Modification of the (Ca2+ + Mg2+)-ATPase protein of sarcoplasmic reticulum with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole.
The (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase (Ca2+-transporting), EC 3.6.1.38) protein of rabbit skeletal sarcoplasmic reticulum (SR) rapidly incorporated 2 mol of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) per 10(5) g of protein with little change in the Ca2+-dependent ATPase activity. When 2 additional mol of the reagent were bound the Ca2+-ATPase, activity was inhibited. The same pattern was found for modified intact SR and the Ca2+ uptake ability was inhibited. MgATP, CaATP and MgADP protected the Ca2+-ATPase activity concurrent with a decrease of about 1 mol of the NBD group per 10(5) g protein, but the Ca2+ uptake ability was not protected. Calcium alone had no effect on the modification. The modified ATPase protein or SR formed non-serial oligomers or aggregates, but the ATPase protein remained the predominant species present. In the presence of MgATP, oligomer formation was reduced partially but the major changes in the Ca2+-ATPase activity were due to the modification of the ATPase monomer. Thiolysis of the NBD-ATPase protein with dithiothreitol did not restore the Ca2+-ATPase activity, although more than 1 mol of the NBD group was removed from cysteine residues. Cysteine residues were modified in the NBD-ATPase protein or SR when the enzyme activity was inhibited. Trypsin digestion of NBD-SR or its ATPase protein released the A, B, A1, and A2 fragments. The A fragment and its subfragment A2 contained most of the label. Substrate MgATP protection studies showed that the A1 and A2 fragments were involved in maintaining the Ca2+-ATPase activity. Reagent-induced conformational changes of these fragments rather than direct active site group labeling accounted for the loss of ATPase activity.
Bailin
G
G
Department of Biochemistry, University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, Piscataway 08854-5635.
Huang
J R
JR
D009327:4-Chloro-7-nitrobenzofurazan / Q000378:metabolism / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D010069:Oxadiazoles / Q000494:pharmacology*; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D010069:Oxadiazoles; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D000255:Adenosine Triphosphate; D014357:Trypsin; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D009327:4-Chloro-7-nitrobenzofurazan; D002118:Calcium; D004229:Dithiothreitol