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M K Jain

Publications and source records attributed to M K Jain.

At least 91 records · Page 5Linked to original sources
PubMed1996

Mitogen-activated protein kinase phosphatase-1 in rat arterial smooth muscle cell proliferation.

Smooth muscle cell proliferation and migration is important in arteriosclerosis. In this process, cytokines and growth factors are upregulated and bind to their respective receptors, which in turn stimulate mitogen-activated protein (MAP) kinases. MAP kinases then relay signals to the nucleus that activate quiescent smooth muscle cells. Phosphatases downregulate MAP kinases. We investigated the role of a dual-specificity tyrosine phosphatase, MAP kinase phosphatase-1 (MKP-1), in smooth muscle cell proliferation. MKP-1 expression was high in arterial tissue by Northern analysis, and MKP-1 message was detected mainly in the arterial smooth muscle layer by in situ hybridization. After balloon injury of the rat carotid artery, expression of MKP-1 decreased greatly, whereas that of MAP kinases, especially p44 MAP kinase, increased. The time course of the reduction in MKP-1 message correlated with increased tyrosine phosphorylation and elevated p44 MAP kinase enzymatic activity. In rat arterial smooth muscle cells overexpressing MKP-1, growth was arrested in the G1 phase and entry into the S phase was blocked. A reduction in MKP-1 expression may contribute in part to proliferation of smooth muscle cells after vascular injury, possibly through a decrease in dephosphorylation of p44 MAP kinase.

Angioplasty, Balloon↗
PubMed1996

Phospholipase A2 engineering. Deletion of the C-terminus segment changes substrate specificity and uncouples calcium and substrate binding at the zwitterionic interface.

It has been suggested [Dijkstra, B. W., Drenth, J., & Kalk, K. H. (1981) Nature 289, 604-606] that the interfacial binding site of phospholipase A2 (PLA2) involves a large number of residues, including a cluster at the N-terminus and another cluster at the C-terminus. The approaches of multiple mutation and deletion were used to test the roles of the C-terminal residues of bovine pancreatic PLA2 overexpressed in Escherichia coli. A double mutant K120A/K121A and a deletion mutant delta 115-123/ C27A were constructed, and structural and functional analyses were performed on both mutants. The double mutant showed little perturbation in the global structure on the basis of proton NMR and X-ray crystallographic analyses. The proton NMR analysis of the deletion mutant suggested that a few residues at the active site, the hydrophobic channel, and the calcium binding loop are perturbed, but the global conformation is not changed. The mutants were then characterized for catalytic and binding properties by use of various kinetic and spectroscopic methods. The double mutant behaved in a manner similar to that of the wild type (WT) PLA2 in every property examined. The deletion mutant was found to show an interesting change of substrate specificity. The kcat,app of the zwitterionic DC8PC micelles but not the anionic DC8PM micelles decreased by a factor of > 100; however, the activity of DC8PC was restored upon addition of 4 M NaCl. The results of fluorescence spectroscopic studies indicate that the deletion mutant behaves in a manner similar to that of WT in the binding to anionic vesicles and to zwitterionic neutral diluent. Thus, the binding affinity of the enzyme to the interface (the E to E* step) should not be the main cause for the change in substrate specificity. The cause lies at least partially in the binding of substrate or inhibitor to the active site of the enzyme at the interface, i.e., the E* to E*L step, as revealed by the results of equilibrium binding studies. The equilibrium dissociation constants of ligands are generally higher for the deletion mutant (relative to WT) at the zwitterionic interface but not at the anionic interface. The cause for the low affinity of an active site-directed ligand to the active site at the zwitterionic interface could be related to the inability of Ca2+ to enhance ligand binding for the deletion mutant. This is in contrast to the WT PLA2 for which Ca2+ binding enhances binding of the substrate to the active site. Overall, the structural and functional perturbations caused by deleting the C-terminal segment are modest, but the changes in substrate specificity and the uncoupling between substrate and calcium binding are interesting and significant.

