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Articles in PresS, published online ahead of print February 26, 2002
Am J Physiol Endocrinol Metab, 10.1152/ajpendo.00428.2001
Submitted on September 24, 2001
Accepted on February 14, 2002
1 Institute of Cell Biology, ETH-Zurich, Zurich, Switzerland
2 Human Genetics, Wellcome Trust Center, Oxford, United Kingdom
3 Human Physiology, University of Copenhagen, Copenhagen, Denmark
4 Biochemistry, University of Oxford, Oxford, United Kingdom
* To whom correspondence should be addressed. E-mail: bernd_walzel{at}cotyinc.com.
Despite the pivotal role of creatine (Cr) and phospho-creatine (PCr) in muscle metabolism surprisingly little is known about sarcolemmal creatine transport, creatine transporter isoforms and subcellular localization of the creatine transporter proteins. To be able to quantify creatine transport across the sarcolemma we have developed a new in vitro assay using rat sarcolemmal giant vesicles. The rat giant sarcolemmal vesicle assay reveals the presence of a specific, high affinity and saturable transport system for Cr in the sarcolemma (Km = 52.4 (±9.4) µM and Vmax value = 17.3 (±3.1) pmoles x mg vesicle protein-1 x min-1), which co-transports Cr into skeletal muscle together with Na+ and Cl- ions. The regulation of Cr transport in giant vesicles by substrates, analogues, and inhibitors, as well as by different effectors and hormones was also studied. Several specific anti-creatine transporter (CRT) peptide antibodies all predominantly recognize two major polypeptides on Western blots with an apparent Mr of 72 kDa and 52 kDa, respectively. The highest CRT expression occurs in heart, brain, and kidney and although creatine kinase is absent in liver cells, CRT is also found in this tissue. Surprisingly, cell fractionation and cell surface biotinylation studies, as well as immunofluorescence staining of cultured adult rat heart cardiomyocytes with specific anti-CRT antibodies revealed that only a minor CRT species of intermediate Mr of ~58 kDa is present in the sarcolemma, whereas the previously identified major CRT protein species of 72 and 52 kDa are specifically located in mitochondria. Our studies show that mitochondria are the major compartment of CRT expression, a fact that provides a new aspect for the current debate about the existence and whereabouts of intracellular Cr and PCr compartments that have been inferred from [14C]-PCr/Cr measurements in vivo, as well as from in vivo NMR techniques.
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