title: Distinguishing between functional monomeric and oligomeric complexes of the Ca,Mg-ATPase in sarcoplasmic reticulum.
abstract: Techniques are described for using blocking agents to distinguish between enzymes which are functional monomers and oligomers. To achieve this distinction the blocking agent must react exclusively at the active site with a stoichiometry of one mole of site per mole enzyme. The effect of the blocking agent on enzymatic activity in oligomers of n = 2 and 4 are described and the optimal degree of blocking is considered for tests of enzyme activity at saturating and less than saturating substrate concentrations. For saturating concentrations and a dimer the distinction between dimer and monomer is best observed with 50 per cent of sites blocked. For a tetramer the distinction is best made at higher degrees of blockade. The use of saturating substrate concentrations is thus limited to small oligomers. If nonsaturating substrate concentrations are used and normalized double reciprocal plots of the dependence of enzyme activity on substrate concentrations are made then the distinction between monomer and oligomer can readily be made for dimers, tetramers, and higher n-mers. The principles developed to distinguished monomeric from oligomeric enzymes are applied to published data obtained with the Ca Mg-ATPase of sarcoplasmic reticulum. Fluorescein isothiocyanate is the blocking agent. Plots of the published data support both the monomeric and tetrameric models for allosteric regulation with the preponderance of the data supporting the monomeric model.
mesh_terms: D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000032:analysis* / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000032:analysis* / Q000378:metabolism; D004789:Enzyme Activation; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
chemical_list: D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases