Assay for phosphatidylserine decarboxylase utilizing DEAE-cellulose column chromatography.
A simple assay for phosphatidylserine decarboxylase is described. Following incubation of a mitochondrial fraction from Saccharomyces cerevisiae with purified, exogenous phosphatidyl[3H]serine, the lipid extract is applied to a small DEAE-cellulose column equilibrated in CHCI3-CH3OH (1:1). The unreacted substrate, phosphatidyl[3H]serine, is quantitatively bound by the ion-exchange column while the product, phosphatidyl[3H]ethanolamine, is eluted by sequential washing with CHCI3-CH3OH (1:1) and CH3OH. The organic solvents are evaporated, and the amount of radiolabeled phosphatidyl[3H]ethanolamine formed by enzymatic decarboxylation is determined by liquid scintillation spectrometry. The reliability of this assay was established by showing that several enzymatic properties of the yeast enzyme, defined by the new assay, were essentially identical to the properties characterized by a more tedious paper chromatographic assay described previously. Virtually identical rates of enzymatic decarboxylation of phosphatidyl[3H]serine were also obtained for mitochondrial fractions from pig brain and rat liver when the activities were compared by the column and paper chromatographic methods.
Overmeyer
J H
JH
Department of Biochemistry, University of Kentucky College of Medicine, A.B. Chandler Medical Center, Lexington 40536.
Waechter
C J
CJ
D000818:Animals; D001921:Brain / Q000201:enzymology; D002262:Carboxy-Lyases / Q000032:analysis*; D002845:Chromatography / Q000379:methods; D002854:Chromatography, Paper; D003636:DEAE-Cellulose; D008928:Mitochondria / Q000201:enzymology; D008930:Mitochondria, Liver / Q000201:enzymology; D051381:Rats; D012441:Saccharomyces cerevisiae; D013552:Swine
D003636:DEAE-Cellulose; D002262:Carboxy-Lyases; C022353:phosphatidylserine decarboxylase