Purification of the CaATPase of sarcoplasmic reticulum by affinity chromatography.
Proteins from sarcoplasmic reticulum vesicles solubilized by a nonionic detergent were fractionated by use of a reactive red-120 agarose column. The Ca-ATPase was obtained in pure form by eluting the column with 400 microM adenyl 5'-yl imidodiphosphate, yielding an enzyme of almost twice the starting specific activity in a fraction containing half the initial protein. The conclusion that the ATPase comprises 50% of the sarcoplasmic reticulum vesicle protein agrees with estimates gained from densitometry using 7 1/2% Laemmli slab gels but not from densitometry using 7% Weber and Osborn slab gels. The mechanism of purification was found to be affinity chromatography, with the ATPase binding the reactive red-120 ligand in its nucleotide-binding site. The steady-state concentration of phosphorylated intermediate relative to the specific activity was found to be lower in the purified enzyme as compared to the starting vesicular enzyme.
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D004396:Coloring Agents / Q000378:metabolism; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000378:metabolism; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012685:Sepharose / Q000378:metabolism; D013861:Thiocyanates / Q000378:metabolism; D014227:Triazines
D004396:Coloring Agents; D005452:Fluoresceins; D013861:Thiocyanates; D014227:Triazines; C042516:reactive red 120 dye; D012685:Sepharose; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate