Rotational dynamics of the single tryptophan of porcine pancreatic phospholipase A2, its zymogen, and an enzyme/micelle complex. A steady-state and time-resolved anisotropy study

The rotational dynamics of the single tryptophan of porcine pancreatic phospholipase A2 and its zymogen (prophospholipase A2) have been studied by polarized fluorescence using steady-state and time-resolved single-photon counting techniques. The motion of Trp-3 in phospholipase A2 consists of a rapid subnanosecond wobble of the indole ring with an amplitude of about +/- 20 degrees accompanied by slower isotropic rotation of the entire protein. The rotational correlation times for overall particle rotational diffusion are consistent with conventional hydrodynamic theory. When phospholipase A2 binds to micelles of n-hexadecylphosphocholine, the amplitude of the fast ring rotation decreases. The whole particle rotational correlation time of the enzyme/micelle complex is smaller than the minimum value calculated from hydrodynamic theory. A similar result is obtained for the micelle itself by using the lipophilic probe transparinaric acid. These low values for the particle correlation times can be understood by postulating that an isotropic motion of the fluorophore in the small detergent particles contributes to the angular reorientation of the fluorophore. The internal reorientational motion of the tryptophan in the zymogen, prophospholipase A2, is of larger amplitude than that observed for the enzyme; specifically, the proenzyme exhibits a motion with a significant amplitude on the nanosecond time scale. This additional freedom of motion is attributed to segmental mobility of the N-terminal residues of prophospholipase A2. This demonstrates that this region of the protein is flexible in the zymogen but not in the processed enzyme. The implications of these findings for the mechanism of surface activation of phospholipase A2 are discussed by analogy with a trypsinogen-trypsin activation model.     

Unusual kinetic behavior of porcine pancreatic (pro)phospholipase A2 on negatively charged substrates at submicellar concentrations

The negatively charged detergents S-n-alka-noylthioglycol sulfates (C8, C9, and C10) are substrates for porcine pancreatic phospholipase A2 and its zymogen. At pH 6.0 and detergent concentrations up to 0.08 X critical micelle concentration (cmc), the activities of active enzyme and zymogen are similar and very low. From 0.08 X cmc to 0.12 X cmc a tremendous increase in activity is observed for phospholipase A2, but not for the zymogen. Concomitant with this increase in activity there is a sharp rise in molecular weight of the substrate-enzyme complex, from 15 000 to 95 000, and in detergent to protein molar ratio of 1:1 to about 7:1. This indicates both substrate and enzyme aggregation. Most probably a lipid-water interface is formed inside the aggregated protein particle by which the enzyme is activated. Although the zymogen also forms high molecular weight complexes with similar molar ratios, no activation is observed probably because of distortion of its lipid binding domain.     

Kinetic analysis of phospholipase A2 activity toward mixed micelles and its implications for the study of lipolytic enzymes.

A detailed kinetic scheme is proposed for the action of phospholipase A2 on mixed micelles of phospholipid and surfactant: see article. where E is the enzyme, A is the mixed micelle, and B is the phospholipid substrate in the mixed micelle. This scheme takes into account quantitatively the involvement of the lipid-water interface in the action of this enzyme toward substrate in macromolecular lipid complexes. The kinetic equation for this scheme is derived and four simplifying assumptions which are necessary for its practical application are described. Kinetic data are reported for the action of cobra venom phospholipase A2 (Naja naja naja) on 1,2-dipalmitosyl-sn-glycero-3-phosphorylcholine in mixed micelles with the nonionic surfactant Triton X-100, and these data are analyzed in terms of the kinetic equation presented. At 40 degrees, pH 8.0, and in the presence of 10 mM Ca2+, V was found to be about 4 X 10(3) mumol min(-1) mg of protein(-1). KsA, which is the dissociation constant for the enzyme-mixed micelle complex, is about 5 X 10(-4) M. KmB, the Michaelis constant for the catalytic step, which is (k-2 + k3)/k2, is 1 to 2 X 10(-10) mol cm-2. This kinetic treatment, together with the fact that the mixed micelle system allows the concentration of the substrate in the lipid-water interface to be varied, has made possible the quantitative separation of the association of a lipolytic enzyme with the lipid-water interface (expressed as KsA) and the binding to the substrate in the interface (reflected in the KmB term). The implications of this kinetic scheme for the analysis of phospholipase A2 from other sources acting on other aggregated forms of phospholipid and for the study of other phospholipases and lipases is considered.     

