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M K Jain

Publications and source records attributed to M K Jain.

At least 127 records · Page 7Linked to original sources
PubMed1993

Relationship of interfacial equilibria to interfacial activation of phospholipase A2.

The equilibrium dissociation constants for the distribution of pig pancreatic phospholipase A2, its competitive inhibitors, and their complexes at the interface of a neutral diluent are determined. The relationship between these parameters and their significance for interfacial catalysis is elaborated in terms of a model based on the relationship between the underlying equilibria. By using a combination of spectroscopic and chemical modification methods, it was possible to determine the equilibrium dissociation constant of an inhibitor bound to the interface (K') or of the inhibitor bound to the enzyme in the aqueous phase (KI) or the interface (KI*). The equilibrium dissociation constant for the free enzyme (Kd) or for the enzyme-inhibitor complex (KdI) from the interface were also obtained. These constants are shown to be thermodynamically related, i.e., K'KdKI* = KIKdI, as predicted on the basis of the cyclic equilibrium scheme (thermodynamic box) describing the distribution of the enzyme and the inhibitor between the aqueous phase and the interface at constant calcium concentration. Results show that (i) calcium is required for the binding of a substrate or inhibitor molecule to the catalytic site; (ii) the effective dissociation constant of the inhibitor-enzyme complex in the aqueous phase is considerably larger than that for the enzyme at the interface, i.e., KI >> effective KI*; (iii) KdI does not depend on the structure of the inhibitor and Kd >> KdI; and (iv) structure-activity correlations suggest that ionic interactions between a ligand and the interfacial recognition site of the enzyme are important for K', which controls the concentration of the bound inhibitor that the enzyme "sees" in the interface. These observations demonstrate that the binding of the enzyme to the interface and the binding of the inhibitor to the active site of the enzyme at the interface are two distinguishable processes. Therefore, binding of a ligand to the active site of the enzyme promotes binding of the enzyme-inhibitor complex to other amphiphiles and the interface with higher affinity. It is suggested that the primary effect of binding the enzyme to the interface is to increase its intrinsic affinity toward the active-site-directed ligands, i.e., the interfacial activation of PLA2 is of K-type.

Animals↗
PubMed1993

The chemical step is not rate-limiting during the hydrolysis by phospholipase A2 of mixed micelles of phospholipid and detergent.

The effect of detergents on the overall catalytic turnover by secreted phospholipase A2 (PLA2) on codispersions of the substrate phospholipid is characterized. The overall rate of interfacial catalytic turnover depends on the effective substrate "concentration" (mole fraction) that the bound enzyme "sees" at the interface. Therefore, besides the intrinsic catalytic turnover rate determined by the Michaelis-Menten cycle in the interface [Berg et al. (1991) Biochemistry 30, 7283], two other interfacial processes significantly alter the overall effective rate of hydrolysis: first, the fraction of the total enzyme at the interface; second, the rate of replenishment of the substrate. At low mole fractions (< 0.3), bile salts promote the binding of pig pancreatic PLA2 to zwitterionic vesicles, and the rate of hydrolysis increases with the fraction of the enzyme in the interface. At higher (> 0.3) mole fractions of the detergent, the bilayer is disrupted, and the rate of hydrolysis decreases by more than a factor of 10. The detergent-dependent decrease in the rate of hydrolysis of the sn-2-oxyphospholipids is much larger than that of sn-2-thiophospholipid, and therefore the element effect (O/S ratio) decreases from about 10 in bilayers to less than 2 in mixed micelles. This loss of the element effect in mixed micelles shows that the chemical step is no longer rate-limiting during the hydrolysis of mixed micelles formed by the disruption of vesicles by the detergent. Such effects were observed with phospholipase A2 from several sources acting on substrates dispersed in a variety of detergents including bile salts, 2-deoxylysophosphatidylcholine, and Triton X-100.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

The divalent cation is obligatory for the binding of ligands to the catalytic site of secreted phospholipase A2.

