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in detail to the point where at a given PC concentration, the micelle size distribution is known, and an area per head group can be estimated. Since they can be presented to enzymes as monomers, micelles, or in bilayer structures, short-chain phospholipids are ideal for examining kinetic preferences of lipolytic enzymes. In the future detailed studies of short-chain phospholipids with different head groups should facilitate kinetics with phospholipases which show a preference for anionic substrates.
[10] P h o s p h o l i p a s e A 2 - C a t a l y z e d H y d r o l y s i s o f Vesicles: U s e s o f I n t e r f a c i a l C a t a l y s i s in t h e S c o o t i n g M o d e
By MAHENDRA KUMAR JAIN and MICHAEL H. GELB Introduction
Interfacial catalysis by phospholipase A2 (PLA2) is adequately described by Scheme I.~ As a first step, the enzyme in the aqueous phase (E) binds to the substrate interface, and the bound enzyme (E*) undergoes the catalytic turnover in the interface according to the classic Michaelis-Menten formalism as shown in the box. Thus, the binding of the enzyme to the interface and the binding of a phospholipid substrate to the active site of the bound enzyme (E*) to produce the enzyme-substrate complex (E'S) are distinct steps. This is implicit in the proposal that the enzyme contains an interfacial binding surface that is topologically and functionally distinct from the catalytic site. This kinetic view of interfacial catalysis has been the working hypothesis in several laboratories. 1-5 A critical experimental test of Scheme I has not been possible until recently. 1 In most studies, especially with micelles3,4 and monolayers, 2 kinetic and equilibrium contributions of the E to E* step are not readily discernible. Even in bilayers, many of the anomalies seen in the kinetics of action of PLA 2 on insoluble lipid substrates are due to a significant, yet variable, contribution of the E to E* step to the steady-state enzymatic turnover.l'2 This is illustrated in the following two examples (see Refs. 1 i M. K. Jain and O. G. Berg, Biochim. Biophys. Acta 1002, 127 (1989). 2 R. Verger and G. H. de Haas, Annu. Rev. Biophys. Bioeng. 5, 77 (1976). 3 H. M. Verheij, A. J. Slotboom, and G. H. de Haas, Rev. Physiol. Biochem. Pharmacol. 91, 91 (1981). 4 E. A. Dennis, in "The Enzymes" (P. D. Boyer, ed.), 3rd Ed., Vol. 16, p. 307. Academic Press, New York, 1983. 5 See other contributions to this volume.
METHODS IN ENZYMOLOGY, VOL. 197
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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and 2 for others). First, a lag phase is seen in the reaction progress curve for the action of porcine pancreatic PLA 2 on vesicles of phosphatidylcholine. This can be explained on the basis of the observation that the enzyme has a very weak affinity for vesicles of zwitterionic phospholipids, 6,7 but it binds several orders of magnitude more tightly to anionic vesicles: Initially, with zwitterionic vesicles, most of the enzyme is in the aqueous phase and the reaction velocity is low. As the anionic fatty acid product accumulates in the interface, the reaction progress curve accelerates as more and more of the enzyme partitions into the interface where the lipolysis occurs. 8-~° The second example relates to the inhibition of PLA 2. Most inhibition studies reported in the literature are difficult to interpret because the effects of inhibitors on the E to E* step are not easy to characterize, l~'12 For example, many of the reported inhibitors of PLA 2 work not by binding tightly to the enzyme, but by promoting the desorption of enzyme from the interface. ~2,~3Thus, in most of the assay procedures used for PLA 2 that have been reported, the fundamental features of the enzymology that occur within the interface are "blurred" by the reversible association of the enzyme with the interface, that is, the E to E* step (see Ref. I for a review and Ref. 13 for a resolution of these difficulties). In order to minimize such kinetic complexities of interfacial catalysis, we have developed a methodology 14-~7for studying the action of PLA2 on substrate vesicles in the "scooting mode,"6 in which all of the enzyme is tightly bound to the interface. Here, after the initial binding of the enzyme to the interface, the E to E* step is no longer part of the catalytic turnover within the interface. In the scooting mode, the anomalous effects associated with the E to E* step, such as the latency period, 6,8 the effect of 6 G. C. Upreti and M. K. Jain, J. Membr. Biol. 55, 113 (1980). 7 M. K. Jain, M. R. Egmond, H. M. Verheij, R. J. Apitz-Castro, R. Dijkman, and G. H. de Haas, Biochim. Biophys. Acta 688, 341 (1982). 8 R. J. Apitz-Castro, M. K. Jain, and G. H. de Haas, Biochim. Biophys. Acta 688, 349 (1982). 9 M. K. Jain and D. V. Jahagirdar, Biochim. Biophys. Acta 814, 313 (1985). 10 M. K. Jain, B. Yu, and A. Kozubek, Biochim. Biophys. Acta 980, 23 (1989). t~ M. K. Jain, M. Streb, J. Rogers, and G. H. de Haas, Biochem. Pharmacol. 33, 2541 (1984). 12 M. K. Jain and D. V. Jahagirdar, Biochim. Biophys. Acta 814, 319 (1985). 13 M. K. Jain, W. Yuan, and M. H. Gelb, Biochemistry 28, 4135 (1989). 14 M. K. Jain, J. Rogers, D. V. Jahagirdar, J. F. Marecek, and F. Ramirez, Biochim. Biophys. Acta 860, 435 (1986). 15 M. K. Jain, B. P. Maliwal, G. H. de Haas, and A. J. Slotboom, Biochim. Biophys. Acta 860, 448 (1986). 16 M. K. Jain, J. Rogers, J. F. Marecek, F. Ramirez, and H. Eibl, Biochim. Biophys. Acta 860, 462 (1986). 17 M. K. Jain, G. H. de Haas, J. F. Marecek, and F. Ramirez, Biochim. Biophys. Acta 860, 475 (1986).
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FIG. 1. Schematic representation of interfacial catalysis phospholipase by A2 in the scooting mode. See text for details.
gel-fluid phase transitions, 6 and the activating or inhibiting effects of amphiphilic solutes, 1'12 are no longer observed. We have found that PLA 2hydrolyzes vesicles of anionic phospholipids, such as dimyristoylphosphatidylmethanol (DMPM), in the scooting mode. Here hydrolysis begins without any noticeable latency period (less than 5 sec), and the enzyme undergoes several thousand catalytic turnovers without leaving the interface. This interfacial catalysis in the scooting mode continues until all of the substrate in the outer layer of the enzymecontaining vesicles is hydrolyzed. Throughout the entire reaction progress, the vesicles retain their overall physical integrity. This is illustrated schematically in Fig. 1. The observation that the enzyme undergoes several thousand catalytic cycles while remaining attached to the same vesicle provides the strongest evidence for interfacial catalysis according to the kinetic Scheme I. A quantitative interpretation of the reaction in the
E E SCHEMEI. Interfacialcatalysis by phospholipase A 2.
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scooting mode I requires an appreciation of the structure and molecular dynamics of the vesicles of anionic phospholipids. ~8,19 The substrate used for these studies are small unilamellar vesicles of DMPM containing no additives; however, the scooting phenomenon is observed with vesicles of a number of different phospholipids as long as they contain a critical mole fraction of anionic phospholipid (typically 5-10 mol %). PLA2 binds essentially irreversibly to vesicles of DMPM (see below). Under the conditions used for such studies, neither the enzyme nor the substrate or products of hydrolysis exchange with other vesicles. In addition, complications arising from fusion of vesicles as well as transbilayer movement of phospholipids have been eliminated. In this chapter, we present a detailed description of the basic protocols for the study of PLA2 in the scooting mode. In addition, we emphasize some applications of this unique technique in studies on lipolytic enzymes.
