Partial purification of trimethylamine-N-oxide (TMAO) demethylase from crude fish muscle microsomes by detergents.
Detergent treatments were examined for their efficacy in purifying trimethylamine-N-oxide (TMAO) demethylase activity from fish muscle microsomes. Tritons X-100 and X-45, deoxycholate, Brijs, Tweens 20, 65, and 80, and SDS were generally ineffective in solubilizing demethylase activity from this membrane fraction, at concentrations up to 10 mg detergent per mg protein. In all of these cases, specific activity became enriched in the particulate fraction obtained post-treatment. Highest fold-purification was achieved by using 10 mg SDS per mg protein in 5 mM histidine, pH 7.0 at 10-14 degrees C. Activity was relatively stable to the presence of SDS at this level, and with this treatment, TMAO demethylase activity became purified in the resultant particulate fraction 28- and 58-fold for activity stimulated by ascorbate-iron-cysteine and FMN-NADH, respectively. The presence of urea or 2-mercaptoethanol, or sonication of the SDS-microsome suspension during purification resulted in significant losses of recovered activity. This partially purified fraction represented about 1% of the original microsomal protein and SDS-PAGE revealed the presence of several protein components. The partially purified demethylase could utilize the same two cofactor systems as the native microsomes. It displayed a curvilinear dependence on iron for activity and a sigmoidal response for cysteine. Utilization of NADH, FMN, and ascorbate differed for the purified fraction as compared to the microsomes. Substrate inhibition by TMAO was observed for the partially purified preparation, whereas saturation kinetics were previously noted for microsomal activity.(ABSTRACT TRUNCATED AT 250 WORDS)
D000446:Aldehyde-Lyases / Q000302:isolation & purification* / Q000378:metabolism; D000693:Anaerobiosis; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D003902:Detergents; D005399:Fishes; D005486:Flavin Mononucleotide / Q000494:pharmacology; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D009243:NAD / Q000494:pharmacology; D010084:Oxidation-Reduction
D003902:Detergents; D009243:NAD; D005486:Flavin Mononucleotide; D000446:Aldehyde-Lyases; C023188:trimethylamine-oxide aldolase; D001205:Ascorbic Acid