Use of hydrophobic chromatography for purification of the membrane-located choline dehydrogenase from a Pseudomonas strain.
Choline dehydrogenase has been purified using hydrophobic chromatography 250-fold from a Pseudomonas strain. Although the enzyme is associated with the cell membrane and could be extracted from membrane preparations, it was best purified from a complete cell extract made with a non-ionic detergent. Only phenazine methosulfate was able to act as electron acceptor; there was no evidence of bound flavin, but there was evidence of pyrroloquinoline quinone cofactor. The purified enzyme had a specific activity of up to 67 units/mg, which is at least ten times higher than the values reported for mitochondrial choline dehydrogenases, and up to 100 times higher than previous reports for the Pseudomonas enzyme. The estimated subunit size of 66 kDa, which corresponds with the native size, is close to that deduced from the gene sequence of the Escherichia coli betA gene, and preliminary N-terminal sequencing shows homology with this deduced sequence. The next enzyme in the degradation pathway of choline, betaine aldehyde dehydrogenase, was also purified from the same extract.
Russell
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Centre for Protein and Enzyme Technology, La Trobe University, Bundoora, V3083, Australia.
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D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology; D002794:Choline / Q000032:analysis; D050545:Choline Dehydrogenase; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003902:Detergents; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D046911:Macromolecular Substances; D008773:Methylphenazonium Methosulfate; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011549:Pseudomonas / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
D003902:Detergents; D046911:Macromolecular Substances; D008773:Methylphenazonium Methosulfate; D000429:Alcohol Oxidoreductases; D050545:Choline Dehydrogenase; D002794:Choline