The multiple active enzyme species of gamma-aminobutyric acid aminotransferase are not isozymes.
Purified gamma-aminobutyric acid aminotransferase (GABA-AT) from pig brain under certain conditions gave a single band on 12% NaDodSO(4)-PAGE, whereas two or three distinct bands were observed on 7.5% native PAGE. These multiple active species were isolated by 5% preparative gel electrophoresis and characterized by N-terminal sequencing and MALDI-TOF mass spectrometry. The results indicate that these active enzyme species are not GABA-AT isozymes in pig brain, but are the products of proteolysis of the N-terminus of GABA-AT, differing by 3, 7, and 12 residues from the full sequence (as deduced from the cDNA), respectively. Conditions for obtaining the nontruncated GABA-AT were found, and the potential cause for the proteolysis was determined. It was found that Na(2)EDTA inhibits the N-terminal cleavage during GABA-AT preparation from pig brain. The presence of Triton X-100 in the homogenization step is partially responsible for this proteolysis, and Mn(2+) strongly enhances the protease activity, suggesting the presence of a membrane-bound matrix metalloprotease that causes the N-terminal cleavage.
Koo
Y K
YK
Department of Chemistry, Northwestern University, Evanston, Illinois 60208-3113, USA.
Nandi
D
D
Silverman
R B
RB
D000612:4-Aminobutyrate Transaminase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013552:Swine
D007527:Isoenzymes; D010446:Peptide Fragments; D017886:Durapatite; D000612:4-Aminobutyrate Transaminase