Glycogen Phosphorylase in the Fat Body of Two Cockroach Species, Periplaneta americana and Nauphoeta cinerea: Isolation, Partial Characterization of Three Forms and Activation by Hypertrehalosaemic Hormones

Abstract

The presence of endogenous phosphorylase kinase and phosphorylase phosphatase in crude extracts of fat bodies from the cockroaches Nauphoeta cinerea and Periplaneta americana is demonstrated in vitro by activation/inactivation of glycogen phosphorylase under appropriate conditions. Fractionation of fat body extracts of both cockroach species on an anion-exchange medium results in the elution of three peaks with phosphorylase activity. According to their AMP dependency these activity peaks are designated as phosphorylase b (inactive without AMP), phosphorylase ab (active without AMP, but several times stimulated with AMP) and phosphorylase a (active without AMP). It is shown chromatographically that incubating crude extracts of fat bodies from both cockroaches, under conditions where the phosphorylase kinase is active, results in all phosphorylase b being converted to the ab- or a-form, whereas under conditions where the phosphorylase phosphatase is active all phophorylase a is converted to the ab- or b-form. Endogenous phosphorylase kinase of N. cinerea crude fat body extract can convert vertebrate phosphorylase b into the a-form, and, conversely, vertebrate muscle phosphorylase kinase and phosphorylase phosphatase, respectively, are able to convert partially purified N. cinerea phosphorylase ab or b and the ab- und a-form, respectively. In resting cockroaches most of the phosphorylase activity resides in the b-form and only a small fraction (10%) in the a-form, whereas between 26% (N. cinerea) and 35% (P. americana) occurs in the ab-form. Injection of endogenous hypertrehalosaemic peptides into N. cinerea (the deca- peptide Bld-HrTH) or P. americana (the two octapeptides Pea-CAH-I and II) causes interconversion of phosphorylase; after injection, mainly (60%) phosphorylase a is present, while 25% and 15% exists in the ab- und b-form, respectively. Purification of the three phosphorylase forms from N. cinerea is achieved by anion-exchange chromatography on DEAE-Sephacel followed by affinity chromatography on AMP-Sepharose. The final specific activities are 2.1, 6.9 and 27.2 U/mg protein for the a-, ab- und b-form. The molecular mass of the active molecules on gel filtration is between 173, 000 and 177, 000, and SDS gel electrophoresis reveals a subunit mass of 87, 100, suggesting a homodimeric structure for all three forms. Kinetic studies show hyperbolic saturation curves for the substrates glycogen and Pi? respectively, with KM-values of 0.021, 0.019 and 0.073% for glycogen and 8.3, 6.3 and 17.9 mM for Pj (a-, ab- and b-form). Phosphorylase a exhibits a more or less hyperbolic response to AMP and needs 70 jim AMP for maximal stimulation. The kinetics for the ab- and b-forms are sigmoidal and maximal activities are displayed at about 3 mM (half-maximum activation as calculated from Hill plots are 55 and 280 pM for the ab- und b-form, respectively). Caffeine is a strong inhibitor of the b-form, but has only a slight inhibiting effect (10-20%) on the ab- and a-form in the presence of AMP. © 1991, Verlag der Zeitschrift für Naturforschung. All rights reserved.