Purification of human platelet monoamine oxidase b by high performance liquid chromatography

G. A.S. Ansari, Nutan T. Patel, Richard R. Fritz, Creed W. Abell

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

Human platelet monoamine oxidase (MAO B), a membrane bound enzyme was purified to homogeneity by DEAE-Sephacel column chrom-atography, chromatofocusing, and high performance liquid chromatography (HPLC). The crucial purification step was HPLC on a anion exchange column (SynChropak AX 300). The HPLC column was eluted initially with potassium phosphate buffer (100 mM, pH 7.4) for 10 min at a flow rate of 1.0 ml/min, followed by a gradient (0-1%) of octyl-B-D-glucopyranoside (octylglucoside) in the same buffer for 10 min, and finally with buffered octylglucoside (1%) for 40 min. The elution of pargyline-bound or active MAO was established by determining either radioactivity in each fraction when MAO B had previously been covalently-labeled with [3H]-pargyline [3H(G)] or catalytic activity using [14C-methylene]-benzylamine as substrate. [3H]-pargyline-bound and active MAO B eluted from the column at approximately 34 min. The extent of homogeneity and the subunit M (approximately 59,000) of MAO B were determined by sodium-dodecylr sulfate polyacrylamide gel electrophoresis followed by silver staining for proteins.

Original languageEnglish (US)
Pages (from-to)1407-1419
Number of pages13
JournalJournal of Liquid Chromatography
Volume6
Issue number8
DOIs
StatePublished - Jun 1 1983

ASJC Scopus subject areas

  • Molecular Medicine

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