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R Majumdar

Publications and source records attributed to R Majumdar.

At least 37 records · Page 2Linked to original sources
PubMed1992

Melting characteristics of highly supercoiled DNA.

The effect of high supercoil densities on the melting characteristics of a supercoiled DNA has been studied. It is found that although the melting temperature increases abruptly on converting a linear DNA merely into the relaxed circular form, it falls back substantially at high supercoil densities. It is further predicted, in such cases, that the number of melted base pairs should be significantly enhanced even at the physiological temperature, which may facilitate the binding of other molecules to the highly supercoiled DNA.

Journal Article↗
PubMed1991

Functional consequences of substitution of the active site (phospho)histidine residue of Escherichia coli succinyl-CoA synthetase.

Succinyl-CoA synthetase (EC 6.2.1.5, succinate:CoA ligase (ADP-forming] of Escherichia coli is an alpha 2 beta 2 tetramer, with the active site believed to be located at the point of contact between the two subunit types. It has been previously established that the reaction involves the intermediate participation of a phosphorylated enzyme form in the process of catalysis. The site of phosphorylation (His-246) and the binding sites for the substrates ADP and ATP are located in the alpha subunit, and the succinate and CoA binding sites are in beta. A mutant form of this enzyme, with the active site histidine residue replaced by aspartate, has been produced in large quantities and purified to homogeneity. This form appears to be indistinguishable from the native enzyme with respect to its subunit assembly, but has no ability to catalyze the overall reaction. As expected, the His-246 alpha----Asp mutant is incapable of undergoing phosphorylation. We have developed an assay based upon the arsenolysis of succinyl-CoA that effectively isolates the partial reaction that occurs in the portion of the active site contributed by the beta subunit; this reaction does not involve covalent participation of His-246 alpha. We have found that the His-246 alpha----Asp mutant is also devoid of activity in this arsenolysis reaction, indicating that an intact His-246 alpha is required for the establishment of the microenvironment in this portion of the active site that is required for the corresponding step of the overall reaction.

Adenosine Diphosphate↗
PubMed1990

Mitochondrial translocation and processing of the precursor to the alpha-subunit of rat liver succinyl-CoA synthetase.

Succinyl-CoA synthetase functions in the mitochondrial matrix as an alpha beta-dimer. Its constitutive subunits are thus expected to be encoded in the nucleus and synthesized in the cytoplasm as precursors containing signal sequences for mitochondrial translocation. We have previously reported the isolation and sequence of a rat liver cDNA clone (lambda SCS19) that apparently encodes the cytoplasmic precursor to the alpha-subunit. Here we report the preparation of mRNA transcripts of this cDNA insert and their in vitro translation to produce labeled protein that can be translocated across the membranes of subsequently added rat liver mitochondria. Translocation is accompanied by proteolytic processing to convert the 34.5-kilodalton precursor to the 32-kilodalton mature form of the subunit. The N-terminal sequence of the mature alpha-subunit from the GTP-specific isozyme has been determined by sequential Edman degradation and compared with the amino acid sequence deduced from the cDNA. This confirms that the cloned sequence encodes the GTP-specific alpha-subunit, and establishes that the point of cleavage is between histidyl and glycyl residues and that the signal sequence consists of 27 residues. The signal sequence shares characteristics of other mitochondrial targeting sequences that have been elucidated (largely of yeast mitochondrial precursors), including the potential to form an amphiphilic helix. Import is dependent upon the presence of ATP and is inhibited by compounds that diminish mitochondrial membrane potential. Translocation of the precursor is effective for precursor produced by the reticulocyte translation system, but is not seen for the product that is translated by a wheat germ extract, indicating that the latter may lack a factor or component that is necessary for the targeting and import process.

Amino Acid Sequence↗
PubMed1988

Cloning and sequencing of the cytoplasmic precursor to the alpha subunit of rat liver mitochondrial succinyl-CoA synthetase.