Activation of porcine pancreatic phospholipase A2 by the presence of negative charges at the lipid-water interface

The effect of surface charge on the porcine pancreatic phospholipase A2 catalyzed hydrolysis of organized substrates was examined through initial rate enzyme kinetic measurements. Two long-chain phospholipid substrates, phosphatidylglycerol (PG) and phosphatidylcholine (PC), were solubilized in seven detergents differing in polar head-group charge. The neutral or zwitterionic detergents selected were Triton X-100, Zwittergent 314, lauryl maltoside, hexadecylphosphocholine (C16PN), and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The negatively and positively charged detergents used were cholate and CTAB, respectively. In general, the negatively charged phospholipid PG was hydrolyzed much more rapidly than the neutral (zwitterionic) phospholipid PC. The rate of hydrolysis of PG was rapid when solubilized in all the neutral detergents and in cholate but was essentially zero in the positively charged CTAB. Conversely, hydrolysis of PC was negligible when solubilized in neutral detergents, except C16PN, and was maximal in the negatively charged detergent, cholate. The rate of hydrolysis of PC solubilized in a neutral detergent became significant only when a negative surface charge was introduced by addition of SDS. Taken together, these kinetic measurements indicate that the surface charge on the lipid aggregates is an important factor in the rate of hydrolysis of phospholipid substrates and the highest activity is observed when the net surface charge is negative. Fluorescence and electron spin resonance (ESR) spectroscopic data provide additional support for this conclusion. The fluorescence emission spectrum of the single tryptophan of phospholipase A2 is a sensitive monitor of interfacial complex formation and shows that interaction of the protein with detergent micelles is strongly dependent on the presence of a negatively charged amphiphile.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adsorption of pancreatic (pro)phospholipase A2 to various physiological substrates

The adsorption of pancreatic phospholipase was studied in vitro in the presence of egg yolk lipoprotein emulsion, Intralipid emulsion, and milk fat globules. When the emulsions are incubated with bile salts, the latter dissociate a considerable fraction of the phospholipids initially associated with the emulsions, leading to the coexistence of an emulsified phase and a phase of mixed micelles. After the addition of pancreatic phospholipase A2, gel filtration shows that the enzyme was more than 90% bound to mixed micelles, regardless of the type of emulsion used. Comparable results were obtained by replacing the bile salts with human gallbladder bile. In parallel, pancreatic zymogen was never found to be bound to any of the lipid structures present (emulsion or mixed micelles). When the catalytic site of pancreatic phospholipase A2 was blocked with 4-bromophenacylbromide, there was no fixation on mixed micelles. Fixation was restored by the presence of lysolecithins and fatty acids in the incubation mixtures. The partial transformation of all emulsified substrates to mixed micelles by bile salts in vivo would thus lead to optimum activity of pancreatic phospholipase A2.     

A Fourier transform infrared spectroscopic (FTIR) study of porcine and bovine pancreatic phospholipase A2 and their interaction with substrate analogues and a transition-state inhibitor

Fourier transform infrared spectroscopy has been used to investigate the secondary structure of porcine and bovine pancreatic phospholipase A2 (PLA2) and the zymogen of porcine PLA2, prophospholipase A2 (proPLA2), in both H2O and D2O media. Detailed qualitative analysis was made of these proteins using second derivative and deconvolution techniques. Quantitative studies of the proteins in solution made using Factor Analysis gave average values of 54% alpha-helix, 15% beta-sheet and 23% beta-turns. These values agree well with the secondary structures deduced from previous studies of single crystals using X-ray techniques. No significant differences in secondary structure were observed for porcine pancreatic (pro)phospholipase A2 in the presence or absence of Ca2+ ions, or in the temperature range 10-45 degrees C. The binding of the non-degradable phospholipid analogue, n-alkylphosphocholine, in monomeric form produced no significant difference in the secondary structure of either enzyme. Conformational differences were, however, observed between the enzyme lyophilised in a solid film and in aqueous solution. The change is probably due to the formation of beta-sheet upon hydration, coupled with a loss of random structures. Conformational differences in both porcine and bovine pancreatic PLA2 were observed on binding to n-alkylphosphocholine micelles. This change may be due to a small increase in the alpha-helical structure and a decrease in the beta-sheet, and/or possibly beta-turn content. Similar conformational changes were observed for the interaction of porcine and bovine PLA2 with the substrate analogue inhibitor 1-heptanoyl-2-heptanoylamino-2-deoxy-sn-glycero-3-phospho glycol in micellar form.     

