Size and shape of rabbit skeletal muscle calsequestrin.
Calsequestrin, a calcium-binding protein isolated from rabbit skeletal muscle sarcoplasmic reticulum, was subjected to physiochemical analysis using sodium dodecyl sulfate gel electrophoresis, gel filtration, sedimentation, viscosity, and circular dichroism techniques. The effects of sodium dodecyl sulfate, alkaline pH, guanidine hydrochloride, and calcium ions on the hydrodynamic properties of the protein were studied. Calsequestrin, in the absence of calcium, had a random coil conformation with an alpha-helical content of 11%. Calsequestrin bound 1.7 mg of sodium dodecyl sulfate per mg of protein resulting in an increase in the alpha-helical content to 20%. The protein was completely random coil in guanidine hydrochloride and had a molecular weight of 42,000 as determined by gel filtration in the presence of this denaturant. Sedimentation equilibrium studies showed that calsequestrin was not subjected to aggregation and had a molecular weight of 38,000. Calsequestrin had a low sedimentation coefficient (2.20 S), a high Stokes radius (45 A), and a high intrinsic viscosity (27.1 ml/g) that increased slightly to 32 ml/g in the presence of guanidine hydrochloride, all indicative of a highly extended structure. Similar studies, performed at pH 9.5, revealed that the protein was even more asymmetric at alkaline pH. Calsequestrin bound 50 mol of calcium ions per mol of protein with an affinity of 1 mM as determined by gel filtration. Calcium binding was accompanied by a change of the protein from a highly extended structure (Rs = 45 A) to a much more compact structure (Rs = 35 A).
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002155:Calsequestrin / Q000302:isolation & purification* / Q000378:metabolism; D002942:Circular Dichroism; D046911:Macromolecular Substances; D008970:Molecular Weight; D009124:Muscle Proteins / Q000302:isolation & purification*; D009132:Muscles / Q000032:analysis*; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D014783:Viscosity
D002155:Calsequestrin; D046911:Macromolecular Substances; D009124:Muscle Proteins; D002118:Calcium