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1.
The effect of fructose 2,6-P2, AMP and substrates on the coordinate inhibition of FBPase and activation of PFK in swine kidney has been examined. Fructose 2,6-P2 inhibits the activity of FBPase and stimulates the activity of PFK in the presence of inhibitory concentrations of ATP. Under similar conditions 2.2 μM fructose 2,6-P2 was required for 50% inhibition of FBPase and 0.04 μM fructose 2,6-P2 restored 50% of the activity of PFK. Fructose 2,6-P2 also enhanced the allosteric activation of PFK by AMP and it increased the extent of inhibition of FBPase by AMP. Fructose 2,6-P2, AMP and fructose 6-P act cooperatively to stimulate the activity of PFK whereas the same latter two effectors and fructose 1,6-P2 inhibit the activity of FBPase. Taken collectively, these results suggest that an increase in the intracellular level of fructose 2,6-P2 during gluconeogenesis could effectively overcome the inhibition of PFK by ATP and simulataneously inactivate FBPase. When the level of fructose 2,6-P2 is low, a glycolytic state would be restored, since under these conditions PFK would be inhibited by ATP and FBPase would be active.  相似文献   

2.
J M Storey  K B Storey 《Cryobiology》1982,19(2):185-194
The kinetic properties of cytoplasmic glycerol-3-P dehydrogenase from the third instar larva of the gall fly, Eurosta solidaginis, were studied with emphasis on temperature effects on the enzyme and the regulation of enzyme activity during the synthesis of the cryoprotectant, glycerol. Isoelectrofocusing revealed one major and two minor forms of the enzyme with no alteration in the pI's or relative activities of the forms in larvae acclimated to 24 versus ?30 °C. Kinetic properties of the enzyme were also the same in larvae acclimated to high and low temperatures. Arrhenius plots were linear over a 30 to 0 °C range with an activation energy of 12,630 ± 185 cal/mol and a Q10 of 2.16. The Km for dihydroxyacetone-P was constant, at 50 μM, between 30 and 10 °C but increased by 75% at 0 °C; this increase may be a factor in the cessation of glycerol synthesis which occurs below 5 °C in this species. The Km(NADH), by contrast, was higher (5–6 μM) at 30 °C but decreased (3 μM) at lower temperatures. In the reverse direction, Km's were 340 μM for glycerol-3-P and 12 μM for NAD+. Effects of most inhibitors (of the forward reaction), glycerol-3-P (Ki = 2.4 mM), NAD+ (Ki = 0.2 mM), ATP, Mg·ATP, and Pi, were unaltered by assay temperature but ADP effects were potentiated by low temperature while citrate inhibition was greatest at high temperatures. Glycerol and sorbitol, which accumulate as cryoprotectants in E. solidaginis, had no significant effects on kinetic constants at any temperature but decreased the Vmax activity of the enzyme. Thermal inactivation studies showed an increased thermal stability of the larval enzyme compared to the homologous enzyme from rabbit muscle while added polyols stabilized enzyme activity, decreasing the rate of enzyme inactivation at 50 °C.  相似文献   

3.
The effects of AMP and fructose 2,6-bisphosphate (Fru-2,6-P2) on porcine fructose-1,6-bisphosphatase (pFBPase) and Escherichia coli FBPase (eFBPase) differ in three respects. AMP/Fru-2,6-P2 synergism in pFBPase is absent in eFBPase. Fru-2,6-P2 induces a 13° subunit pair rotation in pFBPase but no rotation in eFBPase. Hydrophilic side chains in eFBPase occupy what otherwise would be a central aqueous cavity observed in pFBPase. Explored here is the linkage of AMP/Fru-2,6-P2 synergism to the central cavity and the evolution of synergism in FBPases. The single mutation Ser45 → His substantially fills the central cavity of pFBPase, and the triple mutation Ser45 → His, Thr46 → Arg, and Leu186 → Tyr replaces porcine with E. coli type side chains. Both single and triple mutations significantly reduce synergism while retaining other wild-type kinetic properties. Similar to the effect of Fru-2,6-P2 on eFBPase, the triple mutant of pFBPase with bound Fru-2,6-P2 exhibits only a 2° subunit pair rotation as opposed to the 13° rotation exhibited by the Fru-2,6-P2 complex of wild-type pFBPase. The side chain at position 45 is small in all available eukaryotic FBPases but large and hydrophilic in bacterial FBPases, similar to eFBPase. Sequence information indicates the likelihood of synergism in the FBPase from Leptospira interrogans (lFBPase), and indeed recombinant lFBPase exhibits AMP/Fru-2,6-P2 synergism. Unexpectedly, however, AMP also enhances Fru-6-P binding to lFBPase. Taken together, these observations suggest the evolution of AMP/Fru-2,6-P2 synergism in eukaryotic FBPases from an ancestral FBPase having a central aqueous cavity and exhibiting synergistic feedback inhibition by AMP and Fru-6-P.  相似文献   