The divalent cation requirement for partial reactions of the catalytic turnover cycle during interfacial catalysis by pig pancreatic phospholipase A2 (PLA2) is investigated. Results show that the specific role of calcium in all the events of the catalytic cycle at the active site is not shared by other divalent cations. Cations such as calcium, barium, and cadmium bind to the enzyme in the aqueous phase. The active-site-directed ligands (substrate, products, and transition-state mimics) do not bind to the enzyme in the absence of a divalent cation. The synergistic binding of such ligands to the active site of PLA2 bound to the interface is, however, observed only in the presence of isosteric ions like calcium and cadmium, but not with larger ions like strontium or barium. The equilibrium constants for ligands bound to the enzyme in the presence of calcium and cadmium are virtually the same. However, only calcium supports the catalytic turnover; the rate of hydrolysis in the presence of cadmium is less than 1% of that observed with calcium. The role of divalent ions on the interfacial catalytic turnover cycle of PLA2 is not only due to the cation-assisted binding of the substrate but also due to its participation in the chemical step. Other roles of divalent ions in the events of interfacial catalytic turnover are also identified. For example, the binding of the enzyme to the interface is apparently promoted because the divalent cation is required for the sequential step, i.e., the binding of the substrate to the active site of PLA2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

A phospholipase A2 inhibitor decreases generation of thiobarbituric acid reactive substance during lung ischemia-reperfusion.

A novel active-site directed specific inhibitor of phospholipase A2 (PLA2), 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33), administered endotracheally co-dispersed in liposomes, significantly reduced the formation of thiobarbituric acid reactive substances (TBARS) in isolated rat lungs subjected to ischemia-reperfusion. Elevated conjugated dienes were unaffected. This contrasts with the effects of the cyclo-/lipoxygenase inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA), which decreased formation of both TBARS and conjugated dienes (CD). The effects of MJ33 plus ETYA were additive for TBARS but results for CD were similar to ETYA alone. A similar dissociation of inhibition of TBARS and CD formation by MJ33 was observed with t-butyl hydroperoxide induced lipid peroxidation of isolated lung microsomes. Assay of lung homogenate with phosphatidylcholine as substrate showed that MJ33 selectively inhibited the Ca(2+)-independent acidic PLA2. MJ33 had no effect on thromboxane B2 release by the isolated lung, indicating the effects of acidic PLA2 inhibition do not involve the arachidonate cascade. MJ33 also partially prevented lung edema and lactate dehydrogenase release associated with ischemia-reperfusion. The observations show that this PLA2 inhibitor can be delivered to oxidant-sensitive lung sites by its co-dispersal in liposomes, and that oxidant-induced lipid peroxidation in this model of lung injury occurs in a complex lipid prior to PLA2 activity.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1993

Human nonpancreatic secreted phospholipase A2: interfacial parameters, substrate specificities, and competitive inhibitors.

The rate and equilibrium parameters for the interfacial catalysis by recombinant human nonpancreatic secreted phospholipase A2 were determined. Results show that the enzyme binds to anionic interfaces with considerably higher affinity than to zwitterionic interfaces. The extent of hydrolysis per enzyme on anionic vesicles in the processive scooting mode shows that the enzyme is fully catalytically active as a monomer. Among several secreted phospholipases A2 tested, the human nonpancreatic secreted enzyme is unique in its ability to undergo slow intervesicle exchange either by dissociation from the interface followed by binding to a different vesicle or by promoting the fusion of vesicles. The equilibrium dissociation constants for calcium, substrate analogs, reaction products, and several competitive inhibitors bound to the enzyme at the interface were determined by monitoring the ligand-conferred protection of the active site histidine residue from alkylation by phenacyl bromide. The interfacial Michaelis-Menten parameters were determined from the analysis of the entire reaction progress curve and also by monitoring the effect of competitive inhibitors on the initial rate of hydrolysis in the scooting mode. The interfacial Michaelis constant (KM*) for the substrate 1,2-dimyristoylglycero-sn-3-phosphomethanol was determined to be considerably above the maximal attainable mole fraction of unity for the substrate in the bilayer. Substrate specificity studies show that the enzyme does not significantly discriminate between phospholipids that differ in the type of polar head group or in the degree of unsaturation of the fatty acyl chains. Competitive inhibitors are described that display a high degree of selectivity for binding to the nonpancreatic versus pancreatic phospholipase A2. The kinetic properties of the human nonpancreatic secreted phospholipase A2 suggest that the enzyme has evolved to hydrolyze substrates at anionic interfaces and at high calcium concentrations.

Catalysis↗
PubMed1993

Interaction of sporidesmin, a mycotoxin from Pithomyces chartarum, with lipid bilayers.

