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P Datta

Publications and source records attributed to P Datta.

At least 127 records · Page 7Linked to original sources
PubMed1978

Homoserine dehydrogenase of Rhodospirillum rubrum. Physical and chemical characterization.

A detailed physicochemical characterization of purified homoserine dehydrogenase of Rhodospirillum rubrum is presented. The enzyme has a molecular weight of 110000 and consists of two subunits of identical molecular weight of 55000. Depending on the ionic strength and protein concentration it is possible for the native enzyme to dimerize to produce an enzymatically active species of molecular weight 220000. Titrations of the native and detergent-treated enzyme with a variety of sulfhydryl reagents show 2 mol free--SH groups per 110000 g, one of which is buried in the protein interior. L-Threonine and/or high concentrations of salt can expose the buried--SH group, and this--SH group is essential for the catalytic activity of the enzyme. Two independent lines of evidence show that extensive polymerization of the enzyme caused by L-threonine and/or high concentrations of salt does not involve the formation of intermolecular disulfide bonds.

Alcohol Oxidoreductases↗
PubMed1978

Control of DNA synthesis in growing BALB/c 3T3 mouse cells by a fibroblast growth regulatory factor.

A cell surface component from quiescent BALB/c 3T3 mouse cells that inhibits DNA synthesis and cell division when added to a culture of growing 3T3 cells has been detected. The inhibition of DNA synthesis by this factor was dependent on concentration and time of incubation; a transient exposure of cells to the factor followed by incubation in its absence for 20 hr was sufficient to elicit its inhibitory effect. The active component appears to be protein in nature, as judged by heat inactivation and trypsin sensitivity. Extracts obtained in an identical manner from quiescent 3T3 cells that had been preincubated in situ with uridine diphosphate N-acetyl-D-glucosamine (UDP-GlcNAc) did not inhibit DNA synthesis. The effect was specific for UDP-GlcNAc: incubation with three other nucleotide sugars yielded active component. Incubation of the inactive component from UDP-GlcNAc-treated cells with purified N-acetyl-beta-D-glucosaminidase in vitro restored its inhibitory property. Extracts from growing cells failed to inhibit DNA synthesis. These results suggest that reversible glycosylation with N-acetyl-D-glucosamine residues may serve as a regulatory signal for the conversion of the active factor to its inactive form. We propose that the onset of quiescence of 3T3 cells is due to a casual relationship between depletion of growth factors in the culture medium and the presence of the active regulatory factor on the cell surface that inhibits DNA synthesis; conversion of the regulatory factor to its inactive form under favorable nutritional status may be viewed as a switch that allows DNA synthesis to resume.

Acetylglucosamine↗
PubMed1978

Uridine diphosphate N-acetylglucosamine stimulates uptake of nutrients by quiescent BALB/C3T3 cells.

Preincubation of quiescent BALB/C3T3 cells with uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) in conditioned medium resulted in a time- and concentration-dependent stimulation of uptake of 2-deoxyglucose, uridine, and alpha-aminoisobutyric acid over that seen in conditioned medium alone. The apparent Km values for 2-deoxyglucose and uridine uptake were the same for cells incubated with or without UDP-GlcNAc, whereas the Vmax values were higher for cells pretreated with the nucleotide sugar. The stimulation of uptake was specific for UDP-GlcNAc, and the other nucleotide sugars tested were ineffective; incubation of UDP-GlcNAc-pretreated cells with N-acetylglucosaminidase abolished the stimulatory effect. In all cases, the extent of stimulation of nutrient uptake was comparable to that seen with serum-stimulated cells. Incubation of quiescent cells in situ with UDP-[3H]GlcNAc led to incorporation of radioactive N-acetylglucosamine into the acid-precipitable fraction; a large fraction of the labeled amino sugar was found on the cell surface acceptors. We interpret these data to mean that the cellular acceptors of quiescent cells are "under-glycosylated," at least in terms of N-acetylglucosamine, and that stimulation of uptake of nutrients may be a consequence of restoration of the amino sugar residues on the oligosaccharide chains of acceptors on the cell surface.

