ARCHIVES
OF
BIOCHEMISTRY
Analysis
AND
604-609
(1976)
of Phospholipase C (Bacillus Micelles of Phospholipid BARBARA
Department
176,
BIOPHYSICS
of Chemistry,
R. EATON University
AND
of California
Received
February
cereus) Action and Surfactant’
EDWARD at San Diego,
toward
Mixed
A. DENNIS* La
Jolla,
California
92093
23, 1976
The action of phospholipase C (Bacillus cereus) toward mixed micelles of phosphatidylcholine and the nonionic surfactant Triton X-100 is analyzed according to the “surfaceas-cofactor” kinetic scheme recently proposed for characterizing the action of lipolytic enzymes [Deems, R. A., Eaton, B. R., and Dennis, E. A. (1975) J. Biol. Chem. 250,901390201. According to this scheme, the enzyme first associates with the surface or mixed micelles, where the dissociation constant is K sA. The enzyme, now part of the mixed micelle surface, then binds the substrate phospholipid molecule in its active site and this binding is related to the Michaelis constant, KmB. The surface, or mixed micelles in this scheme, behaves kinetically as a cofactor in that, under initial rate conditions, the surface properties of the mixed micelles are virtually unchanged after catalysis. For phospholipase C with egg phosphatidylcholine as substrate, it was found that at pH 6.4 (the pH optimum for the enzyme) and 4O”C, V is about 2 x lo3 pmol min’ (mg of protein)-‘. KSA is about 2 mM and K,,,B is 1 to 2 x 10-l” mol cm~m2. The kinetic constants for phospholipase C are compared with those previously reported for phospholipase A, and the membrane-bound enzyme phosphatidylserine decarboxylase determined under similar conditions.
Enzymes of lipid metabolism normally act on substrates which are part of a macromolecular aggregate, and the site of catalytic action is often at a hydrophobichydrophilic interface. We have recently proposed a general scheme for the kinetic analysis and study of enzymes that act on phospholipids and triglycerides (1). This scheme has been used to analyze the action of phospholipase A2 toward mixed micelles of phosphatidylcholine and the nonionic surfactant Triton X-100 (1) and a solubilized form of the membrane-bound enzyme phosphatidylserine decarboxylase toward mixed micelles of phosphatidylserine and Triton X-100 (2). We wish to report here results of kinetic studies on the action of phospholipase C toward mixed micelles of phosphatidylcholine and Triton X-100.
This study provides details of the action of a phospholipase at another structural site on a phospholipid molecule which is in mixed micelles of the same composition and structure as have been previously studied with phospholipase A,. This allows the testing and elaboration of certain assumptions implicit in the general scheme previously developed. These experiments were conducted with a highly purified preparation of phospholipase C isolated from the growth medium of Bacillus cereus and purified according to the procedure of Zwaal et al. (3). EXPERIMENTAL
’ This work is taken in part from the Ph.D. Dissertation submitted by B. R. Eaton in partial satisfaction of the requirements for the Ph.D. degree, University of California at San Diego, 1975. ’ To whom correspondence should be addressed. 604 Copyright All rights
0 1976 by Academic Press, of reproduction in any form
Inc. reserved.
PROCEDURE
Phospholipase C was isolated from the growth medium of the bacterium Bacillus cereus (ATCC 10987). It was purified by the procedure of Zwaal et al. (3) and had a specific activity of 800 pmol min-’ (mg of protein)-’ at 40°C and pH 8.0 with 40 mM Triton X-100 and 20 mM phosphatidylcholine in a pH-Stat assay described elsewhere (4). Thin-layer chromatography of the reaction products after prolonged hydrolysis (4) confirmed that this enzyme
PHOSPHOLIPASE
C ACTION
has only phospholipase C activity toward phosphatidylcholine. Thin-layer chromatography was conducted on glass plates coated with silica gel G and visualized with iodine vapor or the phosphorus reagent of Dittmer and Lester (5). Using chloroform:methanol:water (65:25:4, v/v/v), as developing solvent, one product, a neutral lipid, ran near the solvent front while the other product, phosphorylcholine, remained at the origin. Using ethyl ether:benzene:ethano1(40:50:2, v/v/v) as solvent, the neutral lipid ran as a diglyceride against appropriate standards. Phosphatidylcholine was purified from fresh chicken egg yolks by a modification of the method of Singleton et al. (6). The final product was judged pure by thin-layer chromatography using chloroform:methanol:water (65:25:4, v/v/v) as developing solvent. Triton X-100 (Rohm and Haas) is a polydisperse mixture of p-tert-octylphenoxypolyethoxyethanols with an average chain length of about 9.5 oxyethylene units. Concentrations are expressed in terms of its average monomer molecular weight of 624. Stock solutions of mixed micelles of a given phospholipid/Triton composition were prepared by adding a solution of Triton X-100 in water to dry phospholipid and mixing briefly with the Teflon pestle of a Potter-Elvehjem homogenization apparatus. The pH was then brought to pH 6.4 with KOH. Phospholipase C assay. Assays were conducted in &ml centrifuge tubes set in a .rotary shaking bath maintained at 40°C. The assay tubes contained mixed micelles and buffer; the reaction was initiated by the addition of enzyme. Reactions were quenched by the addition of 2 ml of methanol followed by vortexing and rapid cooling of the tubes in a dry iceacetone bath. Zero-time controls were included in each experiment. The phosphorylcholine produced by the reaction was isolated by extraction and its amount determined by a phosphorus analysis. To each assay