Fluorescence properties of reduced thionicotinamide--adenine dinucleotide and of its complex with octopine dehydrogenase.
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Reduced 3-thionicotinamide--adenine dinucleotide (sNADH) is shown to be fluorescent, with an emission maximum at 510 nm when excited in the region of the absorption maximum (398 nm), and with a very low quantum yield, (3.4 +/- 0.5) x 10(-4). The interaction between sNADH and octopine dehydrogenase was investigated by ultraviolet-difference spectroscopy and fluorescence. Some surprising fluorescence features were found when sNADH was bound to the enzyme in the presence of D-octopine, as follows. (a) There is an unusually high enhancement of the dinucleotide fluorescence (by at least a factor of 100) attended by a 40-nm blue shift of the emission maximum. (b) The protein fluorescence is quenched almost completely. (c) The bound coenzyme analog undergoes a photoreaction, which proceeds differently from that occurring the free form. These features appear to be unique to the octopine.sNADH complex, as for example they are not present when sNADH is bound to horse liver alcohol dehydrogenase, or when NADH is bound to octopine dehydrogenase. The possible origin of these fluorescence features is discussed. Binding and kinetic studies were carried out with sNAD and sNADH. It was found that sNAD neither binds nor acts kinetically as a coenzyme. sNADH exhibits relatively good binding, with Km and Ki values close to those of the natural coenzyme, but the turnover number is 460 times smaller than that with NADH. The kinetic consequences of these findings are discussed. The sNADH dissociation constants were determined as a function of temperature, and appear to be practically temperature-independent in the range 10--40 degrees C. It seems thus, in agreement with previous studies, that the interaction between octopine dehydrogenase and coenzymes proceeds athermically, regardless of the structure, affinity, and chemical reactivity of the coenzyme. The possible biological and chemical meaning of this finding is discussed. (+info)
Investigation on the kinetic mechanism of octopine dehydrogenase. A regulatory behavior.
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The kinetic scheme of octopine dehydrogenase of Pecten maximus L., a monomeric enzyme obeying a bi-ter sequential mechanism, was completed, essentially in the forward reaction, by steady-state studies over a wide range of substrate concentration at pH 7.0. Deviation from the Michaelis-Menten behavior with respect to NAD+ and other significant kinetic data led us to ascribe for octopine dehydrogenase mechanism the mnemonical enzyme concept. In addition, another regulatory behavior can be envisaged involving the formation of two dead-end complexes enzyme.NADH.D-octopine and enzyme.NAD+.pyruvate.L-arginine. (+info)
Acute hypoxia induces HIF-independent monocyte adhesion to endothelial cells through increased intercellular adhesion molecule-1 expression: the role of hypoxic inhibition of prolyl hydroxylase activity for the induction of NF-kappa B.
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Remnants of an ancient pathway to L-phenylalanine and L-tyrosine in enteric bacteria: evolutionary implications and biotechnological impact.
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The pathway construction for biosynthesis of aromatic amino acids in Escherichia coli is atypical of the phylogenetic subdivision of gram-negative bacteria to which it belongs (R. A. Jensen, Mol. Biol. Evol. 2:92-108, 1985). Related organisms possess second pathways to phenylalanine and tyrosine which depend upon the expression of a monofunctional chorismate mutase (CM-F) and cyclohexadienyl dehydratase (CDT). Some enteric bacteria, unlike E. coli, possess either CM-F or CDT. These essentially cryptic remnants of an ancestral pathway can be a latent source of biochemical potential under certain conditions. As one example of advantageous biochemical potential, the presence of CM-F in Salmonella typhimurium increases the capacity for prephenate accumulation in a tyrA auxotroph. We report the finding that a significant fraction of the latter prephenate is transaminated to L-arogenate. The tyrA19 mutant is now the organism of choice for isolation of L-arogenate, uncomplicated by the presence of other cyclohexadienyl products coaccumulated by a Neurospora crassa mutant that had previously served as the prime biological source of L-arogenate. Prephenate aminotransferase activity was not conferred by a discrete enzyme, but rather was found to be synonymous with the combined activities of aspartate aminotransferase (aspC), aromatic aminotransferase (tyrB), and branched-chain aminotransferase (ilvE). This conclusion was confirmed by results obtained with combinations of aspC-, tyrB-, and ilvE-deficient mutations in E. coli. An example of disadvantageous biochemical potential is the presence of a cryptic CDT in Klebsiella pneumoniae, where a mutant carrying multiple enzyme blocks is the standard organism used for accumulation and isolation of chorismate.(ABSTRACT TRUNCATED AT 250 WORDS) (+info)
Renal protection in chronic kidney disease: hypoxia-inducible factor activation vs. angiotensin II blockade.
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Neuroactive carbamate adducts of beta-N-methylamino-L-alanine and ethylenediamine. Detection and quantitation under physiological conditions by 13C NMR.
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beta-N-Methylamino-L-alanine (BMAA) reacts with dissolved carbon dioxide to form two carbamate compounds at physiological pH, temperature and bicarbonate concentration. The reversible reactions of BMAA with dissolved carbon dioxide were monitored by 13C NMR. At 37 degrees C and pH 7.4, the fraction of BMAA existing as the alpha-N-carboxy adduct is 0.22 while the fraction of BMAA existing as the beta-N-carboxy adduct is 0.09. Although both adducts could be implicated in the bicarbonate-dependent neurotoxicity of BMAA (Weiss, J. H., and Choi, D. W. (1988) Science 241, 973-975; Mroz, E. A., Weiss, J. W., and Choi, D. W. (1989) Science 243, 1613), the beta-N-carboxy adduct shares structural characteristics with the appropriate endogenous ligand, glutamic acid. Analogously, the GABA-mimetic properties of ethylenediamine have been attributed to a carbamate adduct, ethylenediamine monocarbamate (Kerr, D. I. B., and Ong, J. (1987) Br. J. Pharmacol. 90, 763-769). Using the same method, we were able to detect directly and quantify the formation of this carbamate under physiological conditions. Information on the carbamate equilibria of these compounds is essential in order to address questions of their neuroactive potency. (+info)