Fasciola gigantica: enzymes of the ornithine-proline-glutamate pathway--characterization of delta1-pyrroline-5-carboxylate dehydrogenase.
Ornithine aminotransferase (OAT), proline oxidase (PO), Delta 1-pyrroline-5-carboxylate reductase (P5CR), and Delta 1-pyrroline-5-carboxylate dehydrogenase (P5CD) were assessed in Fasciola gigantica. All enzymes are involved in the conversion of ornithine into glutamate and proline. High levels of P5CD suggest that the direction of the metabolic flow from ornithine is more toward glutamate than proline. F. gigantica P5CD1 and P5CD2 were separated from the majority of contaminating proteins in crude homogenate using a CM-cellulose column. A Sephacryl S-200 column was employed for P5CD2 to obtain pure enzyme with increased specific activity. The molecular mass of P5CD2 was estimated to be 50kDa using a Sephacryl S-200 column and SDS-PAGE. It migrated as a single band on SDS-PAGE, indicating a monomeric enzyme. P5CD2 had Km values of 1.44mM and 0.37mM for NAD and P5C, respectively. P5CD2 oxidized a number of aliphatic and aromatic aldehydes, where the aromatic compounds had higher affinity toward the enzyme. All amino acids examined had partial inhibitory effects on the enzyme. While 3mM AMP caused 31% activation of enzyme, 3mM ADP and ATP inhibited activity by 18% and 23%, respectively. Apart from Cu2+, the divalent cations that were studied caused partial inhibitory effects on the enzyme.
Mohamed
Saleh A
SA
Molecular Biology Department, National Research Centre, Cairo, Egypt. saleh38@hotmail.com
Mohamed
Tarek M
TM
Fahmy
Afaf S
AS
El-Badry
Mohamed O
MO
Abdel-Gany
Somia S
SS
D050842:1-Pyrroline-5-Carboxylate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000227:Adenine Nucleotides / Q000494:pharmacology; D000447:Aldehydes / Q000378:metabolism; D000596:Amino Acids / Q000494:pharmacology; D000818:Animals; D002412:Cations / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D005209:Fasciola / Q000201:enzymology*; D018698:Glutamic Acid / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000469:parasitology; D008970:Molecular Weight; D009952:Ornithine / Q000378:metabolism*; D009953:Ornithine-Oxo-Acid Transaminase / Q000378:metabolism; D011392:Proline / Q000096:biosynthesis*; D011394:Proline Oxidase / Q000378:metabolism; D011762:Pyrroline Carboxylate Reductases / Q000378:metabolism; D012756:Sheep; D013379:Substrate Specificity; D000097502:delta-1-Pyrroline-5-Carboxylate Reductase
D050842:1-Pyrroline-5-Carboxylate Dehydrogenase; D000227:Adenine Nucleotides; D000447:Aldehydes; D000596:Amino Acids; D002412:Cations; D018698:Glutamic Acid; D009952:Ornithine; D009953:Ornithine-Oxo-Acid Transaminase; D011392:Proline; D011394:Proline Oxidase; D011762:Pyrroline Carboxylate Reductases; D000097502:delta-1-Pyrroline-5-Carboxylate Reductase