Purification and characterization of rat kidney L-alanine: 4,5-dioxovalerate transaminase and inhibition by hemin.
Rat kidney L-alanine:4,5-dioxovalerate transaminase (EC 2.6.1.43), which may be involved in the formation of aminolevulinic acid in mammalian cells, was purified 82-fold to apparent homogeneity with a 19% yield. Molecular weight of the enzyme, as estimated by gel filtration, was found to be 225 000. In polyacrylamide gel electrophoresis under denaturing conditions, the enzyme moved as a single band corresponding to an Mr of 37 000, suggesting that the enzyme is composed of six identical subunits. The Km values of L-alanine and 4,5-dioxovalerate are 2.9 and 0.25 mM, respectively. The enzyme had an optimum activity at pH 6.6 and was most active at 65 degrees C. Among some amino acids tested, L-alanine proved to be the most efficient amino donor, and the enzyme was also stereospecific for the L-isomer. The effect of intermediate metabolites of heme biosynthesis, for example, delta-aminolevulinic acid, protoporphyrin, hemin and bilirubin has been studied on purified L-alanine:4,5-dioxovalerate transaminase. Amongst these metabolites, hemin and protoporphyrin were found to be effective inhibitors.
D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000031:analogs & derivatives*; D006427:Hemin / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D051381:Rats; D013696:Temperature; D000637:Transaminases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification
D006418:Heme; D006427:Hemin; D000637:Transaminases; C019687:aminolevulinate aminotransferase