tube, 4 ml of chloroform was added, the tube was vortexed and shaken for 2 min and then centrifuged (500 rpm, room temperature, 2 min). Aliquots of the upper methanol/water layers were transferred to acidwashed ignition tubes and analyzed for phosphorus by a modification of the method of Turner and Rouser (7). The samples were brought to dryness in an aluminum heating block maintained at 170°C. To each tube was added 0.65 ml of 70% perchloric acid, and heating was continued an additional 25 min. The tubes were cooled, and 4.3 ml of a solution containing ammonium molybdate and ascorbic acid so that the final concentrations were 0.25 and l%, respectively, were added to the tubes. A marble was placed on each tube and the covered tubes were then placed in a boiling-water bath for 5 min. The amount of phosphorus present was determined on a Perkin-Elmer double beam spectrophotometer at 666 nm with appropriate blanks and standards. A
TO WARD
605
MICELLES
unit of activity is defined as the amount of enzyme that produces 1 pmol/min of phosphorylcholine. The average of duplicate analyses is reported. RESULTS
AND
“Surface-as-Cofactor”
DISCUSSION
Theory
The activity of phospholipase C toward substrate in mixed micelles was determined according to the general theory and methodology developed for the analysis of lipolytic enzymes which was applied previously to phospholipase A, (1). This theory assumes that the enzyme, E, first associates with a surface site, A, as shown in Eq. [ll to form an enzyme-surface complex, EA: E+A
k, =
EA
111
k-1 When the substrate molecule, B, which makes up part of the surface, A, binds in the active site of E, the Michaelis complex, EAB forms. Upon catalysis, the products, Q, are formed and EA is regenerated as indicated in Eq. [21: EA+B
k, Z$ km,
EAB
k, -+ EA + Q
121
In the case of phospholipase C, Q represents two products: phosphorylcholine and diglyceride. If (A) is the concentration of surface sites capable of associating with an enzyme molecule and (B) is the concentration of lipid substrate in the two-dimensional surface, then the rate equation derived for this scheme is given in Eq. [31:
VW (B) ’ = KsAKmB + KmB(A)
+ (A) (B)
[31
KS* = k-,/k, K mB = (k-,
+ kR)/kZ
According to this scheme, the surface, A, behaves kinetically as a cofactor for the reaction, and it emerges essentially unchanged at the end of the reaction. In the case of the mixed micelle system, composed of the nonionic surfactant Triton X-100 and phospholipid, the surface is in the form of mixed micelles which have been previously characterized in detail (8,
606
EATON
AND
9). Since each molecule contributes to the total surface of the aggregate, the surface term, (A), is related to the sum of the concentrations of T&on, T, and phospholipid, P. (B), the term that expresses the concentration of substrate in that surface, is related to the mole fraction of phospholipid present, P/(T + P). The mixed micelle system allows the independent variation of each of these terms. However, in order to apply the above concepts, four assumptions are necessary. These assumptions have been discussed in detail elsewhere (1) and are summarized here. i. The average surface area occupied by a phospholipid molecule and the average surface area occupied by a Triton molecule are constant as (A) is varied at constant (B). ii. The size of the surface (micelle) segment to which one enzyme molecule is bound is constant under all experimental conditions. It is defined as n in square centimeters per mole. The value of n is not known, but certain assumptions (1) allow the calculation of a minimum value of n. For phospholipase AZ, n = 3.6 x 1O’O cm2 mol-’ . A similar calculation of the minimum value of n for phospholipase C gives n = 6.7 x lOlo cm2 mol-’ (10). iii. In a mixed micelle, the average surface area occupied by a phospholipid molecule and that occupied by a Triton molecule are approximately the same. This area is defined by x in square centimeters per mole. Although its value is not known precisely, for the purposes of calculation x is assumed to be 5.1 x log cm2 mol-’ . iv. As the concentration of phospholipid in the surface, (B), is increased, while the sum of the phospholipid and Triton concentrations is kept constant, the average surface area occupied by a phospholipid molecule and a Triton molecule is decreased. This introduces a proportionality factor, s, into the definitions of (A) and (B), and s is the fractional decrease in x at a particular value of P/(P + T). The value of s is not known and would be difficult to determine experimentally. We found with phospholipase A, that, ifs was assumed to vary linearly
DENNIS
between 1.0 and 0.5 as P/(P + T) varies between 0 and 0.33, reasonable fits of the experimental data to theory were obtained. In the experiments reported here, mixed micelles of the same range of compositions, P/(P + T), were employed as with phospholipase A, and the same variation in s with P/(P + T) is assumed. Other assumptions about s are considered elsewhere (10). It should be noted that in our previous work (11, assumption iv was stated differently, and a modification of the assumption was presented in the text. Here, for simplicity, we introduce the proportionality factor, s, and begin with an assumption about its value that was suggested by physical studies on mixed micelles and curve fitting in our previous work. The value of (A) is given in Eq. [41 and the value of (B) in Eq. [5]: (A) = ;
(P + T) = x As n
[41
where A” = s(P + T)
[51
where n, s, n, P, and T are defined above. Equation [3] can be rewritten in a different algebraic form and in terms ofA”, B”, X, and n as shown in Eq. [61.
