Bernd Wurster

Cyclic GMP in Dictyostelium discoideum Oscillations and pulses in response to folic acid and cyclic AMP signals

respond chemo- tactically to.cyclic AMP [l] and to folic acid [3]. Cyclic AMP is a most efficient attractant for cells of the aggregation phase [2], folic acid is more effective with preaggregation cells [4]. Both cyclic AMP and folic acid stimulate also cell development from the preaggregation phase to the aggregation competent state [S-8]. During this process the cells acquire the capacity to synthesize cyclic AMP periodically, and to release it into the extracellular space in form of pulses [9] . The administration of cyclic AMP or folic acid pulses accelerates the onset of sustained oscillations [5,8] . The cyclic-AMP induced responses are known to be mediated by cell-surface receptors [lo-121. The intermediate steps in signal processing from the receptors to the various intracellular targets are unknown. In the present communication we show that upon stimulation of early preaggregation cells with folic acid a rapid increase of the cyclic GMP concentration is induced. A biphasic increase of cyclic GMP is observed in late preaggregation cells stimulated by cyclic AMP. The second cyclic GMP peak is suc- ceeded by a cyclic AMP peak known to be based on the activation of adenylate cyclase [ 131. Free-running oscillations of cyclic GMP were observed together with the periodic formation of cyclic AMP pulses, and the cyclic GMP peaks seemed to occur slightly in advance of the cyclic AMP peaks. 2.

Cyclic GMP regulation and responses of Polysphondylium violaceum to chemoattractants

In cells of the cellular slime mold Polysphondylium violaceum an attractant, which is released during the aggregation stage, causes a transient rise of the cyclic GMP concentration. Cells of this organism develop in shaken suspensions after they have finished growth. Cell development is not accompanied by an increase in the EDTA stability of cell adhesion. Both the developmental regulation and the specificity of chemotactic responses is reflected in the light scattering patterns recorded in cell suspensions: Folic acid causes a strong response in early preaggregation cells and the Polysphondylium attractant does the same in aggregation competent cells, whereas cyclic AMP is inactive in both stages.

Glucose-6-phosphate-1-epimerase from baker's yeast. A new enzyme

Salas et al. [ 1] reported that phosphoglucose isomerase from yeast not only catalyses the isomerization of glucose-6-phosphate to fructose-6-phosphate but also the conversion of a-D-glucopyranose-6-phosphate to the aldehyde form of glucose-6-phosphate. Also Carlson et al. [2] could show that this enzyme accelerates the mutarotation of/3-D-glucopyranose-6sulphate. Investigating this intrinsic anomerase activity of PGI [3] in a quantitative way we discovered another enzyme in the yeast cell which, at the branch point of glucose metabolism, catalyses the equilibrium of the anomeric forms of glucose-6-phosphate. The separation and partial characterization of this glucose6-phosphate-1-epimerase will be described in this paper.

Tautomeric and anomeric specificity of allosteric activation of yeast pyruvate kinase by D-fructose 1, 6-bisphosphate and its relevance in D-glucose catabolism.

In aqueous solution Dfructose 1,6-bisphosphate (FBP)? exists as equilibrium mixture of 20 + 4% a-D-fructofuranose 1,6-bisphosphate, 80 f 10% /3-Dfructofuranose 1,6-bisphosphate, and less than 1.5% of the open chain [l-4] . The two anomeric forms are spontaneously interconvertible via the open chain tautomer. The first order rate constant for the ring opening of the o!-D-fructofuranose 1,6-bisphosphate is 0.55 set-’ at pH 7.6 and 25°C [4]. The terms ‘tautomeric’ and ‘anomeric’ are used here according to the definitions given in a previous report [5]. D-fructose 1,6-bisphosphate takes part in four reactions of D-glucose metabolism: it is the product of D-fructose-6-phosphate kinase, the substrate of

Anomeric specificity of fructosediphosphate aldolase E.C.4.1.2.13 from rabbit muscle

Abstract Fructosediphosphate aldolase from rabbit muscle is shown to accept β-D-fructofuranose-1,6-diphosphate as substrate, whereas α-D-fructofuranose-1,6-diphosphate can only be cleaved by the enzyme after a spontaneous change of configuration. The first order velocity constant of the spontaneous reaction was computed to be 0.55 sec −1 (at 25° C, pH 7.6). The equilibrium mixture of D-fructose-1,6-diphosphate was computed to 26% α- and 74% β-D-fructofuranose-1,6-diphosphate.

