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

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Abstract

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.

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Authors and Affiliations

  1. Departments of Physiology, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands

    Joep F. F. Brinkmann, Frans A. van Nieuwenhoven & Ger J. van der Vusse

  2. Molecular Genetics, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands

    Maurice M. A. L. Pelsers & Jan F. C. Glatz (PhD)

  3. Thrombosis Research Laboratory, Otsuka Maryland Medicinal Laboratories, Rockville, MD, 20850, USA

    Narendra N. Tandon

  4. Molecular Genetics, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, P.O. Box 616, 6200, Maastricht, MD, The Netherlands

    Jan F. C. Glatz (PhD)

Authors
  1. Joep F. F. Brinkmann
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  2. Maurice M. A. L. Pelsers
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  3. Frans A. van Nieuwenhoven
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  4. Narendra N. Tandon
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  5. Ger J. van der Vusse
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  6. Jan F. C. Glatz
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Correspondence to Jan F. C. Glatz.

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Brinkmann, J.F.F., Pelsers, M.M.A.L., van Nieuwenhoven, F.A. et al. Purification, immunochemical quantification and localization in rat heart of putative fatty acid translocase (FAT/CD36). Mol Cell Biochem 284, 127–134 (2006). https://doi.org/10.1007/s11010-005-9033-2

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  • DOI: https://doi.org/10.1007/s11010-005-9033-2

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