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1.
The ability of glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase, pyruvate kinase (PK), and lactate dehydrogenase muscle-type (LDH(M)), to generate interactive microtubule networks was investigated. Bundles have previously been defined as the parallel alignment of several microtubules and are one form of microtubule networks. Utilizing transmission electron microscopy, interactive networks of microtubules as well as bundles were readily observed in the presence of GAPDH, aldolase, or PK. These networks appear morphologically as cross-linked microtubules, oriented in many different ways. Light scattering indicated that the muscle forms of GAPDH, aldolase, PK and LDH(m) caused formation of the microtubule networks. Triose phosphate isomerase (TPI) and lactate dehydrogenase heart-type (LDH(H)), glycolytic enzymes which do not interact with tubulin or microtubules, did not produce bundles, or interactive networks. Sedimentation experiments confirmed that the enzymes that cross-link also co-pellet with the microtubules. Such cross-linking of microtubules indicate that the enzymes are multivalent with the capability of simultaneous binding to more than one microtubule.  相似文献   

2.
The effect of hypoxia on the levels of glycogen, glucose and lactate as well as the activities and binding of glycolytic and associated enzymes to subcellular structures was studied in brain, liver and white muscle of the teleost fish, Scorpaena porcus. Hypoxia exposure decreased glucose levels in liver from 2.53 to 1.70 mumol/g wet weight and in muscle led to its increase from 3.64 to 25.1 mumol/g wet weight. Maximal activities of several enzymes in brain were increased by hypoxia: hexokinase by 23%, phosphoglucoisomerase by 47% and phosphofructokinase (PFK) by 56%. However, activities of other enzymes in brain as well as enzymes in liver and white muscle were largely unchanged or decreased during experimental hypoxia. Glycolytic enzymes in all three tissues were partitioned between soluble and particulate-bound forms. In several cases, the percentage of bound enzymes was reduced during hypoxia; bound aldolase in brain was reduced from 36.4 to 30.3% whereas glucose-6-phosphate dehydrogenase fell from 55.7 to 28.7% bound. In muscle PFK was reduced from 57.4 to 41.7% bound. Oppositely, the proportion of bound aldolase and triosephosphate isomerase increased in hypoxic muscle. Phosphoglucomutase did not appear to occur in a bound form in liver and bound phosphoglucomutase disappeared in muscle during hypoxia exposure. Anoxia exposure also led to the disappearance of bound fructose-1,6-bisphosphatase in liver, whereas a bound fraction of this enzyme appeared in white muscle of anoxic animals. The possible function of reversible binding of glycolytic enzymes to subcellular structures as a regulatory mechanism of carbohydrate metabolism is discussed.  相似文献   

3.
In this study, we demonstrate that the catalytic subunit of rabbit muscle protein phosphatase-1 (PP1) binds to muscle phosphofructokinase (6-phosphofructo-1-kinase, PFK). A protein of 85 kDa was isolated from rat muscle by affinity chromatography on PP1-Sepharose and was identified as phosphofructokinase by partial amino acid sequence analysis. This novel finding of a protein-protein interaction between PP1 and PFK was confirmed by reciprocal experiments in which the binding of PP1 to PFK-agarose was demonstrated. Elution of PP1 from PFK-agarose was maximal at ca. 0.4 M NaCl. The specificity of binding was demonstrated by isolation of PP1 from a partially purified rabbit muscle PP1 preparation. All four known isoforms of PP1 (PP1alpha, PP1gamma1, PP1gamma2, and PP1delta) were shown to bind to PFK-agarose. The activity of PP1 was only partially inhibited by PFK. The preformed complex between PP1 and PFK did not bind to inhibitor-2-Sepharose. The stoichiometry of binding of PP1 to the PFK monomer was found to be 1:1 in the isolated PP1.PFK complex. An interaction between PP1 and PFK in muscle extracts was demonstrated by their coimmunoprecipitation. Our findings raise the interesting possibility that PP1 may be targeted to PFK, and may be physiologically relevant in the context that PFK and other glycolytic enzymes have been shown to be micro-compartmentalized by binding to F-actin. This in turn points to a role for PP1 in control of glycolytic flux by protein phosphorylation-dephosphorylation mechanisms.  相似文献   

