Removal of the growth medium and resuspension of Blastocladiella emersonii vegetative cells in a sporulation medium resulted in an abrupt fall of fructose 2,6-bisphosphate concentration to about 2% of its initial value within 10 min. The concentrations of hexose 6-phosphate and of fructose 1,6-bisphosphate also decreased by, respectively, three and tenfold over the same period. All these values remained at their low level throughout the sporulation phase and during the subsequent germination of zoospores when performed in the absence of glucose. In contrast, the concentration of cyclic AMP was low during the sporulation period and exhibited a transient increase a few minutes after the initiation of germination. Other biochemical events occurring during sporulation were a 70% reduction in glycogen content and the complete disappearance of trehalose. The remaining glycogen was degraded upon subsequent germination of the zoospores. B. emersonii phosphofructo 2-kinase (PFK-2) and fructose-2,6-bisphosphatase (FBPase-2) could not be separated from each other by various chromatographic procedures, suggesting that they were part of a single bifunctional protein. On anion-exchange chromatography, two peaks of PFK-2 and FBPase-2 were resolved. Upon incubation of fractions from the two peaks or of a crude extract in the presence of [2-32P]fructose 2,6-bisphosphate, two radiolabelled subunits with molecular masses close to 90 and 54 kDa were obtained. The labelling of the subunit of higher molecular mass was greater than that of the lower one in extracts prepared in the presence of protease inhibitors and in the first peak of the Mono Q column. PFK-2 and FBPase-2 displayed kinetic properties comparable to those of mammalian enzymes, but no indication of a cyclic AMP-dependent regulation could be obtained. Phosphofructo 1-kinase and fructose-1,6-bisphosphatase from B. emersonii were, respectively, stimulated and inhibited by micromolar concentrations of fructose 2,6-bisphosphate. The physiological significance of these properties is discussed. A simple method for the determination of trehalose is also reported.
AragonJ. J.,
SanchezV.,
BotoL.1986; Fructose 2,6- bisphosphate in Dictyostelium discoideum. Independence of cyclic AMP production and inhibition of fructose-1,6-bisphosphatase. European Journal of Biochemistry 161:757–761
BehrensM. M.,
MaiaJ.C.C.1986; Differentiation-specific activation of cyclic AMP-dependent protein kinase in Blastocladiella emersonii.
. Biochemistry International 12:503–512
BradfordM. M.1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248–254
CzokR.1974; Pyruvate, phosphoenolpyruvate and d-glycerate-2- phosphate. In Methods of Enzymatic Analysis,, 2nd edn.. pp
BergmeyerH. U.
Edited by New York & London: Academic Press;
CourtoisJ. E.,
DemelierJ. F.1966; Repartition de la trehalase chez l’homme et quelques mammifères. Bulletin de la Sociètè de Chimie Biologique 48:277–286
El-MaghrabiM. R.,
ClausT. H.,
PilkisJ.,
FoxE.,
PilkisS. J.1982; Regulation of rat liver fructose-2,6-bisphosphatase. Journal of Biological Chemistry 257:7603–7607
El-MaghrabiM.R.PATE,
MurrayK. J.,
PilkisS. J.1984; Differential effects of proteolysis and protein modification on the activities of 6-phosphofructo 2-kinase/fructose-2,6-bisphospha- tase. Journal of Biological Chemistry 259:13104–13110
FrancoisJ.,
VanSchaftingenE.,
HersH. G.1983; On the mechanism of inhibition of neutral liver fructose-1,6-bisphosphatase by fructose-2,6-bisphosphate. European Journal of Biochemistry 134:269–273
FrancoisJ.,
VanSchaftingenE.,
HersH. G.1984; The mechanism by which glucose increases fructose 2,6-bisphosphate concentration in Saccharomyces cerevisiae. A cyclic AMP-dependent activation of phosphofructokinase 2. European Journal of Biochemistry 145:187–193
FrancoisJ.,
ErasoP.,
GancedoC.1987; Changes in the concentration of cyclic AMP, fructose 2,6-bisphosphate and related metabolites and enzymes in Saccharomyces cerevisiae during growth on glucose. European Journal of Biochemistry 164:369–373
FrancoisJ.,
VanSchaftingenE.,
HersH. G.1988; Characterization of phosphofructokinase 2 and of enzymes involved in the degradation of fructose 2,6-bisphosphate in yeast. European Journal of Biochemistry 171:599–609
GancedoJ. M.,
GancedoC.1971; Fructose-1,6-bisphosphatase, phosphofructokinase and glucose 6-phosphate dehydrogenase from fermenting and non-fermenting yeasts. Archives of Microbiology 76:132–138
HersH. G.,
FrancoisJ.,
VanSchaftingenE.1985; Fructose2,6-bisphosphate versus cyclic AMP in the liver and in lower eucaryotic cells. Current Topics in Cellular Regulation 27:399–410
LamprechtW.,
TrautscholdI.1974; Adenosine-5′-triphos- phate, determination with hexokinase and glucose 6-phosphate dehydrogenase. In Methods of Enzymatic Analysis,, 2nd edn.. pp 2101–2110BergmeyerH. U.
Edited by New York & London: Academic Press;
LangG.,
MichalG.1974; d-Glucose 6-phosphate and d-fructose 6-phosphate. In Methods of Enzymatic Analysis,, 2nd edn.. pp 1238–1242BergmeyerH. U.
