Temperature and the regulation of enzyme activity in poikilotherms. Properties of lungfish fructose diphosphatase.
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ABSTRACT:
SUBMITTER: Behrisch HW
PROVIDER: S-EPMC1187762 | biostudies-other | 1969 May
REPOSITORIES: biostudies-other
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Project description:1. The properties of fructose diphosphatase from the liver of rainbow trout (Salmo gairdnerii) were examined over the physiological temperature range of the organism. 2. Saturation curves for substrate (fructose 1,6-diphosphate) and a cofactor (Mg(2+)) are sigmoidal, and Hill plots of the results suggest a minimum of two interacting fructose 1,6-diphosphate sites and two interacting Mg(2+) sites per molecule of enzyme. 3. Mn(2+)-saturation curves are hyperbolic, and the K(a) for Mn(2+), which inhibits the enzyme at high concentrations, is 50-100-fold lower than the K(a) for Mg(2+). 4. Fructose diphosphatase is inhibited by low concentrations of AMP; this inhibition appears to be decreased and reversed by increasing the concentrations of Mg(2+) and Mn(2+). Higher concentrations of AMP are required to inhibit the trout fructose diphosphatase in the presence of Mn(2+). 5. The affinities of fructose diphosphatase for fructose diphosphate and Mn(2+) appear to be temperature-independent, whereas the affinities for Mg(2+) and AMP are highly temperature-dependent. 6. The pH optimum of the enzyme depends on the concentrations of Mg(2+) and Mn(2+). In addition, pH determines the K(a) for Mg(2+); at high pH, K(a) for Mg(2+) is lowered. 7. The enzyme is inhibited by Ca(2+) and Zn(2+), and the inhibition is competitive with respect to both cations. 8. The possible roles of these ions and AMP in the modulation of fructose diphosphatase and gluconeogenic activity are discussed in relation to temperature adaptation.
| S-EPMC1187510 | biostudies-other
Project description:1. The calculated energy charge of the liver cell from migrating salmon is very low (0.464), in keeping with the extended starvation and high rates of muscular and biosynthetic activity of these organisms. 2. Affinity of fructose 1,6-diphosphatase for substrate increases with a decrease in temperature. 3. Arrhenius plots of the saturation kinetics are complex and suggest an interconversion of one or more forms of the enzyme; this interconversion is dependent on the identity of the cofactor. 4. Affinity of salmon fructose 1,6-diphosphatase for its allosteric inhibitor (AMP) is lower than in other fructose 1,6-diphosphatases and this enzyme-AMP interaction is largely insensitive to temperature. The functional significance of diminished AMP-sensitivity is that it allows normal or high fructose 1,6-diphosphatase activity during a low energy charge. 5. These findings suggest mechanisms for the maintenance of high rates of gluconeogenesis in salmon during spawning migration.
| S-EPMC1185194 | biostudies-other
Project description:1. The properties of fructose diphosphatase from skeletal muscle of the Alaskan king-crab (Paralithodes camtschatica) were examined over the physiological temperature range of the animal. 2. King-crab muscle fructose diphosphatase is first activated by Na(+) and NH(4) (+) and is then partially inhibited by these cations at concentrations higher than 10mm at 0 degrees , 8 degrees and 15 degrees C. Enzyme activity is stimulated by K(+) at 0 degrees C, but is curtailed at 8 degrees C and 15 degrees C, an effect that could render rate independent of temperature. 3. Affinity for substrate increases with decreasing temperature; below the temperature of acclimatization, K(m) for fructose 1,6-diphosphate increases, resulting in a complex U-shaped temperature-K(m) curve. 4. King-crab muscle fructose diphosphatase is inhibited by low concentrations of AMP. As with enzymes of other poikilotherms, inhibition by AMP is sensitive to temperature; the enzyme is least sensitive to inhibition by AMP near the temperature of acclimatization. 5. The affinity of fructose diphosphatase for fructose 1,6-diphosphate is enhanced by phosphoenolpyruvate, and this activation is temperature-sensitive; 0.5mm-phosphoenolpyruvate causes a sevenfold decrease in K(m) for fructose 1,6-diphosphate at 15 degrees C but a 25-fold decrease at 0 degrees C. 6. Phosphoenolpyruvate appears to decrease the affinity of king-crab muscle fructose diphosphatase for AMP at low temperature, whereas at the higher temperature it appears to enhance inhibition by AMP. Phosphoenolpyruvate was not observed to cause a reversal of inhibition of fructose diphosphatase activity by AMP. The identification of phosphoenolpyruvate as an activator of a rate-limiting step in gluconeogenesis permits the suggestion of a coupling of the controlling mechanisms of several steps in the glycolytic and gluconeogenic chains. 7. These findings suggest mechanisms for the maintenance and regulation of control of fructose diphosphatase activity in king-crab skeletal muscle at low temperature and under conditions that favour concomitant activity of phosphofructokinase.