Amino Acid Sequence↗
PubMed1996

Kinetic basis for the substrate specificity during hydrolysis of phospholipids by secreted phospholipase A2.

Kinetics of hydrolysis of aqueous dispersions of arsono-, sulfo-, phosphono- and phospholipids by phospholipase A2 from pig pancreas are characterized in terms of interfacial rate and equilibrium parameters. The enzyme with or without calcium binds with high affinity to the aqueous dispersions of the four classes of anionic lipids and shows the same general kinetic behavior. The rate of hydrolysis of anionic substrates does not show an anomalous change at the critical micelle concentration because the enzyme is present in aggregates even when bulk of the substrate is dispersed as a solitary monomer. Apparent affinities of the enzyme for the interface of different anionic lipids are virtually the same. Also, affinities of these substrates for the active site of the enzyme at the interface are comparable. However, a significant change in the catalytic turnover rate is seen as the sn-3 phosphodiester group is modified; the apparent maximum rate at saturating bulk substrate concentration, V(M)app values, increase in the order: homo- and arsonolipids < sulfo- < phosphono- < phospholipids. Not only the basis for the sn-2 enantiomeric selectivity but also the decrease in the rate of hydrolysis with the increasing chain length is due to a decrease in the value of V(M)app. Results show that even when the bulk concentration of anionic phospholipid is below cmc, hydrolysis occurs in aggregates of enzyme and substrate where the chemical step of the turnover cycle remains rate-limiting, which provides a basis for the assumption that V(M)app is directly related to Kcat. The fact that Kcat depends on the nature of the head group (phosphate, phosphonate, sulfate, arsonate) implies that the head group plays a critical role in the rate-limiting chemical step of the catalytic cycle, possibly during the decomposition of the tetrahedral intermediate. The significance of these results for the microscopic steady-state condition for hydrolysis at the micellar interface, mechanism of esterolysis by phospholipase A2, and inhibitor design are discussed.

Animals↗
PubMed1996

APEG-1, a novel gene preferentially expressed in aortic smooth muscle cells, is down-regulated by vascular injury.

Despite the importance of phenotypic alterations in arterial smooth muscle cells (ASMC) during the pathogenesis of arteriosclerosis, little is known about genes that define differentiated ASMC. Using differential mRNA display, we isolated a novel gene preferentially expressed in the rat aorta and termed this gene APEG-1. The cDNA of rat APEG-1 contained an open reading frame encoding 113 amino acids, which would predict a basic protein of 12.7 kDa. The amino acid sequence of rat APEG-1 was highly conserved among human and mouse homologues (97 and 98%, respectively). Using an APEG-1 fusion protein containing an N-terminal c-Myc tag, we identified APEG-1 as a nuclear protein. By in situ hybridization, APEG-1 mRNA was expressed in rat ASMC. Although APEG-1 was expressed highly in differentiated ASMC in vivo, its expression was quickly down-regulated and disappeared in dedifferentiated ASMC in culture. In vivo, APEG-1 mRNA levels decreased by more than 80% in response to vascular injury as ASMC changed from a quiescent to a proliferative phenotype. Taken together, these data indicate that APEG-1 is a novel marker for differentiated ASMC and may have a role in regulating growth and differentiation of this cell type.

Amino Acid Sequence↗
PubMed1996

Specificity for the exchange of phospholipids through polymyxin B mediated intermembrane molecular contacts.

