Preparation and Partial Characterization of Catalytically Active Tryptic Derivatives of Phosphorylase b

Abstract

Abstract In the presence of 5 mm spermine, glycogen phosphorylase b may be activated by IMP to 70% to 90% of AMP activation. Incubation of the enzyme with trypsin results in a differential loss in IMP and AMP activation of the enzyme. The time course of the trypsin effect on the nucleotide activation is biphasic: a rapid reduction in the ratio of IMP to AMP activation from 0.7 to 0.2, due to a preferential loss in IMP activation, followed by a gradual increase in this ratio to approximately 0.7, due to a preferential loss in AMP activation. The biphasic curve suggests that at least two derivatives, phosphorylases bt and bt' are produced. In contrast to phosphorylase b, the derivatives are not stimulated by spermine. In the absence of spermine, kinetic properties of phosphorylase b and bt are similar in that they are preferentially activated by AMP. This is because AMP, but not IMP, can enhance the apparent affinity of these enzyme species toward glucose 1-phosphate, whereas both nucleotides can enhance Vmax. Phosphorylase bt' however, is activated by the two nucleotides to approximately the same extent since only Vmax is affected by either of the nucleotides. While sedimentation velocity experiments indicate that the molecular weights of the derivatives are similar to that of phosphorylase b, polyacrylamide gel electrophoretic analysis shows clear distinctions among the three derivatives. Sodium lauryl sulfate gel electrophoresis shows that phosphorylases b and bt have similar subunit structure in that they both contain 2 subunits with molecular weights of about 90,000. The sodium lauryl sulfate gel pattern of phosphorylase bt' shows two protein bands with molecular weights 43,500 and 27,000. Gel filtration on Sephadex G-75 in the presence of sodium lauryl sulfate indicates the existence of additional polypeptides with molecular weights of a few thousands in phosphorylase bt'. These peptides constitute 20% of the total proteins. End group analyses show that the NH2 and COOH termini of phosphorylase bt are glycine and arginine, respectively. Several different amino acids at varying amounts have been found to be the NH2 termini of phosphorylase bt'. The results suggest that phosphorylase bt' contains more than one molecular species.