Kinetic analysis of the dual phospholipid model for phospholipase A2 action

A kinetic scheme is proposed for the action of cobra venom phospholipase A2 on mixed micelles of phospholipid and the nonionic detergent Triton X-100, based on the "dual phospholipid model." (formula; see text) The water-soluble enzyme binds initially to a phospholipid molecule in the micelle interface. This is followed by binding to additional phospholipid in the interface and then catalytic hydrolysis. A kinetic equation was derived for this process and tested under three experimental conditions: (i) the mole fraction of substrate held constant and the bulk substrate concentration varied; (ii) the bulk substrate concentration held constant and the Triton X-100 concentration varied (surface concentration of substrate varied); and (iii) the Triton X-100 concentration held constant and the bulk substrate concentration varied. The substrates used were chiral dithiol ester analogs of phosphatidylcholine (thio-PC) and phosphatidylethanolamine (thio-PE), and the reactions were followed by reaction of the liberated thiol with a colorimetric thiol reagent. The initial binding (Ks = k1/k-1) was apparently similar for thio-PC and thio-PE (between 0.1 and 0.2 mM) as were the apparent Michaelis constants (Km = (k-2 + k3)/k2) (about 0.1 mol fraction). The Vmax values for thio-PC and thio-PE were 440 and 89 mumol min-1 mg-1, respectively. The preference of cobra venom phospholipase A2 for PC over PE in Triton X-100 mixed micelles appears to be an effect on k3 (catalytic rate) rather than an effect on the apparent binding of phospholipid in either step of the reaction.     

The interaction between the presynaptic phospholipase neurotoxins beta-bungarotoxin and crotoxin and mixed detergent-phosphatidylcholine micelles. A comparison with non-neurotoxic snake venom phospholipases A2

Certain phospholipase A2 enzymes (E.C.3.1.1.4) selectively inhibit neurotransmitter release from cholinergic nerve terminals. Both specific acceptor proteins and the physical state of nerve terminal phospholipids have been implicated in studies of the mechanism of phospholipase neurotoxin action. Here we have examined the effects of charge on a micellar phospholipid substrate by comparing the enzyme activity and binding of two neurotoxic phospholipases (beta-bungarotoxin and crotoxin) with other non-neurotoxic phospholipases. This has been achieved by altering either the phospholipid or the ionic charge of the detergent in the mixed phospholipid micelle. The neurotoxic phospholipases were only active on negatively charged micelles, whereas the non-neurotoxic enzymes were equally active in hydrolyzing neutral micelles. This distinction was also reflected in binding studies; the non-neurotoxic phospholipases bound to both types of substrate, whereas beta-bungarotoxin and crotoxin selectively bound to negatively charged micellar structures. These experiments suggest that, in addition to the existence of any specific acceptor proteins, neurotoxin binding is also governed by the charge on the lipid phase of the nerve terminal membrane.     

Analysis of the kinetics of phospholipid activation of cobra venom phospholipase A2

A kinetic analysis of the "dual phospholipid model" for cobra venom phospholipase A2 (Hendrickson, H. S., and Dennis, E. A. (1984) J. Biol. Chem. 259, 5734-5739) was applied to the activation of phospholipase A2-catalyzed hydrolysis of a thiol ester analog of phosphatidylethanolamine (thio - PE) in Triton X - 100/phospholipid mixed micelles by various phosphorylcholine-containing activators. Activation of thio-PE hydrolysis by didecanoylphosphatidylcholine (PC) was found to be a function of the surface concentration of activator rather than bulk concentration. Its presence did not affect the initial binding of enzyme to phospholipid in the micelle surface as determined kinetically. After initial binding of enzyme to the surface, the activation appears to be due to enzyme-lipid binding in the surface. Activation does not appear to affect the affinity of the enzyme for phospholipid substrate, but rather affects the catalytic efficiency of the enzyme as characterized by the value of Vmax. The monomeric phospholipid dibutyryl-PC, when used as an activator at 57 mM (bulk concentration), also showed effects of surface dilution with Triton X-100, which would not be expected unless the lipid is incorporated into the micelles to some extent at these high concentrations. A thiol ester analog of phosphatidylcholine, thio-PC, was less effective than didecanoyl-PC as an activator, but appeared to be more effective than decylphosphorylcholine. A conformational change of the enzyme upon binding of the activator, after enzyme is bound to substrate at the interface, is discussed as a possible mechanism for this activation.     

