Crosslinking of sarcoplasmic reticulum ATPase protein with 1,5-difluoro 2,4-dinitrobenzene.
The reacton of 1,5-difluoro-2,4-dinitrobenzene with the ATPase protein of rabbit skeletal sarcoplasmic reticulum caused a marked loss of the Ca2+-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity during an interval when 2 mol of the crosslinking reagent were incorporated/10(5) g of protein. The modified ATPase protein formed non-serial high molecular weight aggregates or oligomers during short (1--5 min) or long exposure (60 min) to the reagent at 25 degrees C or 4 degrees C. The same pattern was found when sarvoplasmic reticulum was treated similarly; only the ATPase protein formed oligomers (homopolymers). In all cases the ATPase protein monomer remained the predominant species present. During the appearance of the high molecular weight oligomers the Ca2+-ATPase activity was unaffected but Ca2+ uptake was inhibited. Major changes in the ATPase activity occurred when the monomeric ATPase protein was modified. Disubstituted dinitrophenylene derivatives of cysteine and tyrosine were found in modified ATPase protein and only a small amount of monosubstituted dinitrophenyl groups were identified. Thiolysis of the modified ATPase protein with 2-mercaptoethanol removed approx. 35% of the incorporated groups, but there was no restoration of the Ca2+-ATPase activity. Substrate MgATP2- protected the Ca2+-ATPase activity of the ATPase protein and sarcoplasmic reticulum but Ca2+ had no effect on the modificaton. Different conformational states of the ATPase protein could be ascertained from a comparison of the effects of Ca2+ and MgATP2- on the bifunctional reagent dinitrophenylation of the ATPOase protein with that of the monofunctional reagent 1-fluoro-2,4-dinitrobenzene (Bailin, G. (1980) Biochim. Biophys. Acta 623, 213-224). Intramolecular crosslinking of the ATPase protein predominated and oligomers which formed during the reaction were not essential for the maintenance of the ATPase activity.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D003432:Cross-Linking Reagents; D004139:Dinitrofluorobenzene / Q000031:analogs & derivatives / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009132:Muscles / Q000201:enzymology; D009578:Nitrobenzenes / Q000494:pharmacology*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D003432:Cross-Linking Reagents; D009578:Nitrobenzenes; D004139:Dinitrofluorobenzene; D000251:Adenosine Triphosphatases; D000252:Calcium-Transporting ATPases; C002041:1,5-difluoro-2,4-dinitrobenzene; D008274:Magnesium