Succinyl-CoA synthetase [succinate-CoA ligase (GDP-forming); EC 6.2.1.4] of rat liver, an alpha beta dimer, is a component of the enzymology of the tricarboxylic acid cycle and functions within the mitochondrial matrix. We have isolated and determined the sequence of a cDNA clone containing the coding sequence of the cytoplasmic precursor to the alpha subunit of this enzyme together with stretches of nontranslated sequence at the 5' and 3' ends. The translated amino acid sequence indicates the presence of a 27-residue N-terminal signal sequence for mitochondrial targeting. The amino acid sequence of the mature alpha subunit shows an extraordinary degree of homology to the alpha subunit of Escherichia coli succinyl-CoA synthetase, with greater than 70% of the residues identical. This suggests that the fundamental differences in the quaternary structures and catalytic functions of the mammalian and bacterial enzymes must be attributable to differences in the beta subunits. mRNA that hybridizes to the cloned DNA is approximately equal to 1800 nucleotide residues in length, confirming that each of the two subunits is encoded separately and does not arise by proteolysis of a primary gene product containing both subunits of the mature protein.

Amino Acid Sequence↗
PubMed1987

Thermodynamics of B-Z transition in supercoiled DNA.

The nature of the possible supercoil-induced B-Z transition has been analyzed from the thermodynamical point of view, by taking into account the effects of the twisting as well as the writhing components of the supercoiling free energy. The cooperative aspects of the transition, as predicted by theory, agrees well with the corresponding experimental data.

Base Composition↗
PubMed1987

The subunits of succinyl-coenzyme A synthetase--function and assembly.

Succinyl-CoA synthetase is made up of two kinds of subunits, designated alpha and beta. The enzyme from Escherichia coli is an alpha 2 beta 2 tetramer (mol. mass. 142 kDa), whereas the mammalian mitochondrial species is an alpha beta dimer. By means of active enzyme centrifugation, we have shown that the active form of the bacterial enzyme is the tetramer even at very low assay concentrations, while the pig heart enzyme is a non-associating dimer over a wide concentration range. The E. coli enzyme shows distinct half-of-the-sites reactivity with respect to the phosphorylation of a histidine residue in the alpha-subunit that represents a step in catalysis. Many lines of evidence (hybrid enzyme formation, oxygen exchange kinetics, 31P-n.m.r. studies) suggest that co-operative interactions between alternatingly functional active sites on the two halves of the E. coli enzyme contribute to its catalytic efficacy. In further refining this model for catalysis, we have shown that the monothiophosphorylated E. coli enzyme does not catalyse exchange of 18O from the beta, gamma-bridge to the beta-non-bridge position of ATP, indicating that the enzyme does not undergo even transient bis-phosphorylation. As a first step in studying the in vivo synthesis and assembly of the enzyme in the mammalian mitochondrial matrix, we have cloned and sequenced a 900 bp cDNA fragment that encodes most of the alpha subunit of rat liver succinyl-CoA synthetase. The derived amino acid sequence shows an impressive degree of homology to that of the alpha subunit of the enzyme from E. coli. We have shown that the alpha subunit in rat liver is a discrete nuclear gene product, complete with cleavable signal sequence to specify mitochondrial targetting.

Animals↗
PubMed1986

A test of the bay-region mechanism in carcinogenesis for monomethyl benz[a]anthracenes in a self-consistent-field molecular orbital theory.

The relative carcinogenic activity of the isomeric monomethyl derivatives of benz[a]anthracene has been studied on the basis of their bay-region reactivity, and the subsequent ease of carbonium ion formation, as obtained from a suitable 'self-consistent-field' molecular orbital theory for the mobile pi-electrons. The predicted order of carcinogenic activity of these molecules is compared with the available experimental data.

Benz(a)Anthracenes↗
PubMed1985

The solubilization of platelet membrane-bound acetylcholinesterase and aryl acylamidase by exogenous or endogenous phosphatidylinositol specific phospholipase C.

Phosphatidylinositol specific phospholipase C from Staphylococcus aureus could solubilize acetylcholinesterase up to 55% from sheep platelets in the presence of ethylenediaminetetra acetic acid (EDTA). The endogenous phosphatidylinositol specific phospholipase C of platelets activated by deoxycholate (at 3-5 mM) could also solubilize the enzyme to a similar extent. The solubilized enzyme could be further purified to apparent homogeneity by affinity chromatography without the use of any detergents. It is suggested that phosphatidylinositol specific phospholipase C will be a useful tool in the solubilization of acetylcholinesterase from mammalian sources and its purification free of detergents. The present study also demonstrates the parallel behaviour of acetylcholinesterase and aryl acylamidase in platelets confirming their identity.

Acetylcholinesterase↗
PubMed1985

Melting transition of covalently closed DNA with supercoil-induced cruciforms.

