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Protein Expr Purif. 2010 Apr;70(2):211-7. doi: 10.1016/j.pep.2009.10.013. Epub 2009 Oct 31.

High-level expression and purification of rat monoamine oxidase A (MAO A) in Pichia pastoris: comparison with human MAO A.

Author information

1
Department of Biochemistry, Emory University, Rollins Research Center, 1510 Clifton Rd., Atlanta, GA 30322, USA.

Abstract

The high-level heterologous expression in Pichia pastoris, purification and characterization of recombinant membrane-bound rat liver monoamine oxidase A (MAO A) are described. A 1-L culture of cells produces approximately 700 U of rat MAO A activity. The rat MAO A activity is found in outer mitochondrial membrane of the cell. Using a modification of the human MAO A purification procedure, approximately 200mg of recombinant rat MAO A is purified in a 43% yield and exhibits a molecular weight of approximately 60,000 kDa on SDS-PAGE. The purified enzyme contains a covalently bound FAD and forms a N(5) flavocyanine adduct on inhibition by clorgyline. Edman sequencing shows that the amino terminus of rat MAO A is blocked at an N-terminal threonyl residue. The purified rat enzyme exhibits a higher thermal stability than does purified human MAO A. Compared with human MAO A, rat MAO A oxidizes serotonin or kynuramine with twofold higher k(cat)/K(m) values, oxidizes phenethylamine with a 6.7-fold higher catalytic efficiency and benzylamine with a approximately 40-fold higher catalytic efficiency. Although approximately 90% identical in sequence to human MAO A, rat MAO A is a more efficient catalyst for amine neurotransmitter oxidation.

PMID:
19883764
PMCID:
PMC2827670
DOI:
10.1016/j.pep.2009.10.013
[Indexed for MEDLINE]
Free PMC Article

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