4.
1. We show here that treatment of diaphragm muscle with 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, abolished the stimulatory action of insulin on binding of the glycolytic enzymes, phosphofructokinase (PFK) and aldolase, to muscle cytoskeleton. This effect was demonstrated with low concentration of DNP, which caused only a small decrease in ATP and did not affect the basic levels of cytoskeleton-bound glycolytic enzymes. 2. Higher concentrations of DNP, which induced a drastic decline in ATP content, caused a decrease in cytoskeleton-bound glycolytic enzymes and damage to myofibrils. 3. These results suggest that mitochondrial ATP is required for both the preservation of the basal levels of cytoskeleton-bound glycolytic enzymes and cell structure, as well as for the expression of the stimulatory action of insulin on glycolytic enzymes' binding to muscle cytoskeleton.  相似文献   

5.
A theoretical metabolic-control-analysis approach has been used to study aspects of glycolytic-flux control and carbon-metabolite regulation, particularly the role of ATP demand (ATPase), in order to determine what general features of the regulation of energy metabolism would be consistent with good carbon-metabolite homeostasis in the face of large changes in carbon flux. On the basis of a semi-quantitative control-analysis model, incorporating estimates of substrate, product and effector actions on the enzymes, the experimentally observed characteristics of glycolytic-flux changes prove to impose constraints on the feasible ranges of these estimates. This leads to the identification of several features of energy metabolism, each of which is necessary but not sufficient to explain the observations; although most of these have been advocated previously (such as AMP activation of phosphofructokinase (PFK), ADP inhibition of ATPase and the role of energy charge or ATP/ADP ratio), our analysis allows their relative importance to be assessed. In the model, the distribution of flux control depends primarily on ADP inhibition of ATPase, and on the activation of PFK by AMP; increase in ADP inhibition of ATPase increases the control on PFK; increase in AMP activation of PFK increases control on ATPase. PFK exerts greater flux control than does ATPase over approximately 50% of the ranges (parameter space) studied, but its control is sufficiently high to achieve sizeable flux increases over less than 20% of the space. Furthermore, control by alteration in PFK activity is shown to result in poor glycolytic metabolite homeostasis over the entire parameter space studied. However, over a large proportion of the parameter space, control by activation of ATPase can lead to large flux changes, i.e. high flux control, coupled with excellent glycolytic-metabolite homeostasis, similar to that observed in working muscle. As well as altering the relative degrees of flux control invested in PFK and ATPase, ADP inhibition of ATPase and AMP activation of PFK have pronounced effects on the homeostatic properties of the system. Stronger ADP inhibition of ATPase results in improved homeostasis of glycolytic metabolites, ATP and ADP in response to PFK activation, whereas stronger activation of PFK by AMP improves the homeostasis of these three quantities in response to ATPase activation. The results are further evidence of the potential for physiological ATP demand to exert control over glycolytic flux, but additionally show that the known effector interactions, in addition to their previously known role in ATP regulation, could contribute to the remarkable homeostasis of glycolytic-metabolite levels observed in vivo. They further indicate that quantitative characterisation of likely domains of behaviour of metabolic systems can be achieved by an algebraic analysis that is not highly dependent on a full and precise knowledge of the molecular details of the kinetic/regulatory properties of the enzymes, but that still allows an assessment of whether hypotheses regarding the system are feasible and sufficient to account for the observations.  相似文献   

