Urea cycle of Fasciola gigantica: purification and characterization of arginase.
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 Km of 6 mM. The optimum temperature of F. gigantica Arg II was 40 degrees C and the enzyme was stable up to 30 degrees C and retained 80% of its activity after incubation at 40 degrees C for 15 min and lost all of its activity at 50 degrees 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 Mn2+, where the other metals Fe2+, Ca2+, Hg2+, Ni2+, Co2+ and Mg2+ had inhibitory effects.
Mohamed
Saleh A
SA
Molecular Biology Department, National Research Centre, Cairo, Egypt. saleh38@hotmail.com
Fahmy
Afaf S
AS
Mohamed
Tarek M
TM
Hamdy
Soha M
SM
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001119:Arginase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001123:Argininosuccinate Lyase / Q000378:metabolism; D001124:Argininosuccinate Synthase / Q000378:metabolism; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000378:metabolism; D002412:Cations / Q000378:metabolism; D005209:Fasciola / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000737:chemistry; D009952:Ornithine / Q000378:metabolism; D009954:Ornithine Carbamoyltransferase / Q000378:metabolism; D012756:Sheep; D013379:Substrate Specificity; D013696:Temperature; D014508:Urea / Q000378:metabolism*
D000596:Amino Acids; D002412:Cations; D008670:Metals; D014508:Urea; D009952:Ornithine; D009954:Ornithine Carbamoyltransferase; D001119:Arginase; D001123:Argininosuccinate Lyase; D002222:Carbamoyl-Phosphate Synthase (Ammonia); D001124:Argininosuccinate Synthase