As and B” for any kinetic point can be determined from the concentrations of P and T and the value of s. Equation [6] shows that if initial rate studies are conducted in which the concentration of phospholipid in the surface, B”, is held constant while the total concentration of surface binding sites, A”, is varied, then plots of l/v versus l/A” at constant B” should give linear plots. These lines should intersect at a point with the coordinates -xlnKsA, l/V. Beplots of the slopes and intercepts would also allow the determination of V, nKsA/x, and xKmB. Specifi-
PHOSPHOLIPASE
C ACTION
TOWARD
607
MICELLES
tally, a plot of the l/u intercept for each value of B” plotted as a function of l/W should be linear, and the intercept of this line on the l/u intercept axis should be l/ V, and on the l/B” axis it should be -11 xK,,~. A plot of the slope of each line in the original graph against l/B” should be linear and pass through the origin; the slope of the resulting line should be nK,*KmBIV so that the value of nK,“/x can be determined from this replot and the values of xK,nB and V. These considerations lead to the determination of the kinetic constants and V defined in Eq. [3] in terms K*, K,rtB, of the additional constants x and n. It is not necessary to know the values ofx and n in order to analyze the kinetic data since they are constants and simply change the units of KS* and KmB. However, with values for x and n, KS* and K,B can be obtained in meaningful units for this system.
lent metal ion required for this enzyme (3, 11, 12). All assays were conducted at 4o”C, which was well below the temperature optimum for the enzyme and is the same temperature employed in previous studies with phospholipase A,. Kinetic experiments outlined above were conducted and the resulting activities are shown in Fig. 2 plotted in terms of
Kinetic
FIG. 1. pH variation of phospholipase C activity. The assay mix consisted of 1.2 Kg of phospholipase C, 40 mM Triton X-100, 20 mM phosphatidylcholine, and 50 mM buffer (a mixture of succinic acid, maleic acid, and Tris adjusted to the designated pH with NaOH and then brought to uniform ionic strength with NaCl). Incubations were conducted for 20 min at 40°C in a total volume of 0.5 ml and the activity analyzed as described in Experimental Procedure.
Results
The pH-rate profile of the enzyme is given in Fig. 1. Subsequent experiments were conducted at pH 6.4. At that pH with 50 mM sodium maleate buffer, the activity was linear with time and protein, and it was not stimulated by the addition of Ca2+, although it is not clear if there is a diva-
1 ,‘,
P-l
1 I q8
I I I 58 68 PH
I I 78
FIG. 2. Activity of phospholipase C toward phosphatidylcholine in mixed micelles with Triton X-100. The assay mix consisted of 0.6 pg of phospholipase C, phosphatidylcholine and Triton X-100 at the designated concentrations, and 50 mM maleate buffer, pH 6.4, in a total volume of 2.0 ml. Incubations were conducted for 5 min at 40°C. The activity was determined as described in Experimental Procedure. Assumptions about the value ofs were: when T/P = 12:1, s = 1.0; T/P = lO:l, s = 0.9; T/P = 8:1, s = 0.8; T/P = 6:1, s = 0.7; T/P = 4:1, s = 0.6; and T/P = 2:1, s = 0.5. The data are plotted as l/u versus l/A” at several values of B”: 0.077 (A), 0.100 (01, 0.139 (01, 0.207 (A), 0.332 (m), and 0.666 (0).
I BB
608
EATON
AND
A” and B”. Replots of the slopes and l/u intercepts versus l/B” are shown in Fig. 3. The approximate values of V, nKsA/x, and xK,nB, along with KsA and K,,tB using the values of x and n stated earlier, are given in Table I along with these constants for phospholipase A, and phosphatidylserine decarboxylase . Action of Phospholytic Mixed Mice&s
Enzymes
toward
It is interesting to note that the V’s for the two extracellular phospholipases acting on phosphatidylcholine in mixed micelles are similar, although this may be coincidence, while the V for the biosynthetic enzyme phosphatidylserine decarboxylase is about 100 times slower. The value of nKsA/x for phospholipase C is somewhat larger than for phospholipase A, but is in a similar range to that found for the decarboxylase. The value ofKSA for
(IX,
FIG. 3. Replot of l/v intercepts obtained from Fig. 2, versus
(0) l/B”.
