Effect of ascorbic acid and some reducing agents on N-nitrosopiperidine metabolism by liver microsomes.

The effect of ascorbic acid on the oxidation of N-nitrosopiperidine by a microsomal preparation obtained from guinea-pig liver has been examined. The metabolites from the oxidation of N-nitrosopiperidine were found to be 5-hydroxypentanal (about 1.93%), N-nitroso-3-hydroxypiperidine (about 0.02%), and N-nitroso-4-hydroxypiperidine (about 0.15%). With microsomes separated from the liver of guinea-pigs pretreated with phenobarbital and 3-methylcholranthrene, the yields of N-nitroso-3-hydroxypiperidine and N-nitroso-4-hydroxypiperidine were found to decrease upon increasing the concentration of ascorbic acid in the reaction mixture. In the presence of small amounts of ascorbic acid, the yields of these compounds with untreated liver microsomes apparently increased, while the presence of larger amounts of ascorbic acid caused a decrease in the yields of these metabolites. In contrast with the yields of N-nitroso-3-hydroxypiperidine and N-nitroso-4-hydroxypiperidine, that of 5-hydroxypentanal decreased with increasing amounts of ascorbic acid.

Inactivation of ribulosebisphosphate carboxylase by modification of arginyl residues with phenylglyoxal.

Phenylglyoxal rapidly and completely inactivates spinach and Rhodospirillum rubrum ribulosebisphosphate carboxylases. Inactivation exhibits pseudo-first-order kinetics and a reaction order of approximately one for both enzymes, suggesting that modification of a single residue per protomeric unit suffices for inactivation. Loss of enzymic activity is directly proportional to incorporation of [14C]phenylglyoxal until only 30% of the initial activity remains. For both enzymes, extrapolation of incorporation to 100% inactivation yields 4-5 mol of [14C]phenylglyoxal per mol protomer. Amino acid analyses confirm the expected 2:1 stoichiometry between phenylglyoxal incorporation and arginyl modification and suggest that other kinds of amino acid residues are not modified. (Thus, inactivation correlates with modification of 2-3 arginyl residues per protomer). The substrate ribulose bis-phosphate and some competitive inhibitors reduce the rates of inactivation of the carboxylases and prevent modification of about 0.5-1.0 arginyl residue per protomer. Inactivation is therefore a consequence of modification of a small number of residues out of the 35 and 29 total arginyl residues per protomer in spinach and R. rubrum carboxylases, respectively.

A new RNA-RNA crosslinking reagent and its application to ribosomal 5S RNA.

The synthesis of a new RNA specific bifunctional crosslinking reagent, 1.4-phenyl-diglyoxal, is described which reacts exclusively with guanosines. The properties of the crosslinked products enabled us to develop a straightforward method for identifying the reacted nucleotides. Results obtained with ribosomal 5S RNA of Escherichia coli demonstrate the formation of an intramolecular crosslink between guanosine-2 and guanosine-112 in the stem region.

Alpha-hydroxylation in the metabolism of N-nitrosopiperidine by rat liver microsomes: formation of 5-hydroxypentanal.

Metabolism of n-nitrosopiperidine by a purified microsomal preparation from rat liver was shown to yield 5-hydroxypentanal. This is the predicted product for a pathway involving an initial oxidation of the nitrosamine at the carbon atom alpha to the nitroso group.

The search for new cancerostatic agents.

Following the lead given by Albert Szent-Györgyi's bioelectronic theory of cancer, work was continued in two major directions: (i) designing new electrophilic molecules, related to methylglyoxal, and (ii) using L-ascorbic acid as a (non-toxic) carrier for methylglyoxal and its derivatives in the form of its acetals. The vinylogue of methylglyoxal, 4-oxopent-2-enal, was expected to be a most reactive electron acceptor, on the basis of quantum mechanical calculations by J.J. Ladik's group. A new reaction, the formation of the ene-2, 3-diol acetal and hemiacetal-hemiketal, was found to occur with 'conjugated' aldehydes, such as methylglyoxal, glyoxal, phenylglyoxal, malealdehyde and acrylaldehyde; the reaction proceeded very smoothly with 4-oxopent-2-enal. The structural determination of these new types of acetals by 1H and 13C n.m.r. spectroscopy and by chemical methods is discussed.

