Rat lenses were cultured 4-24 hr at 37 degrees C in balanced salt medium containing 5 mM 15N-glutamate, 15N-alanine, or amido-15N-glutamine. Free amino acids were extracted with 6% trichloracetic ...acid containing alpha-aminoisobutyrate as an internal standard, and trifluoroacetyl-n-butyl (TAB) derivatives were prepared. Amino acids were quantified by gas chromatography, and 15N enrichment in amino groups of several free amino acids was determined by mass spectrometry. Culture of lenses with either 15N-glutamate or 15N-alanine resulted in 15N-labeling of the glutamate, aspartate, alanine, proline, serine and glycine pools. No detectable amino-15N (less than 5%) was observed in amino acids when lenses were cultured with amido-labeled 15N-glutamine or with 15N-ammonium chloride. It is concluded that the alpha-amino groups of several amino acids are actively involved in lens metabolism. In contrast, although glutamine may serve as the major source of glutamate for lens, the amido nitrogen of glutamine plays a minor role, if any, as a source of alpha-amino groups for amino acid metabolism.
In isolated perfused rat liver, added 4-methyl-thio-2-oxobutyrate and phenylpyruvate are rapidly transaminated to the corresponding amino acids with glutamine, the latter being supplied via the ...portal vein or by endogenous synthesis. With portal glutamine concentrations below 5mM and in the presence of a oxo-acid acceptor, the flux through glutamine transaminases exceeded the ammonium ion-stimulated glutaminase flux. 4-Methylthio-2-oxobutyrate-induced extra glutamine uptake was not dependent on the perfusate pH in the range of pH 7 to 8. During glutamine/4-methylthio-2-oxobutyrate transamination, the amide nitrogen of glutamine is fully recovered as glutamate, ammonia, urea and alanine. Oxoglutarate formed by omega-amidase activity is released as glutamate or oxidized by oxoglutarate dehydrogenase. alpha-Cyanocinnamate, the inhibitor of the monocarboxylate translocator in the mitochondrial membrane inhibited 4-methylthio-2-oxobutyrate-induced glutamine uptake and methionine release by about 30%. This might indicate that about 2/3 of glutamine transaminase flux is cytosolic. alpha-Cyanocinnamate inhibited 4-methylthio-2-oxobutyrate-induced glutamate efflux by about 90%. Stimulation of flux through glutamine transaminases is accompanied by a 70-80% inhibition of glutaminase flux. This is not explained by a direct inhibition of glutaminase by 4-methylthio-2-oxobutyrate but by a substrate competition between glutaminase and glutamine transaminases. 4-Methylthio-2-oxobutyrate decreases glutamine release by the liver due to withdrawal by transamination. The oxo acid itself is without effect on glutamine synthetase flux. With respect to hepatocyte heterogeneity there is no evidence for a zonal distribution of glutamine transaminase activities, as it has been shown for glutamine synthetase and glutaminase activities.
Both enantiomers of methyl (or ethyl) phenyl phosphoramidate (8, 13) were prepared from the corresponding diastereomeric amidates of L-phenylalanine ester (5, 10), via a transaminative reaction ...involving the following sequence : N-chlorination, treatment with sodium methoxide, and mild acid-catalyzed hydrolysis of the resulting amino acetal (7, 12). Stereochemical studies of H+- and BF3-catalyzed alcoholyses of 8 and 13 indicated that the nature of the acid catalyst had a profound effect on the steric course of the reaction. Complete stereospecificity observed in H+ catalysis was ascribed to direct displacement with inversion of configuration at phosphorus (A-2 mechanism). On the other hand, BF3 catalysis, which produced both inversion (70%) and retention product (30%), was explained in terms of (1) a pentacoordinate intermediate mechanism for retention product formation and (2) a dual mechanism, pentacoordinate intermediate and direct displacement A-2 mechanisms, for inversion product formation.
Transaminative metabolism of L-cysteine was investigated using homogenates of guinea pig liver and kidney. L-Cysteine was transaminated in the presence of 2-oxoglutarate and the homogenate of either ...liver or kidney. S-(2-Hydroxy-2-carboxyethylthio)cysteine (HCETC) (3-mercaptolactate-cysteine disulfide) was formed by liver homogenate, but the amount was very small. On the other hand, a relatively large amount of HCETC was formed in the presence of kidney homogenate. Transamination between 3-mercaptopyruvate and certain amino acids was catalyzed actively by both liver and kidney homogenates in the presence of L-glutamate. However, more half-cysteine was formed by liver than kidney, and more HCETC was produced by kidney than liver. L-Glutamate was the most potent amino donor, and L-aspartate strongly inhibited the reaction. Results indicate that L-cysteine can be transaminated both in liver and kidney of the guinea pig, and that kidney is more active than liver. 2-Oxoglutarate is the most active 2-oxo acid for cysteine transamination. Oxaloacetate (and aspartate in the reverse reaction) is inhibitory to the reaction. These results are in agreement with the previous conclusion that cysteine aminotransferase is identical with aspartate aminotransferase.
A procedure for transaminating proteins and removing the transaminated N-terminal residue has been used for studying structure-function relationship of protein (Dixon and Fields 1972, Meth. Enzymol. ...25, 409-419). We show that it is convenient for measuring the relative molecular masses of proteins by measuring the glycine formed from glyoxylate during such transamination. Quinoxaline derivatives have been synthesized by the reaction of 2-oxo acids from amino acids reacting with o-phenylenediamine. 2-Oxo acids transaminated from amino acids fluoresced at around 405 nm, depending on the nature of residual side groups. The emission maximum of 3-benzyl-2-hydroxy-quinoxaline from the reaction of o-phenylenediamine with phenylpyruvate was at 363 nm. The fluorescent derivatives have been used to study conformational changes of peptides and to detect whether or not N-termini of proteins were blocked.
Aluminum can be determined by reaction with ethyl pyridoxyliminopyruvate in methanol. The neutral methanolic solution of pyridoxamine and a methanolic solution of ethyl pyruvate are mixed and heated ...at 70°for 15 min. This Schiff base solution is added to the methanolic solution of a sample containing aluminum nitrate. The time-absorbance curves have to be recorded for each sample, because the coloration of species absorbing at 488 nm is unstable. Calibration curve is obtained by plotting the maximum values in the time-absorbance curve against aluminum concentration. This spectrophotometric method can be used in the concentration range of 0.89 to 2.11 μg/ml of aluminum.
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