Reversible control of oligomeric interaction of the sarcoplasmic reticulum calcium ATPase with the use of a cleavable cross-linking agent.
Reversible cross-linking with a cleavable homobifunctional reagent, dithiobissuccinimidyl propionate, of the Ca2+ ATPase polypeptides of fragmented sarcoplasmic reticulum produces oligomers in the range of 2 to 4 X 100,000 Da (cf. Louis, C. F., and Holroyd, J. A., (1979) Biochim. Biophys. Acta 535, 222-232). The presence of millimolar ATP during the dithiobissuccinimidyl propionate reaction protects the enzyme from inactivation without affecting cross-linking. In the presence of both ATP (e.g. 1 mM) and Ca2+ (e.g. 0.21 mM), there is no inhibition of phosphoenzyme (EP) formation and presteady state Ca2+ translocation, whereas Ca2+-induced conformational changes of the enzyme, and EP decomposition are inhibited. Cleavage of the S-S bond of the cross-links reverses the inhibition of conformational changes but has little effect on the inhibited EP decomposition. This indicates that the inhibition of conformational changes is due to cross-linking, while that of EP decomposition is due to the chemical modification as such. If [Ca2+] is low during the dithiobissuccinimidyl propionate reaction (e.g. pCa 7.6), the Ca2+-induced conformational changes of the enzyme, EP formation, presteady state Ca2+ translocation, and EP decomposition are inhibited, even in the presence of ATP. Cleavage of the cross-links reverses the inhibition of conformational changes and EP formation, but again has little effect on EP decomposition. Thus, the primary effect of cross-linking at high and low [Ca2+] is the inhibition of the Ca2+-induced conformational change, suggesting that extensive interaction of subunits is involved in this reaction step. The fact that EP formation and presteady state Ca2+ translocation are inhibited by cross-linking carried out at low but not at high [Ca2+] suggests that cross-linking takes place at different regions of the enzyme molecule at different [Ca2+].
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D007700:Kinetics; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013388:Succinimides / Q000494:pharmacology*
D003432:Cross-Linking Reagents; D013388:Succinimides; D000252:Calcium-Transporting ATPases; C011240:dithiobis(succinimidylpropionate)