Biochemistry 1980, 19, 1621-1625 Kluetz, M. D., & Schmidt, P. G. (1 977a) Biochemistry 16, 5 191-5 199. Kluetz, M. D., & Schmidt, P. G. (1977b) Biochem. Biophys. Res. Commun. 76, 40-45. Makinen, M. W., & Fink, A. L. (1977) Annu. Rev. Biophys. Bioeng. 6, 301-343.
1621
Mondovi, B., Costa, M. T., Finazzi-Agro, A., & Rotilio, G. (1967) Arch. Biochem. Biophys. 119, 373-381. NylBn, U., & Szybek, P. (1974) Acta Chem. Scund., Ser. B 28, 1153-1 160. Werle, E., & von Pechmann, E. (1949) Justus Liebigs Ann. Chem. 562, 44-60.
Interaction of Phospholipase A2 from Cobra Venom with Cibacron Blue F3GAt Roland E. Barden,$ Paul L. Darke, Raymond A. Deems, and Edward A. Dennis*
ABSTRACT:
Cibacron Blue F3GA has been suggested as a site-specific probe for dinucleotide binding sites and the “dinucleotide fold” in proteins [Stellwagen, E. (1977) Acc. Chem. Res. 10, 921. However, we have found that cobra venom phospholipase A2 (Nuju nuju nuju) reversibly binds the Cibacron Blue dye with a Kd N 2 pM as measured by difference spectroscopy. NADH and NAD’ will not displace the dye from phospholipase A2, but the water-soluble phospholipid dihexanoylphosphatidylcholinewill. The dye inhibits catalysis, and a double-reciprocal plot of inhibition as a function of dye concentration is linear and yields a Kirr, 3.5 pM. p-Bromophenacyl bromide chemically modifies the active site of phospholipase A2, and the Cibacron dye inhibits this
process with an apparent Kd N 7 pM. When the dye-enzyme interaction is monitored at low protein concentrations (less than 2 pM), the difference spectral titrations, inhibition of catalysis, and prevention of chemical modification by p-bromophenacyl bromide all suggest that the dye interacts with a single type of site on the phospholipase A2. However, at higher protein concentrations where cobra venom phospholipase A2 is known to exist as dimers and higher order oligomers, the difference spectra show the appearance of new types of binding sites. These data demonstrate that Cibacron Blue F3GA is not a reliable, specific probe for the dinucleotide fold in proteins and that the dye is a useful probe for exploring the dimerization of phospholipase A2 and phospholipid binding to the enzyme.
s t e l l w a g e n (1977) has reported that many dehydrogenases and kinases bind tightly to immobilized Cibacron Blue F3GA dye. According to Stellwagen (1977), this interaction is sufficiently specific that it constitutes a diagnostic test for a supersecondary structural feature in enzymes known as the “dinucleotide fold”. The reaction catalyzed by phospholipase A2 (EC 1.1.1.4) (Deems & Dennis, 1975) does not involve dinucleotides, mononucleotides, or sugar phosphates, and it is highly unlikely that this enzyme contains a dinucleotide fold. Yet, we have found that cobra venom phospholipase A2 binds quite tightly to Blue Dextran, a conjugate of Cibacron Blue F3GA and dextran used as a void volume marker in gel filtration experiments. We report here an investigation of the binding of the free dye Cibacron Blue F3GA to cobra venom phospholipase A2 by utilizing primarily spectroscopic methods. These studies suggest that the dye binding site on phospholipase A2 includes the active site of the enzyme and that this provides a somewhat different environment for the dye than does the dinucleotide fold of dehydrogenases and kinases.
