Abstract
Membrane-associated phosphatidylserine synthase (CDP-diacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) was purified from the microsomal fraction of Saccharomyces cerevisiae strains S288C and VAL2C(YEpCHO1). VAL2C(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 X-100 extraction of the microsomal membranes, CDP-diacylglycerol-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 sulfate-polyacrylamide gel electrophoresis. Phosphatidylserine synthase was dependent on manganese and Triton X-100 for maximum activity at pH 8.0. The apparent Km values for serine and CDP-diacylglycerol were 0.58 mM and 60 microM, respectively. Thioreactive agents inhibited enzyme activity. The enzyme was thermally labile above 40 degrees C. Results of isotopic exchange reactions between substrates and products suggest that the enzyme catalyzes a sequential Bi Bi reaction.
Highlights
From the Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers, The State University, New Brunswick, New Jersey 08903
These obstacles have been overcome by the developments of a solubilization procedure to release phosphatidylserine synthase fromyeast membranes (5,6, 16), the application of CDP-diacylglycerol-Sepharose affinity chroabove 40 “C. Results of isotopic exchange reactions matography (17,18)for the partialpurification of the enzyme between substrates and products suggest that the en- (7), and thceloning of the CHOl gene on the YEpl3plasmid zyme catalyzes a sequential Bi Bi reaction
We report the purification of phosphatidylserine synthase from S. cereuisiae and our initial studies
Summary
CDP-diacylglycerolderived from egg lecithin was prepared by the All steps were carried out at 5 "C. Microphatidylserine was prepared from CDP-diacylglycerol and L - [ ~ - ~ Hs]omes were collected by differential centrifugation as described by serine with purified phosphatidylserine synthase under standard as- Fischl and Carman (17). CDP-diacylglycerol-SepharoseChromatography-A CDPdiacylglycerol-Sepharose column (0.9 X 4 cm) was equilibrated with 50 mlof chromatography buffer (50 mM Tris-HC1 (pH 8.0), 30 mM tions of Fischl and Carman (17). Yeast Strains andGrowth Conditions affinity column in 1.5-ml aliquots. Strain VALZC(YEpCHOl), which overproduces phosphatidylserine synthase activity (7), wasgrown in synthetic complete medium (1) without leucine. Phosphatidylserine synthase was eluted from the column with this buffer at a flow rate of 1 ml/min. Fractions containing activity werepooled and desalted on a Sephadex G-25 column equilibrated with 20mM Tris-HC1 buffer (pH 8.0) containing
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