Urea cycle of Fasciola gigantica: Purification and characterization of arginase

Abstract

The ornithine–urea cycle has been investigated in Fasciola gigantica. Agrinase had very high activity compared to the other enzymes. Carbamoyl phosphate synthetase and ornithine carbamoyltransferase had very low activity. A moderate enzymatic activity was recorded for argininosuccinate synthetase and argininosuccinate lyase. The low levels of F. gigantica urea cycle enzymes except to the arginase suggest the urea cycle is operative but its role is of a minor important. The high level of arginase activity may benefit for the hydrolysis of the exogenous arginine to ornithine and urea. Two arginases Arg I and Arg II were separated by DEAE-Sepharose column. Further purification was restricted to Arg II with highest activity. The molecular weight of Arg II, as determined by gel filtration and SDS-PAGE, was 92,000. The enzyme was capable to hydrolyze l-arginine and to less extent l-canavanine at arginase : canavanase ratio (> 10). The enzyme exhibited a maximal activity at pH 9.5 and K m of 6 mM. The optimum temperature of F. gigantica Arg II was 40 °C and the enzyme was stable up to 30 °C and retained 80% of its activity after incubation at 40 °C for 15 min and lost all of its activity at 50 °C. The order of effectiveness of amino acids as inhibitors of enzyme was found to be lysine > isoleucine > ornithine > valine > leucine > proline with 67%, 43%, 31%, 25%, 23% and 15% inhibition, respectively. The enzyme was activated with Mn 2+, where the other metals Fe 2+, Ca 2+, Hg 2+, Ni 2+, Co 2+ and Mg 2+ had inhibitory effects.