Acetylglucosamine↗
PubMed1977

Homoserine dehydrogenase: spontaneous reactivation by dissociation of p-mercuribenzoate from an inactive enzyme--p-mercuribenzoate complex.

Incubation of Rhodospirillum rubrum homoserine dehydrogenase (L-homoserine:NAD+ oxidoreductase, EC 1.1.1.3) with p-mercuribenzoate (PMB) in the presence of 0.2 M KCl and 2 mM L-threonine resulted in complete loss of enzyme activity. Upon removal of excess PMB, KCl, and L-threonine, a time-dependent recovery of enzyme activity was observed in 25 mM phosphate/I mM EDTA buffer, pH 7.5. Circular dichroism studies indicated that the transition from inactive to reactivated form of the enzyme was accompanied by a conformational change in the protein. Experiments with [14C]PMB revealed loss of enzyme-bound radioactivity during reactivation. Increase in ionic strength of the phosphate buffer and/or addition of L-threonine, leading to enzyme aggregation, decreased the rate of enzyme reactivation, aggregated enzyme that remained inactive retained [14C]PMB on the enzyme. Sulfhydryl titration of various forms of the enzyme suggested a preferential release of PMB from a sulfhydryl group essential to enzymic activity. We conclude that reactivation of the inactive enzyme is due to dissociation of PMB from an "active-site" sulfhydryl group and that changes in the protein structure influence the rate of dissociation of the enzyme-PMB complex.

Alcohol Oxidoreductases↗
PubMed1975

Catabolite inactivation of biodegradative threonine dehydratase of Escherichia coli.

Incubation of Escherichia coli cells with glucose, pyruvate, and certain other metabolites led to rapid inactivation of inducible biodegradative threonine dehydratase. Analysis with several mutant strains showed that pyruvate, and not a metabolite derived from pyruvate, was capable of inactivating enzyme, and that glucose acted indirectly after being converted to pyruvate. Some other alpha-keto acids such as oxaloacetate and alpha-ketobutyrate (but not alpha-ketoglutarate) were also effective. Inactivation of threonine dehydratase by pyruvate was also observed with purified enzyme preparations. The rates of enzyme inactivation increased with increased concentrations of pyruvate and decreased with increased levels of AMP. Increasing protein concentrations lowered the rates of enzyme inactivation. Dithiothreitol had a large effect on the maximum extent of inactivation of the enzyme by pyruvate; high concentrations of AMP and DTT almost completely counteracted the effect of pyruvate. Gel filtration data showed that pyruvate influenced the oligomeric state of the enzyme by altering the association-dissociation equilibrium in favor of dissociation; the Stokes' radius of the pyruvate-inactivated enzyme was 32 A as compared to 42 A for the untreated enzyme. Reassociation of the dissociated form of the enzyme was achieved by removal of excess free pyruvate by dialysis against buffer supplemented with AMP and DTT. Incubation of threonine dehydratase with [14-C]pyruvate revealed apparent covalent attachment of pyruvate to the enzyme. Strong protein denaturants such as guanidine, urea, and sodium dodecyl sulfate failed to release bound radioactive pyruvate; the molar ratio of firmly bound pyruvate was approximately 1 mol/150,000 g of protein. Pretreatment of the enzyme with p-chloromercuribenzoate and 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) did not reduce the binding of [14-C]pyruvate suggesting no active site SH was involved in the pyruvate-enzyme linkage. Titration of active and pyruvate-inactivated enzyme with Nbs2 indicated that the loss in enzyme activity was not due to oxidation of essential sulfhydryl groups on the enzyme. Based on these data we propose that the mechanism of enzyme inactivation by pyruvate involves covalent attachment of pyruvate to the active oligomeric form of the enzyme followed by dissociation of the oligomer to yield inactive enzyme.

Adenosine Monophosphate↗