Uncoupling of mitochondrial oxidative phosphorylation abolishes the stimulatory action of insulin on the binding of glycolytic enzymes to muscle cytoskeleton

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.     

Triosephosphate isomerase deficiency: predictions and facts

Deficiencies in around 20 enzymes, associated with widely different degrees of severity and complexity, have been identified for human erythrocytes. The fact that glycolysis is crucial for erythrocyte function is reflected by the large number of inherited glycolytic enzymopathies. Triosephosphate isomerase (TPI) deficiency, a rare autosomal disease, is usually associated with nonspherocytic hemolytic anemia, progressive neurologic dysfunction, and death in childhood. The two affected Hungarian brothers studied by us have less than 3% TPI activity and enormously (30-50-fold) increased dihydroxyacetone phosphate (DHAP) concentration in their erythrocytes. The well-established concept of the metabolic control theory was used to test the contribution of TPI and some related enzymes to the control of a relevant segment of the glycolytic pathway in normal and deficient cells. Deviation indices, DEJ = (delta J/delta E) E(r)/J(r), which give a good estimation of flux control coefficients using a single large change in enzyme activity, were determined from the fluxes in the absence and presence of exogeneous enzymes. We found that PFK and aldolase are the enzymes that predominantly control the flux, however, the quantitative values depend extensively on the pH: DEJ values are 0.85 and 0.14 at pH 8.0 and 0.33 and 0.67 at pH 7.2 for aldolase and PFK, respectively. Neither the flux rates nor the capacities of the enzymes seem to be significantly different in normal and TPI deficient cells. There is a discrepancy between DHAP levels and TPI activities in the deficient cells. In contrast to the experimental data the theoretical calculations predict elevation in DHAP level at lower than 0.1% of the normal value of TPI activity. Several possibilities suggested fail to explain this discrepancy. Specific associations of glycolytic enzymes to band-3 membrane proteins with their concomitant inactivation have been demonstrated. We propose that the microcompartmentation of TPI that could further decrease the reduced isomerase activity of the deficient cells, is responsible for the high DHAP level.     

Effect of electrical stimulation post mortem of bovine muscle on the binding of glycolytic enzymes. Functional and structural implications

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.     

Targeting of the catalytic subunit of protein phosphatase-1 to the glycolytic enzyme phosphofructokinase

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.     

Glycolytic enzymes and assembly of microtubule networks

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.     

Inhibitory effects of caffeine on Ca2+ influx and histamine secretion independent of cAMP in rat peritoneal mast cells

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).     

gamma-Interferon produced by CD8+ T cells infiltrating Kaposi''s sarcoma induces spindle cells with angiogenic phenotype and synergy with human immunodeficiency virus-1 Tat protein: an immune response to human herpesvirus-8 infection?

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.     

Crystal structure of fructose-1,6-bisphosphate aldolase from the human malaria parasite Plasmodium falciparum

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.     

Clinical and radiographic diagnosis of occlusal caries: a study in vitro

We report on the autopsy study of a premature boy with multiple joint contractures who died soon after birth of severe lung hypoplasia. Muscle histology showed PAS-positive vacuoles, and electronmicroscopy revealed massive subsarcolemmal and intermyofibrillar accumulation of glycogen. Biochemical analysis of fresh-frozen muscle tissue disclosed increased glycogen content and a complete lack of phosphofructokinase (PFK) activity. The brain showed focal cerebral and diffuse cerebellar white matter gliosis, and patchy loss of internal granular and Purkinje cells in the cerebellar cortex. The spinal cord was normal. This report describes the first case of PFK deficiency, presenting as a lethal fetal akinesia sequence.     

Occurrence of free creatine, phosphocreatine and creatine phosphokinase in adipose tissue

We evaluated brown and white adipose tissues for the presence of creatine, phosphocreatine and creatine phosphokinase activity. In rats 3.6 and 0.4 mumol of total creatine were found per g wet weight of brown and white adipose tissues, respectively. We were able to identify creatine by thin-layer chromatography after a pulse label of [14C]creatine had been given in vivo. Free creatine and phosphocreatine were shown to occur by column chromatography. Of the total creatine of brown adipose tissue, approximately one third to one half were attributable to phosphocreatine. The activity of creatine phosphokinase was demonstrated in both white and brown adipose tissue, the values of the latter prevailing over those of the former by a factor of 200, if based on wet weight, or 50, if expressed as specific enzyme activity. The labeling of total creatine in vivo proceeded much faster in adipose tissue than in skeletal muscle. The results strongly suggest that the energy metabolism of adipose tissue is closely dependent on the presence of creatine. The specific activities of free creatine and phosphocreatine of brown adipose tissue differed strikingly as long as 24 h after radioactive creatine was injected; this difference points to a metabolic or structural compartmentation of creatine.     