and
slopes
TABLE KINETIC
CONSTANTS
FOR THE ACTION
Enzyme
V (pm01 min-’
DENNIS
all three enzymes appears more similar, but, until the value of n is known precisely cannot for each enzyme, comparisons really be made. The value of xK,* is between 0.5 and 1.0 (moles of phospholipid to total moles of Triton plus phospholipid in the mixed micelles) for both phospholipases. Because mixed micelles of phosphatidylcholine and Triton X-100 are not formed above values of B” of 0.33 (with s = 1.0) (9) and the values of s at various values of T/P between 2:l and 12:l are not known accurately, the value of xK,,B cannot be determined precisely at this time. It is clear, however, that saturation of the active site, if possible at all, requires a very high concentration of phospholipid in the mixed micelle, (B). In contrast, it appears that the active site of the decarboxylase can be saturated with phosphatidylserine at a lower substrate concentration in the mixed micelle (xK,* = 0.03 in units of moles of phospholipid to total moles of Triton plus phospholipid). The xKmB values can be converted to KmB values which are in the correct units for a two-dimensional surface, moles per square centimeter. Thus, the KmB of 0.06 x lo-lo mol crnm2 for the decarboxylase is significantly lower than the K,* of l-2 x lo-lo mol cm-’ for the two phospholipases. The kinetic scheme developed for lipolytic enzymes has been applied to three enzymes: phospholipase A, (11, phosphatidylserine decarboxylase (2), and here to phospholipase C. Each of these enzymes acts on a different area of the phospholipid molecule, but in each case the phospholipid is part of a two-dimensional surface I
OF LIP~LYTIC ENZYMES AND SURFACTANT mg-‘)
nK,Alx (KIM)
TOWARD KSA bM)
MIXED
MICELLES
XKmB (mol fraction
P/
OF PHOSPHOLIPID
blol
KlliB cm-*)
IP+Tl)
Phospholipase Phospholipase Phosphatidylserine carboxylase
C A, (1) de-
2 x 103 4 x 103 25
30 4 40
2 0.5 0.3
0.5-l 0.5-l 0.03
l-2 l-2
x 10-10 x 10-10 6 x lo-l2
(2) n
” In Ref. (2) because T/P ratios of 6:l to 128:l were utilized, it was assumed that s = 1 at all values of T/P and for simplicity the symbols KsA and K,nB were used for nKSAIx and xKmB defined here. For the calculation ofK,* here, it was assumed that one decarboxylase molecule binds to one micelle composed of 150 molecules of surfactant and phosphatidylserine giving a value of n = 7.7 x 10” cm2 mol-’ (13).
PHOSPHOLIPASE
C ACTION
rather than existing in bulk solution. The use of the mixed micelle system allows the independent variation of the amount of surface present, A, and the amount of substrate in that surface, B. The “surface-ascofactor” kinetic scheme and equation allow the evaluation of V, the enzyme-surface association constant KS*, and the Michaelis constant KrfiB, as separate entities, which has not been possible previously. The application of this scheme to phospholipase C reported here suggests that this approach may be generally applicable to the consideration of the action of lipolytic enzymes. ACKNOWLEDGMENTS We wish to thank Mr. Raymond A. Deems for helpful discussions. Financial support was provided by a grant from the National Institutes of Health (GM 20,501). B.R.E. was a predoctoral trainee ofthe National Institutes of Health (GM-1045). REFERENCES 1. DEEMS, R. A., EATON, B. R., AND DENNIS, (1975) J. Biol. Chem. 250, 9013-9020.
E. A.
TOWARD
MICELLES
609
2. WARNER, T. G., AND DENNIS, E. A. (1975) J. Biol. Chem. 290, 8004-8009. P., 3. ZWAAL, R. F. A., ROELOFSEN, B., CONFURIUS, AND VAN DEENEN, L. L. M. (1971) Biochim. Biophys. Acta 233, 474-479. 4. DENNIS, E. A. (197315. Lipid Res. 14.152-159. 5. DITTMER, J. C., AND LESTER, R. L. (1964) J. Lipid Res. 5, 126-127. 6 SINGLETON, W. S., GRAY, M. S., BROWN, M. L., AND WHITE, J. L. (1965) J. Amer. Oil Chem. Sot. 42, 53-56. 7. TURNER, J. D., AND ROUSER, G. (1970) Anal. Biochem. 38, 423-436. a. DENNIS, E. A. (1974) J. Supramol. Struct. 2, 686-694. 9. DENNIS, E. A. (1974) Arch. Biochem. Biophys. 165, 764-773. 10. B. R. EATON (1975) Ph.D. Dissertation, University of California at San Diego, La Jolla, Calif.; (1976) Dissertat. Abstr. 36, 3351B. 11. OTNAESS, A.-B., PRYDZ, H., BJBRKLID, E., AND BERRE, A. (1972) Eur. J. Biochem. 27, 23% 243. 12. OTTOLENGHI, A. C. (1965) Biochim. Biophys. Acta 106, 510-518. 13. T. G. WARNER (1975) Ph.D. Dissertation, University of California at San Diego, La Jolla, Calif., (1976) Dissertat. Abstr. 36, 3939B3940B.
