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Biomedical subjects

Y Hatefi

Publications and source records attributed to Y Hatefi.

At least 109 records · Page 6Linked to original sources
PubMed1981

Possible occurrence and role of an essential histidyl residue in succinate dehydrogenase.

Diethylpyrocarbonate (Et2PC) inhibits the succinate dehydrogenase [succinate:(acceptor) oxidoreductase, EC 1.3.99.1] activity of submitochondrial particles, Complex II (succinate:ubiquinone oxidoreductase), and the soluble, pure succinate dehydrogenase. The reaction order with respect to Et2PC concentration is close to unity, suggesting modification of one essential residue per active unit of the enzyme. The pH profile of Et2PC inhibition, the partial reversal of inhibition by hydroxylamine, and the spectral change of the Et2PC-treated enzyme in the UV region suggest modification of a histidyl residue. Succinate dehydrogenase activity can be protected against Et2PC inhibition by succinate, fumarate, malonate, or oxaloacetate (also by activating anions such as ClO4(-) and Br-), suggesting that the Et2PC-modified essential residue might be at the active site. In both submitochondrial particles and the purified enzyme, succinate dehydrogenase activity is highest and relatively constant at pH greater than or equal to 7.0 and diminishes precipitously at pH less than 7.0. By contrast, fumarate reductase activity is highest at pH less than or equal to 7.0 and diminishes at pH greater than 7.0. These results are consistent with the possible participation of the unprotonated and protonated forms of the imidazole moiety of the putative histidyl residue, respectively, in succinate oxidation and fumarate reduction.

Animals↗
PubMed1980

Energy-linked transhydrogenation from NADPH to [14C]NADP.

Submitochondrial particles catalyze transhydrogenation from NADPH to [14C]NADP. This transhydrogenation is energy-linked, since its rate increases several-fold when the system is energized by succinate oxidation in the presence of rotenone (inhibitable by antimycin A or uncouplers), or by ATP hydrolysis (inhibitable by rutamycin or uncouplers). As in the case of transhydrogenation reactions from NAD(P)H to 3-ace-tylpyridine adenine dinucleotide phosphate and to thionicotinamide adenine dinucleotide phosphate, transhydrogenation from NADPH to [14C]NADP is also sensitive to treatment of the particles with trypsin or the arginyl residue modifier, butanedione. However, unlike the former reactions, transhydrogenation from NADPH to [14C]NADP cannot accumulate energy in the concentrations of the products, because, except for radioactivity, the nature and concentrations of the reactants and products remain unchanged throughout the course of the reaction. Therefore, the unrecoverable energy utilization by this region could be ascribed to an entropic component of the process, very likely an enzyme conformation change necessary for facilitation of hydride ion transfer from NADPH to [14C]NADP. This interpretation is in agreement with our previous kinetic evidence for enzyme conformation change associated with energy-linked transhydrogenation from NADH to 3-acetylpyridine adenine dinucleotide phosphate and thionicotinamide adenine dinucleotide phosphate, and with our conclusions regarding the mechanism of action of the transhydrogenase enzyme (Galante, Y.M., Lee, Y., and Hatefi, Y. (1980) J. Biol. Chem. 255, 9641-9646).

Animals↗
PubMed1980

Effect of pH on the mitochondrial energy-linked and non-energy-linked transhydrogenation reactions.

