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

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, γ-glutamyltransferase and leucine amino-peptidase). For adenylate cyclase, the best conditions for solubilization were: pretreatment with 10 mM NaF; 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, 6.8 S; Stokes radius, 7 nm; partial specific volume, 0.82 ml/g; mass, 303000 g/mol; f/f0, 1.5. The high partial specific volume measured in the presence of detergent indicates that the enzyme binds a large quantity of detergent (220 mol Lubrol PX/mol protein). If one assumes a partial specific volume of the enzyme itself of 0.735 ml/g, its mass may be estimated as 183000 g/mol. It would be 207000 g/mol if the partial specific volume of the protein was assumed to be 0.76 ml/g. These calculations are validated by the two following lines of evidence: adenylate cyclase is clearly separated from the four other enzyme activities tested in gel chromatography and sucrose gradient, and does not seem to be part of an aggregated complex. Furthermore, the molecular masses calculated for 5′-nucleotidase (143000), leucine aminopeptidase (259000) and γ-glutamyltransferase (103000), assuming a partial specific volume of 0.71 (as might be expected for glycoproteins), are in agreement with the values reported in the literature. The size and detergent-binding properties of adenylate cyclase from rat liver plasma membranes are similar to those reported for adenylate cyclase from S 49 lymphoma cells and bovine cerebral cortex. Therefore, rat liver adenylate cyclase belongs to the class of cyclases that bind a large amount of detergent and behave as intrinsic membrane proteins.