OF
BIOCHEMISTRY
Analysis
AND
604-609
(1976)
of Phospholipase C (Bacillus Micelles of Phospholipid BARBARA
Department
176,
BIOPHYSICS
of Chemistry,
R. EATON University
AND
of California
Received
February
cereus) Action and Surfactant’
EDWARD at San Diego,
toward
Mixed
A. DENNIS* La
Jolla,
California
92093
23, 1976
The action of phospholipase C (Bacillus cereus) toward mixed micelles of phosphatidylcholine and the nonionic surfactant Triton X-100 is analyzed according to the “surfaceas-cofactor” kinetic scheme recently proposed for characterizing the action of lipolytic enzymes [Deems, R. A., Eaton, B. R., and Dennis, E. A. (1975) J. Biol. Chem. 250,901390201. According to this scheme, the enzyme first associates with the surface or mixed micelles, where the dissociation constant is K sA. The enzyme, now part of the mixed micelle surface, then binds the substrate phospholipid molecule in its active site and this binding is related to the Michaelis constant, KmB. The surface, or mixed micelles in this scheme, behaves kinetically as a cofactor in that, under initial rate conditions, the surface properties of the mixed micelles are virtually unchanged after catalysis. For phospholipase C with egg phosphatidylcholine as substrate, it was found that at pH 6.4 (the pH optimum for the enzyme) and 4O”C, V is about 2 x lo3 pmol min’ (mg of protein)-‘. KSA is about 2 mM and K,,,B is 1 to 2 x 10-l” mol cm~m2. The kinetic constants for phospholipase C are compared with those previously reported for phospholipase A, and the membrane-bound enzyme phosphatidylserine decarboxylase determined under similar conditions.
Enzymes of lipid metabolism normally act on substrates which are part of a macromolecular aggregate, and the site of catalytic action is often at a hydrophobichydrophilic interface. We have recently proposed a general scheme for the kinetic analysis and study of enzymes that act on phospholipids and triglycerides (1). This scheme has been used to analyze the action of phospholipase A2 toward mixed micelles of phosphatidylcholine and the nonionic surfactant Triton X-100 (1) and a solubilized form of the membrane-bound enzyme phosphatidylserine decarboxylase toward mixed micelles of phosphatidylserine and Triton X-100 (2). We wish to report here results of kinetic studies on the action of phospholipase C toward mixed micelles of phosphatidylcholine and Triton X-100.
This study provides details of the action of a phospholipase at another structural site on a phospholipid molecule which is in mixed micelles of the same composition and structure as have been previously studied with phospholipase A,. This allows the testing and elaboration of certain assumptions implicit in the general scheme previously developed. These experiments were conducted with a highly purified preparation of phospholipase C isolated from the growth medium of Bacillus cereus and purified according to the procedure of Zwaal et al. (3). EXPERIMENTAL
’ This work is taken in part from the Ph.D. Dissertation submitted by B. R. Eaton in partial satisfaction of the requirements for the Ph.D. degree, University of California at San Diego, 1975. ’ To whom correspondence should be addressed. 604 Copyright All rights
0 1976 by Academic Press, of reproduction in any form
Inc. reserved.
PROCEDURE
Phospholipase C was isolated from the growth medium of the bacterium Bacillus cereus (ATCC 10987). It was purified by the procedure of Zwaal et al. (3) and had a specific activity of 800 pmol min-’ (mg of protein)-’ at 40°C and pH 8.0 with 40 mM Triton X-100 and 20 mM phosphatidylcholine in a pH-Stat assay described elsewhere (4). Thin-layer chromatography of the reaction products after prolonged hydrolysis (4) confirmed that this enzyme
PHOSPHOLIPASE
C ACTION
has only phospholipase C activity toward phosphatidylcholine. Thin-layer chromatography was conducted on glass plates coated with silica gel G and visualized with iodine vapor or the phosphorus reagent of Dittmer and Lester (5). Using chloroform:methanol:water (65:25:4, v/v/v), as developing solvent, one product, a neutral lipid, ran near the solvent front while the other product, phosphorylcholine, remained at the origin. Using ethyl ether:benzene:ethano1(40:50:2, v/v/v) as solvent, the neutral lipid ran as a diglyceride against appropriate standards. Phosphatidylcholine was purified from fresh chicken egg yolks by a modification of the method of Singleton et al. (6). The final product was judged pure by thin-layer chromatography using chloroform:methanol:water (65:25:4, v/v/v) as developing solvent. Triton X-100 (Rohm and Haas) is a polydisperse mixture of p-tert-octylphenoxypolyethoxyethanols with an average chain length of about 9.5 oxyethylene units. Concentrations are expressed in terms of its average monomer molecular weight of 624. Stock solutions of mixed micelles of a given phospholipid/Triton composition were prepared by adding a solution of Triton X-100 in water to dry phospholipid and mixing briefly with the Teflon pestle of a Potter-Elvehjem homogenization apparatus. The pH was then brought to pH 6.4 with KOH. Phospholipase C assay. Assays were conducted in &ml centrifuge tubes set in a .rotary shaking bath maintained at 40°C. The assay tubes contained mixed micelles and buffer; the reaction was initiated by the addition of enzyme. Reactions were quenched by the addition of 2 ml of methanol followed by vortexing and rapid cooling of the tubes in a dry iceacetone bath. Zero-time controls were included in each experiment. The phosphorylcholine produced by the reaction was isolated by extraction and its amount determined by a phosphorus analysis. To each assay tube, 