Edited by New York & London: Academic Press;
LarondelleY.,
MertensE.,
VanSchaftingenE.,
HersH. G.1986; Purification and properties of spinach leaf phosphofructokinase 2/fructose-2,6-bisphosphatase. European Journal of Biochemistry 161:351–357
LarondelleY.,
CorbineauF.,
DethierM.,
ComeD.,
HersH. G.1987; Fructose 2,6-bisphosphate in germinating oat seeds. A biochemical study of seed dormancy. European Journal of Biochemistry 166:605–610
LodiW. R.,
SonnebornD. R.1974; Protein degradation and protease activity during the life cycle of Blastocladiella emersonii.
. Journal of Bacteriology 117:1035–1042
LovettJ. S.1975; Growth and differentiation of the water mold Blastocladiella emersonii: cytodifferentiation and the role of ribonucleic acid and protein synthesis. Bacteriological Reviews 39:345–404
MacDonaldF. D.,
CsékeC.,
ChouQ.,
BuchananB. B.1987; Activities synthesizing and degrading fructose 2,6-bisphosphate in spinach leaves reside on different proteins. Proceedings of the National Academy of Sciences of the United States of America 84:2742–2747
MichalG.,
BeutlerH. O.1974; d-Fructose 1,6-bisphosphate, dihydroxyacetone phosphate and d-glyceraldehyde 3-phosphate. In Methods of Enzymatic Analysis,, 2nd edn.. pp. 1314–1322BergmeyerH. U.
Edited by New York & London: Academic Press;
PlessmanncamargoE.,
MeuserR.,
SonnebornD.1969; Kinetic analysis of the regulation of glycogen synthetase activity in zoospores and growing cells of the water mold Blastocladiella emersonii.
. Journal of Biological Chemistry 244:5910–5919
SelitrennikoffC. P.,
AllinD.,
SonnebornD. R.1976; Chitin biosynthesis during Blastocladiella zoospore germination: evidence that the hexosamine biosynthetic pathway is posttranslationally activated during cell differentiation. Proceedings of the National Academy of Sciences of the United States of America 73:534–538
SelitrennikoffC. P.,
DalleyN. E.,
SonnebornD. R.1980; Regulation of the hexosamine biosynthetic pathway in the water mold Blastocladiella emersonii: sensitivity to endproduct inhibition is dependent upon the life cycle phase. Proceedings of the National Academy of Sciences of the United States of America 77:5998–6002
SollD. R.,
SonnebornD. R.1971a; Zoospore germination in Blastocladiella emersonii: cell differentiation without protein synthesis?. Proceedings of the National Academy of Sciences of the United States of America 68:459–463
SollD. R.,
SonnebornD. R.1971b; Zoospore germination in Blastocladiella emersonii. III. Structure changes in relation to protein and RNA synthesis. Journal of Cell Science 9:679–699
SuberkroppK. F.,
CantinoE. C.1973; Utilization of endogenous reserves by swimming zoospores of Blastocladiella emersonii.
. Archiv für Mikrobiologie 89:205–221
ToveyK. C.,
OldhamK. G.,
WhelanJ. A.1974; A simple direct assay for cyclic AMP in plasma and biological samples using an improved competitive protein binding technique. Clinica Chimica Acta 56:221–234
VanLaereA.,
VanSchaftingenE.,
HersH. G.1983; Fructose 2.6-bisphosphate and germination of fungal spores. Proceedings of the National Academy of Sciences of the United States of America 80:6601–6605
VanSchaftingenE.,
HersH. G.1981a; Formation of fructose 2.6-bisphosphate from fructose 1,6-bisphosphate by intracellular cyclisation followed by alkaline hydrolysis. European Journal of Biochemistry 117:319–323
VanSchaftingenE.,
HersH. G.1981b; Inhibition of fructose- 1, 6-bisphosphatase by fructose 2,6-bisphosphate. Proceedings of the National Academy of Sciences of the United States of America 78:2861–2863
VanSchaftingenE.,
JettM. F.,
HueL.,
HersH. G.1981; Control of liver 6-phosphofructokinase by fructose-2,6-bisphosphate and other effectors. Proceedings of the National Academy of Sciences of the United States of America 78:3483–3486
VanSchaftingenE.,
HersH. G.1983; Fructose 2,6-bisphosphate in relation with the resumption of metabolic activity in slices of Jerusalem artichoke tubers. FEBS Letters 164:195–200
VanSchaftingenE.,
LedererB.,
BartronsR.,
HersH. G.1982b; A kinetic study of pyrophosphate: fructose 6-phosphate phosphotransferase from potato tubers. European Journal of Biochemistry 129:191–195
VanSchaftingenE.,
OpperdoesF. R.,
HersH. G.1986a; Stimulation of Trypanosoma brucei pyruvate kinase by fructose 2,6- bisphosphate. European Journal of Biochemistry 153:403–406
VanSchaftingenE.,
CoulieP. G.,
VanSnickJ.,
HersH. G.1986b; Reaction of phosphofructokinase 2/fructose-2,6-bisphos- phatase with monoclonal antibodies. A proof of the bifunctionality of the enzyme. European Journal of Biochemistry 159:367–373