Fructose 1, 6-diphosphate oxidation

The long-known stimulating effect of mono- and polynitro com-pounds on the onset of fermentation in yeast maceration juice has been reinvestigated by Vandendriessche. The induction time is shortened significantly by 2,4- or 2,5-dinitrophenol, while 2,6-dinitro-phenol did not show such an effect. The influence is evident when using as substrates the fermentable hexoses and D-fructose-6-phosphate, but not hexose diphosphate. According to MarkoviCev a stimulation of the oxidation processes can be proved thereby. It is probable that these effects are related to the known phytochemical reduction of nitro compounds (see pp. 98 and 99). 

The ozone treatment had no significant effect on the vitro NR activity, indicating that it did not inactivate the NR protein (Table III), Leaf extracts that would couple the oxidation of fructose-1, 6-diphosphate to nitrate reduction were prepared from leaves exposed to either 0 or 980 yg/m ozone ( ), Ozone depressed the in vitro coupled NR activity 58% (Table III), indicating that the observed ozone depression of nitrate reduction in the vivo leaf disk assay resulted from a depression in the rate of NADH formation by GPD,... 

Z F6P + ATP + 2 NADH + H+ <-> 2 glycerol-3-phosphate + ADP + 2 NAD+ where F6P is fructose-6-phosphate, FDP is fructose-1,6-diphosphate, DHA-P is dihydroxyacetone phosphate, TIM is triosephosphate isomerase, and GDH is glycerol-3-phosphate dehydrogenase. The oxidation of NADH is a measure of the 6-PFK activity and is determined photometrically (decrease of OD per minute) [4]. 

Another assay for phosphoffuctokinase involves converting the fructose 1,6-diphosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate with aldolase, equilibrating the triosephosphates with triosephosphate isomerase, and then measuring the production of NADH on the oxidation of the glyceraldehyde phosphate by glyceraldehyde 3-phosphate dehydrogenase. 

L-Lactate dehydrogenase (l-LDH, EC 1.1.1.27) catalyzes the reduction of pyruvate to (S)-lactate with a simultaneous oxidation of NADH. l-LDH is found in all higher organisms. There are two kinds of l-LDHs enzymes from one group are activated by fructose 1,6-diphosphate while the other group stays independent [71]. l-LDH is highly selective for pyruvate, short-chain 2-keto acids and phenylpyruvic acid [80]. All bacterial NAD+-dependent LDHs form lactate from pyruvate in vivo, and there is no evidence at all that they catalyze the other direction as well. The equilibrium constant lies far on the direction of lactate formation, and thus the reaction catalyzed by bacterial LDHs can be considered almost irreversible. LDHs from some lacto-bacilli like Lactobacillus fermentum or L. cellobiosus show no or just poor reaction with lactate [71], whereas mammalian LDHs can be considered as reversible [71]. Well characterized l-LDHs are summarized in Table 2. 

Because LD is present in excess, the rate of NADH oxidation is limited by the activity of PK. The reaction rate is measured by the rate of decrease in absorbance at 340 nm. Assays are performed at low substrate concentration with and without the addition of fructose-1,6-diphosphate, because some PK variants associated with hemolysis have atypical reaction kinetics (and thus may exhibit normal activity at high substrate concentrations but lower than normal activity at lower substrate concentrations) or may show absence of enhancement by fructose-1,6-diphosphate, the allosteric activator of PK. 

Flora F-6-P = fructose-6-phosphat, NADP+/NADPh = nicotinamide adenosindinucleotide phosphate in oxidized and reduced form (under physiological conditions NADPH2 is deprotonized into NADPH + H+), PGA = phosphoglyceric acid, R-5-P = ribulose-5-phosphate, R-1.5-DP = ribulose-1,5-diphosphate,... 

A number of intermediates common to both the hexose monophosphate shunt and the glycolytic pathway are glucose-6-phosphate, fructose-6-phosphate, fructose-6,1-diphosphate, and triose phosphate. Thus, the two pathways can be expected to compete for intermediates, and, indeed, when a reconstituted glycolytic system made of purified enzymes is added to the reconstituted hexose monophosphate shunt, glucose oxidation by the shunt is inhibited by glycolysis. 

Whereas sodium participates in metabolism mainly by its cationic properties, potassium is more directly involved in metabolism. Potassium stimulates the activity of a specific enzyme— pyruvic kinase—and is required for the phosphorylation of fructose-1-phosphate to fructose-1,6-diphosphate. Similarly, potassium stimulates acetyl kinase activity. Many alterations in the bioenergetic pathways of the cell are accompanied by changes in the intracellular concentration of potassium. After insulin administration, some of the potassium of the extracellular fluid is transferred inside the cells. During oxidative phosphorylation, potassium accumulates inside the mitochondria, and dinitrophenol uncouples the ion penetration and the oxidation. 

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