Enzymological evidence for the indispensability of small intestine in the synthesis of arginine from glutamate. II. N-acetylglutamate synthase.
We describe here a concise assay procedure for N-acetylglutamate (AGA) synthase (AGAS) and its application to an extensive study of tissue distribution of AGAS activity. Crude mitochondria from several tissues were incubated in a pair of assay mixtures with [14C]glutamate in the absence and presence of acetyl-CoA at 15 degrees C for 10 min. Anionic components including [14C]AGA were first isolated from glutamate by a cation exchanger column. In order to remove anionic contaminants such as succinate, the AGA was converted to glutamate enzymatically by aminoacylase, and then the glutamate was isolated by cation exchange chromatography and counted. Recoveries were corrected individually. The difference between the pair incubations was taken as the activity. An extensive survey of AGAS activity in rats showed that, although the liver expressed the highest activity, the small intestine, testis, lung and submaxillary gland also exhibited considerable activity. Sexual differences in activity were not found in the liver and small intestine. We also detected activity in the human small intestine for the first time. Optimization of incubation temperature and time and the presence of arginine in an assay mixture was essential and we demonstrated that the AGAS reaction with crude mitochondria as an enzyme source was unstable without arginine and at higher temperatures. This procedure appears suitable for studying the physiological and nutritional role of AGAS in non-hepatic tissues. In the accompanying paper we applied this procedure to study the ontogeny of AGAS in developing rat tissues.
Wakabayashi
Y
Y
Department of Biochemistry, Kyoto Prefectural University of Medicine, Japan.
Iwashima
A
A
Yamada
E
E
Yamada
R
R
D000123:Acetyltransferases / Q000378:metabolism*; D051046:Amino-Acid N-Acetyltransferase; D000818:Animals; D001120:Arginine / Q000096:biosynthesis*; D005260:Female; D005971:Glutamates / Q000378:metabolism*; D007421:Intestine, Small / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D009928:Organ Specificity; D051381:Rats; D011919:Rats, Inbred Strains; D013696:Temperature
D005971:Glutamates; D001120:Arginine; D000123:Acetyltransferases; D051046:Amino-Acid N-Acetyltransferase