Phosphatidylserine Synthesis in Saccharomyces cerevisiae
نویسنده
چکیده
Membrane-associated phosphatidylserine synthase (CDP-diacylglycero1:L-serine O-phosphatidyltransferase, EC 2.7.8.8) was purified from the microsomal fraction of Saccharomyces cerevisiae strains S288C and VALBC(YEpCHO1). VALBC(YEpCHO1) contains a hybrid plasmid bearing the structural gene for phosphatidylserine synthase and overproduces the enzyme 6-7-fold (Letts, V. A., Klig, L. S., Bae-Lee, M., Carman, G. M., and Henry, s. A. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 7279-7283) compared to wild-type S288C. The purification procedure included Triton X100 extraction of the microsomal membranes, CDPdiacylglycerol-Sepharose affinity chromatography, and DE-53 chromatography. The procedure yielded a preparation from each strain containing a major peptide band (Mr = 23,000) upon sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Phosphatidylserine synthase was dependent on manganese and Triton X-100 for maximum activity at pH 8.0. The apparent K,values for serine and CDP-diacylglycerol were 0.58 mM and 60 PM, respectively. Thioreactive agents inhibited enzyme activity. The enzyme was thermally labile above 40 “C. Results of isotopic exchange reactions between substrates and products suggest that the enzyme catalyzes a sequential Bi Bi reaction.
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