Studies on the formation by rat brain preparations of CDP-diglyceride from CTP and phosphatidic acids of varying fatty acid compositions

Abstract

The enzyme, CTP:phosphatidate cytidylyltransferase (EC2.7.7.41) which catalyses formation of CDP-diglyceride from CTP and phosphatidic acid has been studied in rat brain preparations and other tissues. Improvement, as judged by the higher tissue activities obtained, in the assay method for this enzyme was achieved through use of phosphatidic acids sonicated in buffer-detergent solution saturated with ether and containing bovine serum albumin and use of short incubation times which essentially provided a measure of initial rates. The enzyme of rat brain microsomes yielded with 1,2-dioleolphosphatidic acid as substrate a pH optimum of 6.8 with maleate buffer and optimal concentrations of 60mM for MG2+, 6MM for CTP and 250 mug per 0.8 ml for phosphatidic acid. Enzyme activity was mainly located in the 90,000 X g fraction (microsomal) with small but significant activity in the 12,000 X g fraction. Comparison of activities (nanomoles CTP incorporated per milligram protein per minute) amongst tissues showed the following order: brain, 1.87; liver, 1.32; lung, 1.19; small intestine, 1.00; kidney, 0.69; heart, 0.41; diaphragm, 0.07; skeletal muscle, 0.02. Examination of the effect of varying the fatty acid composition in the phosphatidic acids added exogenously gave the following order (activities in parentheses); 1-stearoyl-2-oleoyl- (5.58), 1-oleoyl-2-stearoyl- (5.37), 1,2-dioleoyl- (4.49) 1-palmitoyl-2-oleoyl-(3.85), 1-stearoyl-2-arachidonoyl-(3.31), 1-arachidonoyl-2-stearoyl-(3.16), 1,2-diarachidonoyl-(0.72), 1,2-dicaproyl-(0.67), 1,2-dipalmitoyl-(0.67) and 1,2-distearoyl-(0.18). The single bis- and lysophosphatidic acids tested were inactive as substrates. Apart from a possible preference for one or more unsaturated fatty acids the transferase enzyme showed no selectivity in respect to the fatty acid distribution of phosphatidic acids.

CDP‐diacylglycerol synthesis in rat liver mitochondria

“…The CDP-diacylglycerol synthase of mammalian cells and prokaryotes has been extensively studied; CDPdiacylglycerol synthase has been detected in different animal tissues [4], and it was shown to be a key enzyme for the synthesis of membrane phospholipids in Escherichta coR [20]. Microsomes are definitely the major site for the synthesis of CDP-diacylglycerol in eukaryotes where the a,cailability of CDP-diacylglycerol may control the biosynthesis of phosphatidylinositol.…”

“…The assay condition was a modification of Bishop and Strickland's method 1"4]. The reaction mixture in a final volume of 0.3 ml contained: 0.23 mM phosphatidi¢ acid (prepared according to Bishop and $triekland [4]), 50 mM maleate buffer pH 7.0, 30 mM MgC!,, !6 .uS Atlas G-3634A, 0.5 mM DTT, 2.5 mM 5-[~H]CTP (2/zCi) and 0.4-0.6 mg protein.…”

“…This enzyme activity has been found in both bacteria and eukaryotes [2][3][4]. CDP-diacylglycerol is an obligatory intermediate compound in the biosynthesis of the anionic phospholipids such as cardiolipin, phosphatidylglycerol and phosphatidylinositol [5].…”

CDP-diacylglycerol phospholipid synthesis in detergent-soluble, non-raft, membrane microdomains of the endoplasmic reticulum

“…6 , detail 1). Most strikingly, solubilization is a characteristic property of membrane rafts; hence, we investigated the gradient distribution of CDS activity in membranes prepared in the presence of ␤ -octylglucoside (10 mM) and deoxycholate (4 mM), a detergent mix which, unlike Triton X-100, does not result in signifi cant solubilization of acidic phospholipids ( 52 ) or inhibition of CDS activity ( 53 ). In these gradients, a significant proportion of the cellular caveolin content was detected in the low buoyant density (1.06-1.1 g/ml sucrose) Fig.…”

Isolation and Expression of an Isoform of Human CDP-Diacylglycerol Synthase cDNA

“…Specifically, PIP2 is the substrate for phospholipase C that is activated in response to a wide variety of extracellular stimuli, leading to the generation of two lipid second messengers, namely, DAG for the activation of protein kinase C and IP3 for the release of Ca2+ from internal stores (Kent, 1995). malian liver and brain tissues has been found in membrane fractions from endoplasmic reticulum and mitochondria (Mok et al, 1992; Lin and Strickland, 1991;Bishop and Strickland, 1976). Characterization of the partially purified CDS enzyme from these membrane fractions suggests that there may be at least two forms of mammalian CDS, differing in response to GTP in separation on anion-exchange columns and in response to PA levels (Mok et al, 1992).…”