oleoyl-coenzyme-a and 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate

Developing sunflower seeds exhibit a high diacylglycerol acyltransferase (DAGAT, EC 2.3.1.20) activity. The distribution of the enzyme has been studied in subcellular fractions prepared by differential centrifugation of seed homogenate. Its activity was characterized using [1-(14)C]oleoyl-CoA and diolein dispersed in Tween 20. Some properties of the microsomal fraction of DAGAT were investigated. Hyperbolic kinetics were observed, the apparent K(m) was 60 microM and the specific activity of the reaction 15 pmol/min/mg of protein. Addition of BSA (0.1%) stimulated oleate incorporation, which was not dependent on the presence of exogenous diacylglycerol. Detergents which might solubilize DAGAT, Triton X-100 and CHAPS, were tested for enzyme inhibition, and CHAPS was found to be the least denaturing.

Topics: Acyl Coenzyme A; Acyltransferases; Carbon Radioisotopes; Cholic Acids; Detergents; Diacylglycerol O-Acyltransferase; Diglycerides; Helianthus; Kinetics; Octoxynol; Protein Denaturation; Seeds

The lysophosphatidylcholine:acyl-CoA acyltransferase (LAT, EC 2.3.1.23) is an integral membrane protein participating in the membrane turnover and the T-cell activation process. Here, we present data that crude membranes of pig spleen contain two different LAT enzyme activities based on topological localization studies and the enzyme specificities towards various acyl-CoAs. When crude membranes are washed with solutions of high ionic strength the supernatant contains a distinct LAT activity that we refer to as peripheral LAT (pLAT). The majority of LAT activity is found in the membrane pellet also after treatment with CHAPS. The CHAPS-insoluble LAT activity is named integral LAT (iLAT) accordingly. While pLAT prefers arachidonoyl-CoA rather than oleoyl-CoA, iLAT shows no specificity towards both unsaturated acyl-CoAs. Further investigations reveal that the CHAPS-insoluble LAT activity in the membranes can be solubilized by n-octyl glucoside and restored to original activity by reconstitution with artificial membranes. The reconstituted iLAT prefers arachidonoyl-CoA rather than oleoyl-CoA. Despite a great deal of effort by several groups little progress has been made so far in LAT purification because of the enzyme instability. We establish experimental conditions that enhance the stability of both enzyme activities and, therefore, allow further protein purification.

Topics: 1-Acylglycerophosphocholine O-Acyltransferase; Acyl Coenzyme A; Animals; Cell Membrane; Cholic Acids; Liposomes; Osmolar Concentration; Solubility; Spleen; Substrate Specificity; Swine

Heart muscle microsomes catalyze the transacylation of lysophosphatidylcholine (lyso PC) to produce phosphatidylcholine (PC). The enzyme which catalyzes this reaction, lyso PC:lyso PC transacylase, has been isolated and characterized from bovine heart muscle microsomes. The purification of the enzyme was achieved by a procedure involving extraction with 3-[3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS) detergent and chromatography on DEAE-cellulose, Reactive blue agarose, and Matrex gel green A. The purified enzyme was nearly homogeneous and consisted of a single molecular species of 128 kDa as determined by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. The catalytic activity of the enzyme was dependent on the presence of either CoA or acyl-CoA, both of which maximally stimulated at concentrations of approx. 10 microM. Analysis of the PC produced in the reaction showed that the enzyme catalyzed a transacylation in which both acyl groups arose from lyso PC. Furthermore, the enzyme did not possess acyl-CoA:lyso PC acyltransferase activity, lysophospholipase or acyl-CoA hydrolase activity, nor did it catalyze transacylation from lyso PC to lysophosphatidylethanolamine, lysophosphatidylinositol or lysophosphatidylserine. Although transacylation was highly specific for lyso PC as the substrate, various unsaturated fatty acyl-CoA derivatives served as activators. Palmitoyl-CoA and stearoyl-CoA did not significantly activate, although acetyl-CoA was an effective activator. Further modulation of activity was produced by palmitic acid and PC, both of which further activated the enzyme in the presence of oleoyl-CoA, whereas arachidonic acid, oleic acid, phosphatidylethanolamine and phosphatidylserine had no effect on activity. The high activity of this transacylase and its regulation by lipids suggests an important role for disaturated PC species in membranes and a mechanism for controlling the metabolism of lyso PC.

Topics: Acyl Coenzyme A; Acyltransferases; Animals; Catalysis; Cattle; Cholic Acids; Chromatography, DEAE-Cellulose; Coenzyme A; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Lipids; Lysophosphatidylcholines; Lysophospholipase; Microsomes; Molecular Weight; Multienzyme Complexes; Myocardium; Palmitic Acid; Palmitic Acids; Phosphatidylcholines; Phospholipases