Biochemical characterization and regulation of cardiolipin synthase in Saccharomyces cerevisiae.
Cardiolipin (CL) synthase activity was characterized in mitochondrial extracts of the yeast Saccharomyces cerevisiae and was shown for the first time to utilize CDP-diacylglycerol as a substrate. CL synthase exhibited a pH optimum of 9.0. Maximal activity was obtained in the presence of 20 mM magnesium with a Triton X-100: phospholipid ratio of 1:1. The apparent Km values for phosphatidylglycerol and CDP-diacylglycerol were 1 mM and 36 microM, respectively. CL synthase activity was maximal at 45 degrees C and heat inactivation studies showed that the enzyme retained greater than 75% of its activity at temperatures up to 55 degrees C. To study the regulation of CL synthase, the enzyme was assayed in cells grown under conditions known to affect general phospholipid synthesis. Unlike many phospholipid biosynthetic enzymes including PGP synthase, which catalyzes the initial step in CL biosynthesis, CL synthase was not repressed in cells grown in the presence of the phospholipid precursor inositol. Detailed procedures for the enzymatic synthesis of 32P-labelled substrates are described.
Tamai
K T
KT
Department of Biological Chemistry, University of Michigan, Ann Arbor 48709-0606.
Greenberg
M L
ML
D003567:Cytidine Diphosphate Diglycerides / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007294:Inositol / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008565:Membrane Proteins; D008928:Mitochondria / Q000201:enzymology; D017830:Octoxynol; D010715:Phosphatidylglycerols / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism*; D011092:Polyethylene Glycols; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000648:ultrastructure; D013379:Substrate Specificity; D013696:Temperature; D017855:Transferases (Other Substituted Phosphate Groups)
D003567:Cytidine Diphosphate Diglycerides; D008565:Membrane Proteins; D010715:Phosphatidylglycerols; D011092:Polyethylene Glycols; D007294:Inositol; D017830:Octoxynol; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); C100091:cardiolipin synthetase; D008274:Magnesium