauf Maddy, H. 1964 A fluorescent label for the outer components of the plasma membrane, for reading the manuscript and for their Biochim. Biophys. Acta, 8 8 : 390. comments. Obaid, A. L., A. F. Rega and P. Garrahan 1972 The effects of maleic anhydride on the ionic LITERATURE CITED permeability of red cells. J. Membrane Biol., Bodemann, H., and H. Passow 1972 Factors 9: 385. controlling the resealing of the membrane of Passow, H. 1969a Passive ion permeability of human erythrocyte ghosts after hypotonic the erythrocyte membrane. Progr. Biophys. hemolysis. J. Membr. Biol., 8 : 1. Mol. Biol., 19: 424. 1969b The molecular basis of ion disCabantchik, Z . I., and A. Rothstein 1972 The crimination in the erythrocyte membrane. In: nature of the membrane sites controlling anion The molecular basis of membrane function, permeability of human red blood cells as deD. C. Tosteson, ed., Prentice Hall Inc. Engletermined by studies with disulfonic stilbene wood Cliffs, New Jersey, p. 319. derivatives. J. Membrane Biol., 10: 311. 1971 Effects of pronase on passive ion _ _ ~1974a Membrane proteins related to permeability of the human red blood cell. J. anion permeability of human red blood cells. Membrane Biol., 6: 233. I. Localization of disulfonic stilbene binding sites i n proteins involved i n permeation. J. Passow, H., H. Fasold, L. Zaki, B. Schuhmann and S. Lepke 1975 Membrane proteins and Membrane Biol., 1 5 : 207. anion exchange in human erythrocytes. In: 1974 Membrane proteins related to Biomembranes: Structure and Function. Vol. anion permeability of human red blood cells. 35. G. Gardos and I. Szasz, eds. Federation of 11. Effects of proteolytic enzymes o n disulfonic European Biochemical Societies, Proceedings of stilbene sites of surface proteins. J. Membrane the Ninth Meeting, Budapest, 1974. Publishing Biol., 15: 227. House of the Hungarian Academy of Sciences, Deuticke, B. 1967 Uber die Kinetik der PhosBudapest, pp. 197-214. phat-Permeation in den Menschen-Erythrozyten Poensgen, J., and H. Passow 1971 Action of bei Variation von extrazellularer Phosphatl-Fluoro-2,4-Dinitrobenzene on passive ion per-

membrane surface which control the access to the binding sites on the carrier. If the affinity of DAS would be high for the accessibility controlling sites, and low for the sites on the carrier, DAS would only inhibit at the outer surface. If the affinity of APMB for the carrier would be higher or at least as high as its affinity for the accessibility controlling sites, APMB would act with equal strength at either surface. Different affinities for the two types of sites could easily explain why some agents are more effective at the outer than at the inner surface. However, without further assumptions such model cannot explain all the findings presented in this paper. Thus, the observation that APMB produces little further effect beyond 8 5 % inhibition while DIDS produces more than 98% inhibition, can obviously not be deduced from the model. In summary, the results presented in this paper are compatible with the assumption that amino groups on the protein in band 3 are involved in the control of anion permeability. However, they are by no means conclusive and suggest that more than one type of binding sites participates in anion transfer across the membrane.

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meability of the human red blood cell. J. Membrane Biol., 6: 210. Schnell, K. F. 1972 On the mechanism of inhibition of the sulfate transfer across the human erythrocyte membrane. Biochim. Biophys. Acta, 282: 265. Schnell, K. F., S . Gerhard, S. Lepke and H. Passow 1973 Asymmetric inhibition by phlorizin of halide movements across the red blood cell membrane. Biochim. Biophys. Acta, 318: 474. Schnell, K . F. and H. Passow 1969 Chemical modifiers of passive ion permeability of the erythrocyte membrane. Experientia, 25: 460. Schwoch, G., and H. Passow 1973 Preparation and properties of human erythrocyte ghosts. Mol. Cell Biochem., 2: 1973. Schwoch, G., V. Rudloff, I. Wood-Guth and H. Passow 1974 Effect of temperature o n sulfate movements across chemically or enzymatically modified membranes of human red blood cells. Biochim. Biophys. Acta, 339, 126. Steck, T. L. 1974 The organization of the pro-

teins i n the human red blood cell membrane. Biology, 62: 1. Tanner, M. J. A,, and D. H. Boxer 1972 Separation and some properties of the major proteins of the human erythrocyte membrane. Biochem, J., 129: 333. Wieth, J. 0. 1970 Effect of some monovalent anions o n chloride and sulphate permeability of human red cells. J. Physiol., 207: 581. Zacharius, R. M., T. E. Zell, J. H. Morrison and J. J. Woddlock 1969 Glycoprotein staining following electrophoresis on acrylamide gels. Analyt. Biochem., 30: 148. Zaki, L., C. Gitler and H. Passow 1971 The effect of trinitrobenzene sulfonate on passive ion permeability of the human red blood cell. XXV Intern. Congress Physiol. Sci., Munich, p. 616. Zaki, L., and H. Passow 1973 The binding of 2,4 dinitrofluorobenzene (DNFB) to the proteins of the red blood cell membrane. Abstract 9th Intern. Congress of biochemistry, Stockholm, p. 287.

Chemical modification of membrane proteins in relation to inhibition of anion exchange in human red blood cells.

Mono-, di-, and trisulfonic acids, including 4,4'-diacetamido stilbene-2,2'-disulfonic acid (DAS) and 2-(4'-amino phenyl)-6-methylbenzene thiazol-3',7...
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