Non-linear dynamic phenomena in open reconstituted enzyme systems.

The dynamic pattern of an open reconstituted in-vitro enzyme system containing phosphofructokinase, pyruvate kinase and adenylate kinase has been investigated. The approach is experimentally based on a stirred flow-through reactor. Stationary concentrations of phosphofructokinase and pyruvate kinase are maintained by entrapment in polyacrylamide gel particles. The results fit to a theoretical model based on the initial kinetic responses of the enzymes involved. An S-shaped steady state input characteristic for fructose 6-phosphate has been experimentally obtained.     

Self-stabilization of the energy charge in a reconstituted enzyme system containing phosphofructokinase

The self-stabilization of the energy charge and of ATP was investigated in an open reconstituted enzyme system containing phosphofructokinase, pyruvate kinase, adenylate kinase, and glucose-6-phosphate isomerase. The experiments were performed in a stirred flow-through reactor containing gel-entrapped enzymes. The dynamics of the system were analyzed theoretically by a model based on the kinetic properties of the individual enzymes. The energy charge was identified as one of the essential variables of the system. According to the theoretical prediction, homoeostasis of the energy charge was observed experimentally when either the maximal activity of phosphofructokinase, the energy charge of the influx solution of the flow rate through the reaction chamber was varied. It is shown that the efficiency of stabilization of the energy charge is related to the occurrence of alternative stationary states.     

Dynamic properties of in vitro enzyme systems containing phosphofructokinase.

The dynamic properties of a series of in vitro reaction systems with increasing complexity and containing phosphofructokinase as central enzyme have been investigated. An experimental strategy and a principal mathematical treatment was elaborated to search for the minimum requirements with respect to the enzyme composition of a reaction system for generating limit cycle behaviour. As a criterion, such models have been developed which permit experimental realization by application of a specially designed flow-through equipment. In addition to phosphofructokinase, the following enzymes have been stepwise included into the reaction systems composing the Models 1 through 6: pyruvate kinase, adenylate kinase, hexokinase, and glucose 6-phosphate isomerase. It turned out that only a minimum dynamic system containing phosphofructokinase and pyruvate kinase as well as excesses of adenylate kinase and glucose 6-phosphate isomerase for maintaining equilibrium conditions between the respective reacting species, acquires the property of limit cycle behaviour and, hence, to generate sustained self-oscillations. The approach permits to compute the region of the experimentally variable parameters (influx rates of fructose 6-phosphate and ATP, maximum rate of pyruvate kianse) for which self-oscillatory behaviour can be predicted.     

Sustained oscillations in a reconstituted enzyme system containing phosphofructokinase and fructose 1,6-bisphosphatase

In a reconstituted open and homogeneous enzyme system containing phosphofructokinase, fructose 1,6-bisphosphatase, pyruvate kinase, adenylate kinase, and glucose-6-phosphate isomerase sustained oscillations could experimentally be generated. The approach is based on a stirred flow-through reaction chamber. The periodic motions of the reactants are mainly caused by the antagonistic allosteric effects of the adenine nucleotides on the activities of the phosphofructokinase and fructose 1,6-bisphosphatase.     

A hysteretic cycle in glucose 6-phosphate metabolism observed in a cell-free yeast extract

The dynamics of a partial glycolytic reaction sequence which converts glucose 6-phosphate to triose phosphates is described. The study was performed with cell-free extracts from baker's yeast harvested in the logarithmic and stationary growth phases. The experiments are based on a flow-through reactor supplied with the desalted cell-free extract as well as glucose 6-phosphate, ATP and phosphoenolpyruvate. In the reaction system the quasi-irreversible reactions catalyzed by 6-phosphofructo-1-kinase, pyruvate kinase, and fructose-1,6-bisphosphatase are involved. When substrate is supplied continuously, only stable stationary states can be observed. With transient perturbations of the substrate supply, multiple stationary states appear. Cyclic transitions between unique stable stationary states were induced by appropriate changes of the rate of substrate supply. A hysteretic cycle could then be demonstrated when, during reverse transitions, a parameter region of multistability was passed. The presence (in resting yeast) or absence (in growing yeast) of fructose-1,6-bisphosphatase did not significantly influence the dynamic capabilities of the investigated reaction sequence. The kinetic properties of the cell-free extracts fit mathematical models developed for in vitro systems reconstituted from purified enzymes.     

The in situ assay of Candida albicans enzymes during yeast growth and germ-tube formation

Conditions are described for the preparation of permeabilized cells of Candida albicans. This method has been used for the in situ assay of enzymes in both yeast cells and germ-tube forming cells. A mixture of toluene/ethanol/Triton X-100 (1:4:0.2, by vol.) at 15% (v/v) and 8% (v/v) was optimal for the in situ assay of glucose-6-phosphate dehydrogenase in yeast and germ-tube forming cells, respectively. The concentration of toluene/ethanol/Triton X-100 required for optimal in situ activity of other enzymes was influenced by the cellular location of the enzyme, growth phase and morphology. The membrane-bound enzymes (chitin synthase, glucan synthase, ATPase), cytosolic enzymes (glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, pyruvate kinase, phosphofructokinase, alkaline phosphatase, glucosamine-6-phosphate deaminase and N-acetylglucosamine kinase) and wall enzymes (beta-glucosidase and acid phosphatase) were measured and compared to the activity obtained in cell extracts. The pattern of enzyme induction and the properties of the allosteric enzymes phosphofructokinase and pyruvate kinase were measured in situ. Pyruvate kinase in situ was homotropic for phosphoenolpyruvate with a Hill coefficient of 1.9 and a S0.5 of 0.6 mM, whereas in cell extracts, it had a Hill coefficient of 1.9 and a S0.5 of 1.0 mM. The Km for ATP was 1.6 mM in cell extracts and 1.8 mM in permeabilized cells. In situ phosphofructokinase was homotropic for fructose 6-phosphate (S0.5 of 2.3 mM, Hill coefficient of 4.0). The kinetic properties of pyruvate kinase and phosphofructokinase measured in situ or in vitro were similar for both yeast cells and germ-tube forming cells.     

Regulation by glucagon of hepatic pyruvate kinase, 6-phosphofructo 1-kinase, and fructose-1,6-bisphosphatase

Glucagon stimulates gluconeogenesis in part by decreasing the rate of phosphoenolpyruvate disposal by pyruvate kinase. Glucagon, via cyclic AMP (cAMP) and the cAMP-dependent protein kinase, enhances phosphorylation of pyruvate kinase, phosphofructokinase, and fructose-1,6-bisphosphatase. Phosphorylation of pyruvate kinase results in enzyme inhibition and decreased recycling of phosphoenolpyruvate to pyruvate and enhanced glucose synthesis. Although phosphorylation of 6-phosphofructo 1-kinase and fructose-1,6-bisphosphatase is catalyzed in vitro by the cAMP-dependent protein kinase, the role of phosphorylation in regulating the activity of and flux through these enzymes in intact cells is uncertain. Glucagon regulation of these two enzyme activities is brought about primarily by changes in the level of a novel sugar diphosphate, fructose 2,6-bisphosphate. This compound is an activator of phosphofructokinase and an inhibitor of fructose-1,6-bisphosphatase; it also potentiates the effect of AMP on both enzymes. Glucagon addition to isolated liver systems results in a greater than 90% decrease in the level of this compound. This effect explains in large part the effect of glucagon to enhance flux through fructose-1,6-bisphosphatase and to suppress flux through phosphofructokinase. The discovery of fructose 2,6-bisphosphate has greatly furthered our understanding of regulation at the fructose 6-phosphate/fructose 1,6-bisphosphate substrate cycle.     