Structural specificity for the direct vesicle-vesicle exchange of phospholipids through stable molecular contacts formed by the antibiotic polymyxin B (PxB) is characterized by kinetic and spectroscopic methods. As shown elsewhere [Cajal, Y., Rogers, J., Berg, O.G., & Jain, M.K. (1996) Biochemistry 35, 299-308], intermembrane molecular contacts between anionic vesicles are formed by a small number of PxB molecules, which suggests that a stoichiometric complex may be responsible for the exchange of phospholipids. Larger clusters containing several vesicles are formed where each vesicle can make multiple contacts if sterically allowed. In this paper we show that the overall process can be dissected into three functional steps: binding of PxB to vesicles, formation of stable vesicle-vesicle contacts, and exchange of phospholipids. Polycationic PxB binds to anionic vesicles. Formation of molecular contacts and exchange of monoanionic phospholipids through PxB contacts does not depend on the chain length of the phospholipid. Only monoanionic phospholipids (with methanol, serine, glycol, butanol, or phosphatidylglycerol as the second phosphodiester substituent in the head group) exchange through these contacts, whereas dianionic phosphatidic acid does not. Selectivity for the exchange was also determined with covesicles of phosphatidylmethanol and other phospholipids. PxB does not bind to vesicles of zwitterionic phosphatidylcholine, and its exchange of covesicles is not mediated by PxB. Vesicles of dianionic phospholipids, like phosphatidic acid, bind PxB; however, this phospholipid does not exchange. The structural features of the contacts are characterized by the spectroscopic and chemical properties of PxB at the interface. PxB in intermembrane contacts is readily accessible from the aqueous phase to quenchers and reagents that modify amino groups. Results show that PxB at the interface can exist in two forms depending on the lipid/PxB ratio. Additional studies show that the stable PxB-mediated vesicle-vesicle contacts may be structurally and functionally distinct from "stalks", the putative transient intermediate for membrane fusion. The phenomenon of selective exchange of phospholipids through peptide-mediated contacts could serve as a prototype for intermembrane targeting and sorting of phospholipids during their biosynthesis trafficking in different compartments of a cell. The protocols and results described here also extend the syllogistic foundation in interfacial equilibria and catalysis.

Amino Acid Sequence↗
PubMed1996

Molecular cloning and characterization of SmLIM, a developmentally regulated LIM protein preferentially expressed in aortic smooth muscle cells.

Differentiated, quiescent vascular smooth muscle cells assume a dedifferentiated, proliferative phenotype in response to injury, one of the hallmarks of arteriosclerosis. Members of the LIM family of zinc-finger proteins are important in the differentiation of various cells including striated muscle. We describe here the molecular cloning and characterization of a developmentally regulated smooth muscle LIM protein, SmLIM, that is expressed preferentially in the rat aorta. This 194-amino acid protein has two LIM domains, and comparisons of rat SmLIM with its mouse and human homologues reveal high levels of amino acid sequence conservation (100 and 99%, respectively). SmLIM is a nuclear protein and maps to human chromosome 3. SmLIM mRNA expression was high in aorta but not in striated muscle and low in other smooth muscle tissues such as intestine and uterus. In contrast with arterial tissue, SmLIM mRNA was barely detectable in venous tissue. The presence of SmLIM expression within aortic smooth muscle cells was confirmed by in situ hybridization. In vitro, SmLIM mRNA levels decreased by 80% in response to platelet-derived growth factor-BB in rat aortic smooth muscle cells. In vivo, SmLIM mRNA decreased by 60% in response to vessel wall injury during periods of maximal smooth muscle cell proliferation. The down-regulation of SmLIM by phenotypic change in vascular smooth muscle cells suggests that it may be involved in their growth and differentiation.

Amino Acid Sequence↗
PubMed1996

Intermembrane molecular contacts by polymyxin B mediate exchange of phospholipids.