Calcium ion binding to pancreatic phospholipase A2 and its zymogen: a 43Ca NMR study

Calcium ion binding to phospholipase A2 and its zymogen has been studied by 43Ca NMR. The temperature dependence of the band shape of the calcium-43 NMR signal has been used to calculate the calcium ion exchange rate. The on-rate was calculated to be 5 X 10(6) M-1 s-1, which is 2 orders of magnitude less than the diffusion limit of the hydrated Ca2+ ion in water. The 43Ca quadrupole coupling constant for calcium ions bound to phospholipase, chi = 1.4 MHz, is significantly larger than those found for EF-hand proteins, indicating a less symmetric site. For prophospholipase A2, we found chi = 0.8 MHz, indicating a calcium binding site, which is somewhat more symmetric than the EF-hand sites. The dependence of the 43Ca NMR band shape on the calcium ion concentration showed that there are two cation binding sites on the phospholipase A2 molecule: K1 = 4 X 10(3) M-1 and K2 = 20 M-1. The strong site was found to be affected by a pKa = 6.5 and the weak site by pKa = 4.5.     

Regulation of CTP:phosphocholine cytidylyltransferase by lipids. 1. Negative surface charge dependence for activation.

The activity of phosphocholine cytidylyltransferase (CT), the regulatory enzyme in phosphatidylcholine synthesis, is dependent on lipids. The enzyme, obtained from rat liver cytosol, was purified in the presence of Triton X-100 [Weinhold et al. (1986) J. Biol. Chem. 261, 5104]. The ability of lipids to activate CT when added as Triton mixed micelles was limited to anionic lipids. The relative effectiveness of the lipids tested suggested a dependence on the negative surface charge density of the micelles. The mole percent lipid in the Triton mixed micelle required for activation decreased as the net charge of the lipid varied from 0 to -2. Evidence for the physical association of CT with micelles and vesicles containing phosphatidylglycerol was obtained by gel filtration. The activation by micelles containing PG was influenced by the ionic strength of the medium, with a higher surface charge density required for activation at higher ionic strength. The micelle surface potential required for full activation of CT was calculated to be -43 mV. A specificity toward the structure of the polar group of the acidic lipids was not apparent. CT was activated by neutral lipids such as diacylglycerol or oleyl alcohol when included in an egg PC membrane, but the activities were reduced by dilution with as little as 10 mol % Triton. Thus Triton mixed micelles are not suitable for studying the activation of CT by these neutral lipid activators. We conclude that one way that lipid composition can control CT-membrane binding and activity is by changing the surface potential of the membrane. Other distinct mechanisms involved in the activation by neutral lipids are discussed.     

Conformational changes in phospholipase A2 upon binding to micellar interfaces in the absence and presence of competitive inhibitors. A 1H and 15N NMR study.

An NMR study has been made of porcine pancreatic phospholipase A2 (PLA) in three environments: free in solution, in a binary complex with dodecylphosphocholine micelles, and in a ternary complex with a micelle and the substrate-like inhibitor (R)-1-octyl-2-(N-dodecanoylamino)-2-deoxyglycero-3-phosph oglycol. 1H and 15N chemical shifts, amide exchange rates, and NOE intensities are compared for the enzyme in different environments. From these data, structural differences are found for the N-terminal part, the end of the surface loop at residues Tyr69 and Thr70, and the active site residue His48, and also for the Ca-binding loop (residues 28-32). Specifically, when binding to a micelle, the side chains of residues Ala1, Trp3, and Tyr69, as well as all protons of Thr70, are found to be closer together. After subsequent introduction of the competitive inhibitor, further changes are found for these residues. The N-terminus is flexible in PLA free in solution, in contrast with the crystal structures where it adopts an alpha-helical conformation. According to the NMR data, this helix is rigidly formed only in the ternary complex. Furthermore, in the ternary complex, the N-terminal amino group and the exchangeable hydrogen at N3 of the ring of His48 are observed. We propose that PLA is activated in two steps. An initial conformational change occurs upon binding to a micellar interface. The catalytically active conformation of the enzyme, which has an extensive network of hydrogen bonds, is formed only when binding a substrate or competitive inhibitor at a lipid-water interface.     

