Affinity purification and properties of rat liver mitochondrial L-alanine:4,5-dioxovalerate transaminase and its inhibition by hemin.
The present study describes a new rapid procedure for purification of L-alanine:4,5-dioxovalerate transaminase from rat liver mitochondria which was purified 243-fold with a 32% yield to apparent homogeneity. The purification procedure involved protamine sulfate treatment, followed by phenyl-Sepharose CL-4B column chromatography and alanine-Sepharose 4B affinity chromatography. The Km values for L-alanine and 4,5-dioxovalerate were 3.3 and 0.28 mM, respectively. The enzyme-bound pyridoxal phosphate content was estimated to be two molecules per enzyme molecule. The purified enzyme was inhibited by the reaction product pyruvic acid, substrate analog, methylglyoxal, and sulfhydryl inhibitors. Excess concentrations of 4,5-dioxovalerate was also found to inhibit the enzyme and our experiments failed to demonstrate reversibility of the reaction. Only hemin among the intermediate compounds of heme metabolism tested was shown to be an inhibitor of purified alanine:4,5-dioxovalerate transaminase. Hemin was further shown as an uncompetitive inhibitor of both alanine and dioxovalerate.
D000818:Animals; D001663:Bilirubin / Q000494:pharmacology; D002846:Chromatography, Affinity; D006418:Heme / Q000031:analogs & derivatives*; D006427:Hemin / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D011162:Porphobilinogen / Q000494:pharmacology; D011524:Protoporphyrins / Q000494:pharmacology; D051381:Rats; D013057:Spectrum Analysis; D000637:Transaminases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*
D011524:Protoporphyrins; D006418:Heme; D006427:Hemin; D011162:Porphobilinogen; D000637:Transaminases; C019687:aminolevulinate aminotransferase; D001663:Bilirubin