Comparative study of the kinetic and structural properties of monomeric and oligomeric forms of sarcoplasmic reticulum ATPase.
Sarcoplasmic reticulum (SR) isolated from rabbit muscle was treated with N-ethyl-maleimide (NEM) to specifically inhibit the dephosphorylation step of the Ca2+,Mg2+-dependent ATPase reaction. However, when this membrane was solubilized with dodecyl octaethyleneglycol monoether (C12E8), rapid decomposition of the phosphoenzyme (EP) was observed both in the absence and presence of Mg2+. When the detergent was removed from the reaction mixture, the inhibition of EP decomposition by NEM was observed again. These results support our previous suggestion (1,2) that in the presence of high concentrations of C12E8, EP may be hydrolyzed to produce P1 in a manner different from the reaction in the native SR ATPase. Gel filtration of the solubilized ATPase was performed in the presence of low concentrations of C12E8 to elute ATPase aggregates of various sizes. Two distinct fractions were selected after column chromatography and their physical and kinetic properties were compared. The molecular weights of the ATPase proteins of these two fractions were determined to be about 150 and 360K daltons with Stokes radii of about 5.5 and 8.0 nm, respectively. The Stokes radii agreed with the values obtained from polarization decay measurement data of N-1-pyrene maleimide (N-1-P)-labeled ATPase aggregates separated on the same column. The rate of EP decomposition was determined for the two column fractions described above. After the addition of EDTA the EP decomposition rate of the smaller-sized ATPase was much higher than the EP decomposition rate of the larger-sized ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D046911:Macromolecular Substances; D004533:Egtazic Acid; D004492:Edetic Acid; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases