Identification of a cardiac carnitine binding protein.
The existence of a cardiac carnitine binding protein was demonstrated using an in vitro binding assay. The binding activity was solubilized with Triton X-100 from the pellet following a 59,000 X g centrifugation of rat ventricular homogenates. Preincubation at temperatures above 40 degrees C or treatment with pronase significantly reduced the binding activity, suggesting that the activity was that of a protein. Cell fractionation studies suggested that the cardiac carnitine binding protein was associated with the plasma membrane fraction and that its activity was distinct from carnitine palmitoyl-transferase, carnitine acetyltransferase, and carnitine translocase. Optimal binding required 60 min of incubation at 25 degrees C. The binding of carnitine to the cardiac carnitine binding protein was saturable, and a dissociation constant of 0.7 microM for DL-carnitine was measured. L-Carnitine competed with DL-[methyl-3H]carnitine for competition by D-carnitine was much less effective. Binding was significantly inhibited when N-ethylmaleimide, iodoacetic acid, or mercuric chloride was present. Once DL-[3H]carnitine was bound to the cardiac carnitine binding protein, radioactivity could be dissociated by a variety of mild treatments including dialysis, overnight incubation at 4 degrees C, and application to a gel filtration column.
D015720:5'-Nucleotidase; D000818:Animals; D002332:Carnitine O-Acetyltransferase / Q000378:metabolism; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D006352:Heart Ventricles / Q000378:metabolism; D007700:Kinetics; D008297:Male; D009206:Myocardium / Q000378:metabolism*; D009708:Nucleotidases / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions / Q000378:metabolism; D013385:Succinate Dehydrogenase / Q000378:metabolism
D002352:Carrier Proteins; C035681:carnitine-binding protein, rat; D013385:Succinate Dehydrogenase; D002332:Carnitine O-Acetyltransferase; D009708:Nucleotidases; D015720:5'-Nucleotidase