Immunological and biochemical properties of transverse tubule membranes isolated from rabbit skeletal muscle.
A new method for isolating transverse tubule membranes from rabbit skeletal muscle has been developed. This procedure has the advantage of being mild, fast, and producing with good yields a purified membrane fraction. The transverse tubule membranes are purified by a discontinuous sucrose density centrifugation after loading contaminating light sarcoplasmic reticulum vesicles with calcium phosphate in the presence of ATP. Immunofluorescence staining of cryostat sections of rabbit psoas muscle with purified goat antibodies directed against the purified membranes shows that the reacting antigens are distributed at the boundary of the A and I bands of the myofibrils where transverse tubules are localized in mammalian muscle. The purified antibodies showed no cross-reactivity with sarcoplasmic reticulum, nor did they show any fluorescence staining of the muscle plasma membrane, indicating that the isolated membranes indeed originate from the transverse tubules. The transverse tubule fraction has a characteristic protein composition distinguishable from that of sarcoplasmic reticulum, a much higher cholesterol content than that of the crude microsomes, plasma membrane, and sarcoplasmic reticulum, and a phospholipid content about twice as high as that of sarcoplasmic reticulum and plasma membrane. The purified transverse tubule membrane has a distinct phospholipid composition with high contents of sphingomyelin and phosphatidylserine. A Mg2+-activated ATPase characteristic of the transverse tubule fraction undergoes a 20-30-fold increase in specific activity during purification. The levels of Ca2+-ATPase activity present in the purified transverse tubule fraction remain comparable to those of sarcoplasmic reticulum even after extensive removal of the latter.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D000936:Antigen-Antibody Complex; D000941:Antigens / Q000032:analysis; D002118:Calcium / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient / Q000379:methods; D002784:Cholesterol / Q000032:analysis; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D007425:Intracellular Membranes / Q000378:metabolism* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000032:analysis*; D008870:Microtubules / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism*; D010743:Phospholipids / Q000032:analysis; D011817:Rabbits
D000936:Antigen-Antibody Complex; D000941:Antigens; D008565:Membrane Proteins; D010743:Phospholipids; D002784:Cholesterol; D000251:Adenosine Triphosphatases; D002118:Calcium