Purification, immunochemical quantification and localization in rat heart of putative fatty acid translocase (FAT/CD36).
Evidence is accumulating that the heavily glycosylated integral membrane protein fatty acid translocase (FAT/CD36) is involved in the transport of long-chain fatty acids across the sarcolemma of heart muscle cells. The aim of this study was to analyse the distribution between FAT/CD36 present in cardiac myocytes and endothelial cells. We therefore developed a method to purify FAT/CD36 from total rat heart and isolated cardiomyocytes, and used the proteins as standards in an immunochemical assay. Two steps, chromatography on wheat germ agglutinin-agarose and anion-exchange chromatography on Q-Sepharose fast flow, were sufficient for obtaining the protein in a > 95% pure form. When used to isolate FAT/CD36 from total heart tissue, the FAT/CD36 yield of the method was 9% and the purification factor was 64. Purifying FAT/CD36 from isolated cardiomyocytes yielded the same 88 kDa protein band on SDS-PAGE gels and reactivity of this band on western blots was comparable to that of the FAT/CD36 isolated from total hearts. Quantifying FAT/CD36 contents by western blotting showed that the amounts of FAT/CD36 that are present in isolated cardiomyocytes (10 +/- 3 microg/mg protein) and total hearts (14 +/- 4 microg/mg protein) are of comparable magnitude. Immunofluorescence labelling showed that at least a part of the FAT/CD36 present in the cardiomyocyte is associated with the sarcolemma. This study established that FAT/CD36 is a relatively abundant protein in the cardiomyocyte. In addition, the further developed purification procedure is the first method for isolating FAT/CD36 from rat heart and cardiomyocyte FAT/CD36.
Department of Physiology, Cardiovascular Research Institute Maastricht, CARIM, Maastricht University, Maastricht, The Netherlands.
Joep F F
JF
Brinkmann
Maurice M A L
MM
Pelsers
Frans A
FA
van Nieuwenhoven
Narendra N
NN
Tandon
Ger J
GJ
van der Vusse
Jan F C
JF
Glatz
D000818:Animals; D018955:CD36 Antigens / Q000302:isolation & purification / Q000378:metabolism*; D042783:Endothelial Cells / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D009206:Myocardium / Q000201:enzymology*; D032383:Myocytes, Cardiac / Q000201:enzymology; D051381:Rats; D011917:Rats, Inbred Lew
D018955:CD36 Antigens