Functionally important arginine residues of aspartate transcarbamylase.

The reaction of phenylglyoxal with aspartate transcarbamylase and its isolated catalytic subunit results in complete loss of enzymatic activity (Kantrowitz, E. R., and Lipscomb, W. N. (1976) J. Biol. Chem. 251, 2688-2695). If N-(phosphonacetyl)-L-aspartate is used to protect the active site, we find that phenylglyoxal causes destruction of the enzyme's susceptibility to activation by ATP and inhibition by CTP. Furthermore, CTP only minimally protects the regulatory site from reaction with this reagent. The modified enzyme still binds CTP although with reduced affinity. After reaction with phenylglyoxal, the native enzyme shows reduced cooperativity. The hybrid with modified regulatory subunits and native catalytic subunits exhibits slight heterotropic or homotropic properties, while the reverse hybrid, with modified catalytic subunits and native regulatory subunits, shows much reduced homotropic properties but practically normal heterotropic interactions. The decrease in the ability of CTP to inhibit the enzyme correlates with the loss of 2 arginine residues/regulatory chain (Mr = 17,000). Under these reaction conditions, 1 arginine residue is also modified on each catalytic chain (Mr = 33,000). Reaction rate studies of p-hydroxymercuribenzoate, with the liganded and unliganded modified enzyme suggest that the reaction with phenylglyoxal locks the enzyme into the liganded conformation. The conformational state of the regulatory subunit is implicated as having a critical role in the expression of the enzyme's heterotropic and homotropic properties.

[Mechanism of oxidation reaction of NADH models and phynylglyoxal with hydrogen peroxide. Hypothesis on separate transport of hydrogen and electron atom in certain enzymatic reactions with the participation of NADH and NADPH]. / Mekhanizm reaktsii okisleniia modelei NADH i fenilglioksalia perekis'iu vodoroda. Gipoteza o razdel'nom perenose atoma vodoroda i electrona v nekotorykh fermentativnykh reaktsiiakh s uchastiem NADH NADPH

Kinetics of co-oxidation of 1-benzen-3-carbamido-1,4-dihydropyridine (BDN) and phenylglyoxal (PG) with hydrogen peroxide is studied. Dimeric product (di-e11-benzen-5-carbamido-1,2-dihydropyridyl-2]) is found to be formed at pH 9, and quaternal pyridinium salt (BNA)--at pH 7. Molecular oxigen is determined to participate in the reaction at pH 7. Copper (II) ions catalyze this process. Significant catalytic effect of p-dinitrobenzen (p-DNB) is found. The reaction mechanism is postulated to form hydroperoxide from PG and hydrogen peroxide which are capable to split the hydrogen attom from dihydropyridine, molecular oxigen or p-DNB being an acceptor of the electrone. Hypothesis on separate transfer of hydrogen atom and electrone in biological systems are proposed.

Inhibition of Hageman factor, plasma thromboplastin antecedent, thrombin and other clotting factors by phenylglyoxal hydrate (38500).

Exposure of purified Hageman factor (HF, Factor XII) to phenylglyoxal hydrate (PHG), an agent reacting with arginine residues in protein, inhibited its coagulant properties upon subsequent exposure of negatively charged agents. Once HF had been exposed to kaolin or ellagic acid, however, subsequent addition of PHG was much less inhibitory. PHG had no effect upon the ability of HF to bind to negatively charged surfaces. PGH also inhibited preparations of activated PTA (Factor XI) and thrombin, and, when incubated with plasma, reduced the titer of coagulable fibrinogen, PTA Christmas factor (Factor IX), antihemophilic factor (Factor VIII), Factor VII, Stuart factor (Factor X), proaccelerin (Factor V) and prothrombin (Factor II), and to a lesser degres, HF.