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M Blumenstein

Publications and source records attributed to M Blumenstein.

86 records · Page 5Linked to original sources
PubMed1975

31 P nuclear magnetic resonance studies of the interaction of pyridine nucleotide coenzymes with dehydrogenases.

31P nuclear magnetic resonance spectra of the pyrophosphate group in NAD+ and NADH were recorded in the presence of beef heart lactate dehydrogenase and rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. At high lactate dehydrogenase concentrations (60 mg/ml), two NADH resonances are observed: a slowly exchanging peak which is shifted to 1.9 ppm downfield (relative to free NADH) and a rapidly exchanging peak with a downfield shift of 0.5-0.6 ppm. At lover concentrations (15 mg/ml) only the rapidly exchanging peak is observed thus indicating that the peak observed at-1.9 ppm is due to coenzyme bound to an aggregated enzyme species. With NAD+, rapid exchange and downfield shifts are observed at both enzyme and concentrations, with shifts of about 1.5 ppm and 0.6 ppm at 60 and 15 mg/ml, respectively. In the presence of glyceraldehydephosphate dehydrogenase, the results are independent of enzyme concentration, and slow exchange and upfield shifts of 0.4-0.6 ppm occur with each coenzyme. These data indicate that the environment of the pyrophosphate group of oxidized and reduced coenzyme is the same for a given dehydrogenase, but is different in one enzyme from the other. The resonances observed with glyceraldehydephosphate dehydrogenase are broader than those observed with lactate dehydrogenase. This is indicative of either shorter relaxation times with the former enzyme, or the presence of multiple, unresolved resonances.

Animals↗
PubMed1975

Nonidentical alkylation sites in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase.

These studies establish the specificity of 3,3,3-trifluorobromoacetone for reaction with the active site cysteines of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase and suggest the potential use of trifluoroacetonyl groups as 19F nuclear magnetic resonance probes for study of symmetry relations between the four protomers of the enzyme. The alkylation of the holoenzyme follows biphasic kinetics and indicates either preexistent or induced nonequivalence among the sites; these effects are not predisposed by a low coenzyme/enzyme ratio. Two additional alkylation sites not at the active centers are created by acylation with beta-(2-furyl)acryloyl phosphate: it is concluded that pseudosubstrates cause an intramolecular rearrangement which exposes two sulfhydryl functions besides those of the active site (Cys-149).

Acetone↗
PubMed1975

19F nuclear magnetic resonance studies of structure and function relationships in trifluoroacetonylated rabbit muscle glyceraldehyde-3-phosphate dehydrogenase.

Specific reaction of Cys-149 with 3,3,3-trifluorobromacetone allows one to probe symmetry relation between the active center regions of tetrameric glyceraldehyde-3-phosphate dehydrogenase by 19F nuclear magnetic resonance (nmr) techniques. Nmr titration studies in the pH range of greatest enzymic activity reveal the existence of species with an (alphaalpha')2 structure; this symmetry is not induced by the coenzyme. Addition of NADH to the ketone-labeled protein causes the enzymic reduction of the ligand in a stereospecific manner and is used to demonstrate the functionality of residues other than Cys-149 that are essential for catalysis. The interpretation of chemical shift characteristics found for the trifluoroacetonyl group together with the kinetics of its reduction allows the derivation of a dynamic model for the enzymic structure which may contribute to understanding of the half-of-the-sites phenomenon.

Acetone↗
PubMedDate not supplied

Differential regulation in human amnion epithelial and fibroblast cells of prostaglandin E(2) production and prostaglandin H synthase-2 mRNA expression by dexamethasone but not tumour necrosis factor-alpha.

Previous studies have identified both pro-inflammatory cytokines and glucocorticoids as positive regulators of amnion prostaglandin (PG) biosynthesis. The stimulatory effects of dexamethasone (Dex), a glucocorticoid agonist, on prostaglandin endoperoxide H synthase (PGHS)-2 mRNA expression and PG biosynthesis in amnion have been attributed to an atypical response by the mesenchymal cells of the amnion. The objective of this study was to confirm previous findings concerning cell type-dependant Dex-induced upregulation of PGHS-2 mRNA expression and PG production using separated amnion cell populations, in comparison with the effects of the pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-alpha). Amnion cells from placentae delivered at term by caesarian section were isolated by tryptic digestion and epithelial cells were then separated from mesenchymal cells by differential absorption onto plastic. After 24-72 h, the two cell populations were passaged and sub-cultured. Cells were treated with Dex (10(-9)-10(-6) m) or TNF-alpha (0.1-50 ng/ml) or media alone. Thereafter, PGE(2)production was determined and PGHS-2 mRNA content analysed by a competitive quantitative RT-PCR method established and validated for this study. PGE(2)production in fibroblast-enriched cultures was increased to 310+/-41 per cent (mean+/-sem, n=4 wells per treatment point) of control in the presence of 10(-8) m Dex. Conversely, PGE(2)production in Dex-treated amnion epithelial cells was decreased to 67+/-24 per cent of control. Altered PGE(2)biosynthesis was accompanied by the upregulation of PGHS-2 mRNA in amnion fibroblasts but not in epithelial cells. TNF-alpha increased PG output and PGHS-2 expression independent of cell type. Glucocorticoids therefore appear to have opposing effects on PG biosynthesis in the two major cellular components of the human amnion.

Amnion↗
PubMedDate not supplied

Efficacy and specificity of non-steroidal anti-inflammatory drugs for the inhibition of cytokine-stimulated prostaglandin E(2) secretion by amnion-derived WISH cells.

Prostaglandin H synthase-2 (PGHS-II) specific inhibitors have been proposed as a potential treatment in the prevention of preterm birth. We examined the efficacy of PGHS inhibitors on basal and cytokine-stimulated prostaglandin (PG) production by the amnion-like WISH cell line. WISH cells were treated with interleukin (IL)-1 beta and tumour necrosis factor (TNF)-alpha in the presence or absence of indomethacin, etodolac, 5,5-dimethy-3-(3-fluorophenyl)-4-(4-methlysulphonyl) phenyl-2 (5H)-furanone (DFU) or nimesulide (1.6-1000 nM) for 16 h. PG production was then measured using radioimmunoassay. Nimesulide and DFU were the most selective non-steroidal anti-inflammatory drugs (NSAIDs) of IL-beta-stimulated PG production in these studies with an a IC(50)(basal)/IC(50)(stimulated) ratio of, respectively, 142.2 and 113.8, followed by etodolac (25.3) and indomethacin (2.2). Similar results were obtained when cells were stimulated with TNF-alpha. The results of this study suggest that PGHS-II-selective NSAIDs may be effective in the prevention of cytokine-driven amnion PG production associated with preterm labour.

Amnion↗