Purification and crystallization of the calcium binding protein of sarcoplasmic reticulum from skeletal muscle.
The calcium binding protein of skeletal muscle sarcoplasmic reticulum (also referred to as calsequestrin) was purified by release from the compartment of the vesicles with the detergent octaethyleneglycol mono-n-dodecyl ether (C12E8) and by subsequent precipitation of the calcium binding protein with specific divalent cations. The isolated protein exhibited a single band on NaDodSO4/PAGE and bound 903 nmol of Ca2+ per mg of protein. The calcium binding protein could be crystallized in the presence of Ca2+, Mg2+, Sr2+, or combinations of these three cations used in a narrow concentration range. Needle-shaped crystals of up to 500 X 50 micron were obtained. The removal of the divalent cations resulted in solubilization of the crystals. The spacings and angles of the crystals were obtained by electron microscopy using three different methods of sample preparation. By freeze-drying and negative staining electron microscopy, the spacings along axes a and b were determined to be 10-11 nm each, and the angle between the two axes was 90 degrees. By thin section electron microscopy, the spacing along axis a was 11 nm, along axis c was 15-16 nm, and the angle between the two axes was 75 degrees. This study reports (i) a simple and rapid method for purification of the calcium binding protein; (ii) conditions to crystallize the protein using Ca2+, Mg2+, Sr2+, or combinations of the three; and (iii) some preliminary characteristics of the crystals. The crystalline nature was characterized by electron microscopy and x-ray diffraction. The larger crystals diffracted beyond 3-A Bragg spacing.
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism; D003461:Crystallography; D005260:Female; D007425:Intracellular Membranes / Q000032:analysis / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D008854:Microscopy, Electron; D008970:Molecular Weight; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000032:analysis* / Q000378:metabolism; D014961:X-Ray Diffraction
D002135:Calcium-Binding Proteins; D002413:Cations, Divalent; D008565:Membrane Proteins; D002118:Calcium