Compartmentation and kinetics of urea cycle enzymes in pig enterocytes.
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We recently reported urea synthesis in enterocytes of postweaning pigs (Biochem. J. 312: 717-723, 1995). The objective of this study was to determine the compartmentation and kinetics of intestinal urea cycle enzymes. Jejunal enterocytes were prepared from 60-d-old pigs weaned at 21 d of age. Carbamoylphosphate (CP) synthase I (CPS I) and ornithine carbamoyltransferase (OCT) were located in mitochondria, whereas argininosuccinate (AS) synthase (ASS) and lyase (ASL) were found in the cytosol. Arginase isozymes were present in the cytosol and mitochondria, and differed in their sensitivity to heat inactivation. Vmax values of CPS I, OCT, ASS, ASL, arginase were 6.88 ± 0.43, 713.4 ± 22.3, 1.93 ± 0.07, 3.50 ± 0.23 and 7.13 ± 0.60 nmol.min-1.mg protein-1 (means ± SEM, n=5), respectively. Km values of CPS I for NH4+, HCO3- and Mg-ATP were 1.34 ± 0.11, 58.4 ± 2.4, and 15.2 ± 1.4 mM, respectively. Km values of OCT for ornithine and CP were 5.13 ± 0.24 and 17.1 ± 1.2 mM, respectively. Km values of ASS for Mg-ATP, aspartate and citrulline were 2.86 ± 0.16, 0.054 ± 0.003 and 0.15 ± 0.01 mM, respectively. Km value of ASL for AS was 0.63 ± 0.04 mM. The Km values of cytosolic and mitochondrial arginase ASL for arginine were 7.46 ± 0.52 and 7.09 ± 0.58 mM, respectively. The small intestine is the exclusive organ for citrulline synthesis from glutamine in pigs, and has a relatively low rate of ureagenesis compared with the liver. We suggest that enterocyte urea cycle enzymes function primarily to synthesize citrulline from NH3, HCO3- and ornithine (glutamine metabolites) rather than urea from NH3 and HCO3- in pigs.
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