Sporidesmin, a mycotoxin from Pithomyces chartarum is a hydrophobic molecule. It can therefore be easily incorporated in the cell membrane, where it is likely to cause changes in the bilayer organization and the properties of membrane proteins. In order to understand the redox behaviour of sporidesmin in a hydrophobic environment, we have investigated the effects of oxidized and reduced sporidesmin on the phase transition properties of bilayers and on the susceptibility of bilayers to pancreatic phospholipase A2 (PLA2). The changes induced by sporidesmin in the thermotropic phase transition profiles of dimyristoyl-sn-3-phosphatidyl choline (DMPC) bilayers were similar to those caused by solutes known to localize in the glycerol-backbone region of the lipid bilayer, suggesting a similar localization for oxidized and reduced sporidesmin. Neither form of toxin disrupt the bilayer or membrane organization even at relatively high mole fractions. At concentrations < 10 mole% both forms partitioned equally well in the gel and liquid-crystalline phases, whereas at higher concentrations (approximately 30 mole%) reduced sporidesmin is preferentially localized in the liquid-crystalline phase. These effects of sporidesmin on the phase properties of DMPC vesicles were also reported by the fluorescence behavior of 10-pyrenedecanoic acid (PDA). The effects of oxidized and reduced sporidesmins on PLA2 kinetics are consistent with their ability to perturb bilayer organisation.

Calorimetry, Differential Scanning↗
PubMed1993

Biology, ecology, and biotechnological applications of anaerobic bacteria adapted to environmental stresses in temperature, pH, salinity, or substrates.

Anaerobic bacteria include diverse species that can grow at environmental extremes of temperature, pH, salinity, substrate toxicity, or available free energy. The first evolved archaebacterial and eubacterial species appear to have been anaerobes adapted to high temperatures. Thermoanaerobes and their stable enzymes have served as model systems for basic and applied studies of microbial cellulose and starch degradation, methanogenesis, ethanologenesis, acetogenesis, autotrophic CO2 fixation, saccharidases, hydrogenases, and alcohol dehydrogenases. Anaerobes, unlike aerobes, appear to have evolved more energy-conserving mechanisms for physiological adaptation to environmental stresses such as novel enzyme activities and stabilities and novel membrane lipid compositions and functions. Anaerobic syntrophs do not have similar aerobic bacterial counterparts. The metabolic end products of syntrophs are potent thermodynamic inhibitors of energy conservation mechanisms, and they require coordinated consumption by a second partner organism for species growth. Anaerobes adapted to environmental stresses and their enzymes have biotechnological applications in organic waste treatment systems and chemical and fuel production systems based on biomass-derived substrates or syngas. These kinds of anaerobes have only recently been examined by biologists, and considerably more study is required before they are fully appreciated by science and technology.

Adaptation, Physiological↗
PubMed1992

Phospholipase A2 engineering. The structural and functional roles of aromaticity and hydrophobicity in the conserved phenylalanine-22 and phenylalanine-106 aromatic sandwich.

The highly conserved phenylalanine-22 and phenylalanine-106, arranged as an aromatic sandwich, form part of an invariant hydrophobic wall that shields the active site of bovine pancreatic phospholipase A2 (PLA2) from bulk solvent [Dijkstra, B. W., Drenth, J., & Kalk, K. H. (1981) Nature 289, 604-606]. The residues have also been suggested to interact with the sn-2 acyl chain of bound phospholipid substrate [White, S. P., Scott, D. L., Otwinowski, Z., Gelb, M. H., & Sigler, P. B. (1990) Science 250, 1560-1563]. We now report the importance of these two residues in the structure and function of PLA2 in terms of aromaticity (changing to Ile) and hydrophobic (changing to Ala) and hydrophilic (changing to Tyr) character of these residues. The structural properties of the mutants were analyzed by proton NMR and by guanidine hydrochloride-induced denaturation. The functional properties were determined by measuring kinetic parameters toward various substrates in the forms of monomers, micelles, and vesicles, and by measuring equilibrium dissociation constants at the interface. The results show that (i) The conformational stability of each mutant was as good as that of wild-type PLA2; none of the mutants was significantly perturbed structurally as judged from detailed 1H NMR analysis. These results suggest that neither the Phe-22/Phe-106 face-to-face pair nor the Phe-22/Tyr-111 edge-to-face pair plays a significant structural role. (ii) Mutations to Ile at either position 22 or position 106 resulted in only minor perturbations in activity. This suggests that the aromaticity is not important to the function of these two residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1992

Interfacial catalysis by phospholipase A2: the rate-limiting step for enzymatic turnover.

