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

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

The enzyme acyl-CoA:1-acyl-sn-glycero-3-phosphoinositol acyltransferase (LPI acyltransferase, EC 2.3.1.23) was purified approximately 11,000-fold to near homogeneity from bovine heart muscle microsomes. The purification was effected by extraction with the detergent 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate, followed by chromatography on Cibacron blue agarose, DEAE-cellulose, and Matrex gel green A. The isolated enzyme was a single protein of 58,000 Da as measured by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. This purification procedure also allows isolation of the related enzyme lysophosphatidylcholine (LPC) acyltransferase, which was separated from LPI acyltransferase at the final chromatographic step. The purified LPI acyltransferase exhibits an absolute specificity for LPI as the acyl acceptor. Broader specificity was found for acyl-CoA derivatives as substrates, although the preferred substrates are long-chain, unsaturated derivatives: measured reactivities were in the order arachidonoyl-CoA greater than oleoyl-CoA greater than eicosadienoyl-CoA greater than linoleoyl-CoA. Little activity was found with palmitoyl-CoA or stearoyl-CoA as potential substrates. These properties are consistent with a role of the enzyme in controlling the acyl group composition of phosphoinositides. Comparison of LPC acyltransferase and LPI acyltransferase shows that these two enzymes have distinct kinetic and physical properties and are affected differently by local anesthetics, which are potent inhibitors.

Topics: 1-Acylglycerophosphocholine O-Acyltransferase; Acyl Coenzyme A; Acyltransferases; Anesthetics, Local; Animals; Cattle; Cholic Acids; Chromatography; Electrophoresis, Polyacrylamide Gel; Kinetics; Lysophospholipids; Microsomes; Molecular Weight; Myocardium; Substrate Specificity