Molecular properties and kinetic studies on sphingomyelinase of Bacillus cereus

A sphingomyelinase of Bacillus cereus was purified to a homogeneous state (512 U/mg, 2200-fold) as indicated by SDS-polyacrylamide gel electrophoresis and the molecular weight (23,300) was determined by sedimentation equilibrium. The enzyme contained loosely-bound magnesium atom. The addition of Mg2+ accelerated the enzyme reaction regardless of substrates and their physical state. The addition of Ca2+ also accelerated the enzyme reaction slightly, when water-soluble substrates, i.e., 2-hexadecanoylamino-4-nitrophenylphosphorylcholine and p-nitrophenylphosphorylcholine, were used as substrates. On the other hand, the addition of Ca2+ inhibited enzyme reaction when mixed micelles of either sphingomyelin and Triton X-100 or sodium deoxycholate were used. The surface charge on mixed micelles affected the enzyme reaction. When the mixed micelle of sphingomyelin and Triton X-100 was used as substrate, Ca2+ proved to be a competitive inhibitor against Mg2+, with a Ki value of 33 microM. On the other hand, when the mixed micelle of sphingomyelin and sodium deoxycholate was used as substrate, Ca2+ stimulated the enzyme reaction at lower concentration in the presence of a low concentration of Mg2+, although higher concentrations of Ca2+ were still inhibitory. In this case, added Ca2+ may be used as a substitute of Mg2+ to neutralize the negative charge on the mixed micelle, improving the accessibility of sphingomyelinase to the micellar substrate. A cationic detergent, cetyltrimethylammonium bromide, seemed to denature or inactivate the enzyme.     

Characteristics of kinetics of phospholipid hydrolysis by phospholipase C from Bacillus cereus. Hydrolysis of phosphatidylcholine in the presence of deoxycholate

Using dynamic light scattering and 31P-NMR spectroscopy methods, the reaction of solubilization of phosphatidylcholine by the ionic detergent, sodium deoxycholate, in aqueous solutions was studied. The kinetics of phosphatidylchodine hydrolysis by phospholipase C from B. cereus depending on the size and structural organization of substrate aggregates was investigated. No phosphatidylcholine hydrolysis was observed in the case of lamellar organization of the substrate, the size of lamellas not exceeding 2000-5000 A. The substrate hydrolysis rate within mixed micelles was controlled by the accessibility of the substrate on the surface of micellar aggregates. There was a decrease in the phosphatidylcholine hydrolysis rate at high detergent concentrations in the system. It was concluded that such a decrease in the hydrolysis rate can be due to two reasons, i) the decrease in mixed micelle size with a simultaneous decrease of surface concentration of the substrate, and, ii) the formation of "pure" detergent micelles capable to adsorb the enzyme by decreasing the "effective" concentration of phospholipase C.     

PFG-NMR investigations of the binding of cationic neuropeptides to anionic and zwitterionic micelles

The mechanism by which peptides bind to micelles is believed to be a two-phase process, involving (i). initial electrostatic interactions between the peptide and micelle surface, followed by (ii). hydrophobic interactions between peptide side chains and the micelle core. To better characterize the electrostatic portion of this process, a series of pulse field gradient nuclear magnetic resonance (PFG-NMR) spectroscopic experiments were conducted on a group of neuropeptides with varying net cationic charges (+1 to +3) and charge location to determine both their diffusion coefficients and partition coefficients when in the presence of detergent micelles. Two types of micelles were chosen for the study, namely anionic sodium dodecylsulfate (SDS) and zwitterionic dodecylphosphocholine (DPC) micelles. Results obtained from this investigation indicate that in the case of the anionic SDS micelles, peptides with a larger net positive charge bind to a greater extent than those with a lesser net positive charge (bradykinin > substance P > neurokinin A > Met-enkephalin). In contrast, when in the presence of zwitterionic DPC micelles, the degree of mixed-charge nature of the peptide affects binding (neurokinin A > substance P > Met-enkephalin > bradykinin). Partition coefficients between the peptides and the micelles follow similar trends for both micelle types. Diffusion coefficients for the peptides in SDS micelles, when ranked from largest to smallest, follow a trend where increasing net positive charge results in the smallest diffusion coefficient: Met-enkephalin > neurokinin A > bradykinin > substance P. Diffusion coefficients when in the presence of DPC micelles, when ranked from largest to smallest, follow a trend where the presence of negatively-charged side chains results in the smallest diffusion coefficient: bradykinin > Met-enkephalin > substance P > neurokinin A.     

Amino acid substitutions of the NH2-terminal Ala1 of porcine pancreatic phospholipase A2: a monolayer study

Previously it has been shown that the binding of porcine pancreatic phospholipase A2 to lipid-water interfaces is governed by the pK of the alpha-NH3+ group of the N-terminal alanine. Chemically modified phospholipases A2 in which the N-terminal Ala has been replaced by D-Ala or in which the polypeptide chain has been elongated with DL-Ala no longer display activity toward micellar substrate. The activity of DL-Ala-1-, [D-Ala1]-, and [Gly1]phospholipases A2 on substrate monolayers, which allow a continuous change in the packing density of the lipid molecule, was investigated. At pH 6 [Gly1]phospholipase A2 behaves like the native enzyme on lecithin monolayers. DL-Ala1- and [D-Ala1]phospholipases A2, although they are active in this system, showed a weaker lipid penetration capacity at this pH. Studies on the pH and Ca2+ ion dependency of the pre-steady-state kinetics and of the activity of these radiolabeled proteins showed that [D-Ala1]phospholipase A2 does not possess a second low-affinity site for Ca2+ ions in contrast to the native phospholipase A2. This second low-affinity Ca2+ binding site, which is also absent in [Gly1]phospholipase A2, is induced in the latter enzyme by the presence of lipid-water interfaces.     

On the substrate specificity of rat liver phospholipase A1

The substrate specificity of purified phospholipase A1 was studied using mixed micelles of phospholipid and Triton X-100. The kinetic analysis employed determined Vmax, Ks (a dissociation constant for the phospholipase A1-mixed micelle complex), and Km (the Michaelis constant for the catalytic step which reflects the binding of the enzyme to the substrate in the interface). The order of Vmax values was phosphatidic acid greater than phosphatidylethanolamine greater than phosphatidylcholine greater than phosphatidylserine. The order of Ks values was phosphatidylcholine greater than phosphatidylethanolamine greater than phosphatidic acid greater than phosphatidylserine; the order of Km values was phosphatidic acid greater than phosphatidylethanolamine = phosphatidylserine greater than phosphatidylcholine. When present together, phosphatidylcholine inhibited the hydrolysis of phosphatidylethanolamine but phosphatidylethanolamine did not affect the hydrolysis of phosphatidylcholine. Sphingomyelin, phosphatidylcholine plasmalogen, and phosphatidylethanolamine plasmalogen had no effect on the hydrolysis of phosphatidylethanolamine. The effects of the reaction products, lysolipids and/or fatty acids, were also considered for their influence on phosphatidylethanolamine hydrolysis catalyzed by phospholipase A1. Free fatty acid was found to inhibit, whereas lysophospholipids stimulated hydrolysis of phosphatidylethanolamine. In a mixture of 1,2- and 1,3-diacylglycerides in mixed micelles, only the acyl chain at the sn-1 position of the 1,2 compound was hydrolyzed. Surface charge did not modulate the hydrolysis of phosphatidylcholine vesicles or mixed micelles. In conclusion, it is hypothesized that steric hindrance at position 3 of the glycerol regulates substrate binding in the active site and that an acyl group in position 1 is favored over a vinyl ether linkage for binding.     

