Glycerol formation after the breaking of dormancy of Phycomyces blakesleeanus spores

Abstract

The breaking of dormancy of Phycomyces blakesleeanus spores by a heat shock was followed by a transient production of glycerol, which culminated within 5 -10 min and was terminated at 20 min. Extracts of spores contained a magnesium-dependent glycerol-3-phosphatase active on both L-glycerol 3-phosphate and dihydroxyacetone phosphate but having more affinity for the first substrate than for the second. In extracts from dormant spores, the phosphatase was profoundly inhibited by physiological concentrations of inorganic phosphate, which induced cooperativity for the substrate, whereas the enzyme from heat-activated spores was much less inhibited and this difference in kinetic properties persisted after gel filtration of the enzymic preparation. When measured at 1 mM phosphate and 0.1 mM glycerol 3-phosphate, the phosphatase activity was undetectable in dormant spores, increased sharply during the heat treatment and the following 5 min at 25"C, then fell again to a low value by 20 min. A similar transient activation of the enzyme was observed following the breaking of dormancy by incubation of the spores in the presence of 0.1 M ammonium acetate. Incubation of a cell-free extract or of the partially purified glycerol-3-phosphatase in the presence of ATP-Mg and the catalytic subunit of cyclic-AMP-dependent protein kinase released the enzyme from inhibition by phosphate and endowed it with the same kinetic properties as did the heat treatment of the spores. It appears therefore most likely that phosphorylation of glycerol-3-phosphatase by cyclic-AMP-dependent protein kinase causes its activation and that this transient process explains the equally transient formation of glycerol by the spores after the heat shock.The breaking of dormancy of Phycomyces blakesleeanus spores by a heat shock is directly followed by a 15 -30-min period, called the critical period [l], during which the spores need to be incubated in the presence of glucose or 6-deoxyglucose in order to germinate eventually. This period is characterized by several transient metabolic changes, including the activation of trehalase [2], the formation of hexose 6-phosphates and of fructose 2,6-bisphosphate [l], the formation of cyclic AMP [3] and the production of about 300 pmol glycerol/g spores [4]. On the other hand, a link between the transport of glucose, the formation of cyclic AMP as well as of fructose 2,6-bisphosphate and the activation of trehalase has been recently established in Saccharomyces cerevisiae [5] suggesting that all these metabolic transformations could be connected with cyclic AMP.The present work was undertaken in order to investigate the respective roles of cyclic AMP and of fructose 2,6-bisphosphate in the formation of glycerol which is known to occur during the first minutes after spore activation. It led to the discovery of an interconvertible glycerol-3-phosphatase, which is a substrate for cyclic-AMP-dependent protein kinase,

Purification and properties of an NADPH-dependent dihydroxyacetone reductase fromPhycomycesspores

“…Indeed, the enzyme should represent > 1% of the soluble spore protein. This is consistent with the very high rate of glycerol synthesis in the germinating spores [1,2]. The final specific activity was much higher than that of the purified D. parva enzyme [12] and comparable to that from A. niger [9].…”

“…The equilibrium constant [11,12], the pH curve, the high g m for glycerol and NADP ÷ as well as the fact that NADPH rather than NADH is the coenzyme strongly suggest that in vivo the enzyme is involved in the synthesis of glycerol from dihydroxyacetone and not in the utilization of glycerol. The very high specific activity of the enzyme in the crude spore extracts is consistent with a role, together with glycerolphosphate: NAD + oxydoreductase in the synthesis of large amounts of glycerol known to be produced during early germination [1,2]. Since Phycomyces glycerol phosphatase also attacks dihydroxyacetone phosphate [2], the spores could use both NADPH from the shunt and NADH from glycolysis as reducing equivalents in the rapid synthesis of glycerol.…”

“…The reduction of the glycerol precursor dihydroxyacetone phosphate can be performed on the phosphorylated product, as in yeast [3] and probably also in the green alga Dunaliella [4]. Since the glycerol-3-phosphatase from Phycomyces, which is activated during early germination, also attacks dihydroxyacetone phosphate at a similar rate [2], an alternative route for glycerol synthesis is available if dihydroxyacetone can be reduced in the spores. NAD+-dependent glycerol-2-dehydrogenases (producing dihydroxyacetone, E.C.…”

“…Germinating spores of P. blakesleeanus produce large amounts of glycerol during early germination [1,2]. The reduction of the glycerol precursor dihydroxyacetone phosphate can be performed on the phosphorylated product, as in yeast [3] and probably also in the green alga Dunaliella [4].…”

Molecular characterization of the Aspergillus nidulans treA gene encoding an acid trehalase required for growth on trehalose

“…Activation of the glycerol-3-phosphatase together with the transient accumulation of intracellular glycerol has already been observed during germination of spores of Phycomyces blakesleeanus (Van Schaftingen and Van Laere, 1985). In this species, as in S. cerevisiae, the rapid disappearance of the glycerol pool appears to be mediated by efflux into the extracellular medium (Van Schaftingen and Van Laere, 1985;Luyten et al, 1995). Efflux of glycerol during A. niger growth has been observed (Witteveen and Visser, 1995) and might also be responsible for the disappearance of the glycerol pool during the germination of A. nidulans conidiospores.…”