4.
5.
The cytoplasmic form of fructose 1,6-bisphosphatase (FBPase) was purified over 60-fold from germinating castor bean endosperm (Ricinus communis). The kinetic properties of the purified enzyme were studied. The preparation was specific for fructose 1,6-bisphosphate and exhibited optimum activity at pH 7.5. The affinity of the enzyme for fructose 1,6-bisphosphate was reduced by AMP, which was a mixed linear inhibitor. Fructose 2,6-bisphosphate also inhibited FBPase and induced a sigmoid response to fructose 1,6-bisphosphate. The effects of fructose 2,6-bisphosphate were enhanced by low levels of AMP. The latter two compounds interacted synergistically in inhibiting FBPase, and their interaction was enhanced by phosphate which, by itself, had little effect. The enzyme was also inhibited by ADP, ATP, UDP and, to a lesser extent, phosphoenolpyruvate. There was no apparent synergism between UDP, a mixed inhibitor, and fructose 2,6-bisphosphate. Similarly ADP, a predominantly competitive inhibitor, did not interact with fructose 2,6-bisphosphate. Possible roles for fructose 2,6-bisphosphate and the other effectors in regulating FBPase are discussed.  相似文献   

6.
The rate of cyclic AMP formation by rabbit heart membrane particles decreased at assay temperatures greater than 30 °C. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity (assayed at 24 °C) decreased exponentially with time of preincubation at 30 or 37 °C, providing evidence for the instability of this enzyme. The half-life, t1/2, of the enzyme at 37 °C was 9.9 min in the absence and 4.4 min in the presence of MgCl2. The activity was most labile in the presence of 50 m m Mg2+ and 1 m m ATP, having t1/2 = 1.3min. Prior incubation of membranes with the GTP analog, guanyl-5′-yl imidodiphosphate [Gpp(NH)p], 0.1 m m, for 30 min at 37 °C produced maximal activation of adenylate cyclase; the rate of activation was temperature dependent and was increased in the presence of isoproterenol. The Gpp(NH)p-activated enzyme had increased thermal stability, t1/2 = 170 min, and was also markedly more stable in the presence of Mg-ATP, t1/2 = 72min, than nonactivated enzyme. Preactivation with F? (30 min at 24 °C) also stabilized the activity; t1/2 > 70 min in the absence or presence of Mg-ATP. The Mg2+ concentration required for maximal activity was reduced from approximately 60 m m for nonactivated enzyme to 10 m m for the Gpp(NH)p- and F?activated enzyme.  相似文献   