| S-EPMC1176586 | biostudies-other
Project description:1. The kinetics of the thermally induced enzyme variants of the supernatant NADP-isocitrate dehydrogenase from rainbow-trout liver are investigated. 2. Fish acclimatized to 2 degrees C (cold-adapted enzyme) and 17 degrees C (warm-adapted enzyme) show different relative distributions of the three NADP-isocitrate dehydrogenase isoenzymes; this has been demonstrated with electrophoresis and electrofocusing techniques. 3. Plots of K(m) versus temperature for the cold-adapted and warm-adapted enzyme variants are complex in nature with apparent maximal enzyme-substrate affinity corresponding to the temperature at which the trout is acclimatized. Both substrates, dl-isocitrate and NADP(+), give similar curves although the magnitude of the K(m) change with temperature is much decreased in the case of NADP(+). 4. E(a) values of approx. 18kcal/mol were determined for both the cold-adapted and warm-adapted enzyme variants. 5. In an attempt to determine how velocities can be increased at low temperatures, cation, pH requirements, metabolite and enzyme concentrations were examined. 6. NAD-isocitrate dehydrogenase could not be detected in trout tissues.
| S-EPMC1177070 | biostudies-other
Project description:1. The occurrence of fructose diphosphatase in muscle tissue was investigated with reference to the question whether lactate can be converted into glycogen in muscle, as postulated by Meyerhof (1930), fructose diphosphatase being one of the enzymes required for this conversion. 2. Fructose diphosphatase was found in skeletal muscle of man, dog, cat, rat, mouse, rabbit, guinea pig, cattle, sheep, pigeon, fowl and frog. Under the test conditions between 5 and 60 mumoles of substrate were split/g. fresh wt./hr. at 22 degrees . 3. Like liver fructose diphosphatase, the muscle enzyme is inhibited by substrate concentrations above 0.1 mm, by AMP and by trace quantities of Zn(2+), Fe(2+) and Fe(3+); it is ;activated' by EDTA. Inhibitions by the above agents may account for the failure of previous authors to detect the enzyme. 4. Heart muscle of several vertebrate species and the smooth muscle of pigeon and fowl gizzard had no measurable activity. 5. The presence of fructose diphosphatase and the virtual absence of the enzyme systems converting pyruvate into phosphopyruvate means that lactate and pyruvate cannot be converted into glycogen in muscle, whereas the phosphorylated C(3) compounds can. The reconversion into carbohydrate of lactate (which readily diffuses out of muscle) occurs in liver and kidney only. The reconversion of phosphorylated C(3) intermediates (which cannot diffuse out of the tissue) can occur only within the muscle. 6. alpha-Glycerophosphate is probably the main intermediate requiring conversion into glycogen. The possible role of alpha-glycerophosphate formation in vertebrate muscle, already well established in insect muscle, is discussed.
| S-EPMC1206525 | biostudies-other
Project description:1. Fructose 1,6-diphosphatase has been purified tenfold from rat liver. The final preparation was not contaminated by either glucose 6-phosphatase or phosphofructokinase. The properties of the enzyme have been investigated in an attempt to define factors that could be of revelance to metabolic control of fructose 1,6-diphosphatase activity. 2. The metal ions Fe(2+), Fe(3+) and Zn(2+) inhibited the activity of fructose 1,6-diphosphatase even in the presence of an excess of mercaptoethanol; other metal ions tested had no effect. The inhibition produced by Zn(2+) was reversed by EDTA, but that produced by either Fe(2+) or Fe(3+) was not reversible. 4. The enzyme has a very low K(m) for fructose 1,6-diphosphate (2.0mum). Concentrations of fructose 1,6-diphosphate above 75mum inhibited the activity; however, even at very high fructose 1,6-diphosphate concentrations only 70% inhibition was obtained. 5. The activity was also inhibited by low concentrations of AMP, which lowered V(max.) and increased K(m) for fructose 1,6-diphosphate. Evidence is presented that suggests that AMP can be defined as an allosteric inhibitor of fructose 1,6-diphosphatase. 6. The inhibitions by both fructose 1,6-diphosphate and AMP were extremely specific. Also, the degree of inhibition was not affected by the presence of intermediates of glycolysis, of the tricarboxylic acid cycle, of amino acid metabolism or of fatty acid metabolism. 7. It is suggested that the intracellular concentrations of AMP and fructose 1,6-diphosphate could be of significance in controlling the activity of fructose 1,6-diphosphatase in the liver cell. The possible relationship between these intermediates and the control of gluconeogenesis is discussed.
| S-EPMC1206804 | biostudies-other
Project description:A fructose diphosphatase-phosphofructokinase substrate cycle has been reconstructed in vitro to provide a system that recycles fructose 6-phosphate and hydrolyses ATP to ADP and P(i). The concerted actions of glucose phosphate isomerase, phosphofructokinase, aldolase and triose phosphate isomerase catalysed the loss of (3)H from [5-(3)H,U-(14)C]glucose 6-phosphate. This was used as the basis of a method for the estimation of the fructose diphosphatase-phosphofructokinase substrate cycle. For the reconstructed cycle, the rate of decrease of the (3)H/(14)C ratio in [5-(3)H,U-(14)C]hexose 6-phosphate was proportional to the rate of fructose 6-phosphate substrate cycling. A detailed theoretical treatment of this relationship is developed, which enables the rate of substrate cycling to be determined in vivo.
| S-EPMC1177845 | biostudies-other
Project description:1. The present study extends the finding of Krebs & Woodford (1965) that muscle fructose diphosphatase is more sensitive to AMP inhibition than liver fructose diphosphatase. 2. Hen breast fructose diphosphatase has a K(i) for AMP of 0.1mum; the plot of percentage inhibition is non-sigmoid and the reciprocal plot of activity against AMP concentration is sometimes linear. 3. Percentage inhibition plots for other muscle fructose diphosphatases are sigmoid curves which exhibit different threshold responses to the AMP concentration. 4. The intracellular content of AMP in all muscles tested exceeds the inhibition concentration range of AMP. 5. The sensitivity of muscle fructose diphosphatase to AMP inhibition is decreased by the presence of Mg(2+) or Mn(2+) ions; in the presence of Mn(2+) the inhibition curve for hen breast fructose diphosphatase becomes sigmoid. 6. From the formation constants for the Mg(2+) and Mn(2+) chelates, the effect of these ions in chelation of AMP can be calculated. Although chelation of AMP can explain the Mg(2+) effect, it cannot explain the marked relief of AMP inhibition by Mn(2+). 7. It is suggested that Mn(2+) has a specific effect on this enzyme which reduces the sensitivity to AMP inhibition.
| S-EPMC1270594 | biostudies-other
Project description:1. The maximum catalytic activities of fructose diphosphatase from flight muscles of bumble-bees (Bombus spp.) are at least 30-fold those reported for the enzyme from other tissues. The maximum activity of fructose diphosphatase in the flight muscle of any particular bee is similar to that of phosphofructokinase in the same muscle, and the activity of hexokinase is similar to or greater than the activity of phosphofructokinase. There is no detectable activity of glucose 6-phosphatase and only a very low activity of glucose 6-phosphate dehydrogenase in these muscles. The activities of both fructose diphosphatase and phosphofructokinase vary inversely with the body weight of the bee, whereas that of hexokinase is relatively constant. 2. There is no significant hydrolysis of fructose 1-phosphate, fructose 6-phosphate, glucose 1,6-diphosphate and glycerol 3-phosphate by extracts of bumble-bee flight muscle. 3. Fructose 1,6-diphosphatase from bumble-bee flight muscle and from other muscles is inhibited by Mn(2+) and univalent cations; the potency of inhibition by the latter varies in the order Li(+)>Na(+)>K(+). However, the fructose diphosphatase from bumble-bee flight muscle is different from the enzyme from other tissues in that it is not inhibited by AMP. 4. The contents of ATP, hexose monophosphates, fructose diphosphate and triose phosphates in bumble-bee flight muscle showed no significant changes between rest and flight. 5. It is proposed that both fructose diphosphatase and phosphofructokinase are simultaneously active and catalyse a cycle between fructose 6-phosphate and fructose diphosphate in resting bumble-bee flight muscle. Such a cycle would produce continuous hydrolysis of ATP, with the release of energy as heat, which would help to maintain the thoracic temperature during rest periods at a level adequate for flight.
| S-EPMC1173573 | biostudies-other
Project description:The choanoflagellate Salpingoeca rosetta contains a chimeric rhodopsin protein composed of an N-terminal rhodopsin (Rh) domain and a C-terminal cyclic nucleotide phosphodiesterase (PDE) domain. The Rh-PDE enzyme (SrRh-PDE), which decreases the concentrations of cyclic nucleotides such as cGMP and cAMP in light, is a useful tool in optogenetics. Recently, eight additional Rh-PDE enzymes were found in choanoflagellate species, four from Choanoeca flexa and the other four from other species. In this paper, we studied the molecular properties of these new Rh-PDEs, which were compared with SrRh-PDE. Upon expression in HEK293 cells, four Rh-PDE proteins, including CfRh-PDE2 and CfRh-PDE3, exhibited no PDE activity when assessed by in-cell measurements and in vitro HPLC analysis. On the other hand, CfRh-PDE1 showed light-dependent PDE activity toward cGMP, which absorbed maximally at 491 nm. Therefore, CfRh-PDE1 is presumably responsible for colony inversion in C. flexa by absorbing blue-green light. The molecular properties of MrRh-PDE were similar to those of SrRh-PDE, although the λmax of MrRh-PDE (516 nm) was considerably red-shifted from that of SrRh-PDE (492 nm). One Rh-PDE, AsRh-PDE, did not contain the retinal-binding Lys at TM7 and showed cAMP-specific PDE activity both in the dark and light. These results provide mechanistic insight into rhodopsin-mediated, light-dependent regulation of second-messenger levels in eukaryotic microbes.
| S-EPMC7227045 | biostudies-literature