Intracellular Localization of Enzymes of Arginine Metabolism in Neurospora

Abstract

Abstract The physical basis for the confinement of carbamyl phosphate, ornithine, and arginine to specific anabolic fates has been investigated. Cell extracts of Neurospora grown in minimal medium were fractionated by differential centrifugation to determine the subcellular localization of the relevant enzymes. Carbamyl-P synthase A (arginine-specific; EC 2.7.2.5), ornithine acetyltransferase, and ornithine carbamyltransferase (EC 2.1.2.2) were found to be associated with a particulate (organellar) fraction. All three enzymes co-sediment with mitochondria on linear sorbitol gradients, and appear to be components of the mitochondrial matrix. The mitochondrial membrane limits the ability of exogenous substrates to be metabolized by these enzymes in vitro. Arginine synthesis from citrulline, putrescine synthesis from ornithine, and the catabolism of ornithine and arginine are carried out by cytoplasmic enzymes. The catabolic enzymes are found at significant levels even during growth in minimal medium, but little catabolism occurs in vivo. The results indicate that the anabolic and catabolic pathways of ornithine metabolism are, in part, separated by the mitochondrial membrane. However, the mechanisms responsible for confining carbamyl-P and ornithine to arginine synthesis and arginine to protein synthesis are more complex than the simple physical separation of the anabolic and catabolic enzymes.