Abstract
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.
Highlights
Change enzyme of rat brain microsomeswas copurified to near homogeneity
Sephnrose CL-4BColumn Chromatography-The solubilized enzyme containing extracts was applied on a Sepharose CL-4B column (10 X 100 cm) which was pre-equilibrated with 5 mM HEPES buffer, pH 7.23, containing 20% glycerol, 1mM EDTA, and 1mM mercaptoethanol (GHME buffer), andthe column was eluted with the GHME buffer
The ethanolamine and serine base exchange enzyme activities were present in the solubilized preparation with negligible detectable choline base exchange activity
Summary
CL-Sepharose 4B, phenyl-Sepharose 4B, DEAE-Sephacel, and activated epoxy-Sepharose 4B were purchased from Pharmacia. Nonradioacband on sodium dodecyl sulfate-polyacrylamide gel, tive ethanolamine, serine, and choline were obtained from Sigma. The base exchange enzymes catalyze the incorporation of L-serine, ethanolamine, monomethylethanolamine, and choline into their corresponding phospholipid. These reactions strate, and enzyme preparation in a total volume of0.24 ml. Sephnrose CL-4BColumn Chromatography-The solubilized enzyme containing extracts was applied on a Sepharose CL-4B column (10 X 100 cm) which was pre-equilibrated with 5 mM HEPES buffer, pH 7.23, containing 20% glycerol, 1mM EDTA, and 1mM mercaptoethanol (GHME buffer), andthe column was eluted with the GHME buffer. 10-ml samples were collected, and the fractions from 61 to (Fig. 1)containing high base exchange enzyme activity were pooled. A 2-ml sample was gently layered onto a continuous glycerol gradient which wasprepared according to
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