PURIFICATION AND PROPERTIES OF PORCINE LIVER ORNITHINE TRANSCARBAMYLASE

Orithine transcarbamylase (OTCase) has been purified from porcine live r by a simple four-step procedure that included chromatography on an a ffinity column to which the transition-state analogue, delta-N-phospho nacetyl-L-ornithine (PALO), was covalently bound. The procedures emplo yed yielded an enzyme which was purified some 260-fold and was judged to be homogeneous by nondenaturing- and sodium dodecyl sulfate-polyacr ylamide gel electrophoresis (SDS-PAGE). Apparent homogeneity of the en zyme was confirmed by N-terminal sequence analysis. The molecular weig ht of the porcine enzyme was determined by Sephadex gel exclusion chro matography and sedimentation equilibrium. An approximate molecular wei ght of 107,000 was calculated by both procedures. The single band obta ined by SDS-PAGE indicated a subunit molecular weight of 36,800 +/- 70 0; hence, the enzyme is a trimer of identical subunits. The sedimentat ion coefficient of the native enzyme was determined to be 6.47. At pH 8.0, the K-m values for the substrates are 0.41 and 1.3 mill for ornit hine and carbamyl phosphate, respectively. PALO is a competitive inhib itor and has a K-i of 0.13 mu M, which suggests that it binds with abo ut 10,000 times greater affinity than carbamyl phosphate. Amino acid a nalysis performed on acid hydrolyzed enzyme yielded 323 amino acids pe r monomer. Performic acid oxidation of the enzyme, followed by acid hy drolysis and amino acid analysis, showed three cysteine residues per s ubunit. A partial specific volume of 0.725 cc/g was calculated from th e amino acid composition. Reaction of purified porcine OTCase with phe nylglyoxal, an arginine-specific reagent, results in complete loss of catalytic activity. The decrease in enzymatic activity correlates with the modification of 1 mol of arginine per mole of OTCase monomer. In the presence of 20 mM carbamyl phosphate, 93% of the activity is retai ned during a 1-h reaction time. Other substrates and substrate combina tions offer less protection. (C) 1994 Academic Press, Inc.