Glycogen phosphorylase in Dictyostelium discoideum: Demonstration of two developmentally regulated forms, purification to homogeneity, immunochemical analysis, cAMP induction, in vitro translation, and molecular cloning

Abstract

A key step in the cellular differentiation of Dictyostelium is the degradation of glycogen to provide the precursors for synthesis of the structural end products of development. We have found that the enzyme that initiates this degradative pathway, glycogen phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-glucosyltransferase; EC 2.4.1.1), is developmentally regulated and exists as two forms. During the time course of development, a previously undescribed activity, the "b" form, decreases, while that of the "a" form increases. The "b" form is inactive unless 5'AMP is included in the reaction mixture. The two forms differ in their elution from DE52 cellulose, affinity constants, thermal stability, affinity for 5'AMP Sepharose, subunit molecular weight, and peptide maps. In crude extracts, anti-a antiserum stains a 104-kD protein that is associated with phosphorylase "a" activity and appears late in development, while anti-b antiserum stains a 92-kD protein that is associated with phosphorylase "b" activity and is present throughout development. We have also demonstrated in vitro phosphorylation of the "b" form by an endogenous protein kinase and a corresponding loss of 5'AMP dependence. If intact cells were exposed to exogenous cAMP, "b" activity decreased and was replaced by "a" activity, as well as the 104-kD protein band on SDS-PAGE. In order to determine if the two forms of the enzyme are different gene products, we screened lambda gt11 expression libraries with antibodies against the purified "a" and "b" forms. Three clones were found to be overlapping by Southern analysis. A yeast glycogen phosphorylase cDNA clone (gpy) and a human muscle glycogen phosphorylase clone (HM-11) cross-hybridized with the Dictyostelium inserts, and gpy shared a few common restriction fragments with the Dictyostelium clones on genomic blots. Northern analysis of Dictyostelium total RNA showed that the Dictyostelium inserts and gpy recognize an mRNA of 3.2 kb, while on poly A-enriched RNA, the yeast clone detects preferentially a 3.6-kb message.

Glycogen Phosphorylase and Glycogen Synthase: Gene Cloning and Expression Analysis Reveal Their Role in Trehalose Metabolism in the Brown Planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae)

“…Dodge (de Paula et al 2002) and S. exigua (Tang et al 2012a). GP cDNA was first cloned from human brain (Newgard et al 1988), D. discoideum (Rutherford et al 1988), Escherichia coli (Migula) Castellani & Chalmers (Choi et al 1989), S. cerevisiae (Hwang et al 1989), Dictyostelium (Rutherford et al 1992), D. melanogaster (Gabriella et al 1999), S. exigua (Tang et al 2012a), and O. furnacalis (Guo et al 2016); it was first cloned from human muscle in 1989 and in insects, GP was cloned for the fruit fly D. melanogaster (Tick et al 1999). The analysis of N. lugens GS and GP protein sequences and of the catalytic mechanisms of GS and GP enzymes revealed these were conserved (Fig.…”

SEQUENCING AND CHARACTERIZATION OF GLYCOGEN SYNTHASE AND GLYCOGEN PHOSPHORYLASE GENES FROM Spodoptera exigua AND ANALYSIS OF THEIR FUNCTION IN STARVATION AND EXCESSIVE SUGAR INTAKE

“…Although many GS genes from insects have been cloned or deduced from genomic DNA sequences, there are no reports on the characterization, expression patterns, and function of these genes. GP cDNA has also been cloned from human brain (Newgard et al, ), D. discoideum (Rutherford et al, ), E. coli (Choi et al, ), S. cerevisiae (Hwang et al, ), and Dictyostelium (Rutherford et al, ). This conservation of sequence identity includes the protein sequences and the catalytic mechanisms for enzymes such as GS and GP (Hwang et al, ; Farkas et al, , ).…”

Glycogen Phosphorylase Sequences from the Amitochondriate Protists, Trichomonas vaginalis, Mastigamoeba balamuthi, Entamoeba histolytica and Giardia intestinalis1

“…Although members of this protein superfamily show high amino acid sequence conservation, they exhibit a plethora of different regulatory mechanisms (Buchbinder et al 2001). The mammalian, yeast, and possibly one of the two slime mold ( Dictyostelium discoideum ) GPs are regulated by phosphorylation (Buchbinder et al 2001; Johnson 1992; Rutherford et al 1988), although the mechanism of this regulation differs in the different lineages and is asserted to have arisen in each of them independently (Hudson et al 1993). Plant SPs and the eubacterial MalPs exist only in active form and phosphorylation does not play a role in their regulation (Fukui et al 1982; Preiss, 1982).…”