6.
The extent of binding of glycolytic enzymes to the particulate fraction of homogenates was measured in bovine psoas muscle before and after electrical stimulation. In association with an accelerated glycolytic rate on stimulation, there was a significant increase in the binding of certain glycolytic enzymes, the most notable of which were phosphofructokinase, aldolase, glyceraldehyde 3-phosphate dehydrogenase and pyruvate kinase. From the known association of glycolytic enzymes with the I-band of muscle it is proposed that electrical stimulation of anaerobic muscle increases enzyme binding to actin filaments. Calculations of the extent of enzyme binding suggest that significant amounts of enzyme protein, particularly aldolase and glyceraldehyde 3-phosphate dehydrogenase, are associated with the actin filaments. The results also imply that kinetic parameters derived from considerations of the enzyme activity in the soluble state may not have direct application to the situation in the muscle fibre, particularly during accelerated glycolysis.  相似文献   

7.
In the presence of Uropolinum (sodium and N-methylglucamine diatrizoate) the lactate production of erythrocytes was decreased in half of the cases studied. The addition of albumin showed a protective effect on glycolysis of red blood cells. A decrease in glutathione reductase and increase in glucose-6-phosphate dehydrogenase activity was observed in all cases. Acethylcholinesterase activity was unaffected by Uropolinum. The activities of phosphohexose isomerase, phosphofructokinase. aldolase and ATP-ase were decreased in some subjects and increased in the others. It may be attributed to individual sensitivity of some subjects to diatrizoates and is consistent with clinical observations.  相似文献   

8.
Cleavage of tubulin at tryptophan residues yielded several peptides, one of which strongly interacted with aldolase as determined by inhibition of aldolase activity. This peptide was identified as the C-terminal, residues 408-451, of the alpha-subunit of tubulin. Peptides with identical sequences to the C-terminal regions of the alpha- and beta-subunits of tubulin were synthesized to further characterize interactions with glycolytic enzymes. A 43-amino-acid C-terminal peptide from alpha-tubulin (residues 409-451) was found to have binding properties similar to those of native tubulin and was designated the tubulin glycolytic enzyme binding domain (T-GEBD-43mer).  相似文献   

9.
Osmotically hemolysed pigeon erythrocytes retain a considerable part of the total cell content of aldolase activity. After washing off the ghosts from hemoglobin and removing the nuclei, a considerable portion of aldolase activity is found in the supernatant. The retained part of aldolase is rather firmly bound to plasma membranes (PM), as evidenced by the fact, that double washing with a mixture of 0.3 M sucrose, 0.01 M tris-HCl (pH 7.4) and 0.004 M MgCL2, or with 0.15 M NaCl or H2O does not appreciably decrease the aldolase activity of PM. Only washing of PM with 0.5 M NaCl results in appreciable decrease of aldolase retention by PM. The binding of aldolase proved to be temperature sensitive: after heating the binding of aldolase to PM specifically decreased. These data suggest that the interaction of the enzyme with PM of pigeon erythrocytes occurs in the intact cell and may be of physiological significance.  相似文献   

10.
Physiological acclimation that alters enzyme activity can compensate for the effect of temperature on function and may be achieved by altering enzyme concentration. This study uses phylogenetic analyses to investigate the evolutionary history of and to test several hypotheses about acclimation responses among all the glycolytic enzymes. These hypotheses are that (1) acclimation increases enzyme concentration at lower temperatures to compensate for reduced activity; (2) equilibrium enzymes tend to show acclimation responses; and (3) acclimation responses are more common in species whose populations experience either large temporal or geographical temperature variations. Using maximal activities as indices of enzyme concentration, the presence of acclimation responses in all the glycolytic enzymes in the heart ventricle was determined for five species in the teleost genus Fundulus. Three of these species are distributed along the steep thermal cline of the North American Atlantic coast, and thus these species experience both seasonal and geographical variation in temperature. The other two species are found in the Gulf of Mexico and experience seasonal variation similar to the Atlantic species but no geographical variation in temperature. Two Atlantic coast species, Fundulus heteroclitus and Fundulus majalis, have unique derived acclimation responses. No derived acclimation responses occur in the Gulf species. A conserved response in hexokinase was observed within one subgenus comprising both Atlantic and Gulf species. In F. heteroclitus, enolase responded to acclimation, and in F majalis, aldolase, triphosphate isomerase, and lactate dehydrogenase had acclimation responses. These enzymes are equilibrium enzymes, and the concentrations of all of them increase at lower temperatures, which would compensate for the effect of temperature on enzyme activity. The compensatory changes all occur in the Atlantic species and may be a mechanism for species to expand their ranges. These data suggest that physiological acclimation is evolutionarily labile.  相似文献   

