Purification of the major substrate for palmitoylation in rat adipocytes: N-terminal homology with CD36 and evidence for cell surface acylation.
We previously reported that incubating rat adipocytes with [3H]palmitate predominantly labeled an 85-kDa protein. The labeling was more intensive in the presence of insulin and had characteristics consistent with covalent fatty acylation (Jochen et al. 1991. Biochem. Biophys. Res. Commun. 177: 797-801). In order to determine the significance of this finding we purified the 85-kDa protein, determined its N-terminal sequence, and further characterized its interactions with long-chain fatty acids. The [3H]palmitate-labeled 85-kDa protein was purified from rat adipocyte membranes using the following sequence of procedures: i) affinity chromatography with wheat germ agglutinin-agarose, ii) ion exchange chromatography with DEAE-Sepharose, and iii) SDS-polyacrylamide gel electrophoresis. The resulting labeled protein was sequenced through 30 amino acid residues. With the exception of one conserved substitution, the sequence was identical to CD36 (platelet membrane glycoprotein IV). Further characterization of the 85-kDa protein revealed it was heavily N-glycosylated and possessed a cell surface domain. Labeling of the 85-kDa protein with palmitate was compared in control cells, insulin-treated cells, and cells whose energy was depleted with 2,4-dinitrophenol. Insulin and energy depletion increased labeling approximately 3-fold and 12-fold, respectively. Labeling performed in the presence of energy depletion possessed all of the characteristics of covalent protein acylation. In addition, there was a close association between the ability of energy depletion to increase labeling of the 85-kDa protein and its ability to inhibit depletion of [3H]palmitate from the extracellular incubation media. These results suggest that the major substrate for fatty acylation in adipocytes is a cell surface membrane protein related to CD36 and that this acylation has the unusual properties of being independent of intracellular metabolic energy and of occurring on an exofacial epitope of the protein.
Jochen
A
A
Department of Medicine, Medical College of Wisconsin, Zablocki Veterans Administration Hospital, Milwaukee 53295.
Hays
J
J
D019297:2,4-Dinitrophenol; D000215:Acylation; D000273:Adipose Tissue / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry*; D018955:CD36 Antigens; D002462:Cell Membrane / Q000737:chemistry; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004140:Dinitrophenols / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004734:Energy Metabolism; D006031:Glycosylation; D007328:Insulin / Q000494:pharmacology; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid; D014316:Tritium
D015703:Antigens, CD; D018955:CD36 Antigens; D004140:Dinitrophenols; D007328:Insulin; D008565:Membrane Proteins; D010169:Palmitic Acids; D014316:Tritium; D019308:Palmitic Acid; D019297:2,4-Dinitrophenol