Purification and characterization of the active serine: pyruvate aminotransferase of rat liver mitochondria expressed in Escherichia coli.
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-terminus. The catalytic and physical properties of SPT10, such as substrate specificity, Km for alpha-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.
Oda
T
T
Department of Biochemistry, Hamamatsu University School of Medicine, Shizuoka.
Miyajima
H
H
Suzuki
Y
Y
Ito
T
T
Yokota
S
S
Hoshino
M
M
Ichiyama
A
A
D000595:Amino Acid Sequence; D000818:Animals; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D008099:Liver / Q000648:ultrastructure; D008930:Mitochondria, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010957:Plasmids; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D000637:Transaminases / Q000302:isolation & purification* / Q000378:metabolism
D011994:Recombinant Proteins; D000637:Transaminases; C022638:serine-pyruvate aminotransferase