Purification, immunochemical quantification and localization in rat heart of putative fatty acid translocase (FAT/CD36)

• Published in: Molecular and cellular biochemistry Vol. 284; no. 1-2; pp. 127 - 134
• Main Authors: Brinkmann, Joep F. F, Pelsers, Maurice M. A. L, van Nieuwenhoven, Frans A, Tandon, Narendra N, der Vusse, Ger J. van, Glatz, Jan F. C
• Format: Journal Article
• Language: English
• Published: Netherlands Boston : Springer US 01.03.2006; Springer Nature B.V
• Subjects: Animals; Cardiomyocytes; CD36; CD36 Antigens - isolation & purification; CD36 Antigens - metabolism; Chromatography; endothelial cells; Endothelial Cells - enzymology; Fatty acids; Fluorescent Antibody Technique; Heart; Humans; immunochemistry; In Vitro Techniques; Male; membrane proteins; Myocardium - enzymology; myocytes; Myocytes, Cardiac - enzymology; polyacrylamide gel electrophoresis; Proteins; purification; Rats; Rats, Inbred Lew; Western blotting; yields
• ISSN: 0300-8177; 1573-4919
• DOI: 10.1007/s11010-005-9033-2

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 μg/mg protein) and total hearts (14 ± 4 μg/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.