11.
The electrostatic properties of seven alpha/beta-barrel enzymes selected from different evolutionary families were studied: triose phosphate isomerase, fructose-1,6-bisphosphate aldolase, pyruvate kinase, mandelate racemase, trimethylamine dehydrogenase, glycolate oxidase, and narbonin, a protein without any known enzymatic activity. The backbone of the alpha/beta-barrel has a distinct electrostatic field pattern, which is dipolar along the barrel axis. When the side chains are included in the calculations the general effect is to modulate the electrostatic pattern so that the electrostatic field is generally enhanced and is focused into a specific area near the active site. We use the electrostatic flux through a square surface near the active site to gauge the functionally relevant magnitude of the electrostatic field. The calculations reveal that in six out of the seven cases the backbone itself contributes greater than 45% of the total flux. The substantial electrostatic contribution of the backbone correlates with the known preference of alpha/beta-barrel enzymes for negatively charged substrates.  相似文献   

12.
OBJECTIVE: To observe the effects of 3,4-dihydroxyacetophynone (DHAP), one of the constituents of a traditional Chinese herbal medicine, on the activities of endothelial nitric oxide synthetase (NOS) in vascular endothelial cells (VEC) and vascular smooth muscle cells (VSMC) of placenta and the level of endothelin-1 (ET-1) in the plasma from PIH patients. METHOD: 22 nulliparous PIH patients were randomly divided into PIH group and DHAP treatment group. Each patients in DHAP group received intravenous administration of DHAP (160-240 mg/d). Blood samples were collected before the administration of DHAP and (or) before cesarean section. The placenta were assayed for nitric oxide synthase activity using histochemical analysis; the concentration of ET in plasma was measured by radioimmunoassays. 10 normal pregnant women or 10 nonpregnant women were served as controls. RESULTS: In normal pregnant controls, NOS activity was much higher than that in the PIH group; in DHAP group, there was some recovery of NOS activity after DHAP treatment. The concentration of ET was higher in PIH group than that of normal pregnant controls, but it decreased significantly after DHAP therapy (P < 0.05, vs PIH group or before DHAP therapy). CONCLUSION: This study indicates that DHAP is effective in the treatment of PIH and its mechanism of action may be due to the adjustment of NO/ET imbalance in PIH patients.  相似文献   

13.
3Beta-hydroxysteroid dehydrogenase/steroid delta5-isomerase (3beta-HSD/isomerase) was expressed by baculovirus in Spodoptera fungiperda (Sf9) insect cells from cDNA sequences encoding the human wild-type I (placental) enzyme and the human type I mutant- Y253F. The wild-type and Y253F enzymes were each purified as a single, homogeneous protein from a suspension of the Sf9 cells. Ultraviolet (UV) spectral analyses showed that the wild-type enzyme induced changes in the UV spectrum of the competitive isomerase inhibitor, 19-nortestosterone, and the Y253F mutant did not. The wild-type isomerase required activation by coenzyme to produce the spectral shift. Activation of isomerase by NADH produced a greater change in the 19-nortestosterone spectrum than activation by NAD+. These observations provide direct evidence that Tyr253 functions as the general acid (proton donor) in the isomerase reaction mechanism. Furthermore, the coenzyme-activation profiles support our proposed two-step enzyme mechanism in which NADH produced by the 3beta-HSD activity induces the enzyme to assume the isomerase conformation.  相似文献   