Phospholipase A2 was purified according to the procedure of Deems & Dennis (1975, 1980). The protein concentration was determined by the method of Lowry et al. (1951). Enzymatic activity was measured by the pH stat procedure carried out at 40 OC using mixed micelles of Triton X-100 and egg phosphatidylcholine as substrate (Dennis, 1973). The egg phosphatidylcholine was purified by the method of Singleton et al. (1965). Dihexanoylphosphatidylcholine’and octanoyland myristoyllysophosphatidylcholinewere purchased from Calbiochem. All other chemicals were reagent grade. Cibacron Blue F3GA was a generous gift from Dr. H. Bosshard of Ciba-Geigy, Basel, Switzerland. Weber et al. (1979) have recently reported that commercial samples of Cibacron Blue F3GA contain contaminants, one or more of which may irreversibly inhibit an enzyme. The difference spectroscopy experiments described here were conducted with dye that was purified by chromatography on silica gel (60-200 mesh) as described by Weber et al. (1979). The binding of Cibacron Blue F3GA to the enzyme was detected by measuring difference spectra in the visible region (450-800 nm) following the procedures of Thompson & Stellwagen (1976). Difference spectra were recorded at room temperature with a Cary 219 double-beam spectrophotometer using 1-cm path length cells. When low protein concentrations were used, a Cary 118 double-beam spectrophotometer and 10-cm path length cells were employed. The dye concentration was determined spectrophotometrically at 610 nm by using a molar
Experimental Procedure Lyophilized cobra venom, Nuju nuja naju (Pakistan), Lot NNP8STLZ, was obtained from the Miami Serpentarium. From the Department of Chemistry, University of California at San Diego, La Jolla, California 92093. Received November 2I, 1979. This research was supported by National Science Foundation Grant PCM 76-21552 and National Institutes of Health Grant GM-20,501. *Visiting scientist, Spring Semester, 1979. Permanent address: Departments of Chemistry and Biochemistry, University of Wyoming, Laramie, WY 82071. Recipient of a National Institutes of Health Research Career Development Award (GM-00,246).
0006-2960/80/0419-1621$01.00/0
I Abbreviations used: diacylphosphatidylcholine, 1,2-diacyl-snglycerol-3-phosphorylcholine; acyllysophosphatidylcholine, 1-acyl-snglycerol-3-phosphorylcholine.
0 1980 American Chemical Society
1622
BIOCHEMISTRY
BARDEN, DARKE, DEEMS, A N D DENNIS
,
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1: Typical difference spectra of Cibacron Blue F3GA dye upon binding to phospholipase A2. The sample cuvette contained 30 pM phospholipase A2 while both the sample and the reference cuvettes contained 10 mM Tris-HCI buffer at pH 8.0 and the following dye concentrations: (A) 2.26, (B) 5.94, (C) 12.26, (D) 28.1 pM. FIGURE
FIGURE 2: Titration of phospholipase A2 by Cibacron Blue F3GA dye at X = 676 nm. Both the sample and reference cuvettes contained 10 mM Tris-HCI buffer, pH 8.0, and the sample cuvette also contained 43 pM enzyme. Equal amounts of dye were sequentially added to both cuvettes.
absorption coefficient of 13 600 (Thompson & Stellwagen, 1976). Affi-Gel Blue, 100-200 mesh, was obtained from Bio-Rad Laboratories. The chemical modification of phospholipase A2 with p bromophenacyl bromide was performed in 38 mM Tris-HC1, pH 8.0, as described by Roberts et al. (1977b), except that the reagent was added in methanol and the incubated sample was 20% methanol by volume. Results Analysis of the Dye-Enzyme Interaction by Difference Spectroscopy: Evidence for Different Types of Dye Binding Sites. The absorption spectrum of Cibacron Blue F3GA dye undergoes a red shift when the dye is transferred from a polar environment to a less polar one. Thompson & Stellwagen (1976) have shown that a comparable shift occurs when the dye is bound in the dinucleotide fold of various enzymes. The difference spectra shown in Figure 1 indicate that a similar shift occurs when the dye is added to solutions containing phospholipase A2. While these spectra are similar to those found when the dye is bound