The effects of deoxycholate and trypsin on the cross-linking of rabbit skeletal muscle sarcoplasmic reticulum proteins.
Dithiobis (succinimidyl propionate) has been used to cross-link sarcoplasmic reticulum microsome proteins. Although the 100,000 dalton calcium stimulated ATPase and the 60,000 dalton calcium-binding protein calsequestrin were readily cross-linked to form homopolymers, no heteropolymer formation between these two proteins were detected. The 90,000 dalton protein A1 which is always observed in our preparations appeared to preferrentially form dimers on cross-linking. When calsequestrin was solubilized using 0.1 mg deoxycholate/mg protein, this protein was not cross-linked even at dithiobis(succinimidyl propionate) concentrations ten times those used to cross-link this protein in the intact membrane. In a similar manner the deoxycholate-solubilized ATPase (0.5 mg deoxycholate/mg protein) was not cross-linked by dithiobis (succinimidyl propionate). These results suggest that the state of aggregation of the sarcoplasmic reticulum proteins may be modified when solubilized in detergents such as deoxycholate. When the 100,000 dalton ATPase polypeptide was cleaved with trypsin to two fragments with molecular weights of approximately 55,000, these could be readily cross-linked. The fragments were capable of forming polymers with either other 55,000 dalton fragments or with the 100,000 dalton ATPase. The 29,000 and 22,000 dalton fragments, produced by further tryptic cleavage of the 55,000 dalton fragments, were not cross-linked at dithiobis (succinimidyl propionate) concentrations which readily cross-linked the 55,000 dalton fragments. Thus tryptic cleavage of the ATPase to fragments smaller than 55,000 dalton altered associations made by the ATPase in the membrane.
D000251:Adenosine Triphosphatases; D000818:Animals; D002118:Calcium; D055598:Chemical Phenomena; D002621:Chemistry; D003840:Deoxycholic Acid; D004220:Disulfides; D007202:Indicators and Reagents; D008861:Microsomes; D008970:Molecular Weight; D009124:Muscle Proteins; D010446:Peptide Fragments; D011817:Rabbits; D012519:Sarcoplasmic Reticulum; D013388:Succinimides; D014357:Trypsin
D004220:Disulfides; D007202:Indicators and Reagents; D009124:Muscle Proteins; D010446:Peptide Fragments; D013388:Succinimides; D003840:Deoxycholic Acid; D014357:Trypsin; D000251:Adenosine Triphosphatases; D002118:Calcium