Extraction and stabilization of mammalian CDP-diacylglycerol synthase activity.
CDP-diacylglycerol synthase, also known as CTP: phosphatidic acid cytidylyltransferase (EC 2.7.7.41), is thought to be the rate-limiting enzyme in the synthesis of the inositol phospholipids, phosphatidylglycerol and cardiolipin. Its role in inositol phospholipid synthesis suggests its potential as a regulator of signal transduction as well. Although the mammalian cDNA for the synthase has recently been cloned, attempts to purify this enzyme from a mammalian source have been unsuccessful due to its lability in detergents. We report here the extraction and stabilization of CDP-diacylglycerol synthase from rat liver. Using a buffer containing 2M KCL, we were able to extract virtually all of the activity from microsomal membranes. This extract was stable indefinitely at -72 degrees C and for at least 24 hrs at 4 degrees C. Incubation at room temperature for 24 hours resulted in the loss of mor than half the activity. All detergents tested destroyed the activity. The activity was dependent on both substrates (phosphatidic acid and CTP) as well as on MgCl2, and inhibited by the product, CDP-diacylglycerol. Addition of GTP enhanced the activity approximately 2 fold, and bovine serum albumin increased activity by 6 fold.
Monaco
M E
ME
Department of Veterans' Affairs Medical Center, New York, New York, USA. monacom@is2.nyu.edu
Feldman
M
M
D000818:Animals; D002417:Cattle; D002798:Diacylglycerol Cholinephosphotransferase / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D006160:Guanosine Triphosphate / Q000494:pharmacology; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats; D012995:Solubility
D006160:Guanosine Triphosphate; D002798:Diacylglycerol Cholinephosphotransferase