Kinetic Analysis of Phospholipase A 2 in Scooting Mode
Preparation ofDMPM. DMPM can be easily prepared from commercially available dimyristoylphosphatidic acid as follows. Dimyristoylphosphatidic acid disodium salt (200 mg, 0.31 mmol, Avanti Polar Lipids, Birmingham, AL) is suspended in 16 ml of ether containing 1% (v/v) water in an Erlenmeyer flask with a stir bar. Sufficient HC1 to protonate all of the phosphatidic acid sodium salt (0.15 ml of 6 N HCI) is added, and the mixture is stirred vigorously for several minutes until both layers become clear. Stirring is continued at room temperature, and freshly distilled diazomethane in ether is added dropwise until the characteristic yellow color of the diazomethane persists. An alcohol-flee solution of diazomethane is prepared from Diazald (Aldrich, Milwaukee, WI) according to the manufacturer's directions. The mixture is stirred for an additional 30 min. The solution is then filtered through a pad of silica gel (2 × 3 cm) containing a I cm top layer of Celite in a sintered glass funnel. The silica pad is washed with an additional 50 ml of ether, and the combined solutions are transferred to a tared recovery flask and concentrated on a rotary evaporator at reduced pressure and at room temperature. The flask is attached to a vacuum pump for 30 rain to remove traces of solvent. To the oily residue of the phosphate triester (160 nag, 0.29 mmol) is added reagent-grade acetone (5 ml) and anhydrous LiBr (35 rag, 0.4 mmol, Fluka), and the mixture is refluxed with magnetic stirring for 6 hr. It is not necessary to provide moisture protection. The reaction flask is capped and 18 M. K. Jain, "Introduction to Biological Membranes." Wiley, New York, 1989. 19 H. Eibl, Mernbr. Fluid. Biol. 2, 217 (1983).
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is kept in a freezer at - 2 0 ° overnight. The solid is collected by suction filtration and washed with a few milliliters of cold acetone. The solid is dried in vacuo for 2 hr to give 130 mg of pure DMPM lithium salt. The material can be stored in a desiccator at - 2 0 ° indefinitely. The material shows a single spot (Rf0.5) by thin-layer chromatography on a silica plate with CHCl3/methanol/acetic acid (65/15/2). DMPM can also be prepared from commercially available dimyristoylphosphatidylcholine using phospholipase D in the presence of methanol. 2° In this case, the product is contaminated with a small amount of dimyristoylphosphatidic acid which has little effect on the scooting assay. Most of the DMPM used in our earlier studies 14-17 was prepared from 1,2-dimyristoyl-sn-3glycerol, but this starting material is less readily available. Preparation of DMPM Vesicles. DMPM (20 mg of the Li salt) is weighed into a disposable glass culture tube (10 x 75 mm, Kimball Glass, Vineland, N J). Two milliliters of distilled water is added, and the tube is capped with a rubber septum. For phospholipids containing saturated acyl chains, it is not necessary to keep the solutions under nitrogen. The suspension is briefly sonicated in a bath sonicator (Lab Supplies, Hicksville, NY, Model G112SPIT) for about 5 sec, and the cloudy solution is frozen (these frozen samples can be stored frozen and reused over several months). The frozen suspension is placed in the central cavitation zone of the optimally tuned sonicator bath. Besides the electronic adjustments in the power supply for the sonicator crystal as recommended by the supplier (generally done with the new bath), the sonicator bath is tuned just before use by adjusting the level of water until the energy is focused in a single cavitation zone in the center on the surface of the water. Sonication is allowed to proceed until an almost water-clear dispersion is obtained (typically 50-150 sec, depending on the tuning of the sonicator). The suspension can be kept at room temperature for more than 8 hr, and aliquots are diluted into the reaction solution for the kinetic studies. Kinetic Studies. Hydrolysis of vesicles in the scooting mode is monitored with a pH stat (e.g., Radiometer ETS822 system) equipped with a high-speed mechanical stirrer (e.g., Radiometer TTA60) and a waterjacketed thermostatted vessel maintained at 21 o The corners of the stirrer paddle are rounded a bit with a file so that the stirring is still rapid (tested by injecting a dye solution into the reaction vessel and making sure that the color becomes dispersed within 1 or 2 sec) but not so violent so as to create turbulence, cavitation, or small bubble formation during the reaction progress. Nitrogen is continuously passed over the reaction solution (at a rate of approximately 50 ml/min) to prevent the absorption of carbon 20 H. Eibl and S. Kovatchev, this series, Vol. 72, p. 632.
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dioxide. Prior to a kinetic run, 4 ml of a solution of 0.6 mM CaC12 and 1 mM NaCl in distilled water is placed into the reaction vessel, and the pH is adjusted to 8.0 using the autoburet with 3 mM NaOH as titrant. The stock solution of vesicles in water (typically 0.5 to 1 mg DMPM) is added in one portion, and the pH is adjusted back to 8.0. After the baseline drift subsides (typically within a few minutes if there is no contamination from PLA2), an aliquot of PLA2 (typically 0.05-0.1/zg in 1-10/xl of water) is added in one portion. The reaction is maintained at pH 8.0 by continuous pH-stat titration with 3 mM NaOH. The reaction is allowed to proceed until the rate of consumption of titrant ceases (typically about 15-30 min). Several additional factors are noted. If the solution of the enzyme is acidic or buffered, as is the case with most commercially available preparations, the initial part of the reaction progress curve will have contributions from the pH imbalance. Often, this can be manually subtracted by making a control run in which enzyme is added to vesicles in the absence of calcium. The amount of base used under these conditions corresponds to the amount of acid in the enzyme aliquot. Between kinetic runs, it is sufficient to wash the reaction vessel and pH electrode with distilled water since the enzyme sticks much tighter to the anionic vesicles than to the glass surfaces of the pH electrode or the vessel. In all pH-stat type assays, it is important that the pH electrode be working properly (low pH drift and a quick response time). Occasionally it is useful to clean the electrode surface with a mild detergent such as Micro (International Products Inc., Box 118, Trenton, N J) after several enzymatic runs. Finally, on rare occasion, the observed reaction progress appears more rounded in shape compared to a purely exponential curve (Fig. 2 as discussed below). This is almost always due to polydispersion in the size of the vesicles, and the problem can be easily remedied by freezing and sonicating the stock solution of DMPM a second time. At the end of the enzymatic reaction, the consumption of titrant is very small (less than 3 nmol/min) under the conditions described above. This small (less than 2% of the initial rate) but finite consumption of base is largely due to slow fusion of vesicles and transbilayer movement of phospholipids, and it can be eliminated only at lower calcium concentrations, where other kinetic complications are observed. Quantitative Analysis. A typical reaction progress curve for the hydrolysis of DMPM vesicles by PLA2 is shown in Fig. 2. At 0.6 mM calcium, the reaction progress curve is first-order. Since the contribution of the E to E* step to the overall kinetics is eliminated, ~ this reaction progress curve is interpreted only in terms of the steps in the box in Scheme I. This is just the Michaelis-Menten formalism adopted for catalysis within an interface. The amount (moles) of titrant used at the end of the reaction
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40
30 e'-
.~
O m
20
O O
E
10
0
0
I
I
I
I
10
20
30
40
50
time (min) Fro. 2. Reaction progress curve for the hydrolysis of DMPM vesicles in the scooting mode. Conditions: 0.6 mg of DMPM in 4 ml of 0.6 mM CaC12, 1 mM NaC1, pH 8.0, 21°. The reaction was initiated by the addition of porcine pancreatic PLA 2 (0.1/zg in 2/zl of water). Based on the calibration of the titrant with myristic acid (see text for details), each microliter of titrant corresponds to 3.5 nmol of product.
(Fig. 2) is much less than the total moles of DMPM present in the reaction. This is because the ratio of vesicles to enzyme is large, and the enzyme does not hop to other vesicles. The partial extent of hydrolysis is not due to inactivation of the enzyme. At the end of the reaction, induction of vesicle fusion by raising the calcium concentration ~4or promotion of intervesicle exchange of enzyme by high salt 15 causes immediate resumption of hydrolysis. All other reasonable possibilities for the partial hydrolysis of the substrate were considered and have been ruled out.14 These include severe product inhibition, the formation of multilamellar vesicles, and a decrease in the efficiency of pH titration. As elaborated elsewhere I such curves (Fig. 2) are completely described by Eq. (1), where Pt is the amount of product produced (or titrant utilized) at time t, Pmax is the extent of hydrolysis (the amount of product produced at the end of the reaction), and ki is the first-order relaxation constant.
