Purification and properties of an ethanolamine-serine base exchange enzyme of rat brain microsomes.
The activity of an ethanolamine and serine base exchange enzyme of rat brain microsomes was copurified to near homogeneity. The purification sequence involved detergent solubilization, Sepharose 4B column chromatography, phenyl-Sepharose 4B column chromatography, glycerol gradient sedimentation, and agarose-polyacrylamide gel electrophoresis under non-denaturing conditions. The ratio of the ethanolamine and serine base exchange activities remained almost constant during purification, and both enzyme activities were enriched 25-fold over the initial microsomal suspension. The final enzyme preparation which contained both enzyme activities showed a single protein band on sodium dodecyl sulfate-polyacrylamide gel, having an apparent molecular mass of about 100 kDa. Serine inhibited the ethanolamine incorporation by this preparation and ethanolamine inhibited the serine incorporation. The competitive nature of this inhibition was apparent from Lineweaver-Burk plots, suggesting that the enzyme catalyzes the incorporation of both ethanolamine and serine into their corresponding phospholipids. The Km and Ki values for ethanolamine were quite similar, being 0.02 and 0.025 mM, respectively. The Km and Ki values for serine were also quite similar being 0.11 and 0.12 mM, respectively. The pH optimum was the same at 7.0 with both substrates. The optimum Ca2+ concentration was 8 mM for serine incorporation.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D019856:Ethanolamine; D004983:Ethanolamines / Q000378:metabolism* / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology*; D019884:Nitrogenous Group Transferases; D051381:Rats; D012694:Serine / Q000378:metabolism* / Q000494:pharmacology; D014166:Transferases / Q000302:isolation & purification / Q000378:metabolism*
D004983:Ethanolamines; D012694:Serine; D019856:Ethanolamine; D014166:Transferases; D019884:Nitrogenous Group Transferases; C022370:phospholipid serine base exchange enzyme