Action of the highly purified, membrane-bound enzyme phosphatidylserine decarboxylase Escherichia coli toward phosphatidylserine in mixed micelles and erythrocyte ghosts in the presence of surfactant.
Phosphatidylserine decarboxylase, Escheichia coli, was purified to near-homogeneity by the procedure of Dowhan, W., Wickner, W. T., and Kennedy, E. P. ((1974) J. Biol. Chem. 249, 3079-3084) and assayed by following the production of CO2 using gas chromatography. The purified enzyme has an absolute requirement for the surfactant Triton X-100. The function of Triton in the assay is evaluated and a kinetic scheme describing the action of this membrane-bound enzyme in the micellar system provided by the surfactant is presented. According to this scheme, the enzyme first binds to a mixed micelle, composed of phosphatidylserine and Triton, where the dissociation constant is KSA. The enzyme, now part of the mixed micelle, then binds the substrate phosphatidylserine in its active site and this binding is related to the Michaelis constant, KMB. KSA, expressed as the sum of the molar concentrations of Triton and phosphatidylserine, is about 0.04 M. KMB, expressed as the mole fraction of phosphatidylserine in the mixed micelles, is about 0.03. Phosphatidylserine decarboxylase activity toward phosphatidylserine in human erythrocyte ghosts was also determined. The amount of phsophatidylserine converted to phosphatidylethanolamine and CO2 was found to be related to the amount of phosphatidylserine solubilized from the membrane by Triton X-100. In the absence of Triton, no significant activity of the enzyme toward the ghosts was detected even after subjecting the ghosts to lyophilization, homogenization, or sonication.
D001665:Binding Sites; D002262:Carboxy-Lyases / Q000378:metabolism*; D002462:Cell Membrane / Q000032:analysis; D002850:Chromatography, Gel; D004912:Erythrocytes / Q000032:analysis*; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D008823:Micelles; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines / Q000097:blood; D011092:Polyethylene Glycols / Q000494:pharmacology; D011485:Protein Binding; D000644:Quaternary Ammonium Compounds
D008823:Micelles; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D000644:Quaternary Ammonium Compounds; D011092:Polyethylene Glycols; D002262:Carboxy-Lyases; C022353:phosphatidylserine decarboxylase