14.
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16.
The unidirectional steady state reaction rates of several enzymes and metabolic fluxes of distinct processes were measured simultaneously in hypoxic maize root tips using two-dimensional phosphorus NMR exchange spectroscopy. A single spectrum monitors ATP synthesis and hydrolysis as well as the activities of four enzymes involved in key pathways of central metabolism: UDP-glucose pyrophosphorylase, phosphoglucomutase, hexose-phosphate isomerase, and enolase. The corresponding unidirectional reaction rates and net metabolic fluxes were calculated from spectral intensities. This method provides a unique picture, at enzyme resolution, of how metabolism reacts in a concerted fashion to changes in external parameters such as temperature and oxygen concentration. By increasing hypoxia via an increase in temperature, we measured the expected increase in glycolysis through enolase activity while total ATP synthesis settled. At the same time, we observed a net flux through phosphoglucomutase and UDP-glucose pyrophosphorylase toward carbohydrate synthesis. This result is discussed in relation to the current hypothesis on the turnover of cell walls and sucrose. This reaction also produces a net flux of pyrophosphate, which is needed by pyrophosphate:fructose-6-phosphate 1-phosphotransferase to work as a glycolytic enzyme.  相似文献   

17.
Fifteen red cell enzyme activities of growth-retarded patients with and without growth hormone (GH) deficiency were investigated before and after GH administration. The 15 enzymes were Hexokinase, phosphoglucomutase, glucose phosphate, isomerase, phosphofructokinase, fructose diphosphate aldolase, glyceraldehyde-3-phosphae dehydrogenase, triosephosphate isomerase, 2,3-diphosphoglycerate mutase, 3-phosphoglycerate kinase, 3-phosphoglycerate mutase, enolase, pyruvate kinase, glycose-6-phosphate dehydrogenase, 6-phosphogluconic dehydrogenase, glutathione reducase. Sixty-six subjects were studied: 30 normal control subjects (group N) and 36 patients (aged 5-23 years) with short stature. Complete endocrine evaluation showed 21 (group I) to have GH deficiency (10 patients with isolated GH deficiency) and 15 (group II) to have normal hypothalamic and pituitary function except for two patients with a moderate hypothyroidism. Both had been receiving thyroid hormone treatment for a long time before our studies. All 36 patients were treated with 2 mg human growth hormone intramuscularly for 7 days. Before GH treatment no significant difference was observed between hematologic data in group I (GH deficiency) and group II (no GH deficiency). After GH therapy there was a significant increase in reticulocyte count in both groups of patients with short stature. The mean pretreatment value in group I was 1.294% +/- 0.084 (SEM); the mean post-treatment value was 2.081% +/- 0.287 (SEM)< P less than 0.005. The mean pretreatment value in group II was 1.0% 0.184 (SEM); the mean post-treatment value was 1.407% +/- 0.193 (SEM), P less than 0.01. In group II (no GH deficiency) mean pretreatment erythrocyte enzyme activities were not significantly different from those activities observed in normal control subjects (group N). However, in patients who lacked GH, the pretreatment activities of five red cell enzymes (glucose phosphate isomerase, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, 2,3-diphosphoglycerate mutase, 3-phosphoglycerate kinase) were significantly decreased before GH administration compared with the values in normal control subjects...  相似文献   

18.
19.
The structure of the glycolytic enzyme class I fructose-1, 6-bisphosphate aldolase from the human malaria parasite Plasmodium falciparum has been determined by X-ray crystallography. Homotetrameric P. falciparum aldolase (PfALDO) crystallizes in space group P3221 with one 80 kDa dimer per asymmetric unit. The final refined PfALDO model has an R-factor of 0.239 and an R-free of 0.329 with respect to data from 8 to 3.0 A resolution. PfALDO is potentially a target for antimalarial drug design as the intraerythrocytic merozoite lifestage of P. falciparum is completely dependent upon glycolysis for its ATP production. Thus, inhibitors directed against the glycolytic enzymes in P. falciparum may be effective in killing the parasite. The structure of PfALDO is compared with the previously determined structure of human aldolase in order to determine possible targets for the structure-based design of selective PfALDO ligands. The salient structural differences include a hydrophobic pocket on the surface of PfALDO, which results from some amino acid changes and a single residue deletion compared with human aldolase, and the overall quaternary structure of the PfALDO tetramer, which buries less surface area than human aldolase.  相似文献   

20.
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