in a dinucleotide fold (Thompson & Stellwagen, 1976; Kumar & Krakow, 1977), there are two marked differences. The absorption of the negative peak in the 550-590-nm region is much stronger in the phospholipase A2 spectra and the isosbestic point is shifted from -590 nm in the dinucleotide fold spectra to 645 nm in those of phospholipase AZ. However, the spectra in Figure 1 are similar to the one reported for the complex of dye and yeast glucose-6-phosphate dehydrogenase (Chambers & Dunlap, 1979). For characterization of the dye binding process, phospholipase A2 was titrated with increasing amounts of dye and the intensity of the 675-nm peak of each difference spectrum was plotted vs. the dye concentration as shown in figure 2. Between 0 and -25 pM dye, the curve appears to follow a simple saturation curve; that is, the data appear to be consistent with the titration of a single class of sites that exhibit a relatively strong affinity for the dye. However, at dye concentrations above -35 pM the curve continues to increase slowly, suggesting that a second class of sites with a much weaker affinity for the dye is being titrated. It is clear that the second class of sites is not saturated by the highest dye concentration tested (Figure 2). Binding data are commonly analyzed with a Scatchard plot. This analysis requires that the measured changes in absorbance (Figure 2) be converted to moles of dye bound per mole of enzyme. Such a calculation requires a value for t, the molar absorption coefficient of the change in absorbance in the difference spectra. If the enzyme could be saturated with dye,
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3: Typical difference spectra of dye binding to aggregated forms of phospholipase A% The protein concentration was 340 pM in the sample cuvette, and the buffer was the same as that in Figure 1. The dye concentration in both cuvettes was (A) 9.25 and (B) 134 FIGURE
WM. the molar absorption coefficient would be obtained from the is the change in absorequation t = A675/(nP,/), where bance, n is the number of dye binding sites per protein molecule, P, is the total protein concentration, and l is the path length of the cell. Since the enzyme was not saturated with dye and n was not known, the molar absorption coefficient could not be calculated from the data in Figure 2. In principle, an alternative method of calculating the desired molar absorption coefficient is available. If all of the dye present in the solution is bound to the enzyme, the molar absorption coefficient is given by the equation e = A675/(Dr), where D is the total dye concentration. No assumptions about the number of molecules bound per molecule of protein are required. This method requires high protein concentrations and a very low dissociation constant for the dye. Both requirements were met in an experiment in which a 5 mg mL-' solution of phospholipase A2 was titrated with the dye. The difference spectrum obtained from this experiment is shown as spectrum A in Figure 3. This spectrum differs significantly from those obtained at lower protein concentrations (0.4 mg mL-', Figure 1). The ratio of the intensity of the Assopeak to the A67speak is -0.7, which is much lower than the analogous ratio of 1.3 for spectra D in Figure 1. There is also a flat region between 600 and 625 nm. As the dye concentration was increased above 60 pM, the difference spectra became more and more like those found at lower protein concentrations. Compare spectrum B of Figure 3 with the spectra in Figure 1. Clearly, the bound dye is sensing two different environments in the more concentrated protein solution. Spectrum A of Figure 3 reflects the presence of a different type of binding site with a very strong affinity for the dye. The concentration of this new site is apparently a small fraction of the protein concentration (340 pM in monomer units), since these sites appeared to be saturated when the dye concentration reached
VOL. 19, NO. 8 , 1980
B L U E D Y E B I N D I N G BY P H O S P H O L I P A S E A 2