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Pt = Pmax [1 - e x p ( - kit)]
(1)
This first-order reaction progress curve is typical of the Michaelis-Menten formalism under the condition that the apparent Michaelis constant, /~mpp is much larger than the concentration of substrate. 21'22For interracial catalysis, the concentration of substrate within the interface is expressed as its mole fraction. Since the maximum value of the substrate mole fraction in the vesicle is 1, the first-order nature of the reaction progress curve implies that K ~ p >> 1. The interfacial K ~ p has its normal meaning 1,2,21-23 according to Eq. (2): K~m pp = Kin(1 + 1/Kp)
(2)
where K m = (k_l + k2)/kl. According to Eq. (2), /~mpp differs from the classic Km due to the inhibition by the reaction products. Here, the rate constants that make up/~mpp are given in Scheme I. Kp is the dissociation constant for the product, and, in the case of significant product inhibition (i.e., small Kp), the K~m pp will be magnified according to Eq. (2). The firstorder relaxation constant, ki, in Eq. (1) is given in Eq: (3)1: NTki = k2/I~mpp
(3)
Here, NT is the average number of DMPM molecules in the outer layer of the vesicles and has a value of about 13,000 for the example shown in Fig. 2 (however, see below). NTki is the turnover number for vesicles of DMPM, which has a value of about 2500-3000 min- 1 under the assay conditions described above. Again, Eq. (3) is derived from an extension of the Michaelis-Menten formalism to interfacial catalysis.l In the scooting mode, the enzymes bound to vesicles do not hop to other vesicles. In this case, the moles of product formed after the reaction comes to a halt, Pmax, is simply equal to the moles of enzyme-containing vesicles multiplied by NT. The value of Pmax will be maximal when there is a large excess of vesicles over enzymes so that no vesicle will contain more than one enzyme. According to the Poisson distribution, the probability of having more than one enzyme per vesicle is only 0.5% if the vesicle to enzyme ratio is 6. This is the case for the concentrations of components described above in the assay procedure. As expected, in the presence of excess substrate vesicles, the value of Pmax increases linearly with the amount of the enzyme in the reaction mixture.14 Also, in the presence of a large excess of enzyme over vesicles, 63% of the total 21 I. H. Segel, "Enzyme Kinetics." Wiley, New York, 1975. 2z A. Fersht, "Enzyme Structure and Mechanism," 2nd Ed. Freeman, New York, 1985. 23 A. Pliickthun and E. A. Dennis, J. Biol. Chem. 260, 11099 (1985).
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substrate present in the reaction mixture can be hydrolyzed (this corresponds to the amount of substrate in the outer layer of small unilamellar vesicles). Such experiments demonstrate that only the substrate present in the outer monolayer of a vesicle is accessible for hydrolysis by the bound enzyme. 14 It should also be mentioned that Eq. (3) is valid only under the conditions of at most one enzyme per vesicle. Values ofki and Pmax are easily obtained by fitting the reaction progress curve (Fig. 2) to Eq. (1). In order to convert the value OfPmax in microliters of titrant used to moles of product formed, it is necessary to calibrate the titrant in the following way. A solution of DMPM vesicles in 0.6 mM CaC12, 1 mM NaC1 is prepared as described above and adjusted to pH 8.0 in the pH stat. A solution of myristic acid in ethanol of known concentration (typically 5/zl of a 40 mM solution) is added, and the pH is brought back to 8.0. The amount of titrant used corresponds to the number of moles of myristic acid added. This procedure is more accurate than a simple titration of a strong acid, such as HCI, in water, since it takes into account the fact that the surface pK a of myristic acid in the DMPM vesicle is not far below 8. Such an analysis has shown that the titration efficiency (moles of titrant used per mole of fatty acid added) is 0.90 for myristic acid in DMPM and 0.87 for myristic acid in DMPM vesicles at the end of the reaction progress curve when all of the substrate in the outer layer of the vesicles has been hydrolyzed. Thus, there is no significant change in the titration efficiency during the course of the enzymatic reaction. Comments on Assay of Phospholipase A 2 in Scooting Mode When interfacial catalysis by PLA2 is examined in the scooting mode, many of the previously reported anomalous features of the kinetics are not observed. The hydrolysis of DMPM vesicles begins immediately after the addition of enzyme; the latency period in the reaction progress that is seen in other assays s does not appear with vesicles of DMPM. In this sense, this system with vesicles is also distinctly different than the monolayer system of Verger and de Haas,2 where they assumed that the E to E* step is slow. On the other hand, as shown elsewhere by stopped-flow kinetic analysis, the rate of binding of the enzyme to the miceUe or vesicle interface is rapid. 24Therefore, we believe that the primary mechanism for the origin of the latency period is the much slower buildup of anionic product in the interface, which leads to a time-dependent modulation of the E to E* equilibrium. This is the major difference between our model 1 and the model proposed by Verger and de Haas. 2 24 M. K. Jain, J. Rogers, and G. H. de Haas, Biochim. Biophys. Acta 940, 51 (1988).
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The enzyme is still fully catalytically active at the end of the reaction progress, 14and the cessation of the reaction is due to the consumption of all of the substrate in the outer layer of enzyme-containing vesicles. When the enzyme to vesicle ratio is large enough so that all of the vesicles contain one or more bound enzymes, 63% of the total DMPM present in small sonicated vesicles is hydrolyzed, which corresponds to the fraction of substrate in the outer layer of the vesicles. With large unilamellar vesicles, 50% of the total DMPM is hydrolyzed whereas only small amounts (typically about 8%) of the substrate is hydrolyzed with multilamellar vesicles. The vesicles remain intact, even after all of the substrate in the outer layer has been hydrolyzed. Studies of vesicle fusion by light scattering and fluorescence resonance energy transfer have demonstrated that the rate of fusion of DMPM vesicles, under the above-described conditions, is much too slow to be of any significance except at the end of the first-order reaction progress curve (as discussed above). TM Finally, the rates of transbilayer movement of phospholipids and of intervesicle exchange of enzyme, phospholipid substrate, and the products are also very slow. Although detailed investigations of interfacial catalysis have been carried out with DMPM vesicles, similar results are seen with vesicles of other anionic phospholipids 16'25or with codispersions of zwitterionic and anionic phospholipids. 26 It is particularly striking to note here that over 50 PLA2s tested from different sources exhibit a first-order reaction progress curve of the type shown in Fig. 2. These include not only the enzymes from pancreas and their mutants, but also those from venoms of Apis, Elapidae, Viperidae, and Crotalidae, as well as bacteria like Escherichia coli and Saccharomyces. 27 Thus, the assay in the scooting mode with DMPM vesicles is by far the most generally useful procedure for studying PLA2s reported to date. Other assay procedures for analyzing PLA2s have been described, including the use of mixtures of phospholipids dispersed into detergents, 4,23,2s radiolabeled bacterial membrane fragments, 29 and vesicles of radiolabeled phospholipids.30 These other assays are not generally applicable to all enzymes, and in many cases it is necessary to include a lipophilic additive to promote the binding of the enzyme to the interface. 23 All of these problems stem from the assay-dependent variation in the 25 M. K. Jain and J. Rogers, Biochim. Biophys. Acta 1003, 91 (1989). 26 F. Ghomashchi, B. Yu, O. Berg, M. K. Jain, and M. H. Gelb, in preparation. 27 M. K. Jain, G. N. Ranadive, B.-Z. Yu, and H. M. Verheij, in preparation. 28 W. Niewenhuizen, H. Kunze, and G. H. de Haas, this series, Vol. 32B, p. 147. 29 For example, see R. L. Jesse and R. C. Franson, Biochim. Biophys. Acta 575, 467 (1979). 30 For example, see J. Balsinde, E. Diez, A. Schiiller, and F. Mollinedo, J. Biol. Chem. 263, 1929 (1988).
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amount of enzyme present in the interface (E to E* effects) during the steady-state catalytic turnover. These effects are not observed in the assay with DMPM since the E to E* step has been eliminated. Additional problems with micelle or mixed micelle assays due to substrate replenishment have been discussed. 1 Finally it should be mentioned that although the assay with DMPM described in this chapter makes use of a pH stat to detect the production of product, it is also possible to adopt other methods of product analysis. For example, it is possible to use radiolabeled phospholipids followed by chromatographic separation of the products and analysis of the amount of released radioactive fatty acid by scintillation counting. 26 The important point is that with DMPM as a matrix, the anomalous kinetic effects arising from the E to E* step are avoided.
