Abstract
Several proteins in sarcoplasmic reticulum preparations move in a band with a mobility, in sodium dodecyl sulfate-polyacrylamide gels (0.1 M phosphate buffer, pH 7.0), corresponding to a molecular mass of about 55,000 daltons. Only one of these proteins is the high affinity calcium binding protein. An intrinsic glycoprotein is also present in this band, and it is this glycoprotein which is found in vesicles reconstituted after dissolution of sarcoplasmic reticulum in deoxycholate. Both of these proteins are found in rather constant ratios with the ATPase in light, intermediate, and heavy sarcoplasmic reticulum vesicles. Transverse tubular vesicles can be isolated from the heavy sarcoplasmic reticulum vesicles after disruption of the membrane in a French pressure cell (Lau, Y.H., Caswell, A.H., and Brunschwig, J.P. (1977) J. Biol. Chem. 252, 5565-5574). These vesicles are enriched in their content of the high affinity calcium binding and depleted of the intrinsic glycoprotein. Cycloheptaamylose . fluorescamine complex (CFC) labels the intrinsic glycoprotein heavily indicating that it is at least partially exposed on the cytoplasmic surface of sarcoplasmic reticulum membranes. Since the carbohydrate component of the protein must lie in luminal spaces, it is inferred that the intrinsic glycoprotein is a transmembrane protein. The high affinity calcium binding protein is not labeled by CFC indicating that it is not exposed on the cytoplasmic surface of sarcotubular vesicles. The protein is also not affected by proteolytic digestion of sarcoplasmic reticulum vesicles and can be isolated intact from trypsin-digested vesicles. It is not removed from sarcoplasmic-reticulum vesicles by washing with buffers containing Chelex 100 or ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA). These data show that the high affinity calcium binding protein is localized in the interior of the sarcotubular system and suggest that it might be common to both sarcoplasmic reticulum and transverse tubular membranes.
Highlights
7.0), corresponding to a molecularmass of about 55,000 resis (1)
The high affinity rate thancalsequestrin (8), but thiscould be explained bythe calcium binding protein is not labeled by Cycloheptaamylose fluorescamine complex (CFC) indicat- different capacities and affinities for calcium bindingof these ing that it is neoxt posed onthe cytoplasmic surface of two proteins (5); it is not reactive with antibodies acting a t sarcotubular vesicles
The group of 44,000daltons corresponding to calsequestrin rather than of proteins at 30,000 daltons were separated in this study, and to the 55,000-dalton proteins observed in previous studies (8, we have shown tha5t0% ofthe single protein of 30,000 daltons 16)
Summary
SDS-polyacrylamidegel electrophoresis was carried outusing both high affinity calcium bindingprotein which run as one protein methods of Laemmli (23) and Weber and Osborn (24). Since this procedureled to elution of extrinsic membrane proteins, of these was thehigh affinity calcium bindingprotein, yet,all in somecases (Fig. 7) slab gels werestained withCoomassie blue and moved in the same electrophoretic band in the Weber and destained before treatment with Buffer B. Phosphorylase waslabeledwith '"P by the the high affinity calcium binding protein from their contamimethod of Krebsand Fischer (29) andwasstored a t 4°C in 50% nants, we found it possible to re-examine the questioonf how saturated ammonium sulfate Both substrate and enzyme were dialyzed for 2 h against 10 mM Tris-HCI, pH 7.5, to remove inhibitory salts.
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