Temperature and the regulation of enzyme activity in poikilotherms. Properties of lungfish fructose diphosphatase

1. The properties of fructose diphosphatase from liver of South American lungfish (Lepidosiren paradoxa) were examined. 2. Saturation curves for substrate (fructose diphosphate) and both cofactors (Mn(2+) and Mg(2+)) are sigmoidal and Hill plots of these results suggest about 2 interacting substrate and cofactor sites/molecule of enzyme. 3. Mn(2+) is an efficient positive modulator of the enzyme and K(a) for Mn(2+) is about 20-30-fold lower than the K(a) for Mg(2+). 4. Lungfish fructose diphosphatase is inhibited by low concentrations of AMP, and the affinity of the enzyme for AMP is insensitive to temperature. 5. The affinities of fructose diphosphatase for fructose diphosphate and Mn(2+) appear to be dependent on temperature, whereas affinity for Mg(2+) is temperature-independent. 6. The pH optimum of the enzyme depends on the presence of the particular cofactor. As pH increases, the K(a) values of both cations are lowered, maximum velocities are increased and the saturation curves for cofactor become hyperbolic. 7. The possible roles of these ions, pH and substrate in the modulation of fructose diphosphatase and gluconeogenic activity in the lungfish are discussed in relation to aestivation and temperature adaptation.     

Temperature and the regulation of enzyme activity in poikilotherms. Fructose diphosphatase from migrating salmon

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.     

Regulatory sulfhydryl groups, conformational change, and allosteric inhibition in rabbit muscle fructose diphosphatase

The 16 sulfhydryl groups of native, homogeneous rabbit muscle fructose diphosphatase can all react with 5,5′-dithiobis-(2-nitrobenzoic acid). High concentrations of substrate (1–2 mm) decrease the reaction rate of the sulfhydryl groups, while concentrations up to 70 μm have no effect. After titration of the four most rapidly reacting sulfhydryl groups there is a marked desensitization toward the allosteric inhibitor AMP. In the presence of 30 μm AMP only 4–5 sulfhydryl groups/tetramer react with 5,5′-dithiobis-(2-nitrobenzoic acid), and the enzyme again becomes desensitized toward AMP inhibition. Together with a 3.5-fold increase in the I₅₀ for AMP inhibition, the K_m for Mg²⁺ or Mn²⁺ ions is also increased. In the presence of 7 mm MgCl₂ or 0.28 mm MnCl₂ only 6–8 sulfhydryl groups are modified. The rapid reaction of 4 sulfhydryl groups again results in desensitization. There is neither a protection by the substrate against inactivation, nor a protection by the allosteric inhibitor against desensitization. It is concluded that AMP and the divalent cations induce conformational changes in the protein molecule making 11–12 or 8–10 sulfhydryl groups inert for 5,5′-dithiobis-(2-nitrobenzoic acid), respectively. The K_m for fructose-1,6-diphosphate is not changed after the modification of 4–5 sulfhydryl groups.     

Phosphofructokinase from bumblebee flight muscle. Molecular and catalytic properties and role of the enzyme in regulation of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle

Phosphofructokinase from the flight muscle of bumblebee was purified to homogeneity and its molecular and catalytic properties are presented. The kinetic behavior studies at pH 8.0 are consistent with random or compulsory-order ternary complex. At pH 7.4 the enzyme displays regulatory behavior with respect to both substrates, cooperativity toward fructose 6-phosphate, and inhibition by high concentration of ATP. Determinations of glycolytic intermediates in the flight muscle of insects exposed to low and normal temperatures showed statistically significant increases in the concentrations of AMP, fructose 2,6-bisphosphate, and glucose 6-phosphate during flight at 25 degrees C or rest at 5 degrees C. Measuring the activity of phosphofructokinase and fructose 1,6-bisphosphatase at 25 and 7.5 degrees C, in the presence of physiological concentrations of substrates and key effectors found in the muscle of bumblebee kept under different environmental temperatures and activity levels, suggests that the temperature dependence of fructose 6-phosphate/fructose 1,6-bisphosphate cycling may be regulated by fluctuation of fructose 2,6-bisphosphate concentration and changes in the affinity of both enzymes for substrates and effectors. Moreover, in the presence of in vivo concentrations of substrates, phosphofructokinase is inactive in the absence of fructose 2,6-bisphosphate.     