Uses of Scooting Assay Interfacial catalysis in the scooting mode opens a new dimension in the study of PLA2. The protocols described here can be used as such or adopted appropriately to address questions related to,catalytic turnover, substrate specificity, interfacial rate constants, inhibition, and activation. Detailed studies on many of these topics are reported elsewhere. Because of space limitations, here we present a subset of the uses of the study of PLA2 in the scooting mode. Calibrating Solutions ofPhospholipase A2. Since the extent of hydrolysis, Pmax,under the conditions of excess vesicles to enzyme is proportional to the moles of catalytically active enzyme, the concentration of functional enzyme active sites in a stock solution of enzyme can be determined directly. We have found this protocol to be particularly useful for ascertaining the purity and homogeneity of chemically modified 27 or semisynthetic forms of PLA2 and genetically engineered enzyme mutants. The use of this assay with DMPM vesicles is the only known method for carrying out such a measurement. The usual methods of determining the concentration of PLA2 are based on assays of the total protein content, regardless of whether all of the enzyme in the solution is catalytically active. To obtain the concentration of catalytically active enzyme from the extent of hydrolysis, Pmax, one needs to know the value of NT. We have found that preparations of DMPM, prepared as described above, reproducibly give a value of about 13,000 for N~. However, the value of ArT may vary somewhat in different laboratories. The size of vesicles, prepared by sonication of DMPM, is known to be sensitive to a number of factors including the method of sonication, the presence of fatty acid heterogeneity in the synthesized substrate, and the presence of ions and other impurities. For this reason, it is recommended that the value of Nz be determined
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using a well-characterized PLA2, such as the enzyme from porcine pancreas. This enzyme, when purified by the published procedure, 2s consistently has a specific activity of 1300-1500 ~mol product/min/mg in an assay with egg yolk. 2s The concentration of porcine pancreatic PLA 2 in the stock solution is determined from the absorbance at 280 nm using the published extinction coefficient, 2s El%, of 13.0. The value of NT can then be obtained from a measurement of Pmax, under the conditions of excess vesicles over enzyme, and knowing the amount of porcine pancreatic PLA 2 added to the assay. Once the value of AfT is determined in this manner, it can be used to calibrate solutions of other, less well-characterized PLA2s. Intrinsic Catalytic Activities o f Phospholipases A 2 . The value of NTk i measured in the scooting assay is a characteristic property of a particular PLA2 acting on a particular substrate vesicle. It has the same meaning as the kcat/K m value for an enzyme that operates in water on a particular substrate, except for the fact that rate constants have units which are appropriate for interfacial catalysis) In the past, it has not been possible to obtain accurate values of the turnover numbers for PLA 2 since such calculations are based on maximal velocity measurements using different assays and require the assumptions that all of the enzyme in the solution is fully active and that the exchange of the enzyme, substrate, or products is not part of the catalytic turnover. Detection o f Phospholipase A 2 Impurities in Samples. The detection of trace amounts of PLA2 in biological samples, for example, in preparations of melittin, can be accurately determined using the scooting assay. A second example that comes to mind concerns the activity of unusual or mutant forms of PLA 2 . For example, a PLA 2 from Agkistrodon piscivorus piscivorus has been isolated that contains lysine in place of the catalytically important aspartate residue at position 49. 3~ This enzyme was reported to have significant catalytic activity; however, this result was recently challenged by van den Bergh et a1.,32 who provided strong evidence that the lysine-49 enzyme is contaminated with a very active aspartate-49 PLA2. This problem can be easily and conclusively resolved by analyzing the enzyme in the scooting assay. The moles of catalytically active enzyme in the stock solution can be determined from the values of Pmax and NT since every active molecule of enzyme will eventually hydrolyze all of the substrate in the outer layer of the vesicle to which it is bound. This is true even in the case that the particular P L A 2 under investigation has an 3~ j. M. Maraganore, G. Merutka, W. Cho, W. Welches, F. J. Kezdy, and R. L. Heinrikson, J. Biol. Chem. 259, 13839 (1984). 32 C. J. van den Bergh, A. J. Slotboom, H. M. Verheij, and G. H. de Haas, J. Cell. Biochem. 39, 379 (1989).
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extremely small interfacial turnover number, N T k i . If the absolute number of catalytically active enzyme molecules in the stock solution, as determined using the scooting assay, is much smaller than the total number of enzyme molecules, calculated using a standard protein assay (i.e., absorbance at 280 nm), one would conclude that an active PLA2 impurity is present in a large amount of inactive PLA2 protein. In all of these studies, it is important to check that the sample to be tested does not contain impurities which induce the fusion of vesicles. Other Uses of Scooting Assay and Concluding Remarks In addition to the above-mentioned analyses, the scooting assay has numerous other uses. For example, there has bccn considcrablc controversy in the literature as to whether a particular PLA2 is catalytically active as a dimcr or as a monomer. 4 This issue can bc resolved using the scooting assay.aVIn the presence of excess vesiclesover cnzymc, the value of Pmax will be twicc as largc for a given numbcr of moles of monomeric cnzymc compared to the same number of moles of a dimeric enzyme. Thc scooting assay is also very useful in the discovery of compounds that function as truc competitive inhibitorsof PLA2.13 For example, many of the previously reported P L A 2 inhibitors function by promoting the dcsorption of cnzymc from thc intcrfacc (E to E* effects) rather than by binding directly to the bound enzyme. Thcsc compounds do not inhibit thc action of PLAE in thc scooting mode. 13 In the inhibition studies, thc kinetics wcrc dctcrmincd at higher calcium concentrations (2.5rather than 0.6 raM). Under thcsc conditions the vesicles arc much largcr, and thc shape of thc reaction progress curve is differentthan that shown in Fig. 2 in that itcontains a significantconstant velocity at early times (zero-order phase). This is mainly a result of the fact that the buildup of product and the depletion of substratc take longer with larger vesicles, and the initial velocity is maintaincd for a longer pcriod than in the small vesicles dcscribed hcrc. The fulldctailsof the inhibitionanalysis at high calcium will bc dcscribcd) 3 Perhaps the most important use of the scooting assay will be in the determination of the absolutc substratc spccificiticsand the interracialrate constants of lipolyticenzymes. In previous studies of substratc spccificitics,rclativcvelocitiesfor the action of PLA2s and other lipolyticenzymes on a variety of differentsubstratc vcsiclcs composed of pure phospholipid classes havc bccn reported. In thcse studics, no effort has bccn made to normalize for thc amount of enzyme bound to the interface. The use of 33 M. K. Jain, B.-K. Yu, and O. G. Berg, in preparation.
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the scooting assay, in which all of the enzyme is in the interface, provides an extremely powerful solution to this problem. ~6 At first glance, the scooting assay may appear somewhat unsettling and counterintuitive to traditional enzymologists. The irreversible nature of the interaction of the enzyme with the interface may not be an accurate representation of the action of the enzyme in a physiological environment, such as the inside of a cell. However, this is not a concern, since under physiological conditions all of the fundamental features of interfacial catalysis, whether they involve the selection of various substrates within the cell membrane or the inhibition of the enzyme, will occur within the interface. In other words, only events such as lipid exchange and other processes that occur in parallel to the events within the interface have been constrained in the scooting assay. The mechanism of action of the enzyme within the interface is not altered by this technique. The scooting assay provides an attractive in vitro method for the analysis of lipolytic enzymes that is free from the distortions caused by the relative affinities of the PLA 2 (E to E* effects) for a variety of vesicles composed of pure phospholipids. Acknowledgments This work was supported by Grants HL-36235 (M. H. G.) and GM-29703 (M. K. J.) from the National Institutes of Health and by a grant from Sterling PharmaceuticalCo. (M. K. J.). The authorswouldliketo give specialthanksto FaridehGhomashchifor technical assistance.
[11] A s s a y o f P h o s p h o l i p a s e s C a n d D in P r e s e n c e o f O t h e r Lipid Hydrolases
By KARL Y. HOSTETLER,MICHAEL F. GARDNER,and KATHY A. ALDERN
Introduction Phospholipases C and D were first identified in bacteria I and plants, 2 respectively, but they are now known to be present in mammalian cells. These activities are generally not difficult to measure, and a variety of methods are available. The methods are usually based on the release of a i M. G. Macfarlane and B. C. J. Knight, Biochem. J. 35, 884 (1941). 2 D. J. H a n a h a n and I. L. Chaikoff, J. Biol, Chem. 169, 669 (1947).
