[93] Phosphorylase phosphatase from rabbit muscle

From results of disc gel electrophoresis, the purified fraction is not homogeneous; direct assays of the bands indicate that the phosphatase represents less than 50% of the purified material.

Abstract

This chapter discusses the synthesis of phosphorylase phosphatase from rabbit muscle. Phosphorylase phosphatase catalyzes the conversion of phosphorylase a to phosphorylase b with the release of inorganic phosphate. The reaction may be followed by the disappearance of phosphorylase a activity or by release of radioactive inorganic phosphate from 32P-labeled phosphorylase a. The reagents used, procedure followed, and the steps involved in the purification are also described in the chapter. Two procedure followed are disappearance of phosphorylase a activity (indirect method) and release of radioactivity (direct method). The purified fraction is enriched about 2000-fold from the crude extract and has a Km for phosphorylase a of about 3 × 10-6M. The molecular weight as determined by sucrose density gradient centrifugation is approximately 50,000. From results of disc gel electrophoresis, the purified fraction is not homogeneous; direct assays of the bands indicate that the phosphatase represents less than 50% of the purified material. Crude preparations of the enzyme are activated threefold by small amounts of trypsin and inhibited by adenosine monophosphate (AMP) at concentrations as low as 10-5 M.