Anomeric specificity of enzymes of D-glucose metabolism

Aldose 1-epimerase (EC 5.1.3.3); Fructosediphosphatase, D-fructose-l ,6-bisphosphate l-phosphohydrolase (EC 3.1.3.11); Fructosediphosphate aldolase, D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase (EC 4.1.2.13); Fructosep Galactokinase, ATP:D-galactose 1-phosphotransferase (EC 2.7.1.6); Galactose dehydrogenase, D-galactose: NAD+ 1-oxidoreductase (EC 1.1.1.48); Glucokinase, ATP: D-glucose 6-phosphotransferase (EC 2.7.1.2); Glucose dehydrogenase, p-D-glucose: NAD(P)+ l-oxidoreductase (EC 1.1.1.47); Glucose oxidase, p-D-glucose: oxygen oxidoreductase (EC 1.1.3.4); Glucose-6-phosphatase, D-glucosedphosphate phosphohydrolase (EC 3.1.3.9); Glucosedphosphate dehydrogenase, D-glucosep Glucosed-phosphate 1-epimerase, not listed; Glucosephosphate isomerase, D-glucosep Hexokinase, ATP:D-hexose 6-phosphotransferase (EC 2.7.1.1); Mannosephosphate isomerase, D-mannosed-phosphate ketol-isomerase (EC 5.3.1.8); Phosphoglucomutase, a-D-glucose-1,6_bisphosphate: cY-D-glucose-l-phosphate phosphotransferase (EC 2.7.5.1); p-Phosphoglucomutase, not listed; Xylose isomerase, D-xylose ketol-isomerase (EC 5.3.1.5).

Enzyme-catalyzed anomerization of D-glucose-6-phosphate

Recently we reported the discovery, of the enzyme glucose-6-phosphate 1-epimerase in bakers yeast [1]. This enzyme catalyzes the equilibration of the anomeric forms of D-glucose-6-phosphate at the branch point of D-glucose metabolism. In addition following the experimental results of Salas et al. [2] and Carlson et al. [3], it was shown, that glucosephosphate isomerase catalyzes not only the isomerization of aand /3-D-glucopyranose-6-phosphate to t~and ~-D-fructofuranose-6-phosphate [4, 5] but also the anomerization of t~to/3-D-glucopyranose-6-phosphate [4, 5] and the anomerization of ctto/3-D-fructofuranose-6phosphate [5]. From these observations arose the questions whether glucose-6-phosphate 1-epimerase is present in other organisms as well, and whether glucosephosphate isomerase from other organisms will also catalyze the anomerization of D-glucose-6-phosphate. In this paper the results of an investigation of the occurrence of enzyme catalyzed anomerization of D-glucose-phosphate in E. coli, Rhodotorula gracilis, potato tubers, rat muscle, rat liver and rat kidney are described. In E. coli and Rhodotorula gracilis glucose-6-phosphate 1-epimerases with molecular weights of about 30 000 were detected, and in potato tubers two glucose-6-phosphate 1-epimerases with molecular weights of about 30 000 and 45 000 respectively were discovered. In rat muscle, rat liver and rat kidney the occurrence of glucose-6-phosphate 1-epimerase could not be demonstrated. Glucosephosphate isomerases from all six biological sources catalyze the anomerization of D-glucose-6-phosphate. 2. Materials and methods

[12] Quantitative determination of the anomerase activity of glucosephosphate isomerase from baker's yeast

Publisher Summary Glucosephosphate isomerase from yeast not only catalyzes the isomerization of D-glucose 6-phosphate to D-fructose 6-phosphate, but also the anomerization of D-glucose 6-phosphate, 1-3 D-glucose 6-sulfate, 4 D-fructose 6-phosphate, 3 and D-mannose 6-phosphate. This chapter discusses the quantitative determination of the anomerase activity of glucosephosphate isomerase from bakers yeast. Because of the specificity of glucose-6-phosphate dehydrogenase for β-D-glucopyranose 6-phosphate, the spontaneous and enzyme-catalyzed anomerization of α- to β-D-glucopyranose 6-phosphate can be determined quantitatively in preparations of glucosephosphate isomerase, which contain very little activity of the enzymes: (1) hexokinase, which is used to produce a constant reaction velocity of the test system; (2) ATPase, which causes hydrolysis of the substrate ATP during the incubation period; (3) aldose 1-epimerase, which gives rise to anomerization of α- to β-D-glucopyranose; (4) 6-phosphogluconolactonase, which accelerates the hydrolysis of 6-phosphogluconic acid δ-lactone to 6-phosphogluconate, which inhibits glucosephosphate isomerase; (5) 6-phosphogluconate dehydrogenase, which causes a consecutive reduction of NADP+; (6) fructose-6-phosphate kinase, which traps D-fructose 6-phosphate; and (7) glucose-6-phosphate 1-epimerase2. The reactions are initiated by the addition of freshly dissolved α-D-glucopyranose. In the hexokinase reaction, α-D-glucopyranose 6-phosphate is produced with constant reaction velocity; in the following anomerization reaction, spontaneous and catalyzed by glucosephosphate isomerase, α-D-glucopyranose 6-phosphate is converted to β-D-glucopyranose 6-phosphate, and finally β-D-glucopyranose 6-phosphate is oxidized in the glucose-6-phosphate dehydrogenase reaction. The isomerase activity of glucosephosphate isomerase is determined under identical conditions of buffer and temperature at 2 mM D-fructose 6-phosphate, 2 mi NADP+, and 10 units of glucose-6-phosphate dehydrogenase per milliliter.