Adenylate Cyclase from Brevibacterium liquefaciens

Abstract

Abstract Adenylate cyclase from Brevibacterium liquefaciens was purified to apparent homogeneity and crystallized. The purification was achieved by conventional procedures starting from crude extracts obtained by the digestion of bacteria with lysozyme. The over-all purification was 4500-fold with a yield of 10%. The purified enzyme was activated by pyruvate, and catalyzed the conversion of 30 µmoles of ATP to adenosine 3' : 5'-monophosphate and PPi per min per mg of protein under the standard assay conditions. The purified enzyme was apparently homogeneous as judged by polyacrylamide gel electrophoreses and immunological criteria. Polyhedral crystals were obtained from an ammonium sulfate solution of the enzyme. A minimum weight average molecular weight of the native enzyme was estimated to be 92,400 by sedimentation equilibrium ultracentrifugation using a partial specific volume of 0.728 ml per g. Gel filtration experiments on columns of Sephadex G-200 indicated a Stokes radius of about 49 A. Ultracentrifugation in sucrose density gradients revealed that the purified enzyme has a sedimentation coefficient of about 4.5 S which was not affected by the presence of pyruvate and Mg2+. On the basis of these physical parameters, a frictional ratio of about 1.6 was calculated. The enzyme was dissociated into subunits by treatment with sodium dodecyl sulfate. Polyacrylamide gel electrophoresis showed that in gels containing 0.1% sodium dodecyl sulfate, the two subunits had the same molecular weight of 46,000 and in gels containing 8 m urea, the denatured enzyme migrated as a single species. These results indicated that the native enzyme consists of two polypeptide chains, identical or very similar in size and charge. Certain other properties of the enzyme are also described.