Reaction of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole with the (Ca2+ + Mg2+)- ATPase protein of sarcoplasmic reticulum at low temperature.
Modification of the (Ca2+ + Mg2+)-ATPase protein of rabbit skeletal sarcoplasmic reticulum (SR) with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, NBD-Cl, at 4 degrees C for 5 min caused a 63% loss of the Ca(2+)-dependent ATPase activity when 1 mol of the adenine analog was incorporated per 10(5) g of protein. At 25 degrees C, above the lipid phase transition, the extent of labeling was 3-fold higher although the Ca(2+)-ATPase activity was inhibited to the same extent. MgATP protected the ATPase activity at 4 degrees C and 25 degrees C but there was little change in the extent of labeling at 4 degrees C suggesting that changes in the fluidity of the lipid moiety made different sites on the ATPase protein accessible to the reagent. At 4 degrees C, addition of sodium deoxycholate enhanced the inactivation (6% ATPase activity remained) but the labeling of the SR-ATPase protein did not increase significantly. Incubation with MgATP prior to solubilization with deoxycholate resulted in the protection of the Ca(2+)-ATPase activity and only a small decrease in the labeling occurred. At 25 degrees C, a similar pattern was found with deoxycholate but the loss of ATPase activity was less dramatic and the extent of labeling by NBD-Cl was greater than that at 4 degrees C. MgATP induced changes in the conformation of the ATPase protein protecting essential cysteine residues while shifting the reaction of NBD-Cl with the ATPase protein to non-essential sites in the absence or presence of deoxycholate. An analysis of tryptic digests of the NBD-ATPase protein showed that MgATP shifted the labeling from the A2 subfragment to the A1 subfragment in the absence of deoxycholate and from the A1 subfragment to the A2 subfragment in the presence of deoxycholate. The reagent, NBD-Cl, can distinguish between different temperature dependent conformational states of the ATPase protein.
Bailin
G
G
Department of Molecular Biology, University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, Stratford 08084.
D009327:4-Chloro-7-nitrobenzofurazan / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000737:chemistry*; D003080:Cold Temperature; D003840:Deoxycholic Acid / Q000494:pharmacology; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014357:Trypsin
D003840:Deoxycholic Acid; D000255:Adenosine Triphosphate; D014357:Trypsin; D017301:Ca(2+) Mg(2+)-ATPase; D009327:4-Chloro-7-nitrobenzofurazan