Regulatory properties of a fructose 1,6-bisphosphatase from the cyanobacterium Anacystis nidulans.

A fructose 1,6-bisphosphatase (EC 3.1.3.11) (FBPase) was purified over 100-fold from Anacystis nidulans. At variance with a previous report (R. H. Bishop, Arch. Biochem. Biophys. 196:295-300, 1979), the regulatory properties of the enzyme were found to be like those of chloroplast enzymes rather than intermediate between chloroplast (photosynthetic) and heterotrophic FBPases. The pH optimum of Anacystis FBPase was between 8.0 and 8.5 and shifted to lower values with increasing Mg2+ concentration. Under the experimental conditions used by Bishop, we found the saturation curve of the enzyme to be sigmoidal for Mg2+ ions and hyperbolic for fructose 1,6-bisphosphate. The half-maximal velocity of the Anacystis FBPase was reached at concentrations of 5 mM MgCl2 and 0.06 mM fructose 1,6-bisphosphate. AMP did not inhibit the enzyme. The activity of the FBPase was found to be under a delicate control of oxidizing and reducing conditions. Oxidants like O2, H2O2, oxidized glutathione, and dehydroascorbic acid decreased the enzyme activity, whereas reductants like dithiothreitol and reduced glutathione increased it. The oxido-reductive modulation of FBPase proved to be reversible. Reduced glutathione stimulated the enzyme activity at physiological concentrations (1 to 10 mM).l The reduced glutathione-induced activation was higher at pH 8.0 than at pH 7.0.     

NAD+-malic enzyme. Regulatory properties of the enzyme from Ascaris suum.

The regulatory properties of the NAD-dependent malic enzyme from the mitochondria of Ascaris suum have been studied. The malate saturation curve exhibits sigmoidicity and the degree of this sigmoidicity increases as the pH is increased. Fumarate was the only compound tested that stimulated the enzyme activity, whereas oxalacetate was the most powerful inhibitor. Activation by low levels of fumarate was found to be competitive with malate. It is proposed that this stimulation has physiological significance in controlling the dismutation reaction in the parasite. The branched-chain volatile fatty acid excretion products, tiglate, 2-methylbutanoate, and 2-methylpentanoate, inhibited the enzyme activity and this inhibition was competitive with malate. The Ki values for these compounds are in the physiological range of their concentrations; therefore, it is suggested that they may aid in controlling the malic enzyme activity in vivo. Oxalacetate inhibition of malic enzyme activity was competitive with malate, and the Ki values decreased with an increase in pH. Two alternatives are proposed which could account for the lack of oxalacetate decarboxylation by the ascarid malic enzyme.     

Regulatory properties of 14-day embryo and adult hen skeletal muscle AMP-deaminase. The influence of pH on the enzyme activity

The variations of kinetic parameters with pH of the activity of 14-day-old chicken embryo and adult hen skeletal muscle AMP-deaminase in the presence and in the absence of adenine nucleotide effectors have been examined. The results obtained indicate that the kinetic and regulatory properties of the two developmental forms of AMP-deaminase are different.     

Possible regulation of thiamine diphosphatase activity in rat brain microsomes by lipids.

The effects of various treatments, which affect membrane structure, on microsomal thiamine diphosphatase and thiamine triphosphatase activities of rat brain, were examined. The treatment of micorosomes at alkaline pH caused a 2-fold activation of the thiamine diphosphatase, this being related to a change in membrane structure which was evidenced by a decrease of the turbidity of the microsomal suspension. Repeated freezing and thawing after hypo-osmotic treatment also increased the activity of microsomal thiamine diphosphatase. In addition, the thiamine diphosphatase activity was enhanced by treatment of the microsomes with phospholipase C or acetone. This lipid depletion resulted in a marked reduction in the apparent Km value of the thiamine diphosphatase with a corresponding loss in heat stability of the enzyme. We found further that brain thiamine diphosphatase was solubilized by Triton X-100. This decreased the phospholipid content in the preparation, but did not affect the apparent Km value and heat stability of the enzyme. In contrast with thiamine diphosphatase, thiamine triphosphatase was inactivated by treatment at alkaline pH or with acetone. However, treatment with phospholipase C did not affect the activity of thiamine triphosphatase.     