The melting curve for covalently closed supercoiled DNA has been studied by assuming the existence of cruciforms as significant structural perturbations in the pre-melting region. The statistical mechanical treatment used incorporates these cruciform structures through an appropriate sequence generating function. The variation of the effective hydrogen bond energy with temperature is taken into account by an empirical procedure. The results obtained are in close agreement with the corresponding experimental data in TEA solution where the effect of heterogeneity of the base pairs is minimized.

Base Composition↗
PubMed1984

Chemical modification of acetylcholinesterase from eel and basal ganglia: effect on the acetylcholinesterase and aryl acylamidase activities.

The effect of chemical modification on the acetylcholinesterase and the aryl acylamidase activities of purified acetylcholinesterase from electric eel and basal ganglia was investigated in the presence and absence of acetylcholine, the substrate of acetylcholinesterase, and 1,5-bis[4-(allyldimethylammonium)phenyl]pentan-3-one dibromide (BW284C51), a reversible competitive inhibitor of acetylcholinesterase. Trinitrobenzenesulfonic acid, pyridoxal phosphate, acetic anhydride, diethyl pyrocarbonate, and 2-hydroxy-5-nitrobenzyl bromide under specified conditions inactivated both acetylcholinesterase and aryl acylamidase in the absence of acetylcholine and BW284C51. Chemical modifications in the presence of acetylcholine and BW284C51 by all the above except diethyl pyrocarbonate selectively prevented the loss of acetylcholinesterase but not aryl acylamidase activity; modification by diethyl pyrocarbonate in the presence of acetylcholine and BW284C51 prevented the loss of both acetylcholinesterase and aryl acylamidase activities. Treatment with N-acetylimidazole resulted in the inactivation of acetylcholinesterase and the activation of aryl acylamidase. These changes in both the activities could be prevented by acetylcholine and BW284C51. Modification by phenylglyoxal, 2,4-pentanedione, or N-ethylmaleimide did not affect the enzyme activities. Indophenylacetate hydrolase activity followed a pattern similar to that of acetylcholinesterase in all the above modification studies. The results suggested essential lysine, tyrosine, tryptophan, and histidine residues for the active center of acetylcholinesterase and essential lysine, histidine, and tryptophan residues for the active center of aryl acylamidase.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗
PubMed1982

Serotonin-sensitive aryl acylamidase activity of platelet acetylcholinesterase.

Serotonin-sensitive aryl acylamidase (AAA, EC 3.5.1.13) was purified to apparent homogeneity from sheep platelets by affinity chromatography and it was shown to be associated with the platelet acetylcholinesterase (AChE, EC 3.1.1.7). The basis for the association of the two enzymes was the following. Both enzyme activities co-eluted from the affinity columns with constant ratios of specific activities and percentage recoveries. Both enzymes co-migrated on gel electrophoresis. Both enzymes co-eluted during sepharose 6B gel filtration. Potent inhibitors of AChE such as bis(4-allyldimethyl ammoniumphenyl) pentan-3-one dibromide (BW 284C51), neostigmine and eserine also inhibited AAA potently. Both enzymes lost significant activity on treatment with deoxycholate or taurodeoxycholate and the loss could be partly restored by a mixture of phospholipids. The platelet AAA was specifically inhibited by serotonin and to a lesser extent by tryptamine but not by several other amines. It was also inhibited by acetylcholine and several of its analogues and homologues. It is suggested that in the platelets the two enzymes (AAA and AChE) are probably identical.

Acetylcholinesterase↗
PubMed1980

Relationship between mitochondrial ATPase and membrane lipids from developing human placenta.

Mitochondrial lipids and ATPase activity have been studied in developing human placenta. The enzyme activity in the placenta increases steadily with gestational age until the 18th to 21st week, and decreases in the second half of pregnancy. Both Mg2+ and Ca2+ ions have been found to activate the enzyme system. The solubilization of membrane lipids by deoxycholate inhibits the enzyme activity. Total lipid and total phospholipid contents of the mitochondrial membranes also increase during the early development of the placenta with peaks in the 14th to 17th week, after which the levels decrease. However, the percentage composition of individual phospholipids remains almost the same throughout pregnancy. The profile of the ATPase activity exhibits a correlation with that of membrane lipids in human placenta during intrauterine development of the tissue.

Adenosine Triphosphatases↗