Purification and properties of porcine liver ornithine transcarbamylase.
Ornithine transcarbamylase (OTCase) has been purified from porcine liver by a simple four-step procedure that included chromatography on an affinity column to which the transition-state analogue, delta-N-phosphonacetyl-L-ornithine (PALO), was covalently bound. The procedures employed yielded an enzyme which was purified some 260-fold and was judged to be homogeneous by nondenaturing- and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Apparent homogeneity of the enzyme was confirmed by N-terminal sequence analysis. The molecular weight of the porcine enzyme was determined by Sephadex gel exclusion chromatography and sedimentation equilibrium. An approximate molecular weight of 107,000 was calculated by both procedures. The single band obtained by SDS-PAGE indicated a subunit molecular weight of 36,800 +/- 700; hence, the enzyme is a trimer of identical subunits. The sedimentation coefficient of the native enzyme was determined to be 6.47. At pH 8.0, the Km values for the substrates are 0.41 and 1.3 mM for ornithine and carbamyl phosphate, respectively. PALO is a competitive inhibitor and has a Ki of 0.13 microM, which suggests that it binds with about 10,000 times greater affinity than carbamyl phosphate. Amino acid analysis performed on acid hydrolyzed enzyme yielded 323 amino acids per monomer. Performic acid oxidation of the enzyme, followed by acid hydrolysis and amino acid analysis, showed three cysteine residues per subunit. A partial specific volume of 0.725 cc/g was calculated from the amino acid composition. Reaction of purified porcine OTCase with phenylglyoxal, 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 retained during a 1-h reaction time. Other substrates and substrate combinations offer less protection.
Koger
J B
JB
Department of Animal Science, North Carolina State University, Raleigh 27695-7621.
Howell
R G
RG
Kelly
M
M
Jones
E E
EE
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001667:Binding, Competitive; D002846:Chromatography, Affinity; D002956:Citrulline / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009952:Ornithine / Q000031:analogs & derivatives / Q000494:pharmacology; D009954:Ornithine Carbamoyltransferase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010658:Phenylglyoxal / Q000494:pharmacology; D010710:Phosphates / Q000494:pharmacology; D017421:Sequence Analysis; D013552:Swine; D014461:Ultracentrifugation
D010710:Phosphates; D002956:Citrulline; C020464:N(delta)-(phosphonoacetyl)-L-ornithine; D001120:Arginine; D009952:Ornithine; D009954:Ornithine Carbamoyltransferase; D010658:Phenylglyoxal