title: 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 residues 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. cinerae (the decapeptide 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 Pi (a-, ab- and b-form). Phosphorylase a exhibits a more or less hyperbolic response to AMP and needs 70 microM AMP for maximal stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Zoology Department, University of Cape Town, Rondebosch, Republic of South Africa.
authors.forename: G
authors.initials: G
authors.lastname: Gäde
mesh_terms: D000273:Adipose Tissue / Q000201:enzymology; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D003058:Cockroaches / Q000201:enzymology*; D004789:Enzyme Activation; D007301:Insect Hormones / Q000494:pharmacology*; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D009479:Neuropeptides / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology*; D010762:Phosphorylase a / Q000302:isolation & purification / Q000378:metabolism; D010763:Phosphorylase b / Q000302:isolation & purification / Q000378:metabolism; D006005:Phosphorylases / Q000302:isolation & purification* / Q000378:metabolism; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives; D013045:Species Specificity
chemical_list: D007301:Insect Hormones; D007527:Isoenzymes; D009479:Neuropeptides; D009842:Oligopeptides; C065316:Tem-HrTH; D002110:Caffeine; C043192:glutamyl-valyl-asparaginyl-phenylalanyl-seryl-prolyl-asparaginyl-tryptophanamide; D010762:Phosphorylase a; D010763:Phosphorylase b; D006005:Phosphorylases; D011761:Pyrrolidonecarboxylic Acid