Purification and Characterization of the Active SerineiPyruvate Aminotransferase of Rat Liver Mitochondria Expressed in Escherichia coli1

Abstract

In the previous study (Oda, T., et al. (1985) Eur. J. Biochem. 150, 415–421), we isolated a cDNA clone which expressed in Escherichia coli a specific size of product having the activity of rat liver serine: pyruvate aminotransferase (SPTm). This specific product (SPT10) was purified to homogeneity through three different column chromatographies. The amino acid composition and N-terminal amino acid sequence of the purified enzyme agreed with those predicted from the nucleotide sequence of cDNA and showed that SPT10 consists of the whole amino acid sequence of mature SPTm and several extra amino acid residues at the N-termlnus. The catalytic and physical properties of SPT10, such as substrate specificity, K_m for α-keto acids, electric charge, and quaternary structure, were all very similar to those of SPTm. Using several cDNA clones which lack a 5′-terminal sequence corresponding to a portion of the N-terminal amino acid sequence of SPTm, we examined the expression profile of the specific product in bacteria transformed with each cDNA clone. The products encoded by these cDNAs were segregated into inclusion bodies and were neither catalytically active nor easily solubilized by sonication. In contrast, the inclusion bodies were not formed in the bacteria transformed with the cDNA clone for SPT10.