Direct intermembrane exchange of dimyristoylphosphatidylmethanol is mediated by polymyxin B (PxB), a cationic amphipathic cyclic decapeptide. The possibility that the phospholipid exchange is mediated by solubilization of phospholipids or by fusion of vesicles is ruled out. By kinetic and spectroscopic methods it is shown that the exchange occurs directly through vesicle-vesicle contacts formed by a few PxB molecules. The contact is stable on the time scale of several minutes such that neither PxB nor the vesicles in the pair forming a contact exchange with excess vesicles. Several contacts may be formed on a vesicle, which leads to the formation of a cluster of vesicles, and the lipid molecules on the outer monolayers of vesicles exchange throughout the cluster. Kinetics of substrate replenishment during processive interfacial catalysis suggests that the exchange of anionic lipids over the contact occurs at a rate considerably faster than 300 s-1. The exchange through the contact is specific for certain lipids, and phospholipids with a modified head group or phospholipase A2 bound to a vesicle are not transferred to the other vesicle in contact. Since this phenomenon has not been described before, possible implications of direct vesicle-vesicle exchange of phospholipids through peptide-mediated molecular contacts are discussed. Such a mechanism for intermembrane transfer of phospholipids could be responsible for intracellular trafficking and sorting of phospholipids; it could be a necessary first step for the sequence of events leading to budding, vesiculation, and secretion; and PxB-mediated transfer between the inner and outer membranes of Gram-negative bacteria could also account for its antibiotic action.

Cell Membrane↗
PubMed1996

Formation of Fatty Acid-degrading, anaerobic granules by defined species.

An endospore-forming, butyrate-degrading bacterium (strain BH) was grown on butyrate in monoxenic coculture with a methanogen. The culture formed dense aggregates when Methanobacterium formicicum was the methanogenic partner, but the culture was turbid when Methanospirillum hungatei was the partner. In contrast, a propionate-degrading, lemon-shaped bacterium (strain PT) did not form aggregates with Methanobacterium formicicum unless an acetate-degrading Methanosaeta sp. was also included in the culture. Fatty acid-degrading methanogenic granules were formed in a laboratory-scale upflow reactor at 35(deg)C fed with a medium containing a mixture of acetate, propionate, and butyrate by using defined cultures of Methanobacterium formicicum T1N, Methanosaeta sp. strain M7, Methanosarcina mazei T18, propionate-degrading strain PT, and butyrate-degrading strain BH. The maximum substrate conversion rates of these granules for acetate, propionate, and butyrate were 43, 9, and 17 mmol/g (dry weight)/day, respectively. The average size of the granules was about 1 mm. Electron microscopic observation of the granules revealed that the cells of Methanobacterium formicicum, Methanosaeta sp., butyrate-degrading, and propionate-degrading bacteria were dispersed in the granules. Methanosarcina mazei existed inside the granules as aggregates of its own cells, which were associated with the bulk of the granules. The interaction of different species in aggregate formation and granule formation is discussed in relation to polymer formation of the cell surface.

Journal Article↗
PubMed1995

Continuous, vesicle-based fluorimetric assays of 14- and 85-kDa phospholipases A2.

This paper describes the synthesis and analysis of new substrates for the 85-kDa, mammalian, cytosolic phospholipase A2 (cPLA2) and the 14-kDa, human nonpancreatic, secreted phospholipase A2 (sPLA2). Phosphatidylcholines containing an arachidonyl chain at the sn-2 position and either a 10-pyrenedecyl or a 10-pyrenedecanoyl chain at the sn-1 position were synthesized and shown to be substrates for cPLA2 in a fluorescence-based assay. Most of the assays make use of small and large unilamellar vesicles of substrate phospholipid, although the assay also works when the substrate is dispersed in Triton X-100 mixed-micelles. The cPLA2 assays can be carried out in a fixed time-point mode in which one of the products, the pyrene-containing lysophospholipid, is detected by rapid HPLC. Alternatively, the assay becomes continuous when bovine serum albumin is present in the aqueous phase; this protein extracts the pyrene-containing lysophospholipid from the vesicle, and this leads to the fluorescence of monomeric pyrene label. These assays are capable of detecting subnanogram amounts of cPLA2. The ester formed between gamma-linolenic acid and 7-hydroxycoumarin is also a substrate for cPLA2, and when incorporated into vesicles of the anionic phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphomethanol, provides an assay in which the enzyme does not leave the vesicle surface (scooting mode). Unlike all of the previously reported, vesicle-based cPLA2 assays, a prolonged linear reaction progress is seen with the DOPM-based assay. An assay of sPLA2 with subnanogram sensitivity was developed which makes use of the substrate 1-palmitoyl-2-(10-pyrenedecanoyl)-sn-glycero-3-phosphomethanol and a lipid sink. The latter is composed of phosphatidylcholine vesicles, in excess of substrate vesicles, which do not bind sPLA2 but provide a trap for enzyme-produced 10-pyrenedecanoic acid. The fluorescence of monomeric pyrene label in sink vesicles is detected. A second sPLA2 assay using a single type of vesicle was developed based on the substrate 1,2-di(10-pyrenedecanoyl)-sn-glycero-3-phosphocholine present at 10 mol% in vesicles of the nonhydrolyzable anionic phospholipid 1,2-ditetradecyl-sn-glycero-3-phosphomethanol. The action of sPLA2 on this fluorescent substrate leads to a separation of the pyrene chains resulting in fluorescence emission from monomeric pyrene. These cPLA2 and sPLA2 assays are ideal for inhibitor screening and analysis, and for studying the interfacial kinetics of these enzymes.