a level that was -2096 of the protein concentration in this experiment. Evidently, both spectrum B of Figure 3, and the spectra in Figure 1 arise from the same type of binding site, and under the conditions investigated this site appears to be the predominant dye binding site on phospholipase A2. The existence of two types of dye binding sites prevented the calculation of the molar absorption coefficient from the data obtained at high protein concentrations. At a concentration of 5 mg mL-', phospholipase A2 exists as a mixture of dimers and higher order aggregates (Deems & Dennis, 1975). Presumably, the different types of dye binding sites detected in the experiments cited above have their origin in different states of aggregation. However, it was also possible that there was a dye binding impurity in the phospholipase Az preparation. Affinity Chromatography of Phospholipase A2 on Affi-Gel Blue. Previous studies indicated that cobra venom phosphclipase A2 was homogeneous by the usual biochemical criteria (Deems & Dennis, 1975, 1980); thus, the presence of a dye binding impurity seemed to be a remote possibility. Nonetheless, a sample of the enzyme was subjected to chromatography on an Affi-Gel Blue column (1 X 3 cm) (Thompson et al., 1975) to further check the purity. Affi-Gel Blue has the dye Cibacron Blue F3GA covalently attached to a cross-linked agarose support. Phospholipase A2, sp act. = 600 pmol m i d mg-', was loaded in 50 mM sodium phosphate, pH 6.0, and the column was washed with 2 column volumes of this buffer (no protein was recovered). Subsequently, two protein peaks were eluted. Peak I was eluted with 50 mM ammonium bicarbonate, pH 8.0, and it contained 20% of the protein (sp act. -200 pmol min-' mg-'). Peak I1 was eluted with 50 mM ammonium carbonate, pH 10.5, and it contained -6096 of the protein (sp act. -900 pmol m i d mg-I). About 2096 of the protein was not recovered.2 Thus, it appeared that a form of phospholipase A2 (peak I) with both a decreased catalytic capability and a lesser affinity for the dye could be separated from the enzyme preparation. Yet, when titration experiments were conducted with the higher activity peak (peak 11),the results were identical with those obtained in the experiment with the original protein preparation. The second type of dye binding site detected at high protein concentrations (spectrum A, Figure 3) was therefore not due to an impurity. This new type of site appears to arise solely as a function of protein concentration and the resultant changes in aggregation. A Single Type of Binding Site at Very Dilute Enzyme Concentration. If the heterogeneity in dye binding sites is due to aggregation of phospholipase A2, then titration experiments performed under conditions where a single enzyme form exists should exhibit simple binding curves. Phospholipase A2 exists predominantly as a monomer only at concentrations below -0.03 mg mL-' (2 pM) (Deems & Dennis, 1975). Dye titrations at these low protein concentrations had to be performed in cuvettes with 10-cm path lengths. The difference spectra from these experiments were identical with those shown in Figure 1, except that in this case the plot of absorbance vs. dye concentration appears to follow a simple saturation curve (Figure 4A). The data in Figure 4 were obtained with enzyme purified on Affi-Gel Blue. However, when enzyme that had not been chromatographed on Affi-Gel Blue was titrated at these low protein concentrations, the results were identical with those shown in Figure 4. Thus, removal of the enzyme form with Other preparations of the enzyme have yielded greater total recoveries and a smaller proportion of peak I.
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FIGURE4: Titration of monomeric phospholipase A, with Cibacron Blue F3GA dye at X = 670 nm. The conditions were the same as those in Figure 2, except that the enzyme was first passed through Affi-Gel (see text), the protein concentration in the sample cuvette was only 1.4 pM,and cuvettes with IO-cm light paths were used. (A) Absorbance vs. dye concentration. (B) Scatchard plot of the data, assuming 1 binding site/mol of enzyme.