METHODS IN ENZYMOLOGY,VOL. 197
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in detail to the point where at a given PC concentration, the micelle size distribution is known, and an area per head group can be estimated. Since they can be presented to enzymes as monomers, micelles, or in bilayer structures, short-chain phospholipids are ideal for examining kinetic preferences of lipolytic enzymes. In the future detailed studies of short-chain phospholipids with different head groups should facilitate kinetics with phospholipases which show a preference for anionic substrates.
[10] P h o s p h o l i p a s e A 2 - C a t a l y z e d H y d r o l y s i s o f Vesicles: U s e s o f I n t e r f a c i a l C a t a l y s i s in t h e S c o o t i n g M o d e
By MAHENDRA KUMAR JAIN and MICHAEL H. GELB Introduction
Interfacial catalysis by phospholipase A2 (PLA2) is adequately described by Scheme I.~ As a first step, the enzyme in the aqueous phase (E) binds to the substrate interface, and the bound enzyme (E*) undergoes the catalytic turnover in the interface according to the classic Michaelis-Menten formalism as shown in the box. Thus, the binding of the enzyme to the interface and the binding of a phospholipid substrate to the active site of the bound enzyme (E*) to produce the enzyme-substrate complex (E'S) are distinct steps. This is implicit in the proposal that the enzyme contains an interfacial binding surface that is topologically and functionally distinct from the catalytic site. This kinetic view of interfacial catalysis has been the working hypothesis in several laboratories. 1-5 A critical experimental test of Scheme I has not been possible until recently. 1 In most studies, especially with micelles3,4 and monolayers, 2 kinetic and equilibrium contributions of the E to E* step are not readily discernible. Even in bilayers, many of the anomalies seen in the kinetics of action of PLA 2 on insoluble lipid substrates are due to a significant, yet variable, contribution of the E to E* step to the steady-state enzymatic turnover.l'2 This is illustrated in the following two examples (see Refs. 1 i M. K. Jain and O. G. Berg, Biochim. Biophys. Acta 1002, 127 (1989). 2 R. Verger and G. H. de Haas, Annu. Rev. Biophys. Bioeng. 5, 77 (1976). 3 H. M. Verheij, A. J. Slotboom, and G. H. de Haas, Rev. Physiol. Biochem. Pharmacol. 91, 91 (1981). 4 E. A. Dennis, in "The Enzymes" (P. D. Boyer, ed.), 3rd Ed., Vol. 16, p. 307. Academic Press, New York, 1983. 5 See other contributions to this volume.
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and 2 for others). First, a lag phase is seen in the reaction progress curve for the action of porcine pancreatic PLA 2 on vesicles of phosphatidylcholine. This can be explained on the basis of the observation that the enzyme has a very weak affinity for vesicles of zwitterionic phospholipids, 6,7 but it binds several orders of magnitude more tightly to anionic vesicles: Initially, with zwitterionic vesicles, most of the enzyme is in the aqueous phase and the reaction velocity is low. As the anionic fatty acid product accumulates in the interface, the reaction progress curve accelerates as more and more of the enzyme partitions into the interface where the lipolysis occurs. 8-~° The second example relates to the inhibition of PLA 2. Most inhibition studies reported in the literature are difficult to interpret because the effects of inhibitors on the E to E* step are not easy to characterize, l~'12 For example, many of the reported inhibitors of PLA 2 work not by binding tightly to the enzyme, but by promoting the desorption of enzyme from the interface. ~2,~3Thus, in most of the assay procedures used for PLA 2 that have been reported, the fundamental features of the enzymology that occur within the interface are "blurred" by the reversible association of the enzyme with the interface, that is, the E to E* step (see Ref. I for a review and Ref. 13 for a resolution of these difficulties). In order to minimize such kinetic complexities of interfacial catalysis, we have developed a methodology 14-~7for studying the action of PLA2 on substrate vesicles in the "scooting mode,"6 in which all of the enzyme is tightly bound to the interface. Here, after the initial binding of the enzyme to the interface, the E to E* step is no longer part of the catalytic turnover within the interface. In the scooting mode, the anomalous effects associated with the E to E* step, such as the latency period, 6,8 the effect of 6 G. C. Upreti and M. K. Jain, J. Membr. Biol. 55, 113 (1980). 7 M. K. Jain, M. R. Egmond, H. M. Verheij, R. J. Apitz-Castro, R. Dijkman, and G. H. de Haas, Biochim. Biophys. Acta 688, 341 (1982). 8 R. J. Apitz-Castro, M. K. Jain, and G. H. de Haas, Biochim. Biophys. Acta 688, 349 (1982). 9 M. K. Jain and D. V. Jahagirdar, Biochim. Biophys. Acta 814, 313 (1985). 10 M. K. Jain, B. Yu, and A. Kozubek, Biochim. Biophys. Acta 980, 23 (1989). t~ M. K. Jain, M. Streb, J. Rogers, and G. H. de Haas, Biochem. Pharmacol. 33, 2541 (1984). 12 M. K. Jain and D. V. Jahagirdar, Biochim. Biophys. Acta 814, 319 (1985). 13 M. K. Jain, W. Yuan, and M. H. Gelb, Biochemistry 28, 4135 (1989). 14 M. K. Jain, J. Rogers, D. V. Jahagirdar, J. F. Marecek, and F. Ramirez, Biochim. Biophys. Acta 860, 435 (1986). 15 M. K. Jain, B. P. Maliwal, G. H. de Haas, and A. J. Slotboom, Biochim. Biophys. Acta 860, 448 (1986). 16 M. K. Jain, J. Rogers, J. F. Marecek, F. Ramirez, and H. Eibl, Biochim. Biophys. Acta 860, 462 (1986). 17 M. K. Jain, G. H. de Haas, J. F. Marecek, and F. Ramirez, Biochim. Biophys. Acta 860, 475 (1986).
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FIG. 1. Schematic representation of interfacial catalysis phospholipase by A2 in the scooting mode. See text for details.
gel-fluid phase transitions, 6 and the activating or inhibiting effects of amphiphilic solutes, 1'12 are no longer observed. We have found that PLA 2hydrolyzes vesicles of anionic phospholipids, such as dimyristoylphosphatidylmethanol (DMPM), in the scooting mode. Here hydrolysis begins without any noticeable latency period (less than 5 sec), and the enzyme undergoes several thousand catalytic turnovers without leaving the interface. This interfacial catalysis in the scooting mode continues until all of the substrate in the outer layer of the enzymecontaining vesicles is hydrolyzed. Throughout the entire reaction progress, the vesicles retain their overall physical integrity. This is illustrated schematically in Fig. 1. The observation that the enzyme undergoes several thousand catalytic cycles while remaining attached to the same vesicle provides the strongest evidence for interfacial catalysis according to the kinetic Scheme I. A quantitative interpretation of the reaction in the
E E SCHEMEI. Interfacialcatalysis by phospholipase A 2.
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scooting mode I requires an appreciation of the structure and molecular dynamics of the vesicles of anionic phospholipids. ~8,19 The substrate used for these studies are small unilamellar vesicles of DMPM containing no additives; however, the scooting phenomenon is observed with vesicles of a number of different phospholipids as long as they contain a critical mole fraction of anionic phospholipid (typically 5-10 mol %). PLA2 binds essentially irreversibly to vesicles of DMPM (see below). Under the conditions used for such studies, neither the enzyme nor the substrate or products of hydrolysis exchange with other vesicles. In addition, complications arising from fusion of vesicles as well as transbilayer movement of phospholipids have been eliminated. In this chapter, we present a detailed description of the basic protocols for the study of PLA2 in the scooting mode. In addition, we emphasize some applications of this unique technique in studies on lipolytic enzymes.