7.
Metabolic regulation of gluconeogenesis and glycogenolysis by two phosphorylated derivatives of glycerol, G3P, and DHAP, and by F2,6BP, was assessed in vitro in liver homogenates obtained from Chinese hamsters (C. griseus) of two types: diabetic animals from sublines with consistent glycosuria and hyperglycemia, and normoglycemic controls. Only FBPase was sensitive to inhibition by the phosphorylated metabolites. G3P was weakly inhibitory of FBPase. Addition of 7 X 10(-3) M DHAP halved FBPase activity in the diabetic hamsters and 4 X 10(-3) M DHAP produced the same effect in the controls. The other gluconeogenic enzymes and phosphorylase a were only negligibly inhibited. In contrast, F2,6BP inhibited FBPase at concentrations in the micromolar range. Liver homogenates from diabetic hamsters appeared significantly more sensitive to F2,6BP inhibition of FBPase than those from controls at concentrations 0.6 X 10(-6) M and higher. These data indicate that in well-fed hamsters phosphorylated glycerol derivatives are unlikely to regulate hepatic gluconeogenesis at physiologic concentrations. However, the effects of F2,6BP on gluconeogenesis and glycolysis may be linked to those mediated by insulin. Thus, the deficiency of insulin, elevated end-organ insulin resistance, the alteration in the glucagon-insulin interaction, or a combination of these possible causes can be involved in an abnormal regulation of glycolysis and gluconeogenesis at the FBPase step, associated with changes in F2,6BP concentration.  相似文献   

8.
The activity and thermal stability of α-amylase were studied in the presence of different concentrations of trehalose, sorbitol, sucrose and glycerol. The optimum temperature of the enzyme was found to be 50 ± 2°C. Further increase in temperature resulted in irreversible thermal inactivation of the enzyme. In the presence of cosolvents, the rate of thermal inactivation was found to be significantly reduced. The apparent thermal denaturation temperature (T m )app and activation energy (E a ) of α-amylase were found to be significantly increased in the presence of cosolvents in a concentration-dependent manner. In the presence of 40% trehalose, sorbitol, sucrose and glycerol, increments in the (T m )app were 20°C, 14°C, 13°C and 9°C, respectively. The E a of thermal denaturation of α-amylase in the presence of 20% (w/v) trehalose, sorbitol, sucrose and glycerol was found to be 126, 95, 90 and 43 kcal/mol compared with a control value of 40 kcal/mol. Intrinsic and 8-anilinonaphathalene-1-sulphonic acid (ANS) fluorescence studies indicated that thermal denaturation of the enzyme was accompanied by exposure of the hydrophobic cluster on the protein surface. Preferential interaction parameters indicated extensive hydration of the enzyme in the presence of cosolvents.  相似文献   

9.
To understand the physiological functions of thermostable fructose-1,6-bisphosphatase (TNA1-Fbp) from Thermococcus onnurineus NA1, its recombinant enzyme was overexpressed in Escherichia coli, purified, and the enzymatic properties were characterized. The enzyme showed maximal activity for fructose-1,6-bisphosphate at 95°C and pH 8.0 with a half-life (t 1/2) of about 8 h. TNA1-Fbp had broad substrate specificities for fructose-1,6-bisphosphate and its analogues including fructose-1-phosphate, glucose-1-phosphate, and phosphoenolpyruvate. In addition, its enzyme activity was increased five-fold by addition of 1 mM Mg2+, while Li+ did not enhance enzymatic activity. TNA1-Fbp activity was inhibited by ATP, ADP, and phosphoenolpyruvate, but AMP up to 100 mM did not have any effect. TNA1-Fbp is currently defined as a class V fructose-1,6-bisphosphatase (FBPase) because it is very similar to FBPase of Thermococcus kodakaraensis KOD1 based on sequence homology. However, this enzyme shows a different range of substrate specificities. These results suggest that TNA1-Fbp can establish new criterion for class V FBPases.  相似文献   

10.
Mucosal acidification (from pH 8.1 to 6.0) reversibly inhibited the hydroosmotic responses to oxytocin, cyclic AMP and 8-bromo-cyclic AMP in frog urinary bladder. These inhibitory effects were only observed in the presence of a permeant buffer in the apical medium and could also be elicited by CO2 bubbling, even when the mucosal pH was clamped at 8.1. Acid pH reduced the oxytocin-induced net water flux faster than norepinephrine or oxytocin removal and the difference was especially important at low temperature. The time course of recovery from acid pH inhibition was, at 20°C, similar to that of the hormonal action, but when the medium temperature was reduced to 6–7°C, the recovery from acid pH inhibition paradoxically became faster while the oxytocin action was markedly slowed down (t12 of changes in net water fluxes (expressed in min): oxytocin addition at 20°C, 6.2 ± 0.9; at 6°C, 24 ± 3; oxytocin removal at 20°C, 4.7 ± 0.8; at 6°C, 22 ± 3; pH inhibition at 20°C, 2.6 ± 0.2; at 6°C 2.5 ± 0.2; recovery from pH 6 at 20°C, 6.5 ± 0.9; at 6°C, 2.7 ± 0.3). These results can be explained by accepting two main loci sensitive to medium acidification: (1) the cyclase system and (2) an intracellular, temperature-independent, post-cyclic AMP site. The fact that the intramembranous particle aggregates associated with the oxytocin-induced water permeability increase did not disappear after the flow inhibition by acid pH at low temperature suggests that the second effect could be located at the water channel itself.  相似文献   