the decreased catalytic capability had no detectable effect on the results of the titration experiments. Since the enzyme was saturated as the dye concentration was raised, the data in Figure 4A were analyzed with a Scatchard plot. The value of n was assumed to be 1. Thus, the molar absorption coefficient is given by the equation E = A670/(Ptl)at saturation, and i j , the fraction of protein with dye bound, is given by the equation i j =A670/(tPtl).In Figure 4B, the Scatchard plot for the titration of a 1.4 pM solution of phospholipase A2 is shown. A Kd of 2 pM was obtained, and the linearity of the plot strongly suggests the presence of a single type of binding site. Overall, the analyses of the dye-enzyme interaction by difference spectroscopy indicate that there is one predominant type of dye binding site at all protein concentrations tested. When phospholipase A2 is very dilute so that it exists in the monomeric state, only this one type of dye binding site exists (Figure 4). However, at higher protein concentrations phospholipase A2 aggregates into dimers and higher order forms, and this behavior correlates closely with the appearance of new types of binding sites (Figures 2 and 3). Titration of the Dye-Enzyme Complex with Competing Ligands. Spectral difference titration experiments were conducted to determine whether certain ligands could displace Cibacron Blue F3GA from the dye-enzyme complex (Thompson & Stellwagen, 1976; Chambers & Dunlap, 1979). Initially, phospholipase A2 (10 pM, 0.135 mg mL-') and dye (10 pM) were added to the sample cuvette and dye only was added to the reference cuvette. The buffer was 10 mM Tris-HC1, pH 8.0. Difference spectra were obtained sequentially after the addition to both cuvettes of equal, small volumes of a pH 8.0 solution containing the ligand under investigation. Neither NADH nor NAD+ (up to 1.4 mM) displaced the dye. Difference spectra obtained in the presence of high levels of enzyme-Ba2+ complex (8 mM Ba2+, which is 20 times &) show that the dye binds to the enzyme-BaZf complex, although the presence of Ba2+appears to decrease slightly the affinity of phospholipase A2 for the dye. Also, the difference spectra from 500 to 800 nm for the phospholiphase A2-Ba2+-dye
1624
B I oc H E M I S T R Y
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BARDEN, DARKE, DEEMS, A N D DENNIS
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Reversible inhibition of phospholipase A2by Cibacron Blue F3GA. Catalytic activity was measured as a function of dye concentration by using 0.35 pg of enzyme per assay. Inset: doubleFIGURE5 :
reciprocal plot of the data.
complex (not shown) have a slightly different shape than those shown in Figure 1. Cibacron Blue F3GA shows a pronounced spectral change in the presence of Triton X-100 micelles. This is an expected result since following the spectral change in a “reporter” dye is a common method of determining a critical micelle concentration (Mukerjee & Mysels, 1970). As a consequence of the spectral change exhibited by the dye in the presence of micelles, it was difficult to assess the ability of micellized substrates (phospholipid) and products (lysophospholipid, fatty acid) to displace the dye from the dye-enzyme complex. In studies with myristoyllysophosphatidylcholine,no displacement of dye was detected at concentrations up to 0.05 mM (at which point micelles begin to form). Octanoyllysophosphatidylcholine, in the presence or absence of 1.7 mM Ba2+,displaced less than 20% of the dye at concentrations up to 7.7 mM. A synthetic, short-chain phospholipid, dihexanoylphosphatidylcholine, did displace the dye from the dye-enzyme complex. The concentration of this phospholipid required to displace 50% of the dye was 3.7 mM without BaZ+and 1.4 mM with 8 mM Ba2+. Since previous studies have shown that Ca2+or BaZf must be present before phospholipase A2 can bind to micellized substrate (Roberts et al., 1977a), the enhanced effect of dihexanoylphosphatidylcholinein the presence of Ba2+ is an expected result. Effect of Cibacron Blue F3GA on Catalytic Activity. When added to the standard assay mixture, Cibacron Blue F3GA is a potent reversible inhibitor of phospholipase A2 (Figure 5). An apparent Ki of 3.5 pM was obtained, which shows that the dye binds tightly to the enzyme. The apparent Ki measured in this experiment may represent an upper limit, because in the assay mix the dye partitions between the bulk solution and the mixed micelles of phosphatidylcholine and Triton X-100, and it is possible that one of the “pools” of dye does not contribute to the observed inhibition. The linear double-reciprocal plot shown in Figure 5 suggests that only one dye molecule interacts with each active site. Cibacron Blue F3GA Protection against Irreversible Inhibition by p-Bromophenacyl Bromide. Phospholipase A2 can be chemically modified and fully inactivated by treatment with p-bromophenacyl bromide (Roberts et al., 1977b). The reagent is site specific and a histidine is derivatized. A detailed analysis of the kinetics of irreversible inactivation of phospholipase A2 by p-bromophenacyl bromide was performed, following the method of Kitz & Wilson (1962). The rate of inactivation followed pseudo-first-order kinetics at each reagent concentration tested: 0.05, 0.1, 0.2, and 0.4 mM (data not shown). A secondary plot of kobsd-‘vs. Ip-bromophenacyl bromide]-’ indicated that the chemical modification was a simple bimolecular process, since the straight line extrapolated through the origin. Thus, there was no indication that a
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HGURE 6: Cibacron Blue F3GA protection of phospholipase A, from irreversible inhibition by pbromophenacyl bromide. Each incubated sample (0.5 mL) contained 0.02 mM p-bromophenacyl bromide, 11 pg of enzyme, and the following dye concentrations: 9 mM (m), 6 mM (A),3 mM ( O ) , and none (0). Inset: plot of 2.3(kobd)-lvs. [dye] used to determine the apparent Kdfor Cibacron Blue F3GA.