The kinetics of the phospholipase A2-catalyzed hydrolysis of bilayer vesicles and mixed micelles of several oxyglycero and thioglycero analogues of phospholipids have been studied. The results with vesicles show that, depending on the source of the enzyme, the rates of hydrolysis of the oxy-containing long-chain phosphatidylmethanols are 2.5- to 28-fold higher compared to the rates of hydrolysis of the analogous thio substrates. The oxygen to sulfur substitution does not significantly alter the affinities of the enzymes for the reaction products or calcium. Since it is unlikely that sulfur substitution changes the rate constants for the formation and dissociation of the enzyme-product complex by the same factor, the element effects seen in the rates of hydrolysis of the oxy- and thioester phospholipids in vesicles are primarily due to a change in the rate constant for the chemical step of the catalytic turnover cycle. For bovine pancreatic phospholipase A2, various mutants with lower catalytic activity were used to show that the value of the element effect does not increase in the mutants. These results establish that, for the pancreatic phospholipase A2, the element effect is fully expressed, and the chemical step is fully rate-limiting for both oxyglycero and thioglycero phospholipids in vesicles. It was found that the element effect decreases from 7 to 1 when long-chain phosphatidylmethanols are present in micelles of a neutral diluent. This result suggests that the chemical step is not rate-limiting during the hydrolysis of these mixed micelle substrates.

Animals↗
PubMed1992

Phospholipase A2 engineering. Structural and functional roles of highly conserved active site residues tyrosine-52 and tyrosine-73.

Site-directed mutagenesis was used to probe the structural and functional roles of two highly conserved residues, Tyr-52 and Tyr-73, in interfacial catalysis by bovine pancreatic phospholipase A2 (PLA2, overproduced in Escherichia coli). According to crystal structures, the side chains of these two active site residues form H-bonds with the carboxylate of the catalytic residue Asp-99. Replacement of either or both Tyr residues by Phe resulted in only very small changes in catalytic rates, which suggests that the hydrogen bonds are not essential for catalysis by PLA2. Substitution of either Tyr residue by nonaromatic amino acids resulted in substantial decreases in the apparent kcat toward 1,2-dioctanoyl-sn-glycero-3-phosphocholine (DC8PC) micelles and the v(o) (turnover number at maximal substrate concentration, i.e., mole fraction = 1) toward 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DC14PM) vesicles in scooting mode kinetics [Berg, O. G., Yu, B.-Z., Rogers, J., & Jain, M. K. (1991) Biochemistry 30, 7283-7297]. The Y52V mutant was further analyzed in detail by scooting mode kinetics: the E to E* equilibrium was examined by fluorescence; the dissociation constants of E*S, E*P, and E*I (KS*, KP*, and KI*, respectively) in the presence of Ca2+ were measured by protection of histidine-48 modification and by difference UV spectroscopy; the Michaelis constant KM* was calculated from initial rates of hydrolysis in the absence and presence of competitive inhibitors; and the turnover number under saturating conditions (kcat, which is a theoretical value since the enzyme may not be saturated at the interface) was calculated from the vo and KM* values. The results indicated little perturbation in the interfacial binding step (E to E*) but ca. 10-fold increases in KS*, KP*, KI*, and KM* and a less than 10-fold decrease in kcat. Such changes in the function of Y52V are not due to global conformational changes since the proton NMR properties of Y52V closely resemble those of wild-type PLA2; instead, it is likely to be caused by perturbed enzyme-substrate interactions at the active site. Tyr-73 appears to play an important structural role. The conformational stability of all Tyr-73 mutants decreased by 4-5 kcal/mol relative to that of the wild-type PLA2. The proton NMR properties of Y73A suggested significant conformational changes and substantially increased conformational flexibility. These detailed structural and functional analyses represent a major advancement in the structure-function study of an enzyme involved in interfacial catalysis.

Animals↗
PubMed1992

Basis for the anomalous effect of competitive inhibitors on the kinetics of hydrolysis of short-chain phosphatidylcholines by phospholipase A2.

The effect of four specific competitive inhibitors on the kinetics of hydrolysis of short-chain diacyl-sn-glycero-3-phosphocholines below their critical micelle concentrations was examined. The kinetics of hydrolysis of short-chain substrates dispersed as solitary monomers were generally consistent with the classical Michaelis-Menten formalism; i.e., hydrolysis began without any latency period, the steady-state rate was observed at higher substrate concentrations, the steady-state initial rate showed a linear dependence on the enzyme concentration, and the hyperbolic dependence of the initial rate on the substrate concentration could be described in terms of KM and Vmax parameters. The competitive nature of the inhibitors used in this study has been established by a variety of techniques, and the equilibrium dissociation constants for the inhibitors bound to the enzyme were measured by the protection method [Jain et al. (1991) Biochemistry 30, 7306-7317]. The kinetics of hydrolysis in the presence of competitive inhibitors could be described by a single dissociation constant. However, the value of the dissociation constant obtained under the kinetic conditions was comparable to that obtained by the protection method for the inhibitor-enzyme complex bound to a neutral diluent, rather than to the value of the dissociation constant obtained with solitary monomeric inhibitors and the enzyme in the aqueous phase. Spectroscopic methods showed that the effectively lower dissociation constant of an inhibitor bound to PLA2 at the interface is due to the stabilization of the enzyme-inhibitor complex by interaction with other amphiphiles present in the reaction mixture.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1992