Inactivation of phosphofructokinase by glucagon in rat hepatocytes.

Kinetic evidence of a time- and dose-dependent inactivation of phosphofructokinase by glucagon in isolated rat hepatocytes is reported. This inactivation, which persists after gel filtration of a cell-free extract on Sephadex G-25 and after 400-fold purification of the enzyme on agarose-ATP, is observed when the enzyme activity is measured at subsaturating concentrations of fructose 6-phosphate, while there is no change in Vmax. Phosphofructokinase inactivation by glucagon parallels the known inactivation of pyruvate kinase L and activation of glycogen phosphorylase alpha. Exogenous cyclic AMP mimics the effect of this hormone. Half-maximal effect for both phosphofructokinase and pyruvate kinase L is caused by a similar dose of glucagon (1 x 10(-10) M). The inactivation of phosphofructokinase by nonsaturating concentration of glucagon is reversed spontaneously within 40 min of incubation and this reversion is accelerated by insulin.     

Decreased phosphofructokinase activity in skeletal muscle of diabetic rats

The activities of phosphofructokinase, aldolase and pyruvate kinase were diminished in extracts from skeletal muscle of streptozotocin diabetic rats, whereas the activities of glucose phosphate isomerase and phosphoglucomutase were not changed. Treatment of diabetic rats with insulin restored the activity of phosphofructokinase to normal. A kinetic study of the partially purified enzyme from normal and diabetic rats showed identical Michaelis constants for ATP and equal sensitivity to inhibition by excess of this substrate. Extracts of quick frozen muscle from diabetic rats had higher levels of citrate (an inhibitor of phosphofructokinase) and lower levels of D-fructose-1,6-bisphosphate and D-glucose-1,6-bisphosphate (activators of this enzyme). The levels of D-fructose-6-phosphate, D-glucose-6-phosphate, ATP, ADP and AMP were the same for the two groups. Our data suggest that the in vivo decrease of phosphofructokinase activity in skeletal muscle of diabetic rats is due to a decrease in the level of the enzymatically active protein as well as to an unfavorable change in the level of several of its allosteric modulators.     

Undifferentiated patterns of key carbohydrate-metabolizing enzymes in injured livers. I. Acute carbon-tetrachloride intoxication of rat.

In acute CCL4 intoxication of rats significantly increased activities of hepatic low-Km hexokinases, glucose-6-phosphate dehydrogenase, phosphofructokinase, aldolase A and pyruvate kinase M2 with concurrently decreased activities of glucokinase, glucose-6-phosphatase, fructose-1,6-diphosphatase, aldolase B and pyruvate kinase L were observed. The resulting enzyme pattern was apparently different from that in dietary induction. Principal component analysis revealed that the degree of enzyme deviation in the injured liver was much greater than that in the regenerating liver after partial hepatectomy and was closer to that in fetal liver or hepatoma tissue.     

The effect of sugar phosphates, phosphoenolpyruvate and adenylic acid on muscle, brain and heart creatine kinases]

The effect of adenylic acid, glucose-6-phosphate, fructose-1,6-diphosphate and phosphoenolpyruvate on creatine kinase isoenzymes (brain extract, muscle and heart extracts and purified muscle enzyme) was studied. These effectors, especially phosphoenolpyruvate, are shown to inhibit in different degree the reaction of ATP formation catalysed by creatine kinase from all tissues. The effectors do not inhibit the creatine phosphate synthesis in extracts, but depress purified creatine kinase. The interrelationship of the creatine kinase system and the key glycolytic enzymes (phosphofructokinase, hexokinase, pyruvate kinase) is discussed.     

Control in vivo of rat liver phosphofructokinase by glucagon and nutritional changes.

Glucagon (250 microgram/kg body wt.) intravenously injected into normal fed rats produces within 5 min a marked inactivation of liver phosphofructokinase, only observed when the enzyme activity is measured at subsaturating concentrations of fructose 6-phosphate. Since half-maximal inactivation is observed at a dose of glucagon of 0.32 microgram/body wt., a dose within the range of the physiological concentrations of the hormone, the inactivation of phosphofructokinase can occur in vivo in response to physiological changes in the concentration of glucagon. In gluconeogenic conditions (starved rats or high-protein-diet-fed rats), there is a marked inactivation of liver phosphofructokinase at subsaturating concentrations of fructose 6-phosphate similar to that found in normal fed rats after glucagon treatment. In these gluconeogenic conditions a 50% decrease in the Vmax. of the enzyme is also observed. No significant changes in phosphofructokinase activity either at subsaturating concentrations of fructose 6-phosphate or in the Vmax. of the enzyme are observed when rats are fed on a high-carbohydrate diet. In the last dietary condition, glucagon treatment produces similar effects to that described in the normal fed rats. Similar results have been obtained in the above condtions for pyruvate kinase L activity when measured at subsaturating concentrations of phosphoenolpyruvate.     

Regulation of fitness in yeast overexpressing glycolytic enzymes: responses to heat shock and nitrogen starvation.

Current models based on the analysis of linear metabolic pathways at steady-state predict that large increases over wild type in the activity of one enzyme will not alter an organism's fitness. This prediction is tested at steps in a highly branched pathway under two conditions known to alter steady-state: heat shock and nitrogen starvation. Saccharomyces cerevisiae transformants overproducing 1 of 4 enzymes in glycolysis (hexokinase B, phosphoglucose isomerase, phosphofructokinase, or pyruvate kinase) were subjected to heat shock in both exponential and stationary phases of growth. In neither phase does enzyme overexpression alter heat shock sensitivity. When starved for nitrogen in acetate medium, transformants overproducing hexokinase, phosphoglucose isomerase, and phosphofructokinase sporulate at the same rate and with the same frequency as cells harbouring only the plasmid vector. Current models therefore correctly predict the relationship between activity and components of fitness for 3 of 4 enzymes. By contrast, cells overexpressing pyruvate kinase sporulate poorly. This defect is not observed among cells transformed with a plasmid containing a Tn5 disrupted copy of the PYK gene. These findings are consistent with reports that implicate the PYK locus in yeast cell cycle control and suggest that it may be challenging to model relations between fitness and activity for multifunctional proteins.     

Enzymes of glucose metabolism in Frankia sp.

Enzymes of glucose metabolism were assayed in crude cell extracts of Frankia strains HFPArI3 and HFPCcI2 as well as in isolated vesicle clusters from Alnus rubra root nodules. Activities of the Embden-Meyerhof-Parnas pathway enzymes glucokinase, phosphofructokinase, and pyruvate kinase were found in Frankia strain HFPArI3 and glucokinase and pyruvate kinase were found in Frankia strain HFPCcI2 and in the vesicle clusters. An NADP+-linked glucose 6-phosphate dehydrogenase and an NAD-linked 6-phosphogluconate dehydrogenase were found in all of the extracts, although the role of these enzymes is unclear. No NADP+-linked 6-phosphogluconate dehydrogenase was found. Both dehydrogenases were inhibited by adenosine 5-triphosphate, and the apparent Km's for glucose 6-phosphate and 6-phosphogluconate were 6.86 X 10(-4) and 7.0 X 10(-5) M, respectively. In addition to the enzymes mentioned above, an NADP+-linked malic enzyme was detected in the pure cultures but not in the vesicle clusters. In contrast, however, the vesicle clusters had activity of an NAD-linked malic enzyme. The possibility that this enzyme resulted from contamination from plant mitochondria trapped in the vesicle clusters could not be discounted. None of the extracts showed activities of the Entner-Doudoroff enzymes or the gluconate metabolism enzymes gluconate dehydrogenase or gluconokinase. Propionate- versus trehalose-grown cultures of strain HFPArI3 showed similar activities of most enzymes except malic enzyme, which was higher in the cultures grown on the organic acid. Nitrogen-fixing cultures of strain HFPArI3 showed higher specific activities of glucose 6-phosphate and 6-phosphogluconate dehydrogenases and phosphofructokinase than ammonia-grown cultures.     

Highly sensitive enzyme thermistor determination of ADP and ATP by multiple recycling enzyme systems

An enzyme thermistor method for the determination of ADP and/or ATP with signal amplification by recycling procedures is described. Pyruvate kinase (PK) and hexokinase (HK) coimmobilised on aminopropyl-controlled pore glass were applied in a column reactor. Addition of an excess of phosphoenolpyruvate (PEP) and glucose leads to cofactor recycling and production of glucose-6-phosphate and pyruvate. In presence of PEP an amplification of the sensitivity up to 30 times was reached as compared with the HK-catalysed reaction alone. An additional signal amplification was accomplished by recycling the pyruvate leaving the first enzyme reactor in a second reactor containing L-lactate dehydrogenase, lactate oxidase and catalase. In the presence of NADH an overall amplification of the sensitivity for ATP or ADP up to 1700 times was found. The limits of detection were 6 × 10⁻⁵ M cofactor without recycling at all, 2 × 10⁻⁶M with recycling in the kinase bienzyme reactor and 1 × 10⁻⁸M with the dual recycling system.     

Endogenous phosphorylation of soluble enzymes in human red cells. Cyclic 3',5'-AMP-dependent phosphorylation of phosphofructokinase without detectable regulatory effect

ATP-depleted human red cells have been incubated in a glucose-containing medium with [32P]orthophosphate in the presence and in the absence of cyclic 3',5'-AMP and dibutyril cyclic 3',5'-AMP. Spectrin, pyruvate kinase, phosphofructokinase, glucose-6-phosphate dehydrogenase and hemoglobin A1 have been purified and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein-bound radioactivity has been measured from the sodium dodecyl sulfate polyacrylamide gels and the trichloroacetic acid-precipitated proteins. In the cytosol, the most intense phosphorylation was found for pyruvate kinase whose, in the presence of cyclic AMP, specific radioactivity was comparable to that of the membrane protein and spectrin. In the absence of cyclic nucleotides it was five times less phosphorylated. Phosphofructokinase was only phosphorylated when the red cells were incubated with cyclic nucleotides; the extent of phosphorylation was four times less than for pyruvate kinase. Hemoglobin, glucose-6-phosphate dehydrogenase and a contaminant protein copurified with phosphofructokinase were not phosphorylated: the 'background' of the radioactivity found for these proteins was 100 times less than for pyruvate kinase and spectrin, and 20 times less than for phosphofructokinase (+cyclic AMP).     

Activity and androgenic control of glycolytic enzymes in the epididymis and epididymal spermatozoa of the rat.

1. Procedures were developed for the extraction and assay of glycolytic enzymes from the epididymis and epididymal spermatozoa of the rat. 2. The epididymis was separated into four segments for analysis. When rendered free of spermatozoa by efferent duct ligation, regional differences in enzyme activity were apparent. Phosphofructokinase, glycerol phosphate dehydrogenase and glucose 6-phosphate dehydrogenase were more active in the proximal regions of the epididymis, whereas hexokinase, lactate dehydrogenase and phosphorylase were more active in the distal segment. These enzymes were less active in the epididymis of castrated animals and less difference was apparent between the proximal and distal segments. However, the corpus epididymidis from castrated rats had lower activities of almost all enzymes compared with other epididymal segments. 3. Spermatozoa required sonication to obtain satisfactory enzyme release. Glycolytic enzymes were more active in spermatozoa than in epididymal tissue, being more than 10 times as active in the case of hexokinase, phosphoglycerate kinase and phosphoglycerate mutase. 4. The specific activities of a number of enzymes in the epididymis were dependent on the androgen status of the animal. These included hexokinase, phosphofructokinase, aldolase, glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, glycerol phosphate dehydrogenase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and phosphorylase. 5. The caput and cauda epididymidis differed in the extent to which enzyme activities changed in response to an altered androgen status. The most notable examples were hexokinase, phosphofructokinase, aldolase, phosphoglycerate kinase, 6-phosphogluconate dehydrogenase and phosphorylase.     

Self-association of human erythrocyte phosphofructokinase. Kinetic behaviour in dependence on enzyme concentration and mode of association.

The kinetic behaviour of human erythrocyte phosphofructokinase has been analyzed over a relative wide range of enzyme concentration (0.01 -- 1.7 mug/ml). The kinetic cooperativity which becomes apparent when the enzymic reaction rate is plotted versus the fructose 6-phosphate concentration decreases with increasing enzyme concentration. Simultaneously, a decrease of the half-saturation concentration for fructose 6-phosphate [S]0.5 is observed. Maximum velocity passes through a maximum at increasing enzyme concentrations. Sets of curves representing specific enzymic activity of phosphofructokinase versus enzyme concentration obtained at various fixed concentrations of fructose 6-phosphate and ATP are analyzed. The shapes of these curves are interpreted in terms of an association model of human erythrocyte phosphofructokinase, in which an inactive dimer (Mr 190000) and active multimers of the dimeric form are involved. The conclusion is drawn that the sigmoidal shape of the plots of the enzymic reaction rate versus fructose 6-phosphate concentration is partially caused by a displacement of the equilibrium between different states of association of phosphofructokinase to multimers by this substrate. On the other hand, the inhibition of the enzyme by high concentrations of ATP may be partially caused by a shift of this equilibrium to the state of the inactive dimer.     

Phosphorylation of the α and β polypeptides of the light-harvesting complex I (B870) of Rhodobacter capsulatus in an in vitro translation system

Abstract Sporozoites and unsporulated oocysts of Eimeria tenella were shown to contain a pyrophosphate-dependent phosphofructokinase (PP_i-PFK) but apparently lack an ATP-specific activity. The PP_i-PFK resembles those that occur in a number of other protists in being reversible and not subject to metabolic control. In contrast, the ADP-utilising pyruvate kinase, present in two developmental stages of the parasite, exhibited strong positive cooperativity with respect to its substrate, phosphoenolpyruvate, and was shown to be allostetically activated by glucose 6-phosphate, fructose 6-phosphate and AMP. It is suggested that the PP_i-PFK represents an adaptation of the parasite towards life in an environment containing only low concentrations of oxygen and that the unusual allosteric regulation of pyruvate kinase evolved to compensate for glycolysis not being controlled at the PP_i-PFK step.     

Identification of the regulatory steps in glycolysis in potato tubers

The aim of this work was to identify the regulatory reactions of glycolysis in potato tubers. The amounts of glycolytic intermediates in aerobic and anoxic tubers were measured in freeze-clamped samples of tissue. Comparison of mass—action ratios with apparent equilibrium constants showed that in vivo the reactions catalysed by glucosephosphate isomerase, phosphoglycerate mutase and enolase were close to equilibrium. The ratios fructose-1,6-bisphosphate:fructose 6-phosphate, and pyruvate:phosphoenolpyruvate, respectively, showed that the reactions catalysed by phosphofructokinase and pyruvate kinase were considerably displaced from equilibrium. Stimulation of glycolysis by placing tubers in an atmosphere of nitrogen led to significant declines in their contents of fructose-6-phosphate and phosphoenolpyruvate. It is concluded that phosphofructokinase plays a dominant role in regulating entry into glycolysis, and that pyruvate kinase may regulate exit from glycolysis and the oxidative pentose phosphate pathway. Cold-induced sweetening of the tubers is discussed in the light of the above conclusions.