Biomedical subjects
Publications and source records attributed to A J Cooper.
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Following cryoablation of one kidney in Strain-2 guinea-pigs, the viable opposite kidney was assessed by functional, histological and immunopathological techniques for evidence of immunologically mediated damage. The autoantibody content of postoperative sera was also examined. Control operations involved ligation, surgical nephrectomy and the production of hepatic cryolesions. The results indicate that autoantibody production and glomerular deposition of immune complexes can occur and may cause abnormal passage of hippuran through the kidney. There is no evidence for autoallergy against highly tissue-specific determinants; the specificity, strength and time course of the reactions obtained was dependent on the site of the lesion, whether or not freezing was accompanied by ligation.
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Asparagine transaminase has been purified about 200-fold from rat liver. The enzyme has a broad specificity toward both amino acids and alpha-keto acids. Thus, amino acids substituted in the beta position such as asparagine, S-methylcysteine, phenylalanine, cysteine, serine, and aspartate are substrates. The enzyme is also active with alanine, methionine, homoserine, alpha-aminobutyrate, glutamine, and leucine. The enzyme has a high affinity for glyoxylate but the affinity falls off markedly through the series glyoxylate, pyruvate, alpha-ketoburyrate, alpha-Keto acids substituted in the beta or gamma position, such as alpha-ketosuccinamate, phenylpyruvate, p-hydroxyphenylpyruvate, alpha-keto-gamma-methiolburyrate, and alpha-keto-gamma-hydroxybutyrate, are substrates for the enzyme. Amino acids or alpha-keto acids possessing a branch point at the beta carbon are inactive. Kinetic analysis of the asparagine glyoxylate transamination reaction is consistent with a ping-pong mechanism.
Pulsed Fourier transform proton magnetic resonance was used to study the labilization of protons of various L-amino acids by the enzyme gamma-cystathionase. In the course of the normal reaction, the enzyme labilizes the alpha and beta protons of the substrate, L-homoserine, and promotes elimination of the gamma substituent. It was found that gamma-cystathionase also catalyzes the exchange of the alpha and beta protons of L-amino acids which cannot undergo elimination reactions, but are competitive inhibitors of the enzyme. Both beta protons of L-alpha-aminobutyrate, although not stereochemically equivalent, were exchanged at equal rates, whereas selectivity was shown for one of the beta hydrogens when the carbon length was increased. The data also show that beta-proton exchange cannot occur without alpha-proton exchange. The rate of alpha-proton exchange from amino acids containing a terminal hydroxyl group at the beta, gamma, or lambda carbon is greater than from the corresponding unsubstituted amino acid. Exchange rates of the alpha proton for the inhibitors examined vary from one-seventh that of the normal enzymatic reaction to approximately the same rate as that for the elimination reaction with homoserine. An active site with two areas of substrate-enzyme interaction is proposed. One site contains pyridoxal 5'-phosphate and the base or bases involved in alpha- and beta-proton exchange; the second site contains a base which normally facilitates removal of the gamma substituent and can interact with the gamma and lambda carbons of the substrate molecule.
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L-Methionine sulfoximine is a substrate of L-amino acid oxidase (Crotalus adamanteus), glutamine transaminase, and gamma-cystathionase. In the reaction catalyzed by L-amino acid oxidase, methionine sulfoximine is converted to aplph-imino-gamma-methylsulfoximinylbutyrate, which undergoes rapid gamma elimination yielding methane sulfinimide and 2-imino-3-butenoic acid. Methane sulfinimide is converted to methane sulfonamide, methane sulfinic acid, and methane sulfonic acid; 2-imino-3-butenoic acid is hydrolyzed to vinylglyoxylate, which polymerizes to an insoluble product. When the reaction is carried out in the presence of semicarbazide, the imine formed initially is quantitatively trapped as alpha-keto-gamma-methylsulfoximinylbutyrate semicarbazone, from which the free alpha-keto acid may be obtained. When the reaction is carried out in the presence of a mercaptan (RSH), a gamma exchange reaction occurs leading to formation of a new alpha-keto acid substituted in the gamma position by an SR-group; thus, alpha-keto-gamma-(beta-hydroxyethiol)butyric acid (S-(hydroxyethyl)-2-keto-4-mercaptobutyric acid) was obtained when L-methionine sulfoximine was oxidized in the presence of 2-mercaptoethanol, and enzymatic transamination of this alpha-keto acid with L-glutamine gave the new amino acid, L-omega-hydroxyethionine. The reaction of L-methionine sulfoximine with gamma-cystathionase yields 1 mol each of alpha-ketobutyrate and methane sulfinimide; the latter is hydrolyzed almost exclusively to methane sulfinic acid. Transamination of L-methionine sulfoximine yields the corresponding alpha-keto acid (alpha-keto-gamma-methylsulfoximinylbutyrate), which is stable. Some of these reactions may occur in vivo, and thus contribute to the toxicity of L-methionine sulfoximine.
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Pulsed Fourier transform proton magnetic resonance spectroscopy was used to study the glutamate-alanine transaminase-catalyzed incorporation of deuterium from solvent deuterium oxide into the alpha and beta positions of L-alanine. It was found that the beta proton resonance signal initially disappears slightly faster than the signal due to the alpha proton, but whereas the alpha proton signal decays exponentially, that due to the beta proton signal does not. Eventually, the rate of decrease of the alpha proton signal becomes greater than that for the beta proton. This change in the relative rates is ascribed to a deuterium isotope effect upon substitution of an alpha proton by a deuteron. Furthermore, as deuterium begins to replace hydrogen, two classes of alanine become distinguishable, i.e. alanine which contains deuterium in the alpha position and hydrogen in the beta position, and alanine which contains hydrogen in the alpha position and deuterium in the beta position. Thus, removal of all 3 beta protons is not contingent upon loss of an alpha proton from the same molecule. The two classes of deuterated alanine may conceivably arise by a scrambling mechanism in which protons are transferred from the alpha to the beta position and vice versa. Present evidence excludes this scramblong mechanism and leads to the conclusion that deuterium incorporation into L-alanine involves, (a) the reversible enzymatic conversion of the classical ketimine enzymes intermediate to an enaminetype structure, and (b) considerable conservation of label during the prototropic shift from the alpha carbon of L-alanine to the C4-position of pyridoxal 5'-phosphate. It is also postulated that alanine binds at the active site in such a way as to bring the beta protons into close contact with a basic group on the enzyme surface. This group is distinct from that used in abstraction of an alpha proton. The beta protons of glutamate are not enzymatically removed; presumably glutamate binds in such a way that the beta protons cannot effectively interact with an enzyme base. Similar studies were carried out on soluble glutamate-aspartate transaminase; no evidence was found for significant enzyme-catalyzed deuterium incorporation into the beta position of L-glutamate, L-aspartate, and L-alanine.
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Bilateral lesions of the olfactory bulbs in 1-day-old mice led to a slow rate of growth both before and after weaning and the growth changes were positively associated with the size of the lesion. Mothers continued to care for the bulbectomised mice which showed no evidence of retarded motor development though their eyes opened later and they were less active when exposed to an artificial perfume. Bulbectomised mice failed to survive in litters of two where both infants were bulbectomised and in litters with a large number of unoperated littermates. Keeping litters small and having both bulbectomised and control mice present enhanced the chances of survival and the presence of infant control mice is a necessary condition for the maintenance of lactation and the survival of the bulbectomised babies. Failure to respond to the odours( pheromones) of the mothers may be responsible for the infrequent suckling and the slow growth and development.
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