reversible complex of enzyme and reagent formed prior to the chemical modification step(s). Apparently, the modification of the enzyme proceeds by a direct chemical reaction between p-bromophenacyl bromide and an unusually reactive histidine in the active site. This type of active-site modification has been observed frequently in the past with serine proteases and esterases, e.g., the modification of acetylcholinesterase with methanesulfonyl fluoride (Kitz & Wilson, 1962). When Cibacron Blue F3GA was added to an incubated solution of enzyme and p-bromophenacyl bromide, protection against irreversible inhibition was observed (Figure 6). Further analysis of these data (inset, Figure 6) reveals that k--’ is a linear function of the dye concentration,as predicted by a simple model which assumes that the reaction between enzyme and pbromophenacyl bromide is a simple bimolecular process and that the reversible binding of one dye molecule per molecule of enzyme protects the enzyme from inactivation. An apparent Kd of 7 pM is estimated for the dye from this analysis (inset, Figure 6). Interestingly, if the dye is added to enzyme which has already been chemically modified with p-bromophenacyl bromide, spectra indicative of complex formation are still observed. Increasing the dye concentration results in an increase in absorbance at 670 nm. An estimate of Kd in this case is 8 pM. Discussion Cibacron Blue F3GA binds to cobra venom phospholipase A,, and this observation weakens the argument of Stellwagen (1977) that Cibacron Blue F3GA is a specific probe for a dinucleotide fold in proteins. The dinucleotide fold is composed of four to six parallel /3 strands connected by helical loops. It takes a continuous polypeptide sequence of 150 residues to construct a dinucleotide fold (Stellwagen, 1977). Phospholipase A2 from cobra venom cannot contain the complete dinucleotide fold since it is a very small enzyme ( M , 13 500) containing only 120 amino acid residues (A. Jarvis and E. A. Dennis, unpublished experiments; Deems & Dennis, 1980). In addition, the nucleotides tested here do not perturb dye binding to phospholipase A2. Other groups have also disputed the contention that Cibacron Blue F3GA is highly specific for the dinucleotide fold. Beissner & Rudolph (1978) have shown that this dye binds to dihydrofolate reductase and is a competitive inhibitor with respect to dihydrofolate but noncompetitive with respect to NADP’. Furthermore, Beissner & Rudolph (1978) and Ashton & Polya (1978) have shown that a variety of sulfonated aromatic dyes bind competitively at nucleotide sites, demonstrating that Cibacron Blue F3GA is
-
-
BLUE DYE B I N D I N G BY PHOSPHOLIPASE A2
not a unique nonnucleotide ligand for the dinucleotide fold. The Kd for the dye-phospholipase A2 complex (-2 pM) is comparable to Kd values reported for some kinases and dehydrogenases (Thompson & Stellwagen, 1976), indicating that the interaction between phospholipase A2 and the dye is as favorable. Beissner & Rudolph (1 978) have argued that binding of the dye and analogues to dinucleotide binding sites is in part due to hydrophobic interactions. Thus, the lack of specificity for a particular protein structure, the so-called dinucleotide fold, is clearly and unambiguously revealed in the binding of Cibacron Blue F3GA to phospholipase A2, an enzyme known to interact with hydrophobic ligands. Our studies suggest that new types of dye binding sites appear as the enzyme concentration is increased and oligomers are formed (Figures 1 and 3). Although two forms of phospholipase A2 with different affinities for the dye were identified by affinity chromatography on Affi-Gel Blue, removal of the small fraction of protein