The Multiple Active Enzyme Species of γ-Aminobutyric Acid Aminotransferase Are Not Isozymes

• Published in: Archives of biochemistry and biophysics Vol. 374; no. 2; pp. 248 - 254
• Main Authors: Koo, Yumee Kim, Nandi, Dhirendra, Silverman, Richard B.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.02.2000
• Subjects: 4-Aminobutyrate Transaminase - chemistry; 4-Aminobutyrate Transaminase - isolation & purification; 4-Aminobutyrate Transaminase - metabolism; Amino Acid Sequence; Animals; Brain - enzymology; Chromatography; Chromatography, Gel; Chromatography, Ion Exchange; Durapatite; Electrophoresis, Polyacrylamide Gel; enzyme purification; Isoenzymes - chemistry; isozymes; Molecular Sequence Data; Molecular Weight; multiple active forms; N-terminal cleavage; Peptide Fragments - chemistry; proteolytic fragments; Sequence Alignment; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Swine; γ-aminobutyric acid aminotransferase; γ-aminobutyric acid aminotransferase; enzyme purification; N-terminal cleavage; multiple active forms; isozymes; proteolytic fragments
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1999.1623

Purified γ-aminobutyric acid aminotransferase (GABA-AT) from pig brain under certain conditions gave a single band on 12% NaDodSO4–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 Na2EDTA 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 Mn2+ strongly enhances the protease activity, suggesting the presence of a membrane-bound matrix metalloprotease that causes the N-terminal cleavage.