11.
A propionitrile-induced nitrile hydratase (NHase), a promising biocatalyst for synthesis of organic amides has been purified from cell-free extract of Rhodococcus rhodochrous PA-34. About 11-fold purification of NHase was achieved with 52% yield. The SDS-PAGE of the purified enzyme revealed that it consisted of two subunits of 25.04 kD and 30.6 kD. However, the molecular weight of holoenzyme was speculated to be 86 kD by native-PAGE. This NHase exhibited maximum activity at pH 8.0 and temperature 40°C. Half-life was 2 h at 40°C and 0.5 h at 50°C. The Km and Vmax were 167 mM and 250 μmole/min/mg using 25 mM 3-cyanopyridine as substrate. AgNO3, Pb(CH3COO)2 and HgCl2 inhibited the NHase to extent of 89–100%.  相似文献   

12.
β-N-Acetyl-D-glucosaminidase was purified from viscera of green crab (Scylla serrata) by extraction with 0.01 M Tris-HCl buffer (pH 7.5) containing 0.2 M NaCl, ammonium sulfate fractionation, and then chromatography on Sephadex G-100 and DEAE-cellulose (DE-32). The purified enzyme showed a single band on polyacrylamide gel electrophoresis, and the specific activity was determined to be 7990 U/mg. The molecular weight of the whole enzyme was determined to be 132.0 kD, and the enzyme is composed of two identical subunits with molecular mass of 65.8 kD. The optimum pH and optimum temperature of the enzyme for the hydrolysis of p-nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-NAG) were found to be at pH 5.6 and at 50°C, respectively. The study of its stability showed that the enzyme is stable in the pH range from 4.6 to 8.6 and at temperatures below 45°C. The kinetic behavior of the enzyme in the hydrolysis of pNP-NAG followed Michaelis-Menten kinetics with Km of 0.424 ± 0.012 mM and Vmax of 17.65 ± 0.32 µmol/min at pH 5.8 and 37°C, and the activation energy was determined to be 61.32 kJ/mol. The effects of some metal ions on the enzyme were surveyed, and the results show that Na+ and K+ have no effects on the enzyme activity; Mg2+ and Ca2+ slightly activate the enzyme, while Ba2+, Zn2+, Mn2+, Hg2+, Pb2+, Cu2+, and Al3+ inhibit the enzyme to different extents.  相似文献   

13.
We focused on the influence of different temperature amplitudes on development and reproduction of the two-spotted spider mite, Tetranychus urticae Koch, at a 16:8 (L:D) h photoperiod and 60–95 % RH. The temperature amplitudes varied from 0 to 24 °C in steps of 6 °C; i.e. 22 ± 0, 22 ± 3, 22 ± 6, 22 ± 9 and 22 ± 12 °C. Temperature changed every 24 h between a low and an upper value, but without changing the average temperature (22 °C). The number of eggs laid by five females for 24 h was slightly lower at 22 ± 12 °C than at constant temperature (22 ± 0 °C), and egg hatchability differed among the five temperature regimes. Developmental time at 22 ± 0 °C was shorter than that at 22 ± 3 and 22 ± 6 °C, but longer than that at 22 ± 9 and 22 ± 12 °C. The oviposition period, total fecundity per female and adult longevity gradually decreased with increasing amplitudes. Sex ratio was similar at all five temperature regimes. The intrinsic rate of natural increase (r m) was affected by temperature amplitude and the r m-values at all amplitudes except 22 ± 12 °C were higher than that at constant temperature. Thus, this study showed that variable temperature regimes influence population growth rates of T. urticae and that large amplitude regimes are stressful for this species.  相似文献   