Calcium↗
PubMed1995

Phospholipase A2 engineering. Probing the structural and functional roles of N-terminal residues with site-directed mutagenesis, X-ray, and NMR.

The N-terminal residues of phospholipase A2 (PLA2) are believed to be involved in the hydrogen-bonding network, the interfacial binding site, or the hydrophobic channel. Site-directed mutants of bovine pancreatic PLA2 with substitutions at positions 2, 3, 4, 5, 6, and 9 were constructed to test the roles of these residues in the structure and function of PLA2. Nonconservative mutations of Phe-5 and Ile-9, which are located inside the hydrophobic channel, led to significant perturbations in the conformation and conformational stability. Kinetic studies also indicated that mutations at Ile-9 and Phe-5 caused significant decreases in the rate of hydrolysis toward micellar and vesicle substrates. Scooting mode kinetic analysis showed that the binding step of the mutant enzymes to the DC14PM (1,2-dimyristoyl-sn-glycero-3-phosphomethanol) vesicle interface is not significantly affected and that the perturbations in catalysis occur mainly in kcat at the interface. The results taken together suggest that the residues Ile-9 and Phe-5 are important for both structure and catalysis. The mutant W3A (Trp-3 to Ala) also showed decreased rates of hydrolysis but to a lesser extent than Ile-9 and Phe-5 mutants. In addition, the binding affinity of W3A to the surface of the vesicles (i.e., the E to E* step) has been perturbed to the extent that hopping between anionic vesicles has been observed. On the other hand, the mutants of Gln-4 and Asn-6, which are located at or near the surface, displayed structural and kinetic properties similar to those of the wild-type PLA2 with the exception of the highly hydrophilic lysine mutant. The X-ray structure of the Q4E mutant indicates that the overall structure, the catalytic triad, and the link between residue 4 and Asp-99 via hydrogen bonding through Ala-1 and the structural water remain the same as in the WT. Substitutions for Leu at position 2 showed an acyl chain length discrimination toward different substrates, which may reflect the contacting position(s) of the substrate acyl chain with Leu-2.

Amino Acid Sequence↗
PubMed1995

Direct vesicle-vesicle exchange of phospholipids mediated by polymyxin B.

Direct and rapid intermembrane exchange of phospholipids without fusion is shown to occur across stable vesicle-vesicle contacts formed by stoichiometric amounts of polymyxin B. The exchange is selective for monoanionic, but not dianionic, glycerophospholipids irrespective of their chain length or phase properties. Selective transfer mediated by protein contacts between membranes could serve as a model for stable fusion intermediates and provide a structural and organizational basis for direct phospholipid transfer as implicated in entry of enveloped viruses, secretion, endocytosis, and intracellular transport and targeting of lipids. It is also proposed that the loss of the specificity of phospholipid composition in the inner and outer membranes of gram negative bacteria mediated by polymyxin B could be the basis for its bactericidal action.

