Ornithine synthesis from glutamate in rat small intestinal mucosa

Abstract

The de novo synthesis of ornithine from glutamate by the rat small intestine has been investigated in order to establish conditions necessary for the reaction to proceed optimally. The formation of [ 14C]ornithine from [ 14C]glutamate was found in a subcellular fraction enriched in mitochondria. Ornithine formation required glutamate, ATP, NADPH, and Mg 2+; phosphocreatine in the presence of endogenous creatine phosphokinase was stimulatory. Glutamate and ATP exhibited typical saturation kinetics with apparent K m values of about 8 m m and 60 μ m, respectively. Ornithine synthesis was increased by NADPH concentrations up to 0.2 m m but higher concentrations were inhibitory. The maximal rate observed was 1 to 4 nmol of ornithine/min · mg protein. Synthesis of ornithine from glutamate was not observed with liver or kidney homogenates. Although the intestinal mitochondrial fraction contained ornithine aminotransferase (OAT), partially purified rat liver OAT stimulated ornithine synthesis. In the absence of exogenous liver OAT, pyridoxal 5′-phosphate or pyridoxamine 5′-phosphate stimulated both ornithine synthesis and endogenous OAT activity. These observations support the hypothesis that ATP and NADPH were utilized to reduce glutamate to glutamate-γ-semialdehyde which was then aminated by OAT to yield ornithine [Ross, G., Dunn, D., and Jones, M. E. (1978) Biochem. Biophys. Res. Commun. 85, 140–147]. Pyridoxal 5′-phosphate concentrations greater than 0.01 m m inhibited ornithine formation whereas pyridoxamine 5′-phosphate showed no inhibitory effect at this or higher concentrations. Neither coenzyme inhibited endogenous OAT activity. These results suggest that pyridoxal 5′-phosphate inhibits the synthesis of glutamate-γ-semialdehyde.