The multiple active enzyme species of γ-aminobutyric acid aminotransferase are not isozymes

Yumee Kim Koo, Dhirendra Nandi, Richard B. Silverman*

*Corresponding author for this work

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

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. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)248-254
Number of pages7
JournalArchives of biochemistry and biophysics
Volume374
Issue number2
DOIs
StatePublished - Feb 15 2000

Keywords

  • Enzyme purification
  • Isozymes
  • Multiple active forms
  • N-terminal cleavage
  • Proteolytic fragments
  • γ-aminobutyric acid aminotransferase

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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