Kinetic Analysis of Phospholipase A 2 in Scooting Mode
Preparation ofDMPM. DMPM can be easily prepared from commercially available dimyristoylphosphatidic acid as follows. Dimyristoylphosphatidic acid disodium salt (200 mg, 0.31 mmol, Avanti Polar Lipids, Birmingham, AL) is suspended in 16 ml of ether containing 1% (v/v) water in an Erlenmeyer flask with a stir bar. Sufficient HC1 to protonate all of the phosphatidic acid sodium salt (0.15 ml of 6 N HCI) is added, and the mixture is stirred vigorously for several minutes until both layers become clear. Stirring is continued at room temperature, and freshly distilled diazomethane in ether is added dropwise until the characteristic yellow color of the diazomethane persists. An alcohol-flee solution of diazomethane is prepared from Diazald (Aldrich, Milwaukee, WI) according to the manufacturer's directions. The mixture is stirred for an additional 30 min. The solution is then filtered through a pad of silica gel (2 × 3 cm) containing a I cm top layer of Celite in a sintered glass funnel. The silica pad is washed with an additional 50 ml of ether, and the combined solutions are transferred to a tared recovery flask and concentrated on a rotary evaporator at reduced pressure and at room temperature. The flask is attached to a vacuum pump for 30 rain to remove traces of solvent. To the oily residue of the phosphate triester (160 nag, 0.29 mmol) is added reagent-grade acetone (5 ml) and anhydrous LiBr (35 rag, 0.4 mmol, Fluka), and the mixture is refluxed with magnetic stirring for 6 hr. It is not necessary to provide moisture protection. The reaction flask is capped and 18 M. K. Jain, "Introduction to Biological Membranes." Wiley, New York, 1989. 19 H. Eibl, Mernbr. Fluid. Biol. 2, 217 (1983).
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is kept in a freezer at - 2 0 ° overnight. The solid is collected by suction filtration and washed with a few milliliters of cold acetone. The solid is dried in vacuo for 2 hr to give 130 mg of pure DMPM lithium salt. The material can be stored in a desiccator at - 2 0 ° indefinitely. The material shows a single spot (Rf0.5) by thin-layer chromatography on a silica plate with CHCl3/methanol/acetic acid (65/15/2). DMPM can also be prepared from commercially available dimyristoylphosphatidylcholine using phospholipase D in the presence of methanol. 2° In this case, the product is contaminated with a small amount of dimyristoylphosphatidic acid which has little effect on the scooting assay. Most of the DMPM used in our earlier studies 14-17 was prepared from 1,2-dimyristoyl-sn-3glycerol, but this starting material is less readily available. Preparation of DMPM Vesicles. DMPM (20 mg of the Li salt) is weighed into a disposable glass culture tube (10 x 75 mm, Kimball Glass, Vineland, N J). Two milliliters of distilled water is added, and the tube is capped with a rubber septum. For phospholipids containing saturated acyl chains, it is not necessary to keep the solutions under nitrogen. The suspension is briefly sonicated in a bath sonicator (Lab Supplies, Hicksville, NY, Model G112SPIT) for about 5 sec, and the cloudy solution is frozen (these frozen samples can be stored frozen and reused over several months). The frozen suspension is placed in the central cavitation zone of the optimally tuned sonicator bath. Besides the electronic adjustments in the power supply for the sonicator crystal as recommended by the supplier (generally done with the new bath), the sonicator bath is tuned just before use by adjusting the level of water until the energy is focused in a single cavitation zone in the center on the surface of the water. Sonication is allowed to proceed until an almost water-clear dispersion is obtained (typically 50-150 sec, depending on the tuning of the sonicator). The suspension can be kept at room temperature for more than 8 hr, and aliquots are diluted into the reaction solution for the kinetic studies. Kinetic Studies. Hydrolysis of vesicles in the scooting mode is monitored with a pH stat (e.g., Radiometer ETS822 system) equipped with a high-speed mechanical stirrer (e.g., Radiometer TTA60) and a waterjacketed thermostatted vessel maintained at 21 o The corners of the stirrer paddle are rounded a bit with a file so that the stirring is still rapid (tested by injecting a dye solution into the reaction vessel and making sure that the color becomes dispersed within 1 or 2 sec) but not so violent so as to create turbulence, cavitation, or small bubble formation during the reaction progress. Nitrogen is continuously passed over the reaction solution (at a rate of approximately 50 ml/min) to prevent the absorption of carbon 20 H. Eibl and S. Kovatchev, this series, Vol. 72, p. 632.
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dioxide. Prior to a kinetic run, 4 ml of a solution of 0.6 mM CaC12 and 1 mM NaCl in distilled water is placed into the reaction vessel, and the pH is adjusted to 8.0 using the autoburet with 3 mM NaOH as titrant. The stock solution of vesicles in water (typically 0.5 to 1 mg DMPM) is added in one portion, and the pH is adjusted back to 8.0. After the baseline drift subsides (typically within a few minutes if there is no contamination from PLA2), an aliquot of PLA2 (typically 0.05-0.1/zg in 1-10/xl of water) is added in one portion. The reaction is maintained at pH 8.0 by continuous pH-stat titration with 3 mM NaOH. The reaction is allowed to proceed until the rate of consumption of titrant ceases (typically about 15-30 min). Several additional factors are noted. If the solution of the enzyme is acidic or buffered, as is the case with most commercially available preparations, the initial part of the reaction progress curve will have contributions from the pH imbalance. Often, this can be manually subtracted by making a control run in which enzyme is added to vesicles in the absence of calcium. The amount of base used under these conditions corresponds to the amount of acid in the enzyme aliquot. Between kinetic runs, it is sufficient to wash the reaction vessel and pH electrode with distilled water since the enzyme sticks much tighter to the anionic vesicles than to the glass surfaces of the pH electrode or the vessel. In all pH-stat type assays, it is important that the pH electrode be working properly (low pH drift and a quick response time). Occasionally it is useful to clean the electrode surface with a mild detergent such as Micro (International Products Inc., Box 118, Trenton, N J) after several enzymatic runs. Finally, on rare occasion, the observed reaction progress appears more rounded in shape compared to a purely exponential curve (Fig. 2 as discussed below). This is almost always due to polydispersion in the size of the vesicles, and the problem can be easily remedied by freezing and sonicating the stock solution of DMPM a second time. At the end of the enzymatic reaction, the consumption of titrant is very small (less than 3 nmol/min) under the conditions described above. This small (less than 2% of the initial rate) but finite consumption of base is largely due to slow fusion of vesicles and transbilayer movement of phospholipids, and it can be eliminated only at lower calcium concentrations, where other kinetic complications are observed. Quantitative Analysis. A typical reaction progress curve for the hydrolysis of DMPM vesicles by PLA2 is shown in Fig. 2. At 0.6 mM calcium, the reaction progress curve is first-order. Since the contribution of the E to E* step to the overall kinetics is eliminated, ~ this reaction progress curve is interpreted only in terms of the steps in the box in Scheme I. This is just the Michaelis-Menten formalism adopted for catalysis within an interface. The amount (moles) of titrant used at the end of the reaction
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40
30 e'-
.~
O m
20
O O
E
10
0
0
I
I
I
I
10
20
30
40
50
time (min) Fro. 2. Reaction progress curve for the hydrolysis of DMPM vesicles in the scooting mode. Conditions: 0.6 mg of DMPM in 4 ml of 0.6 mM CaC12, 1 mM NaC1, pH 8.0, 21°. The reaction was initiated by the addition of porcine pancreatic PLA 2 (0.1/zg in 2/zl of water). Based on the calibration of the titrant with myristic acid (see text for details), each microliter of titrant corresponds to 3.5 nmol of product.
(Fig. 2) is much less than the total moles of DMPM present in the reaction. This is because the ratio of vesicles to enzyme is large, and the enzyme does not hop to other vesicles. The partial extent of hydrolysis is not due to inactivation of the enzyme. At the end of the reaction, induction of vesicle fusion by raising the calcium concentration ~4or promotion of intervesicle exchange of enzyme by high salt 15 causes immediate resumption of hydrolysis. All other reasonable possibilities for the partial hydrolysis of the substrate were considered and have been ruled out.14 These include severe product inhibition, the formation of multilamellar vesicles, and a decrease in the efficiency of pH titration. As elaborated elsewhere I such curves (Fig. 2) are completely described by Eq. (1), where Pt is the amount of product produced (or titrant utilized) at time t, Pmax is the extent of hydrolysis (the amount of product produced at the end of the reaction), and ki is the first-order relaxation constant.
