Preparation and properties of ornithine-oxo-acid aminotransferase of rat kidney. Comparison with the liver enzyme.

Ornithine-oxo-acid aminotransferase (EC 2.6.1.13) from rat kidney was prepared as a single homogeneous protein as judged by polyacrylamide gel electrophoresis, ultracentrifuge analysis and double diffusion precipitin test. Content of pyridoxal phosphate, light absorption spectra, circular dicroism spectra, Km values, inhibitors, and electrophoretic mobilities of the proteins after reactions with group modifying reagents were similar for the ornithine-oxo-acid aminotransferases of rat kidney and liver. Rates of reaction with group modifying reagents, stabilities to storage at -15 degrees C, and stabilities to temperatures above 55 degrees C differed significantly for the two enzymes. The liver enzyme contained two more cysteine residues than the kidney enzyme as determined by three different methods. Heating the liver enzyme at 66-67 degrees C at pH 5.9 for 1 h decreased the thiol groups titratable by 5,5'-dithio-bis(2-nitrobenzoic acid) (Nbs2). Uncer the same conditions titratable thiol groups of the kidney enzyme were not decreased. Amino acid analysis revealed probably significant differences in tyrosine and isoleucine content in addition to cysteine. It was concluded that the primary structures of ornithine-oxo-acid aminotransferases of rat liver and kidney are not fully identical.

Synthesis of proline and hydroxyproline in human lung (WI-38) fibroblasts.

Human lung fibroblasts (WI-38) in late exponential phase of growth, in stationary phase after confluency was reached, and at high or low number of population doublings were used to investigate the synthesis of proline and hydroxyproline from glutamate or arginine. Glutamate was from two to five times as effective a precursor as arginine; glutamine did not seem to be involved in these metabolic pathways. Accumulation of protein-bound hydroxyproline in cell layers was observed only after confluency. Confluent cells synthesized more proline from glutamate than did cells in late exponential growth. Conversion of glutamate into intracellular free proline was conducted also to a greater extent in confluent cells at a high number of population doublings. Conversion of glutamate into proline or hydroxyproline in cell-layer protein was not affected significantly by the number of population doublings. Less total protein as well as less hydroxyproline accumulated with cells at a high number of population doublings.

Purification and properties of arginase of rat kidney.

l-Arginase from rat kidney was partially purified and some properties were compared with those of l-arginase of rat liver. The kidney enzyme was firmly bound to the mitochondrial fraction and after solubilization required arginine or an unknown factor in tissue extracts for stabilization after dialysis. The two enzymes differed also in stability with respect to acetone treatment, heating or freezing. In further contrast with liver arginase, arginase from kidney was not adsorbed to CM-cellulose at pH7.5 and its activity was not increased by incubation with Mn(2+). Other differences were seen in relative specificities for substrates, ratio of hydrolysis rates with high and low concentrations of arginine and effects of certain inhibitors. Antisera prepared to pure liver arginase did not cross-react with partially purified kidney arginase.

The formation of ornithine from proline in animal tissues.

1. Homogenates of liver or kidney from rat, mouse, dog and guinea pig formed ornithine from proline but not from glutamate. Rat kidney was most active in this reaction and was used for further studies. 2. The overall reaction was found to be catalysed by proline oxidase to yield glutamic gamma-semialdehyde, followed by transamination of this product with glutamate as catalysed by ornithine-keto acid aminotransferase. 3. The unfavourable equilibrium of the ornithine-keto acid aminotransferase reaction was overcome chiefly by glutamate dehydrogenase in the tissue, which removed the alpha-oxoglutarate produced, by reduction with endogenous ammonia and NADH. 4. Aspartate aminotransferase in these preparations also aided in the removal of alpha-oxoglutarate. In this case the overall reaction was driven also by the rapid decarboxylation of oxaloacetate. 5. No evidence could be found for a pathway of ornithine synthesis involving acylated intermediates as has been observed in some micro-organisms. 6. The rate of ornithine synthesis in homogenates of several rat tissues paralleled the activity of ornithine-keto acid aminotransferase in these tissues, indicating that this enzyme was rate-determining for the synthesis. 7. The possible influence of these reactions on urea synthesis is discussed.