4 ml of chloroform was added, the tube was vortexed and shaken for 2 min and then centrifuged (500 rpm, room temperature, 2 min). Aliquots of the upper methanol/water layers were transferred to acidwashed ignition tubes and analyzed for phosphorus by a modification of the method of Turner and Rouser (7). The samples were brought to dryness in an aluminum heating block maintained at 170°C. To each tube was added 0.65 ml of 70% perchloric acid, and heating was continued an additional 25 min. The tubes were cooled, and 4.3 ml of a solution containing ammonium molybdate and ascorbic acid so that the final concentrations were 0.25 and l%, respectively, were added to the tubes. A marble was placed on each tube and the covered tubes were then placed in a boiling-water bath for 5 min. The amount of phosphorus present was determined on a Perkin-Elmer double beam spectrophotometer at 666 nm with appropriate blanks and standards. A
TO WARD
605
MICELLES
unit of activity is defined as the amount of enzyme that produces 1 pmol/min of phosphorylcholine. The average of duplicate analyses is reported. RESULTS
AND
“Surface-as-Cofactor”
DISCUSSION
Theory
The activity of phospholipase C toward substrate in mixed micelles was determined according to the general theory and methodology developed for the analysis of lipolytic enzymes which was applied previously to phospholipase A, (1). This theory assumes that the enzyme, E, first associates with a surface site, A, as shown in Eq. [ll to form an enzyme-surface complex, EA: E+A
k, =
EA
111
k-1 When the substrate molecule, B, which makes up part of the surface, A, binds in the active site of E, the Michaelis complex, EAB forms. Upon catalysis, the products, Q, are formed and EA is regenerated as indicated in Eq. [21: EA+B
k, Z$ km,
EAB
k, -+ EA + Q
121
In the case of phospholipase C, Q represents two products: phosphorylcholine and diglyceride. If (A) is the concentration of surface sites capable of associating with an enzyme molecule and (B) is the concentration of lipid substrate in the two-dimensional surface, then the rate equation derived for this scheme is given in Eq. [31:
VW (B) ’ = KsAKmB + KmB(A)
+ (A) (B)
[31
KS* = k-,/k, K mB = (k-,
+ kR)/kZ
According to this scheme, the surface, A, behaves kinetically as a cofactor for the reaction, and it emerges essentially unchanged at the end of the reaction. In the case of the mixed micelle system, composed of the nonionic surfactant Triton X-100 and phospholipid, the surface is in the form of mixed micelles which have been previously characterized in detail (8,
606
EATON
AND
9). Since each molecule contributes to the total surface of the aggregate, the surface term, (A), is related to the sum of the concentrations of T&on, T, and phospholipid, P. (B), the term that expresses the concentration of substrate in that surface, is related to the mole fraction of phospholipid present, P/(T + P). The mixed micelle system allows the independent variation of each of these terms. However, in order to apply the above concepts, four assumptions are necessary. These assumptions have been discussed in detail elsewhere (1) and are summarized here. i. The average surface area occupied by a phospholipid molecule and the average surface area occupied by a Triton molecule are constant as (A) is varied at constant (B). ii. The size of the surface (micelle) segment to which one enzyme molecule is bound is constant under all experimental conditions. It is defined as n in square centimeters per mole. The value of n is not known, but certain assumptions (1) allow the calculation of a minimum value of n. For phospholipase AZ, n = 3.6 x 1O’O cm2 mol-’ . A similar calculation of the minimum value of n for phospholipase C gives n = 6.7 x lOlo cm2 mol-’ (10). iii. In a mixed micelle, the average surface area occupied by a phospholipid molecule and that occupied by a Triton molecule are approximately the same. This area is defined by x in square centimeters per mole. Although its value is not known precisely, for the purposes of calculation x is assumed to be 5.1 x log cm2 mol-’ . iv. As the concentration of phospholipid in the surface, (B), is increased, while the sum of the phospholipid and Triton concentrations is kept constant, the average surface area occupied by a phospholipid molecule and a Triton molecule is decreased. This introduces a proportionality factor, s, into the definitions of (A) and (B), and s is the fractional decrease in x at a particular value of P/(P + T). The value of s is not known and would be difficult to determine experimentally. We found with phospholipase A, that, ifs was assumed to vary linearly
DENNIS
between 1.0 and 0.5 as P/(P + T) varies between 0 and 0.33, reasonable fits of the experimental data to theory were obtained. In the experiments reported here, mixed micelles of the same range of compositions, P/(P + T), were employed as with phospholipase A, and the same variation in s with P/(P + T) is assumed. Other assumptions about s are considered elsewhere (10). It should be noted that in our previous work (11, assumption iv was stated differently, and a modification of the assumption was presented in the text. Here, for simplicity, we introduce the proportionality factor, s, and begin with an assumption about its value that was suggested by physical studies on mixed micelles and curve fitting in our previous work. The value of (A) is given in Eq. [41 and the value of (B) in Eq. [5]: (A) = ;
(P + T) = x As n
[41
where A” = s(P + T)
[51
where n, s, n, P, and T are defined above. Equation [3] can be rewritten in a different algebraic form and in terms ofA”, B”, X, and n as shown in Eq. [61.