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Pt = Pmax [1 - e x p ( - kit)]
(1)
This first-order reaction progress curve is typical of the Michaelis-Menten formalism under the condition that the apparent Michaelis constant, /~mpp is much larger than the concentration of substrate. 21'22For interracial catalysis, the concentration of substrate within the interface is expressed as its mole fraction. Since the maximum value of the substrate mole fraction in the vesicle is 1, the first-order nature of the reaction progress curve implies that K ~ p >> 1. The interfacial K ~ p has its normal meaning 1,2,21-23 according to Eq. (2): K~m pp = Kin(1 + 1/Kp)
(2)
where K m = (k_l + k2)/kl. According to Eq. (2), /~mpp differs from the classic Km due to the inhibition by the reaction products. Here, the rate constants that make up/~mpp are given in Scheme I. Kp is the dissociation constant for the product, and, in the case of significant product inhibition (i.e., small Kp), the K~m pp will be magnified according to Eq. (2). The firstorder relaxation constant, ki, in Eq. (1) is given in Eq: (3)1: NTki = k2/I~mpp
(3)
Here, NT is the average number of DMPM molecules in the outer layer of the vesicles and has a value of about 13,000 for the example shown in Fig. 2 (however, see below). NTki is the turnover number for vesicles of DMPM, which has a value of about 2500-3000 min- 1 under the assay conditions described above. Again, Eq. (3) is derived from an extension of the Michaelis-Menten formalism to interfacial catalysis.l In the scooting mode, the enzymes bound to vesicles do not hop to other vesicles. In this case, the moles of product formed after the reaction comes to a halt, Pmax, is simply equal to the moles of enzyme-containing vesicles multiplied by NT. The value of Pmax will be maximal when there is a large excess of vesicles over enzymes so that no vesicle will contain more than one enzyme. According to the Poisson distribution, the probability of having more than one enzyme per vesicle is only 0.5% if the vesicle to enzyme ratio is 6. This is the case for the concentrations of components described above in the assay procedure. As expected, in the presence of excess substrate vesicles, the value of Pmax increases linearly with the amount of the enzyme in the reaction mixture.14 Also, in the presence of a large excess of enzyme over vesicles, 63% of the total 21 I. H. Segel, "Enzyme Kinetics." Wiley, New York, 1975. 2z A. Fersht, "Enzyme Structure and Mechanism," 2nd Ed. Freeman, New York, 1985. 23 A. Pliickthun and E. A. Dennis, J. Biol. Chem. 260, 11099 (1985).
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substrate present in the reaction mixture can be hydrolyzed (this corresponds to the amount of substrate in the outer layer of small unilamellar vesicles). Such experiments demonstrate that only the substrate present in the outer monolayer of a vesicle is accessible for hydrolysis by the bound enzyme. 14 It should also be mentioned that Eq. (3) is valid only under the conditions of at most one enzyme per vesicle. Values ofki and Pmax are easily obtained by fitting the reaction progress curve (Fig. 2) to Eq. (1). In order to convert the value OfPmax in microliters of titrant used to moles of product formed, it is necessary to calibrate the titrant in the following way. A solution of DMPM vesicles in 0.6 mM CaC12, 1 mM NaC1 is prepared as described above and adjusted to pH 8.0 in the pH stat. A solution of myristic acid in ethanol of known concentration (typically 5/zl of a 40 mM solution) is added, and the pH is brought back to 8.0. The amount of titrant used corresponds to the number of moles of myristic acid added. This procedure is more accurate than a simple titration of a strong acid, such as HCI, in water, since it takes into account the fact that the surface pK a of myristic acid in the DMPM vesicle is not far below 8. Such an analysis has shown that the titration efficiency (moles of titrant used per mole of fatty acid added) is 0.90 for myristic acid in DMPM and 0.87 for myristic acid in DMPM vesicles at the end of the reaction progress curve when all of the substrate in the outer layer of the vesicles has been hydrolyzed. Thus, there is no significant change in the titration efficiency during the course of the enzymatic reaction. Comments on Assay of Phospholipase A 2 in Scooting Mode When interfacial catalysis by PLA2 is examined in the scooting mode, many of the previously reported anomalous features of the kinetics are not observed. The hydrolysis of DMPM vesicles begins immediately after the addition of enzyme; the latency period in the reaction progress that is seen in other assays s does not appear with vesicles of DMPM. In this sense, this system with vesicles is also distinctly different than the monolayer system of Verger and de Haas,2 where they assumed that the E to E* step is slow. On the other hand, as shown elsewhere by stopped-flow kinetic analysis, the rate of binding of the enzyme to the miceUe or vesicle interface is rapid. 24Therefore, we believe that the primary mechanism for the origin of the latency period is the much slower buildup of anionic product in the interface, which leads to a time-dependent modulation of the E to E* equilibrium. This is the major difference between our model 1 and the model proposed by Verger and de Haas. 2 24 M. K. Jain, J. Rogers, and G. H. de Haas, Biochim. Biophys. Acta 940, 51 (1988).
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The enzyme is still fully catalytically active at the end of the reaction progress, 14and the cessation of the reaction is due to the consumption of all of the substrate in the outer layer of enzyme-containing vesicles. When the enzyme to vesicle ratio is large enough so that all of the vesicles contain one or more bound enzymes, 63% of the total DMPM present in small sonicated vesicles is hydrolyzed, which corresponds to the fraction of substrate in the outer layer of the vesicles. With large unilamellar vesicles, 50% of the total DMPM is hydrolyzed whereas only small amounts (typically about 8%) of the substrate is hydrolyzed with multilamellar vesicles. The vesicles remain intact, even after all of the substrate in the outer layer has been hydrolyzed. Studies of vesicle fusion by light scattering and fluorescence resonance energy transfer have demonstrated that the rate of fusion of DMPM vesicles, under the above-described conditions, is much too slow to be of any significance except at the end of the first-order reaction progress curve (as discussed above). TM Finally, the rates of transbilayer movement of phospholipids and of intervesicle exchange of enzyme, phospholipid substrate, and the products are also very slow. Although detailed investigations of interfacial catalysis have been carried out with DMPM vesicles, similar results are seen with vesicles of other anionic phospholipids 16'25or with codispersions of zwitterionic and anionic phospholipids. 26 It is particularly striking to note here that over 50 PLA2s tested from different sources exhibit a first-order reaction progress curve of the type shown in Fig. 2. These include not only the enzymes from pancreas and their mutants, but also those from venoms of Apis, Elapidae, Viperidae, and Crotalidae, as well as bacteria like Escherichia coli and Saccharomyces. 27 Thus, the assay in the scooting mode with DMPM vesicles is by far the most generally useful procedure for studying PLA2s reported to date. Other assay procedures for analyzing PLA2s have been described, including the use of mixtures of phospholipids dispersed into detergents, 4,23,2s radiolabeled bacterial membrane fragments, 29 and vesicles of radiolabeled phospholipids.30 These other assays are not generally applicable to all enzymes, and in many cases it is necessary to include a lipophilic additive to promote the binding of the enzyme to the interface. 23 All of these problems stem from the assay-dependent variation in the 25 M. K. Jain and J. Rogers, Biochim. Biophys. Acta 1003, 91 (1989). 26 F. Ghomashchi, B. Yu, O. Berg, M. K. Jain, and M. H. Gelb, in preparation. 27 M. K. Jain, G. N. Ranadive, B.-Z. Yu, and H. M. Verheij, in preparation. 28 W. Niewenhuizen, H. Kunze, and G. H. de Haas, this series, Vol. 32B, p. 147. 29 For example, see R. L. Jesse and R. C. Franson, Biochim. Biophys. Acta 575, 467 (1979). 30 For example, see J. Balsinde, E. Diez, A. Schiiller, and F. Mollinedo, J. Biol. Chem. 263, 1929 (1988).
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amount of enzyme present in the interface (E to E* effects) during the steady-state catalytic turnover. These effects are not observed in the assay with DMPM since the E to E* step has been eliminated. Additional problems with micelle or mixed micelle assays due to substrate replenishment have been discussed. 1 Finally it should be mentioned that although the assay with DMPM described in this chapter makes use of a pH stat to detect the production of product, it is also possible to adopt other methods of product analysis. For example, it is possible to use radiolabeled phospholipids followed by chromatographic separation of the products and analysis of the amount of released radioactive fatty acid by scintillation counting. 26 The important point is that with DMPM as a matrix, the anomalous kinetic effects arising from the E to E* step are avoided.