Four weeks' inhalation exposure of rats to p-cymene affects regional and synaptosomal neurochemistry

Long-lasting effects of inhalation exposure to p-cymene (p-isopropyl-toluene; CAS No. 99-87-6) on regional and subcellular brain neurochemistry were studied. Male Long-Evans rats were exposed to 0, 50, or 250 p.p.m. p-cymene 6 hr/day, 5 days/week for four weeks followed by an exposure-free period of 8 weeks. Synaptosomes were isolated from whole brain minus cerebellum and used as an ex situ model for in situ conditions at the level of the presynaptic nerve terminal. There was no persistent effect on wet weight (regional) or regional noradrenaline (NA), dopamine (DA), or 5-hydroxytryptamine (5-HT) concentrations owing to exposure. Yield of synaptosomal protein was statistically significantly reduced in an exposure concentration-related manner (Control: 16.6 +/- 3.1; 50 p.p.m.: 9.2 +/- 2.1; 250 p.p.m.: 8.6 +/- 1.7 mg protein/g tissue, mean +/- I.S.D.). Synaptosomal NA and DA concentrations and acethycholinesterase, butyrylcholinesterase, and lactate dehydrogenase activities were statistically significantly increased when expressed relative to synaptosomal protein. It is hypothesized that a reduced density and number of synapses in situ are functionally compensated for by increased NA and DA release from noradrenergic and dopaminergic presynaptic nerve terminals. The applicability of the synaptosome as an ex situ neurochemical research model for the presynaptic CNS nerve terminal in situ for the study of solvent neurotoxicity in rats was further supported.     

Study of platelet-activating factor acetylhydrolase in the perioperative period of patients undergoing cardiac surgery

This study was conducted to determine whether brief, intermittent exposure to hypoxia with little change in nutrient intake would affect fetal growth. Pregnant rats were exposed to 1 or 2 h of hypoxia (FiO2 = 0.09-0.095) from days 15 to 19 of gestation. Exposure to 1 h of hypoxia decreased fetal body weight and length, liver weight and increased the brain/liver weight ratio (p < 0.05) as compared to controls. Two hours of hypoxia decreased fetal body weight and length, and heart, lung, kidney, gut, brain and liver weights (p < 0.01), but did not affect the brain/liver weight ratio. Two hours of hypoxia decreased maternal food intake and weight gain (p < 0.05), but fetal growth was not significantly altered in pair-fed controls. These data demonstrate that brief, intermittent periods of intrauterine hypoxia have significant effects on fetal growth.     

Heat production and chemical change during isometric contraction of rat soleus muscle

1. Methods are described whereby the soleus muscle of the rat may be used for the investigation of initial processes in the absence of oxidative recovery. 2. The anaerobic conditions employed had no effect on the concentration of phosphocreatine in resting muscle or the mechanical response during contraction. 3. Muscles were stimulated tetanically for 10 s at 17-18 degrees C. Measurements were made of the heat production and metabolic changes that occurred during a 13 s period following the first stimulus. 4. There was no detectable change in the concentration of ATP. Neither was there detectable activity of adenylate kinase or adenylate deaminase. The changes in the concentration of glycolytic intermediaries were undetectable or very small. 5. The change in the concentration of phosphocreatine was large and amounted to -127 +/- 11-4 mumol/mmol Ct (mean and S.E. of the mean, negative sign indicates break-down, Ct = free creatine + phosphocreatine) which is equivalent to about -2-13 mumol/g wet weight of muscle. The heat production was 6549 +/- 408 mJ/mmol Ct (mean and S.E. of mean) which is equivalent to about 110 mJ/g. 6. About 30% of the observed energy output is unaccounted for by measured metabolic changes. 7. The ratio of heat production (corrected for small amounts of glycolytic activity) to phosphocreatine hydrolysis was -49-7 +/- 5-6 kJ/mol (mean and S.E. of mean), in agreement with previous results using comparable contractions of frog muscle, but different from the enthalpy change associated with phosphocreatine hydrolysis under in vivo conditions (-34 kJ/mol). 8. The results support the notion that the discrepancy between energy output and metabolism is an indication of an unidentified process of substantial energetic significance that is common to a number of species.     

Phylogenetic analysis of thermal acclimation of the glycolytic enzymes in the genus Fundulus

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.     

Alterations of antioxidant enzymes and oxidative damage to macromolecules in different organs of rats during aging

Oxygen free radicals have been hypothesized to play an important role in the aging process. To investigate the correlation between the oxidative stress and aging, we have determined the levels of oxidative protein damage and lipid peroxidation in the brain and liver, and activities of antioxidant enzymes in the brain, liver, heart, kidney, and serum from the Fisher 344 rats at ages of 1, 6, 12, 18, and 24 months. The results showed that the level of oxidative protein damage (measured as carbonyl content) in the brain and liver was significantly higher in older animals than in young animals. No statistical difference was observed in the lipid peroxidation of the liver and brain between young and old animals. The activities of antioxidant enzymes in most tissues displayed an age-dependent decline. Superoxide dismutases in the heart, kidney, and serum, glutathione peroxidase activities in the serum and kidney, and catalase activities in the brain, liver, and kidney, significantly decreased during aging. Cytochrome c oxidase, an enzyme involved in electron transport in mitochondria, initially increased, but subsequently decreased in the aged brain, whereas no significant alteration was observed in the liver mitochondrial antioxidant enzymes. The present studies suggest that the accumulation of oxidized proteins during aging is most likely to be linked with an age-related decline of antioxidant enzyme activities, whereas lipid peroxidation is less sensitive to predict the aging process.