Purification and characterization of the Mg2+-ATPase from rabbit skeletal muscle transverse tubule.
Transverse tubule membranes isolated from rabbit fast skeletal muscle contain a very active Mg2+-ATPase (ATP phosphohydrolase, EC 3.6.1.3). This enzyme is very sensitive to inactivation by most detergents. However, after solubilization with either lysolecithin or digitonin, the Mg2+-ATPase can be purified in active form. Using a combination of selective solubilization followed by lectin affinity chromatography, ion-exchange chromatography, and native gel electrophoresis, the Mg2+-ATPase has been purified to near homogeneity. A prominent band with molecular mass of 105 kDa is observed when the purified protein is analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified 105-kDa Mg2+-ATPase protein is not structurally similar to the sarcoplasmic reticulum Ca2+-ATPase protein, as evidenced by very different cyanogen bromide peptide maps and amino acid compositions. The structural dissimilarities are complemented by functional differences observed between the Ca2+- and Mg2+-ATPases, including differential susceptibility to proteases, chemical modification reagents, and inactivation by fluorescein isothiocyanate and vanadate. All these data taken together demonstrate that the Mg2+-ATPase is a unique protein with little, if any, structural similarity to the sarcoplasmic reticulum Ca2+-ATPase or to other related enzymes such as mammalian kidney (Na,K)-ATPase or gastric mucosal (H,K)-ATPase.
Kirley
T L
TL
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Ohio 45267-0575.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D055598:Chemical Phenomena; D002621:Chemistry; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D003208:Concanavalin A / Q000494:pharmacology; D003488:Cyanogen Bromide; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009132:Muscles / Q000201:enzymology / Q000648:ultrastructure*; D010447:Peptide Hydrolases / Q000378:metabolism; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology
D000596:Amino Acids; D003208:Concanavalin A; D010447:Peptide Hydrolases; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D004047:Diethyl Pyrocarbonate; D003488:Cyanogen Bromide