Direct photoaffinity labeling of the high affinity nitrendipine-binding site in subcellular membrane fractions isolated from canine myocardium.
[3H]Nitrendipine and high intensity ultraviolet irradiation have been used to photoaffinity label the protein component of the high affinity nitrendipine-binding site in subcellular membrane fractions from canine cardiac muscle. Irradiation of isolated cardiac membranes in the presence of [3H]nitrendipine resulted in the covalent labeling of a protein component that migrated on sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular weight of 32,000. Incorporation of [3H]nitrendipine did not occur in the absence of irradiation. The photoaffinity labeling of the 32,000-Da protein by [3H]nitrendipine was inhibited by excess unlabeled nitrendipine, nifedipine, or verapamil. EDTA, ATP, and La3+, which are known to reduce high affinity nitrendipine binding, also inhibited the photoaffinity labeling of this membrane protein by [3H]nitrendipine. The 32,000-Da [3H]nitrendipine-labeled protein was found to be enriched in the ryanodine-sensitive fraction of cardiac sarcoplasmic reticulum and absent from the ryanodine-insensitive fraction of cardiac sarcoplasmic reticulum which is known to lack high affinity nitrendipine binding. Therefore, the 32,000-Da photoaffinity-labeled [3H]nitrendipine-binding protein exhibits properties identical to those expected for the protein component of the high affinity nitrendipine-binding site in isolated cardiac membranes.
D000345:Affinity Labels; D000818:Animals; D002121:Calcium Channel Blockers / Q000378:metabolism*; D015220:Calcium Channels; D004285:Dogs; D007425:Intracellular Membranes / Q000378:metabolism*; D008027:Light; D009206:Myocardium / Q000378:metabolism*; D009543:Nifedipine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D009568:Nitrendipine; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology*; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D012508:Sarcolemma / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013450:Sulfones / Q000494:pharmacology*; D014466:Ultraviolet Rays; D014700:Verapamil / Q000494:pharmacology
D000345:Affinity Labels; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D011978:Receptors, Nicotinic; D013450:Sulfones; D010664:Phenylmethylsulfonyl Fluoride; D009568:Nitrendipine; D014700:Verapamil; D009543:Nifedipine