with a low affinity for the immobilized Cibacron Blue F3GA had no effect on spectral titrations. The dye binding of this low-activity protein apparently was not titrated at dye concentrations tested in this study. Thus, the appearance of more than one dye binding site at high protein concentrations can be attributed to the formation of phospholipase A2 oligomers which have previously been demonstrated to exist at higher protein concentrations (Deems & Dennis, 1975 ) . Several experiments in this study suggest that Cibacron Blue F3GA binds to the active site of phospholipase A2. The competitive inhibition of enzyme catalysis and the ability of the monomeric substrate dihexanoylphosphatidylcholine to displace the dye are data consistent with this idea. The p bromophenacyl bromide modification of phospholipase A2, which is thought to be active-site directed (Roberts et al., 1977b), is also prevented by the reversible binding of Cibacron Blue F3GA, although once the enzyme is covalently modified the dye is still able to bind, making the precise definition of the dye binding site less clear-cut. Recent studies in this laboratory have produced evidence that cobra venom phospholipase A2 may bind two molecules of phospholipid prior to the hydrolytic step (Roberts et al., 1979; Adamich et al., 1979). One molecule of phospholipid appears to act as an activator and it induces the catalytically competent conformation at the catalytic site. This activation process shows a strong preference for a choline-containing phospholipid. The second molecule of phospholipid becomes the substrate for the hydrolytic reaction. In catalyzing this
VOL. 19, NO. 8, 1980
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reaction, the “activated” enzyme apparently shows little preference for the composition of the ionic portion of the substrate phospholipid. Cibacron Blue F3GA can be bound to the p-bromophenacyl bromide modified enzyme, but it is not clear to what extent this represents binding to the activator and/or the catalytic site. The potential of Cibacron Blue F3GA as a probe for the choline-lipid activation of phospholipase A2 and the aggregation state of this enzyme is currently under investigation. References Adamich, M., Roberts, M. F., & Dennis, E. A. (1979) Biochemistry 18, 3308. Ashton, A. R., & Polya, G. M. (1978) Biochem. J. 175, 501. Beissner, R. S., & Rudolph, F. B. (1978) Arch. Biochem. Biophys. 189, 76. Chambers, B. B., & Dunlap, R. B. (1979) J. Biol. Chem. 254, 65 15. Deems, R. A., & Dennis, E. A. (1975) J . Biol. Chem. 250, 9008. Deems, R. A., & Dennis, E. A. (1980) Methods Enzymol. (in press). Dennis, E. A. (1973) J . Lipid Res. 14, 152. Kitz, R., & Wilson, I. B. (1962) J . Biol. Chem. 237, 3245. Kumar, S. A., & Krakow, J. S . (1977) J . Biol. Chem. 252, 5724. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951) J. Biol. Chem. 193, 265. Mukerjee, Po,& Mysels, K. J. (1973) Critical Micelle Concentrations of Aqueous Systems, p 10, US.Government Printing Office, Washington, D.C. Roberts, M. F., Deems, R. A., & Dennis, E. A. (1977a) J . Biol. Chem. 252, 601 1. Roberts, M. F., Deems, R. A., Mincey, T. C., & Dennis, E. A. (1977b) J . Biol. Chem. 252, 2405. Roberts, M. F., Adamich, M., Robson, R. J., & Dennis, E. A. (1979) Biochemistry 18, 3301. Singleton, W. S., Gray, M. S . , Brown, M. L., & White, J. L. (1 965) J . Am. Oil Chem. SOC.42, 53. Stellwagen, E. (1977) Ace. Chem. Res. 10, 92. Thompson, S. T., & Stellwagen, E. (1976) Proc. Natl. Acad. Sei. U.S.A. 73, 361. Thompson, S . T., Cass, K. H., & Stellwagen, E. (1975) Proc. Natl. Acad. Sei. U.S.A. 72, 669. Weber, B. H., Willeford, K., Moe, J. G., & Piszkiewicz, D. (1979) Biochem. Biophys. Res. Commun. 86, 252.