Uses of Scooting Assay Interfacial catalysis in the scooting mode opens a new dimension in the study of PLA2. The protocols described here can be used as such or adopted appropriately to address questions related to,catalytic turnover, substrate specificity, interfacial rate constants, inhibition, and activation. Detailed studies on many of these topics are reported elsewhere. Because of space limitations, here we present a subset of the uses of the study of PLA2 in the scooting mode. Calibrating Solutions ofPhospholipase A2. Since the extent of hydrolysis, Pmax,under the conditions of excess vesicles to enzyme is proportional to the moles of catalytically active enzyme, the concentration of functional enzyme active sites in a stock solution of enzyme can be determined directly. We have found this protocol to be particularly useful for ascertaining the purity and homogeneity of chemically modified 27 or semisynthetic forms of PLA2 and genetically engineered enzyme mutants. The use of this assay with DMPM vesicles is the only known method for carrying out such a measurement. The usual methods of determining the concentration of PLA2 are based on assays of the total protein content, regardless of whether all of the enzyme in the solution is catalytically active. To obtain the concentration of catalytically active enzyme from the extent of hydrolysis, Pmax, one needs to know the value of NT. We have found that preparations of DMPM, prepared as described above, reproducibly give a value of about 13,000 for N~. However, the value of ArT may vary somewhat in different laboratories. The size of vesicles, prepared by sonication of DMPM, is known to be sensitive to a number of factors including the method of sonication, the presence of fatty acid heterogeneity in the synthesized substrate, and the presence of ions and other impurities. For this reason, it is recommended that the value of Nz be determined
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using a well-characterized PLA2, such as the enzyme from porcine pancreas. This enzyme, when purified by the published procedure, 2s consistently has a specific activity of 1300-1500 ~mol product/min/mg in an assay with egg yolk. 2s The concentration of porcine pancreatic PLA 2 in the stock solution is determined from the absorbance at 280 nm using the published extinction coefficient, 2s El%, of 13.0. The value of NT can then be obtained from a measurement of Pmax, under the conditions of excess vesicles over enzyme, and knowing the amount of porcine pancreatic PLA 2 added to the assay. Once the value of AfT is determined in this manner, it can be used to calibrate solutions of other, less well-characterized PLA2s. Intrinsic Catalytic Activities o f Phospholipases A 2 . The value of NTk i measured in the scooting assay is a characteristic property of a particular PLA2 acting on a particular substrate vesicle. It has the same meaning as the kcat/K m value for an enzyme that operates in water on a particular substrate, except for the fact that rate constants have units which are appropriate for interfacial catalysis) In the past, it has not been possible to obtain accurate values of the turnover numbers for PLA 2 since such calculations are based on maximal velocity measurements using different assays and require the assumptions that all of the enzyme in the solution is fully active and that the exchange of the enzyme, substrate, or products is not part of the catalytic turnover. Detection o f Phospholipase A 2 Impurities in Samples. The detection of trace amounts of PLA2 in biological samples, for example, in preparations of melittin, can be accurately determined using the scooting assay. A second example that comes to mind concerns the activity of unusual or mutant forms of PLA 2 . For example, a PLA 2 from Agkistrodon piscivorus piscivorus has been isolated that contains lysine in place of the catalytically important aspartate residue at position 49. 3~ This enzyme was reported to have significant catalytic activity; however, this result was recently challenged by van den Bergh et a1.,32 who provided strong evidence that the lysine-49 enzyme is contaminated with a very active aspartate-49 PLA2. This problem can be easily and conclusively resolved by analyzing the enzyme in the scooting assay. The moles of catalytically active enzyme in the stock solution can be determined from the values of Pmax and NT since every active molecule of enzyme will eventually hydrolyze all of the substrate in the outer layer of the vesicle to which it is bound. This is true even in the case that the particular P L A 2 under investigation has an 3~ j. M. Maraganore, G. Merutka, W. Cho, W. Welches, F. J. Kezdy, and R. L. Heinrikson, J. Biol. Chem. 259, 13839 (1984). 32 C. J. van den Bergh, A. J. Slotboom, H. M. Verheij, and G. H. de Haas, J. Cell. Biochem. 39, 379 (1989).
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extremely small interfacial turnover number, N T k i . If the absolute number of catalytically active enzyme molecules in the stock solution, as determined using the scooting assay, is much smaller than the total number of enzyme molecules, calculated using a standard protein assay (i.e., absorbance at 280 nm), one would conclude that an active PLA2 impurity is present in a large amount of inactive PLA2 protein. In all of these studies, it is important to check that the sample to be tested does not contain impurities which induce the fusion of vesicles. Other Uses of Scooting Assay and Concluding Remarks In addition to the above-mentioned analyses, the scooting assay has numerous other uses. For example, there has bccn considcrablc controversy in the literature as to whether a particular PLA2 is catalytically active as a dimcr or as a monomer. 4 This issue can bc resolved using the scooting assay.aVIn the presence of excess vesiclesover cnzymc, the value of Pmax will be twicc as largc for a given numbcr of moles of monomeric cnzymc compared to the same number of moles of a dimeric enzyme. Thc scooting assay is also very useful in the discovery of compounds that function as truc competitive inhibitorsof PLA2.13 For example, many of the previously reported P L A 2 inhibitors function by promoting the dcsorption of cnzymc from thc intcrfacc (E to E* effects) rather than by binding directly to the bound enzyme. Thcsc compounds do not inhibit thc action of PLAE in thc scooting mode. 13 In the inhibition studies, thc kinetics wcrc dctcrmincd at higher calcium concentrations (2.5rather than 0.6 raM). Under thcsc conditions the vesicles arc much largcr, and thc shape of thc reaction progress curve is differentthan that shown in Fig. 2 in that itcontains a significantconstant velocity at early times (zero-order phase). This is mainly a result of the fact that the buildup of product and the depletion of substratc take longer with larger vesicles, and the initial velocity is maintaincd for a longer pcriod than in the small vesicles dcscribed hcrc. The fulldctailsof the inhibitionanalysis at high calcium will bc dcscribcd) 3 Perhaps the most important use of the scooting assay will be in the determination of the absolutc substratc spccificiticsand the interracialrate constants of lipolyticenzymes. In previous studies of substratc spccificitics,rclativcvelocitiesfor the action of PLA2s and other lipolyticenzymes on a variety of differentsubstratc vcsiclcs composed of pure phospholipid classes havc bccn reported. In thcse studics, no effort has bccn made to normalize for thc amount of enzyme bound to the interface. The use of 33 M. K. Jain, B.-K. Yu, and O. G. Berg, in preparation.
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the scooting assay, in which all of the enzyme is in the interface, provides an extremely powerful solution to this problem. ~6 At first glance, the scooting assay may appear somewhat unsettling and counterintuitive to traditional enzymologists. The irreversible nature of the interaction of the enzyme with the interface may not be an accurate representation of the action of the enzyme in a physiological environment, such as the inside of a cell. However, this is not a concern, since under physiological conditions all of the fundamental features of interfacial catalysis, whether they involve the selection of various substrates within the cell membrane or the inhibition of the enzyme, will occur within the interface. In other words, only events such as lipid exchange and other processes that occur in parallel to the events within the interface have been constrained in the scooting assay. The mechanism of action of the enzyme within the interface is not altered by this technique. The scooting assay provides an attractive in vitro method for the analysis of lipolytic enzymes that is free from the distortions caused by the relative affinities of the PLA 2 (E to E* effects) for a variety of vesicles composed of pure phospholipids. Acknowledgments This work was supported by Grants HL-36235 (M. H. G.) and GM-29703 (M. K. J.) from the National Institutes of Health and by a grant from Sterling PharmaceuticalCo. (M. K. J.). The authorswouldliketo give specialthanksto FaridehGhomashchifor technical assistance.
[11] A s s a y o f P h o s p h o l i p a s e s C a n d D in P r e s e n c e o f O t h e r Lipid Hydrolases
By KARL Y. HOSTETLER,MICHAEL F. GARDNER,and KATHY A. ALDERN
Introduction Phospholipases C and D were first identified in bacteria I and plants, 2 respectively, but they are now known to be present in mammalian cells. These activities are generally not difficult to measure, and a variety of methods are available. The methods are usually based on the release of a i M. G. Macfarlane and B. C. J. Knight, Biochem. J. 35, 884 (1941). 2 D. J. H a n a h a n and I. L. Chaikoff, J. Biol, Chem. 169, 669 (1947).
METHODS IN ENZYMOLOGY,VOL. 197
Copyright © 1991 by Academic Press, Inc. All rightsof reproduction in any form reserved.
