Phosphatidylserine synthesis in Saccharomyces cerevisiae. Purification and characterization of membrane-associated phosphatidylserine synthase

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 phospha...

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Bibliographic Details
Published in:The Journal of biological chemistry 1984-09, Vol.259 (17), p.10857-10862
Main Authors: Bae-Lee, M S, Carman, G M
Format: Article
Language:English
Subjects:
Analytical, structural and metabolic biochemistry
Biological and medical sciences
Cations, Divalent
CDPdiacylglycerol-Serine O-Phosphatidyltransferase - isolation & purification
CDPdiacylglycerol-Serine O-Phosphatidyltransferase - metabolism
Enzymes and enzyme inhibitors
Fundamental and applied biological sciences. Psychology
Intracellular Membranes - enzymology
Kinetics
Microsomes - enzymology
Phosphatidylserines - biosynthesis
Phosphotransferases - isolation & purification
Saccharomyces cerevisiae - enzymology
Species Specificity
Transferases
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1016/s0021-9258(18)90592-2