AMINO ACID METABOLISM IN LEAF PEROXISOMES AND MITOCHONDRIA IN PHOTORESPIRATION (TRANSPORT, AMINOTRANSFERASES).

摘要

Photorespiration is a wasteful respiration occurring in the green leaves of many agriculturally important plants. To reduce or eliminate photorespiration will increase plant productivity. The phororespiratory pathway of glycolate metabolism occurs largely in the peroxisomes, and the remaining steps in the mitochondria. This thesis work was designed to study the pathway in isolated peroxisomes and mitochondria.; Peroxisomes had not been previously isolated in dilute osmotic solution for in vitro metabolic studies. A procedure for isolating intact spinach (Spinacia oleracea L.) leaf peroxisomes in 0.25 molar sucrose solution by Percoll density gradient centrifugation was established.; The transamination of glyoxylate derived from glycolate in peroxisomes were studied by supplying isolated intact peroxisomes with glycolate and one to three of the amino acids serine, glutamate, and alanine. The three amino acids exhibited mutual inhibition to one another due to the competition for the supply of glyoxylate. In addition, competitive inhibition at the active site of enzymes occurred between glutamate and alanine, but not between serine and glutamate or alanine.; The glycerate pathway of converting glycerate to serine in the presence of NAD and alanine in intact peroxisomes was studied. Addition of oxaloacetate or -ketoglutarate plus aspartate enhanced the conversion about three-fold. The amino group donor could be alanine (half-saturation constant, 0.33 mM), glycine (0.45 mM), or asparagine (0.67 mM); the three amino acids produced roughly similar V(,max) values. The results indicate that the transamination is catalyzed by a hydroxypyruvate aminotransferase with characteristics unknown among all other studied leaf peroxisomal aminotransferases. The participation of the peroxisomal malate dehydrogenase in an electron shuttle system across the membrane in the regeneration of NAD/NADH is suggested.; The transport of radioactive glycine, serine and proline into the matrix of spinach leaf mitochondria was studied. The uptake of all three amino acids showed a biphasic characteristic. At concentrations higher than 0.5 mM, an apparent diffusion process dominated. At concentrations lower than 0.5 mM, an active uptake system that accumulated amino acid in the matrix became apparent. The results of mutual inhibitory studies suggest that the active uptake system consists of at least two components with different degrees of amino acid specificity.