14.
Previous kinetic characterization of Escherichia coli fructose 1,6-bisphosphatase (FBPase) was performed on enzyme with an estimated purity of only 50%. Contradictory kinetic properties of the partially purified E. coli FBPase have been reported in regard to AMP cooperativity and inactivation by fructose-2,6-bisphosphate. In this investigation, a new purification for E. coli FBPase has been devised yielding enzyme with purity levels as high as 98%. This highly purified E. coli FBPase was characterized and the data compared to that for the pig kidney enzyme. Also, a homology model was created based upon the known three-dimensional structure of the pig kidney enzyme. The kcat of the E. coli FBPase was 14.6 s−1 as compared to 21 s−1 for the pig kidney enzyme, while the Km of the E. coli enzyme was approximately 10-fold higher than that of the pig kidney enzyme. The concentration of Mg2+ required to bring E. coli FBPase to half maximal activity was estimated to be 0.62 mM Mg2+, which is twice that required for the pig kidney enzyme. Unlike the pig kidney enzyme, the Mg2+ activation of the E. coli FBPase is not cooperative. AMP inhibition of mammalian FBPases is cooperative with a Hill coefficient of 2; however, the E. coli FBPase displays no cooperativity. Although cooperativity is not observed, the E. coli and pig kidney enzymes show similar AMP affinity. The quaternary structure of the E. coli enzyme is tetrameric, although higher molecular mass aggregates were also observed. The homology model of the E. coli enzyme indicated slight variations in the ligand-binding pockets compared to the pig kidney enzyme. The homology model of the E. coli enzyme also identified significant changes in the interfaces between the subunits, indicating possible changes in the path of communication of the allosteric signal.  相似文献   

15.
Euglena gracilis has the ability to accumulate a storage polysaccharide, a β-1,3-glucan known as paramylon, under aerobic conditions. Under anaerobic conditions, E. gracilis cells degrade paramylon and synthesize wax esters. Cytosolic fructose-1,6-bisphosphatase (FBPase) appears to be a key enzyme in gluconeogenesis and position branch point of carbon partitioning between paramylon and wax ester biosynthesis. We herein identified and characterized cytosolic FBPase from E. gracilis. The Km and Vmax values of EgFBPaseIII were 16.5 ± 1.6 μM and 30.4 ± 7.2 μmol min?1 mg protein?1, respectively. The activity of EgFBPaseIII was not regulated by AMP or reversible redox modulation. No significant differences were observed in the production of paramylon in transiently suppressed EgFBPaseIII gene expression cells by RNAi (KD-EgFBPaseIII); nevertheless, FBPase activity was markedly decreased in KD-EgFBPaseIII cells. On the other hand, the growth of KD-EgFBPaseIII cells was slightly higher than that of control cells.  相似文献   

16.
Leaf cytosolic fructose-1,6-bisphosphatase (FBPase), partially purified from both spinach (Spinacia oleracea, var Hipack) and peas (Pisum sativum, var Progress No. 9), is reversibly inactivated by exposure to low temperature. Thus, even though assays were conducted at 22°C, samples incubated at 0 to 12°C had greatly reduced activity relative to controls maintained at 22°C. Following incubation at 22°C prior to assay, the inactivated samples regained their initial activity. Chloroplast FBPase, by contrast, was unaffected by low temperature treatment. This feature as well as lack of a response of cytosolic FBPase to thioredoxins f or cf and to chloroplast FBPase antibody indicate that the FBPase isozymes of leaves are different proteins.  相似文献   