Amino Acid Sequence↗
PubMed1995

Intramolecular domain-domain interactions and intermolecular self-association in bovine prothrombin. A potentiometric and laser light-scattering study.

The interaction of bovine prothrombin with Ca2+ and Mg2+ ions was investigated by following H+ release as a function of metal ion concentration at pH 6 and pH 7.4 at high and low ionic strength. Prothrombin Ca2+ and Mg2+ binding is characterized by high- and low-affinity sites. M2+ binding at these sites is associated with intramolecular conformational changes and also with intermolecular self-association. The pH dependence of H+ release by M2+ is bell shaped and consistent with controlling pKa values of 4.8 and 6.5. At pH 6 and low ionic strength, both Ca2+ and Mg2+ titrations following H+ release clearly show independent low- and high-affinity binding sites. Laser light scattering reveals that at pH 7.4 and low ionic strength, and at pH 6.0 and high ionic strength, the prothrombin molecular weight is between 73 and 98 kD. At pH 7.4 and high ionic strength, prothrombin is monomeric in the absence of metal ions, but appears to dimerize in the presence of M2+. At pH 6.0 and low ionic strength prothrombin exists as a dimer in the absence of metal ions and is tetrameric in the presence of Ca2+ and remains dimeric in the presence of Mg2+. These results and those for metal ion-dependent H+ release indicate that H+ release occurs concomitantly with association processes involving prothrombin.

Animals↗
PubMed1995

Specific competitive inhibitor of secreted phospholipase A2 from berries of Schinus terebinthifolius.

Two structurally related triterpenoids 1 and 2 from pink peppercorn (berries of Schinus terebinthifolius) are identified and characterized as active site-directed specific competitive inhibitors of the three classes of secreted 14 kDa phospholipase A2. The inhibitors not only protect the active site histidine from alkylation but also inhibit the action of secreted phospholipase A2 from pig pancreas, human synovial fluid, and bee venom. Detailed X-ray crystallographic results on the structures of the inhibitors are provided. By physical methods and X-ray crystallography the triterpenoids were identified as masticadienoic acid and masticadienolic acid (schinol). Several other triterpenoids were ineffective as inhibitors of phospholipase A2; however certain ganoderic acid derivatives showed noticeable inhibition. Results show that the side chain of these acidic tetracyclic terpenoids can access the catalytic-site region of phospholipase A2, whereas the acyclic nucleus is at the interfacial recognition region. The selectivity of the assay protocol used here is demonstrated by the fact that the original screen of ethyl acetate extracts of 60 commercially available spices and herbs was carried out with phospholipase A2 from pig pancreas, and only one extract showed inhibitory action on the hydrolytic activity in the scooting mode. Under such assay conditions the enzyme remains tightly bound to the surface of the substrate vesicles. In this way, nonspecific effects of additives that promote desorption of the enzyme from the substrate vesicle surface, under conditions in which the binding of the enzyme to the vesicle is weak, are avoided. The assay protocol is useful for the kinetic characterization of the inhibitors of phospholipase A2, and it does not give false positive results with amphiphilic and hydrophobic compounds, as is the case with virtually all assay systems in use.

Animals↗
PubMed1995

Interfacial enzymology of glycerolipid hydrolases: lessons from secreted phospholipases A2.

Interfacial enzymes operate at an organized interface such as lipid aggregates in contact with the aqueous phase. The enzyme phospholipase A2 is a well studied interfacial enzyme, and a discussion of its behavior at interfaces is the topic of this review. Knowledge gained from studies of phospholipases A2 can be applied toward the quantitative analysis of other interfacial enzymes. The kinetic analysis of these enzymes is greatly simplified if one establishes certain experimental conditions that limit the exchange of enzyme and substrate between different substrate aggregates. With such constraints, the kinetics can be analyzed in terms of classical Michaelis-Menten theory adopted for the action of enzymes at interfaces. It is also possible to describe other enzyme properties such as inhibition and substrate preferences in a meaningful way using formalism that is well known in solution-phase enzymology.

Animals↗