Solubilization, partial purification and photolabeling of the integral membrane protein lysophospholipid:acyl-CoA acyltransferase (LAT).

In the present study, we defined experimental conditions that allowed the extraction of the integral membrane protein lysophospholipid:acyl-CoA acyltransferase (LAT, EC 2.3.1.23) from membranes while maintaining the full enzyme activity using the nonionic detergent n-octyl glucopyranoside (OGP) and solutions of high ionic strength. We found that the optimal OGP concentration depended on the ionic strength of the solubilization buffer. Fluorescence measurements with 1,6-diphenyl-1,3,5-hexatriene indicated that the critical micellar concentration (CMC) of OGP decreased with increasing salt concentrations. Analogous studies revealed that the zwitterionic detergent Chaps was ineffective in extracting LAT from membranes in the absence of salt, whereas its solubilization efficiency increased with increasing salt concentrations. Detailed lipid analysis of the different protein/lipid/detergent mixed micelles showed that the protein/lipid/OGP mixed micelles were relatively enriched with sphingomyelin (SPM) compared to protein/lipid/Chaps mixed micelles, indicating that the differences in the solubilization efficiency may be due to the ability to extract more SPM from membranes. When the protein/lipid/OGP mixed micelles were dissociated into protein/detergent and lipid/detergent complexes by the addition of increasing Chaps concentrations, one-tenth of the LAT enzyme activity was preserved making the enzyme accessible to protein purification. Analysis by native PAGE revealed that in the presence of excess Chaps a high molecular mass protein complex migrated into the gel which could be photolabeled by 125I-labelled-18-(4'-azido-2'-hydroxybenzoylamino)-oleyl-CoA. This fatty acid analogue has been shown to be a competitive inhibitor of LAT enzyme activity in the dark, and an irreversible inhibitor after photolysis. Therefore, this protein complex is assumed to contain the LAT enzyme.