Purification and properties of debranching enzyme from dogfish muscle.

Glycogen debranching enzyme (4-alpha-glucanotransferase amylo-1,6-glucosidase, EC 2.4.1.25 + 3.2.1.33) was purified 140-fold from dogfish muscle in a rapid, high-yield procedure that takes advantage of a strong binding of the enzyme to glycogen, and its quantitative adsorption to concanavalin A-Sepharose only when the polysaccharide is present. The final product was hrophoresis in the presence and absence of dodecyl sulfate. A molecular weight of 162,000 +/- 5000 was determined by sedimentation equilibrium analysis in good agreement with the value of 160,000 estimated by gel electrophoresis, but a low-sedimentation constant of 6.5 S suggests that the enzyme is asymmetric. The molecule appears to be made up of a single polypeptide chain with no evidence for multiple repeating sequences: it could not be dissociated into smaller fragments by dodecyl sulfate even after complete carboxymethylation; tryptic cleavage of the native protein yielded only two fragments of molecular weight 20,000 and 140,000 without loss of enzymatic activity. The amino acid composition of the enzyme is reported; no covalently bound phosphate or carbohydrate could be detected. All 32 sulfhydryl groups present were titrated with 5,5'-dithiobis(2-nitrobenzoic acid) under denaturing conditions; eight reacted readily in the native enzyme without loss of catalytic activity, while substitution of eight additional ones lowered the activity by 50%. Inactivation was greatly reduced by glycogen; the polysaccharide also influenced markedly the electrophoretic behavior of the enzyme and large filamentous aggregates were formed when solutions of both were mixed. Purified debranching enzyme releases 3 mumol of glucose min-1 mg-1 at 19 degrees C, pH 6.0, from a glycogen limit dextrin and one-tenth this amount when the native polysaccharide is used as substrate; glycogen is quantitatively degraded in the presence of phosphorylase. None of the usual sugar phosphates or nucleotide effectors of glycolysis affected enzymatic activity. No phosphorylation by either dogfish or rabbit skeletal muscle protein kinase or phosphorylase kinase could be demonstrated, nor any direct interaction with phosphorylase as measured by SH-group reactivity, enzymatic activity, or rate of phosphorylase b to a conversion. Purification of the 160,000 molecular weight M-line protein of skeletal muscle resulted in the quantitative removal of debranching enzyme, indicating that the two proteins are different.     

Regulatory properties of phosphorylase from chicken skeletal muscle

The main kinetic parameters for purified phosphorylase kinase from chicken skeletal muscle were determined at pH 8.2: Vm = 18 micromol/min/mg; apparent Km values for ATP and phosphorylase b from rabbit muscle were 0.20 and 0.02 mM, respectively. The activity ratio at pH 6.8/8.2 was 0.1-0.4 for different preparations of phosphorylase kinase. Similar to the rabbit enzyme, chicken phosphorylase kinase had an absolute requirement for Ca2+ as demonstrated by complete inhibition in the presence of EGTA. Half-maximal activation occurred at [Ca2+] = 0.4 microM at pH 7.0. In the presence of Ca2+, the chicken enzyme from white and red muscles was activated 2-4-fold by saturating concentrations of calmodulin and troponin C. The C0.5 value for calmodulin and troponin C at pH 6.8 was 2 and 100 nM, respectively. Similar to rabbit phosphorylase kinase, the chicken enzyme was stimulated about 3-6-fold by glycogen at pH 6.8 and 8.2 with half-maximal stimulation occurring at about 0.15% glycogen. Protamine caused 60% inhibition of chicken phosphorylase kinase at 0.8 mg/ml. ADP (3 mM) at 0.05 mM ATP caused 85% inhibition with Ki = 0.2 mM. Unlike rabbit phosphorylase kinase, no phosphorylation of the chicken enzyme occurred in the presence of the catalytic subunit of cAMP-dependent protein kinase. Incubation with trypsin caused 2-fold activation of the chicken enzyme.