Identification of two different lysophosphatidylcholine:acyl-CoA acyltransferases (LAT) in pig spleen with putative distinct topological localization.
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.
Kerkhoff
C
C
Institut für Molekularpharmakologie, Medizinische Hochschule Hannover, Germany.
Gehring
L
L
Habben
K
K
Resch
K
K
Kaever
V
V
D008243:1-Acylglycerophosphocholine O-Acyltransferase / Q000032:analysis*; D000214:Acyl Coenzyme A / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology*; D002793:Cholic Acids; D008081:Liposomes; D009994:Osmolar Concentration; D012995:Solubility; D013154:Spleen / Q000201:enzymology*; D013379:Substrate Specificity; D013552:Swine
D000214:Acyl Coenzyme A; D002793:Cholic Acids; D008081:Liposomes; C026563:arachidonyl-coenzyme A; C017585:oleoyl-coenzyme A; D008243:1-Acylglycerophosphocholine O-Acyltransferase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate