Biochimica et Biophysica Acta 827 (1985) 305-309 305 Elsevier BBA32135 Purification and characterization of rat kidney L-alanine: 4,5-dioxovalerate transaminase and inhibition by heroin Nishi K. Singh and K. Datta School of Environmental Sciences, Jawaharlal Nehru University, New Delhi - 110067 (India) (Received June 14th, 1984) (Revised manuscript received November 20th, 1984) Key words: 4,5-Dioxovaleric acid; L-Alanine: 4,5-dioxovaleric transaminase; Hemin inhibition; (Rat kidney) Rat kidney L-alanine:4,5-dioxovalerate transaminase (EC 2.6.1.43), which may be involved in the formation of anfinolevulinic acid in mammalian ceils, 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 225000. 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 K= values of I.-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°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 heine biosynthesis, for example, ~-aminolevulinic acid, protoporphyrin, hemin and bilirubin has been studied on purified L-alanine:4,5-dioxovalerate transaminase. Amongst these metabolites, bemin and protoporphyrin were found to be effective inhibitors. Introduction In a number of recent reports [1-5] it has been suggested that in the mammalian system the en- zyme L-alanine : 4,5-dioxovalerate transaminase (EC 2.6.1.43) in addition to 8-aminolevulinate syn- thetase (EC 2.3.1.37) provides another route for aminolevulinic acid formation through a trans- amination reaction between L-alanine and 4,5-di- oxovalerate. This enzyme has been purified from bovine liver [3,4] and rat liver mitochondria [5]. In addition, the enzymatic biosynthesis of 4,5-di- oxovalerate by 4-oxo-5-hydroxyvalerate dehydro- genase has also been established recently in rat liver and kidney by Okuno et al. [6], suggests the importance of transaminase route to aminolevu- linic acid synthesis. Furthermore, incorporation of radioactive 4,5-dioxovaleric acid in porphyrin and hemin by intact respiring rat hepatocytes [2] indi- cates that L-alanine:4,5-dioxovalerate trans- aminase may play a role in heme biosynthesis. Prelinfinary study of this enzyme in our labora- tory [7] shows that L-alanine:4,5-dioxovalerate transarnlnase is present not only in liver, but also in other tissues particularly in kidney with high specific activity which has high content of hemeproteins, for example, cytochrome/'45o [8]. It is also well established that the primary site for the regulation of overall activity of heme biosynthesis in animals is at the level of aminolevnlinlc acid formation in mitochondria [9,10]. Though the reg- ulatory role of aminolevnlinic acid synthetase on heine biosynthesis has been studied extensively [11-13], nothing is known about the nature of L-alanine: 4,5-dioxovalerate transaminase. There- fore, as a part of our programme to study the role of L-alanine:4,5-dioxovalerate transaminase on heme biosynthesis, we have purified and char- 0167-4838/85/$03.30 © 1985 Elsevier Science Publishers B.V.