Properties of digitonin-solubilized calmodulin-dependent guanylate cyclase from the plasma membranes of Tetrahymena pyriformis NT-1 cells.
Calmodulin-dependent guanylate cyclase from Tetrahymena plasma membranes was solubilized in about a 22% yield by using digitonin in the presence of 0.2 mM CaCl2 and 20% glycerol. The detergent, when present in the assay at concentrations above 0.05%, diminished the basal and calmodulin-stimulated activity of the enzyme. Guanylate cyclase solubilized with digitonin was eluted from DEAE-cellulose with 200 mM KCl in a yield of 50%. Properties of the solubilized enzyme were similar to those of the native membrane-bound enzyme. The Kms for Mg-GTP and Mn-GTP were 140 and 30 microM, respectively. The enzyme required Mn2+ for maximum activity, the relative activity in the presence of Mg2+ being 30% of the activity with Mn2+. The solubilized enzyme retained the ability to be activated by calmodulin, with its extent being reduced as compared to the membrane-bound enzyme. The presence of a Ca2+-dependent calmodulin-binding site on the solubilized enzyme was shown by the Ca2+-dependent retention of the enzyme on a calmodulin-Sepharose-4B column.
D000818:Animals; D002122:Calcium Chloride / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology*; D002462:Cell Membrane / Q000201:enzymology; D002845:Chromatography; D003902:Detergents / Q000494:pharmacology; D004072:Digitonin / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D006162:Guanylate Cyclase / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D012995:Solubility; D013769:Tetrahymena pyriformis / Q000201:enzymology*
D002147:Calmodulin; D003902:Detergents; D008345:Manganese; D006162:Guanylate Cyclase; D008274:Magnesium; D004072:Digitonin; D002122:Calcium Chloride