Uncoupling of Ca2+ transport from ATP hydrolysis activity of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase.
In reconstituted rabbit skeletal muscle (Ca2+ + Mg2+)-ATPase proteoliposomes, Ca(2+)-uptake is decreased by more than 90% with T2 cleavage (Arg-198). However, no difference in the ATP dependence of hydrolysis activity is seen between SR and trypsin-treated SR. A large decrease in E-P formation and hydrolysis activity of the enzyme appear only at T3 cleavage, which represents the cleavage of A1 fragment to A1a + A1b forms. The disappearance of hydrolysis activity due to digestion is prior to the disappearance of E-P formation. No significant difference is found in the passive Ca2+ efflux between control SR and tryptically digested SR in the absence of Mg2+ + ruthenium red or in the presence of ATP. However, the passive Ca2+ efflux rate for tryptically digested SR is much larger than control SR in the presence of Mg2+ + ruthenium red. These results show that the Ca2+ channel cannot be closed after trypsin digestion of SR membranes by the presence of the Ca2+ channel inhibitors, Mg2+ and ruthenium red. In the reconstituted proteoliposomes, the Ca2+ efflux rates are the same regardless of digestion (T2); also, efflux is not affected by the presence or absence of Mg2+ + ruthenium red. These results indicate that T2 cleavage causes 'uncoupling' of the 'Ca(2+)-pump' from ATP hydrolytic activity. A theoretical model is developed in order to fit the extent of tryptic digestion of the A fragment of the (Ca2+ + Mg2+)-ATPase polypeptide with the loss of Ca(2+)-transport. Fits of the theoretical equations to the data are consistent with that Ca(2+)-transport system appears to require a dimer of the polypeptide (Ca2+ + Mg2+)-ATPase.
Cao
C J
CJ
Department of Biological Chemistry, School of Medicine, Baltimore, Maryland 21201.
Lockwich
T
T
Scott
T L
TL
Blumenthal
R
R
Shamoo
A E
AE
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001693:Biological Transport, Active; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D003124:Colorimetry; D004591:Electrophoresis, Polyacrylamide Gel; D008081:Liposomes / Q000378:metabolism; D008433:Mathematics; D008954:Models, Biological; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology* / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D015220:Calcium Channels; D008081:Liposomes; D000255:Adenosine Triphosphate; D014357:Trypsin; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D002118:Calcium