Kinetic analysis of a high molecular weight phospholipase A2 from rat kidney: divalent metal-dependent trapping of enzyme on product-containing vesicles.

The kinetics of hydrolysis of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine vesicles catalyzed by the high molecular weight phospholipase A2 from rat kidney show an anomalous behavior. The reaction progress lasts for several minutes and then stops after only 5-10% of the available substrate has been hydrolyzed. Addition of more enzyme but not more substrate leads to a new round of hydrolysis. Although this initially suggested that the enzyme becomes inactivated during the turnover, such a conclusion could not be substantiated. Addition of buffer containing 0.15 M NaCl and bovine serum albumin to the reaction after the progress ceased leads to the re-initiation of the lipolysis. The enzyme is not strongly inhibited by the reaction products. Although the enzyme does not bind irreversibly to vesicles composed of pure 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine, it does become irreversibly trapped on vesicles that contain a critical mole percentage of reaction products. This trapping is the most likely explanation for the cessation of the reaction progress. Both the binding of enzyme to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine vesicles and the hydrolysis of 1-stearoyl-2-[3H]arachidonyl-sn-glycerophosphocholine contained in these vesicles require the presence of products. Furthermore, the trapping of enzyme is independent of catalytic turnover. The trapping is sensitive to the structure of the fatty acid present in the vesicles and requires the presence of divalent metals (either Ca2+, Sr2+, Ba2+, or Mg2+). Since the concentrations of the metals needed for the enzymatic activity correlate with the amounts needed to promote the trapping, it is suggested that the role of the metal is only to promote the interfacial binding of the enzyme.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1992

Substituted methyl benzimidazole carbamate: efficacy against experimental cysticercosis.

Methyl [5-([4-(2-pyridinyl)-1-piprazinyl] carbonyl)-1H benzimidazole-2-yl] carbamate, a broad spectrum anthelmintic, was tested against Cysticercus fasciolaris in rats and the cysticercoids of Hymenolepis nana in beetles. A dose of 50 mg kg-1, given intraperitoneally (i.p.) to infected rats on 10 alternate days, was found most effective, killing all mature cysticerci. The same dose, given on each of five days, either from the fifth day prior to infection or the sixth day following infection, fully arrested the establishment of cysticerci. A single prophylatic dose of 500 mg kg-1, given intramuscularly (i.m.), protected rats against challenge with Taenia taeniaeformis eggs for 10 days. The test compound, mebendazole and praziquantel were all totally ineffective against mature cysticercoids in grain beetles but, of the beetles fed the drugs in flour, 66.3%, 50% and 83.8%, respectively, failed to support the development of immature cysticercoids. The effective cysticidal action of the compound makes it a candidate medical anthelmintic.

Animals↗
PubMed1992

Alterations in metabolic activity of Cysticercus fasciolaris on some anthelmintic treatments.

Effect of candidate compounds 81-470 i.e. methyl [5[4-(2-pyridinyl)-1-piperazinyl]carbonyl]-1H-benzimidazole-2-yl]- carbamate and 86-162 i.e. methyl-5(6)-(alpha-hydroxyphenyl methyl) benzimidazole-2-carbamate along with reference drugs mebendazole and praziquantel on energy metabolism of C. fasciolaris recovered from rats treated with single dose of 500 mg/kg, ip was investigated. All the drugs significantly lowered the rate of uptake of glucose and alanine by the parasite. Suppression in the formation of lactate, the major end-product, was also noticed. Nonetheless the ratio of lactate produced versus the substrates consumed was not substantially affected. The recovered cysticerci also possessed less glycogen and ATP compared to the normal parasites. Although the effects exerted by the drugs were of the identical nature, they significantly differed in the magnitude of their action. Mebendazole followed by praziquantel maximally affected all the above metabolic activities while 86-162 proved to be the weakest in action. The results suggest that the examined drugs exert their chemotherapeutic activity by interfering with uptake of glucose and alanine but do not significantly alter their catabolism.

Adenosine Triphosphate↗