Solubilization and Physical Characterization of the Adenylate Cyclase from Rat-Liver Plasma Membranes

Abstract

The aim of the present study was to determine the hydrodynamic parameters of rat liver adenylate cyclase under the optimal conditions of solubilization and to validate the use of physical methods to estimate the partial specific volume of the enzyme detergent complex and the mass of the enzyme itself. This was done by solubilizing four other enzyme activities from the same membrane in parallel (Mg2 +-ATPase, 5'-nucleotidase, y-glutamyltransferase and leucine aminopeptidase). For adenylate cyclase, the best conditions for solubilization were : pretreatment with 10 mM N a F ; use of 0.5% Lubrol PX for 2.5 mg protein/ml; enzymatic assay at 20°C and in the presence of a saturating concentration of substrate. Under these conditions, the yield of solubilization was 100% and the final specific activity was 100 to 150% of that of the membrane enzyme. The other enzymes were optimally solubilized at the same final Lubrol PX concentration. The physical properties of adenylate cyclase have been determined. These are : sedimentation coefficient,

Hydrodynamic characterization of the photoaffinity-labeled insulin receptor solubilized in Triton X-100

“…The use of a combination of gel filtration and sucrose density gradient centrifugation to determine the molecular weight of a protein in a mixture of proteins was first suggested and applied by Siegel & Monty (1966) and has since been applied to several solubilized membrane proteins (e.g., Neer, 1974;Stengel & Hanoune, 1979;Costrini et al, 1979). When applied to detergent-solubilized proteins, however, the method suffers from the inherent assumption, pointed out by Tanford et al (1974), that the molecular parameters determined (a, 520,w.…”