As and B” for any kinetic point can be determined from the concentrations of P and T and the value of s. Equation [6] shows that if initial rate studies are conducted in which the concentration of phospholipid in the surface, B”, is held constant while the total concentration of surface binding sites, A”, is varied, then plots of l/v versus l/A” at constant B” should give linear plots. These lines should intersect at a point with the coordinates -xlnKsA, l/V. Beplots of the slopes and intercepts would also allow the determination of V, nKsA/x, and xKmB. Specifi-
PHOSPHOLIPASE
C ACTION
TOWARD
607
MICELLES
tally, a plot of the l/u intercept for each value of B” plotted as a function of l/W should be linear, and the intercept of this line on the l/u intercept axis should be l/ V, and on the l/B” axis it should be -11 xK,,~. A plot of the slope of each line in the original graph against l/B” should be linear and pass through the origin; the slope of the resulting line should be nK,*KmBIV so that the value of nK,“/x can be determined from this replot and the values of xK,nB and V. These considerations lead to the determination of the kinetic constants and V defined in Eq. [3] in terms K*, K,rtB, of the additional constants x and n. It is not necessary to know the values ofx and n in order to analyze the kinetic data since they are constants and simply change the units of KS* and KmB. However, with values for x and n, KS* and K,B can be obtained in meaningful units for this system.
lent metal ion required for this enzyme (3, 11, 12). All assays were conducted at 4o”C, which was well below the temperature optimum for the enzyme and is the same temperature employed in previous studies with phospholipase A,. Kinetic experiments outlined above were conducted and the resulting activities are shown in Fig. 2 plotted in terms of
Kinetic
FIG. 1. pH variation of phospholipase C activity. The assay mix consisted of 1.2 Kg of phospholipase C, 40 mM Triton X-100, 20 mM phosphatidylcholine, and 50 mM buffer (a mixture of succinic acid, maleic acid, and Tris adjusted to the designated pH with NaOH and then brought to uniform ionic strength with NaCl). Incubations were conducted for 20 min at 40°C in a total volume of 0.5 ml and the activity analyzed as described in Experimental Procedure.
Results
The pH-rate profile of the enzyme is given in Fig. 1. Subsequent experiments were conducted at pH 6.4. At that pH with 50 mM sodium maleate buffer, the activity was linear with time and protein, and it was not stimulated by the addition of Ca2+, although it is not clear if there is a diva-
1 ,‘,
P-l
1 I q8
I I I 58 68 PH
I I 78
FIG. 2. Activity of phospholipase C toward phosphatidylcholine in mixed micelles with Triton X-100. The assay mix consisted of 0.6 pg of phospholipase C, phosphatidylcholine and Triton X-100 at the designated concentrations, and 50 mM maleate buffer, pH 6.4, in a total volume of 2.0 ml. Incubations were conducted for 5 min at 40°C. The activity was determined as described in Experimental Procedure. Assumptions about the value ofs were: when T/P = 12:1, s = 1.0; T/P = lO:l, s = 0.9; T/P = 8:1, s = 0.8; T/P = 6:1, s = 0.7; T/P = 4:1, s = 0.6; and T/P = 2:1, s = 0.5. The data are plotted as l/u versus l/A” at several values of B”: 0.077 (A), 0.100 (01, 0.139 (01, 0.207 (A), 0.332 (m), and 0.666 (0).
I BB
608
EATON
AND
A” and B”. Replots of the slopes and l/u intercepts versus l/B” are shown in Fig. 3. The approximate values of V, nKsA/x, and xK,nB, along with KsA and K,,tB using the values of x and n stated earlier, are given in Table I along with these constants for phospholipase A, and phosphatidylserine decarboxylase . Action of Phospholytic Mixed Mice&s
Enzymes
toward
It is interesting to note that the V’s for the two extracellular phospholipases acting on phosphatidylcholine in mixed micelles are similar, although this may be coincidence, while the V for the biosynthetic enzyme phosphatidylserine decarboxylase is about 100 times slower. The value of nKsA/x for phospholipase C is somewhat larger than for phospholipase A, but is in a similar range to that found for the decarboxylase. The value ofKSA for
(IX,
FIG. 3. Replot of l/v intercepts obtained from Fig. 2, versus
(0) l/B”.
and
slopes
TABLE KINETIC
CONSTANTS
FOR THE ACTION
Enzyme
V (pm01 min-’
DENNIS
all three enzymes appears more similar, but, until the value of n is known precisely cannot for each enzyme, comparisons really be made. The value of xK,* is between 0.5 and 1.0 (moles of phospholipid to total moles of Triton plus phospholipid in the mixed micelles) for both phospholipases. Because mixed micelles of phosphatidylcholine and Triton X-100 are not formed above values of B” of 0.33 (with s = 1.0) (9) and the values of s at various values of T/P between 2:l and 12:l are not known accurately, the value of xK,,B cannot be determined precisely at this time. It is clear, however, that saturation of the active site, if possible at all, requires a very high concentration of phospholipid in the mixed micelle, (B). In contrast, it appears that the active site of the decarboxylase can be saturated with phosphatidylserine at a lower substrate concentration in the mixed micelle (xK,* = 0.03 in units of moles of phospholipid to total moles of Triton plus phospholipid). The xKmB values can be converted to KmB values which are in the correct units for a two-dimensional surface, moles per square centimeter. Thus, the KmB of 0.06 x lo-lo mol crnm2 for the decarboxylase is significantly lower than the K,* of l-2 x lo-lo mol cm-’ for the two phospholipases. The kinetic scheme developed for lipolytic enzymes has been applied to three enzymes: phospholipase A, (11, phosphatidylserine decarboxylase (2), and here to phospholipase C. Each of these enzymes acts on a different area of the phospholipid molecule, but in each case the phospholipid is part of a two-dimensional surface I
OF LIP~LYTIC ENZYMES AND SURFACTANT mg-‘)
nK,Alx (KIM)
TOWARD KSA bM)
MIXED
MICELLES
XKmB (mol fraction
P/
OF PHOSPHOLIPID
blol
KlliB cm-*)
IP+Tl)
Phospholipase Phospholipase Phosphatidylserine carboxylase
C A, (1) de-
2 x 103 4 x 103 25
30 4 40
2 0.5 0.3
0.5-l 0.5-l 0.03
l-2 l-2
x 10-10 x 10-10 6 x lo-l2
(2) n
” In Ref. (2) because T/P ratios of 6:l to 128:l were utilized, it was assumed that s = 1 at all values of T/P and for simplicity the symbols KsA and K,nB were used for nKSAIx and xKmB defined here. For the calculation ofK,* here, it was assumed that one decarboxylase molecule binds to one micelle composed of 150 molecules of surfactant and phosphatidylserine giving a value of n = 7.7 x 10” cm2 mol-’ (13).
PHOSPHOLIPASE
C ACTION
rather than existing in bulk solution. The use of the mixed micelle system allows the independent variation of the amount of surface present, A, and the amount of substrate in that surface, B. The “surface-ascofactor” kinetic scheme and equation allow the evaluation of V, the enzyme-surface association constant KS*, and the Michaelis constant KrfiB, as separate entities, which has not been possible previously. The application of this scheme to phospholipase C reported here suggests that this approach may be generally applicable to the consideration of the action of lipolytic enzymes. ACKNOWLEDGMENTS We wish to thank Mr. Raymond A. Deems for helpful discussions. Financial support was provided by a grant from the National Institutes of Health (GM 20,501). B.R.E. was a predoctoral trainee ofthe National Institutes of Health (GM-1045). REFERENCES 1. DEEMS, R. A., EATON, B. R., AND DENNIS, (1975) J. Biol. Chem. 250, 9013-9020.
E. A.
TOWARD
MICELLES
609
2. WARNER, T. G., AND DENNIS, E. A. (1975) J. Biol. Chem. 290, 8004-8009. P., 3. ZWAAL, R. F. A., ROELOFSEN, B., CONFURIUS, AND VAN DEENEN, L. L. M. (1971) Biochim. Biophys. Acta 233, 474-479. 4. DENNIS, E. A. (197315. Lipid Res. 14.152-159. 5. DITTMER, J. C., AND LESTER, R. L. (1964) J. Lipid Res. 5, 126-127. 6 SINGLETON, W. S., GRAY, M. S., BROWN, M. L., AND WHITE, J. L. (1965) J. Amer. Oil Chem. Sot. 42, 53-56. 7. TURNER, J. D., AND ROUSER, G. (1970) Anal. Biochem. 38, 423-436. a. DENNIS, E. A. (1974) J. Supramol. Struct. 2, 686-694. 9. DENNIS, E. A. (1974) Arch. Biochem. Biophys. 165, 764-773. 10. B. R. EATON (1975) Ph.D. Dissertation, University of California at San Diego, La Jolla, Calif.; (1976) Dissertat. Abstr. 36, 3351B. 11. OTNAESS, A.-B., PRYDZ, H., BJBRKLID, E., AND BERRE, A. (1972) Eur. J. Biochem. 27, 23% 243. 12. OTTOLENGHI, A. C. (1965) Biochim. Biophys. Acta 106, 510-518. 13. T. G. WARNER (1975) Ph.D. Dissertation, University of California at San Diego, La Jolla, Calif., (1976) Dissertat. Abstr. 36, 3939B3940B.