17.
R F Cox  J G Baust 《Cryobiology》1979,16(2):166-170
Exposure of canine myocardial tissue homogenates to Me2SO glycerol (20 to 60%) for periods up to 8 hr resulted in significant alterations in enzyme activity at 0 °, 18 °, and 37 °C. Both CPK and Na+-K+ ATPase demonstrate anomalous enhancement of activity at each temperature with glycerol. Me2SO provides a similar enhancement of Na+-K+ ATPase activity at hypothermic temperatures up to 40%. Thereafter, nearly complete inhibition resulted. Under normothermic conditions complete Me2SO inhibition occurred at 40 °. CPK activity diminished in a linear fashion after 4 hr at 18 ° and 37 ° but was unaffected by up to 40% Me2SO at 0 °C. The results suggest that disruption of the CPK-Na+-K+ ATPase systems may be minimized by hypothermic perfusion at low cryoprotectant concentrations.  相似文献   

18.
The enzyme inulinase (2,1-β-d-fructan fructanohydrolase, EC 3.2.1.7), prepared from Kluyveromyces marxianus has been immobilized using an inorganic solid support, molecular sieve 4A via the metal link method. The immobilized enzyme had around 22 units of inulinase activity per g of the support with retention of 72% of the original activity. The optimum protein to molecular sieve ratio for the maximum retention of inulinase activity was 9 mg/g molecular sieve. The properties of soluble and immobilized enzyme differed in many respects. The optimum pH of the enzyme shifted from 6 to 5 and the optimum temperature of enzyme activity changed from 50 to 55°C. Km values were 6.7 mM for soluble enzyme and 10 mM for immobilized enzyme. The heat stability of the enzyme was improved by immobilization. Immobilized enzyme retained about 76% of the original activity after 40 days of storage at room temperature (30±2°C).  相似文献   

19.
The effect of temperature, pH, different inhibitors and additives on activity and stability of crude laccase obtained from repeated-batch culture of white rot fungus Funalia trogii ATCC 200800 was studied. The crude enzyme showed high activity at 55–90°C, which was maximal at 80–95°C. It was highly stable within the temperature intervals 20–50°C. The half life of the enzyme was about 2 h and 5 min at 60°C and 70°C, respectively. pH optimum of fungal laccase activity was revealed at pH 2.5. The enzyme from F. trogii ATCC 200800 was very stable between pH values of 3.0–9.0. NaN3 and KCN were detected as the most effective potent enzyme inhibitors among different compounds tested. The fungal enzyme was highly resistant to the various metal ions, inorganic salts, and organic solvents except propanol, at least for 5 min. Because of its high stability and efficient decolorization activity, the use of the crude F. trogii ATCC 200800 laccase instead of pure enzyme form may be a considerably cheaper solution for biotechnological applications.  相似文献   

20.
Water temperature is known to be a particularly important environmental factor that affects fish swimming performance, but it is unknow how acute temperature changes affect the fish performance of Ptychobarbus kaznakovi. P. kaznakovi in the Lancang River have declined quickly in recent years, and this species was used to examine the effects of acute temperature changes on swimming abilities and oxygen consumption in a Brett‐type swimming tunnel respirometer. The standard metabolic rate (SMR) and routine metabolic rate (RMR) showed 216% and 134% increases, respectively, at 22°C (an acute increase from 17 to 22°C) compared to those at 12°C (an acute decrease from 17 to 12°C). Moreover, the RMR was approximately 1.7, 1.6 and 1.3 times the value of the SMR at 12°C, 17°C and 22°C, respectively. The critical swimming speed (Ucrit) of P. kaznakovi at 22°C was 5.45 ± 0.45BL/S, which was 45% higher than that at 12°C (3.77 ± 0.92BL/S). The oxygen consumption rates (MO2) reached their maximum values at swimming speeds near the Ucrit for all the temperature treatments. The maximum metabolic rate (MMR) values at 12°C, 17°C and 22°C were 274.53 ± 142.60 (mgO2 kg?1 hr?1), 412.85 ± 216.34 (mgO2 kg?1 hr?1) and 1,095.73 ± 52.50 (mgO2 kg?1 hr?1), respectively. Moreover, there was a narrow aerobic scope at 12°C compared to that at 17°C and 22°C. The effect of acute temperature changes on the swimming abilities and oxygen consumption of P. kaznakovi indicated that water temperature changes caused by dam construction could directly affect energy consumption during the upstream migration of fish.  相似文献   

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