The effect of pH on the kinetics of the following transhydrogenation reactions catalyzed by energized and nonenergized submitochondrial particles has been studied: NADH leads to 3-acetylpyridine adenine dinucleotide phosphate (AcPyADP), NADH leads to thionicotinamide adenine dinucleotide phosphate (thioNADP), NADPH leads to AcPyADP, and NADPH leads to thioNADP. The effect of membrane energization on reaction rates can be approximated in the case of NADH leads to AcPyADP and thioNADP transhydrogenations, or equaled in the case of NADPH leads to AcPyADP and thioNADP transhydrogenations by lowering the assay pH to less than or equal to 6.0. For the reactions NADH leads to AcPyADP and thioNADP under energy-linked conditions, substrate Km values are lowest and Vmax values are highest at pH 7 to 7.5, the optimum pH for mitochondrial energy transduction processes. Under non-energy-linked conditions, however, Km values were lowest and Vmax values were highest at the most acid conditions (pH = 5.5) examined. Plots of ln (Vmax/Km) (an index of enzyme-substrate affinity to form a complex) versus pH showed the highest affinity at pH 7 to 7.5 for energy-linked conditions. Similar plots for non-energy-linked conditions. Similar plots for non-energy-linked conditions. Similar plots for non-energy-linked conditions showed a sharp and linear increase in the value of ln (Vmax/Km) as the assay pH was lowered from 8.5 to 6 to 6.5. This was followed by a less steep line down to pH 5.5, with a clear break at pH 6 to 6.5. These results suggested the involvement of ionizable group(s) with pK value(s) at pH 6 to 6.5 affecting enzyme-substrate binding under non-energy-linked conditions. An analogous mechanism, possibly by way of proton-induced conformation change of the transhydrogenase enzyme (EC 1.6.1.1), might be involved in increasing enzyme-substrate affinity and consequently the rate of transhydrogenation under energy-linked conditions.roton-induced conformation change of the transhydrogenase enzyme (EC 1.6.1.1), might be involved in increasing enzyme-substrate affinity and consequently the rate of transhydrogenation under energy-linked conditions.

Animals↗
PubMed1980

Energy-linked mitochondrial transhydrogenation from NADPH to NADP analogs.

The mitochondrial energy-linked transhydrogenase enzyme catalyzes hydride ion transfer between NAD and HADP, of which the reaction NADH leads to NADP is slow in the absence of energy and is accelerated 10-fold or more when the mitochondrial membrane is energized by ATP hydrolysis or respiration. The enzyme is a proton pump and effects proton translocation coupled to hydride ion transfer from NADPH to NAD (Earle, S.R., and Fisher, R.R. (1980) J. Biol Chem. 255, 827-830). The present studies have shown that submitochondrial particles also catalyze transhydrogenation from NADPH to two NADP analogs, namely 3-acetylpyridine adenine dinucleotide phosphate (AcPyADP) and thionicotinamide adenine dinucleotide phosphate (thioNADP). Both reaction rates are greatly accelerated when the system is energized by ATP hydrolysis (inhibitable by uncouplers or rutamycin) or succinate oxidation (inhibitable by uncouplers or antimycin A). As in the case of NAD(H) in equilibrium with NADP(H) reactions, the transhydrogenations from NADPH to AcPyADP and thioNADP are inhibited by treatment of submitochondrial particles with trypsin or the arginyl residue modifier, butanedione. The Km values of the above substrates and the Vmax values under energy-linked conditions have been determined. The finding that the mitochondrial energy-linked transhydrogenase enzyme catalyzes transhydrogenation from NADPH to NADP analogs has revealed features regarding substrate site specificities and the effect of substrates on the directionality of proton translocation by the enzyme.

Animals↗
PubMed1979

Radiochemical synthesis and photochemical properties of the uncoupler 2-azido-4-nitrophenol, a versatile photoaffinity labeling reagent.

2-Amino-4-nitrophenol was tritiated in an acid-catalyzed hydrogen exchange reaction. Radioactive 2-azido-4-nitrophenol with a specific radioactivity up to 21 mCi/mmol was synthesized from 2-amino-4-nitrophenol by diazotization and azide coupling. The photochemical properties of the uncoupler, 2-azido-4-nitrophenol, were studied as free solute and as ligand bound to uncoupler binding sites in bovine serum albumin and mitochondria. Based on product analyses, irradiation of free or bound 2-azido-4-nitrophenolate with visible light results in the formation of nitrene intermediates with a singlet to triplet ratio of 6:1 to 9:1. 2-Azido-4-nitrophenolate and bovine serum albumin form a strong 1:1 complex (KD = 0.7 micron) which can be converted into a photoproduct with a covalent bond between the label and the protein. The acid dissociation constant of the protein-bound 2-amino-4-nitrophenol moiety is strongly pH dependent. Photoaffinity labeling of mitochondria by 2-azido-4-nitrophenolate follows a pattern expected from equilibrium binding studies using normal and lipid-depleted particles: polypeptides were found to bear 90-95% of the radioactive label, and 5-10% of the latter was bound to phospholipids. Two polypeptides (approximately 56 000 and 31 000 daltons) were associated with 60% of the label, indicating a high degree of specific photochemical labeling.

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