Glycogen phosphorylase in Dictyostelium discoideum: demonstration of two developmentally regulated forms, purification to homogeneity, immunochemical analysis, cAMP induction, in vitro translation, and molecular cloning.
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.
Rutherford
C L
CL
Biology Dept., Virginia Tech University, Blacksburg 24061.
Naranan
V
V
Brickey
D A
DA
Sucic
J F
JF
Rogers
P V
PV
Selmin
O
O
D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000494:pharmacology*; D004271:DNA, Fungal / Q000235:genetics; D004023:Dictyostelium / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D004790:Enzyme Induction; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D006005:Phosphorylases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics
D004271:DNA, Fungal; D007527:Isoenzymes; D012333:RNA, Messenger; D000242:Cyclic AMP; D006005:Phosphorylases