Isolation and characterization of recombinant mitochondrial 4-aminobutyrate aminotransferase.
4-Aminobutyrate aminotransferase, which catalyzes the conversion of 4-aminobutyrate to succinic semialdehyde, is a key enzyme of the 4-aminobutyrate shunt. The amino acid sequence predicted from the cDNA sequence shows that the precursor protein consists of the mature enzyme of 473 amino acid residues and an amino-terminal segment of 27 amino acids (Kwon, O. S., Park, J., and Churchich, J. E. (1992) J. Biol. Chem. 267, 7215-7216). A recombinant 4-aminobutyrate aminotransferase has been expressed in Escherichia coli using pETIId as expression vector. The protein has been purified and characterized as a dimer (2 x 55 kDa). NH2-terminal sequence analysis has revealed the presence of an extra amino-terminal segment (signal peptide) predicted from the cDNA sequence. The isolated precursor of 4-aminobutyrate aminotransferase contains pyridoxal 5-phosphate and exhibits catalytic activity (18 units/mg) comparable to that of the mature enzyme (20 units/mg). The presequence peptide of the precursor of mitochondrial 4-aminobutyrate aminotransferase does not interfere with the folding and functional properties of the mature moiety of the aminotransferase.
Department of Biochemistry, University of Tennessee, Knoxville 37996.
J
J
Park
Y D
YD
Osei
J E
JE
Churchich
D000612:4-Aminobutyrate Transaminase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017886:Durapatite; D004926:Escherichia coli / Q000235:genetics; D006882:Hydroxyapatites; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D015183:Restriction Mapping; D013552:Swine
D006882:Hydroxyapatites; D011994:Recombinant Proteins; D017886:Durapatite; D000612:4-Aminobutyrate Transaminase