Catalytic Ca2+-binding site of pancreatic phospholipase A2: laser-induced Eu3+ luminescence study

7F0----5D0 excitation spectroscopy of Eu3+ has been used to study the catalytic Ca2+-binding site of pancreatic phospholipases A2. Eu3+ binds competitively with Ca2+ to the enzyme with retention of about 5% of the activity found with Ca2+. The dissociation constants for the Eu3+-enzyme complexes of bovine phospholipase A2 and porcine isophospholipase A2 are 0.22 mM and 0.16 mM, respectively. Results obtained with the porcine phospholipase A2 at neutral pH indicate aggregation of this enzyme at protein concentrations above 0.18 mM. The Eu3+ bound at the catalytic site of pancreatic phospholipase A2 is coordinated to four or five water molecules, which, in conjunction with binding constant data, suggests the involvement of two or three protein ligands. Addition of a monomeric substrate analogue to the enzyme-Eu3+ complex results in the loss of an additional water molecule from the first coordination sphere of the bound Eu3+. This result suggests an interaction between the negative charge of the polar head group of the substrate analogue and the Eu3+. Binding of the enzyme-Eu3+ complex to micelles results in a nearly complete dehydration of the Eu3+ bound to the catalytic center. In the phospholipase A2-Eu3+-micelle complex, only one H2O molecule is coordinated to Eu3+. This dehydration at the active site of phospholipase A2 in the protein-lipid complex can be an important reason for the enhanced activity of this enzyme at lipid-water interfaces.     

Light-scattering from detergent-complexed biological macromolecules

A theory is described for Rayleigh light-scattering from solutions of detergent-complexed macromolecules applicable to measurements carried out under conditions of Donnan equilibrium. The theory shows that when scattering measurements are made on detergent-solubilized macromolecules in the presence of detergent micelles the apparent Mr is dependent on the extent of detergent binding and effective charge on the detergent-macromolecule complex and the micellar charge and aggregation number. Equations are given for the apparent Mr of the macromolecule under limiting conditions of high salt and low salt concentration. Low-angle laser-light-scattering measurements were made on lysozyme complexed with sodium n-dodecyl sulphate both in the absence and in the presence of detergent micelles. These experimentally obtained data were used in conjunction with the detergent-binding isotherm to test the theory at high ionic strength. Light-scattering measurements were also made on detergent-saturated complexes as a function of ionic strength and pH. The results are in reasonable accord with both the qualitative and the quantitative predictions of the theory.     

Specific transformations at the N-terminal region of phospholipase A2.

Treatment of porcine pancreatic prophospholipase A2 with methyl acetimidate converted all lysine residues into epsilon-acetimidolysine residues. Enzymatically active epsilon-amidinated phospholipase A2 (AMPA) was obtained from the epsilon-amidinated zymogen by limited tryptic proteolysis cleaving the Arg7-Ala8 bond. AMPA was used to prepare des-Ala8-, des-(Ala8,Leu9)- and des-(ALa8),Leu9,Trp10)-AMP by successive Edman degradations, and des-(A la 8-Arg13)-AMPA by selective splitting of the Arg13-Ser14 bond by trypsin. Structural analogues of AMPA with different N-terminal amino acid residues, viz., D-Ala, beta-Ala, and Gly, have been prepared by reacting des-Ala8-AMPA with the corresponding N-t-Boc-N-hydroxysuccinimide esters of these amino acids. Similarly, the only Trp10 residue has been substituted for Phe by coupling of des-(Ala8-,Leu9,Trp10)-AMPA with N-t-Boc-L-Ala-L-Leu-L-Phe-N-hydroxysuccinimide ester. The feasibility of these substitutions has been proven unambiguously by the retroconversion of des-Ala8-AMPA and of [Ala7]AMPA into AMPA having identical enzymatic activity as the starting AMPA. The single Trp10 residue in native phospholipase A2 and its zymogen was specifically sulfenylated using 0-nitrophenyl-sulfenyl chloride. The homogenous proteins were kinetically analyzed using short-chain lecithins in the monomeric and micellar region. All modified AMPA analogues, except those in which two or more of the N-terminal amino acid residues are removed, show enzymatic activities toward monermic substrate comparable to that of AMPA, indicating that the active site region is still intact. Only [Gly8]-, [beta-Ala8]-, and [Ala8,Leu9,Phe10]AMPA exhibit a dramatic increase in enzymatic activity similar to that of AMPA upon passing the critical micellar concentration (cmc) of the substrate. From these results it can be concluded that the N-terminal region of the enzyme requires a very precise architecture in order to interact with lipid-water interfaces and consequently to display its full enzymatic activity.