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Biomedical subjects

R N Puri

Publications and source records attributed to R N Puri.

44 records · Page 3Linked to original sources
PubMed1983

Transport of glutathione, as gamma-glutamylcysteinylglycyl ester, into liver and kidney.

Administration of gamma-glutamylcysteinylglycyl monomethyl (or monoethyl) ester to mice leads to substantial increases in the levels of glutathione in the liver and kidney. Mice depleted of glutathione by treatment with buthionine sulfoximine, a potent inhibitor of gamma-glutamylcysteine synthetase, exhibited about a 4-fold increase in liver and kidney glutathione levels after administration of glutathione monomethyl ester. This ester also prevented the marked decline in liver glutathione level found after giving mice acetaminophen, and it protected mice from toxicity due to this compound. The findings indicate that the monomethyl and monoethyl esters of glutathione are transported into cells and hydrolyzed to glutathione. Such esters may be useful in experimental work on glutathione metabolism and function and may provide a relatively safe method for protecting cells against damage by toxic compounds, oxygen, and radiation.

Animals↗
PubMed1982

Liberation, purification, and properties of thioesterase component of pigeon liver fatty acid synthetase complex.

Proteolysis of pigeon liver fatty acid synthetase with elastase results in the quantitative cleavage of the thioesterase component from the enzyme complex. This thioesterase component is two or three times more active catalytically in the isolated state than in the native fatty acid synthetase, and its activity is not affected by the presence or absence of reducing thiols. The proteolytically cleaved thioesterase is separated from the core enzyme in one step by size-exclusion chromatography on a Sephadex G-75 column. The peptide obtained by gel permeation is homogeneous with respect to size and charge, as shown by polyacrylamide gel electrophoresis in the presence and absence of SDS. Size-exclusion chromatography on Bio-Gel A 0.5 m and Sephadex G-75 columns, sucrose density gradient ultracentrifugation, and N-terminal amino acid analysis also indicate that the proteolytically cleaved thioesterase is homogeneous. The sedimentation coefficient of the thioesterase is approximately 2.9 S. Proteolytic cleavage with elastase also quantitatively releases the [1,3-14C]- or [1,3-3H]diisopropylphosphofluoridate-labeled thioesterase component from the correspondingly labeled fatty acid synthetase. Binding studies with 14C- or 3H-labelled diisopropylphosphofluoridate and fatty acid synthetase show that 2 mol of the label are bound per mol of the enzyme when complete loss of fatty acid-synthesizing activity occurs. The molecular weight of the thioesterase component is estimated to be 36000 by size-exclusion chromatography, SDS-polyacrylamide gel electrophoresis and amino acid analysis.

Amino Acids↗
PubMed1982

Isolation and characterization of an acyl carrier protein from pigeon liver fatty acid synthetase by controlled proteolysis with elastase.

Controlled proteolytic cleavage of 4'-phospho[14C]pantetheine-labeled pigeon liver fatty acid synthetase generates two 4'-phospho[14C]pantetheine-labeled peptides, Ec1 and Ec2. These are separated from each other and the core enzyme by gel permeation chromatography on a Sephadex G-75 column. The two radioactively labeled peptides constitute 50% of the radioactivity initially present in the 4'-phospho[14C]pantetheine-labeled fatty acid synthetase. The remaining label in the core enzyme is released quantitatively by proteolytic cleavage with trypsin. The molecular weights of Ec1 and Ec2 peptides, as determined by size exclusion chromatography and SDS-polyacrylamide gel electrophoresis, are 12000 and 6000, respectively. Both the higher and lower molecular weight peptides are homogeneous with respect to size and charge, as shown by polyacrylamide gel electrophoresis in the presence and absence of SDS. The higher molecular weight peptide, Ec1, is characterized as an acyl carrier protein by the transacylation reaction between the unlabeled Ec1 peptide and radioactively labeled acetyl- and malonyl-CoA. Since Ec2 peptide also contains the prosthetic group present in the Ec1 peptide, the Ec2 peptide appears to result from the proteolytic cleavage of the higher molecular weight peptide, Ec1. Amino acid composition of the acyl carrier protein shows the presence of 1 mol of 4'-phosphopantetheine per mol of protein. 2 mol of acyl carrier protein are present per mol of the fatty acid synthetase. The amino acid analysis is in good agreement with the molecular weight of the Ec1 peptide, as determined by gel filtration and SDS-polyacrylamide gel electrophoresis. N-Terminal amino acid analysis of this peptide shows the presence of an arginine residue.

Acyl Carrier Protein↗
PubMed1981

Demonstration of the occurrence of inactive fatty acid synthetase in rat liver by immunotitration and its in vitro partial activation.

Direct immunotitrations of rat liver fatty acid synthetase in crude tissue homogenates with monospecific rabbit anti-rat liver fatty acid synthetase antibody enabled us to make a comparison of fatty acid synthetase protein and activity (percentage of maximal activity) as a function of the nutritional state in normal, diabetic, and insulin- and glucagon-insulin treated animals. Previous results, in which large changes in fatty acid synthetase activity were related to protein synthesis and degradation rather than to enzyme activation, were confirmed. It was also shown that fatty acid synthetase activation does not occur immediately on synthesis but follows the synthesis of fatty acid synthetase protein. In order to characterize the enzymatically inactive protein found on immunotitration and to develop an in vitro system for fatty acid synthetase activation, conditions were sought to obtain large amounts of fatty acid synthetase protein free from, or low in, activity. It was found that treatment of hypophysectomized rats with triiodothyronine meets these requirements, yielding milligram quantities of inactive fatty acid synthetase protein with less than 2% of maximal activity. A part of the inactive fatty acid synthetase was found to be the apoenzyme as indicated by beta-ketoreductase and thioesterase activities, by its ability to incorporate label from [G3H]CoA, and by its partial in vitro activation, which led to an increase in overall synthetase activity in crude and partially purified cell-free systems. The components required for activation include magnesium ion and a transferase fraction prepared from livers of 48-h fasted, 12-h refed rats.

Animals↗
PubMedDate not supplied

Design and synthesis of a kininogen-based selective inhibitor of thrombin-induced platelet aggregation.

Thrombin-induced platelet aggregation has been suggested to play an important role in reocclusion following thrombolytic therapy or angioplasty for treatment of myocardial infarction. We previously demonstrated that thrombin-induced platelet aggregation is indirectly mediated by intracellularly activated calpain expressed on the platelet surface through the cleavage of aggregin, a putative ADP-receptor, and that high molecular weight kininogen (HK), a naturally occurring thiol protease inhibitor, modulates thrombin-induced platelet aggregation. Considering the substrate specificity of calpain and the conserved sequence in HK, we studied selective inhibitors of thrombin-induced platelet aggregation by the affinity labeling approach with an S-3-nitro-2-pyridinesulfenyl (Npys) group. H-Phe-Gln-Val-Val-Cys (Npys)-Gly-NH2, which combines chemical and structural features of calpain substrate specificity and the conserved sequence in HK, selectively inhibited thrombin-induced platelet aggregation. It did not inhibit the aggregatory effects of other platelet agonists, and did not inhibit amidolytic activity of thrombin and thrombin-induced platelet shape change. The design and synthesis of such inhibitors could lead to the development of a new class of inhibitors that selectively block thrombin-induced platelet aggregation.

Amino Acid Sequence↗