The effect of dietary and hormonal conditions on the activities of glycolytic enzymes in rat epididymal adipose tissue

1. Measurements were made of the activities of nine glycolytic enzymes in epididymal adipose tissues obtained from rats that had undergone one of the following treatments: starvation; starvation followed by re-feeding with bread or high-fat diet; feeding with fat without preliminary starvation; alloxan-diabetes; alloxan-diabetes followed by insulin therapy. 2. In general, the activities of the glycolytic enzymes of adipose tissue, unlike those of liver, were not greatly affected by the above treatments. 3. The ;key' glycolytic enzymes, phosphofructokinase and pyruvate kinase, were generally no more adaptive in response to physiological factors than other glycolytic enzymes such as glucose phosphate isomerase, fructose diphosphate aldolase, triose phosphate isomerase, glycerol 3-phosphate dehydrogenase, phosphoglycerate kinase and lactate dehydrogenase. 4. Adiposetissue pyruvate kinase did not respond to feeding with fat in a manner similar to the liver enzyme. 5. Glyceraldehyde phosphate dehydrogenase had a behaviour pattern unlike the other eight glycolytic enzymes studied in that its activity was depressed by feeding with fat and was not restored to normal by re-feeding with a high-fat diet after starvation. These results are discussed in relation to the requirements of adipose tissue for glycerol phosphate in the esterification of fatty acids. 6. A statistical analysis of the results permitted the writing of linear equations describing the relationships between the activities of eight of the enzymes studied. 7. Evidence is presented for the existence of two constant-proportion groups amongst the enzymes studied, namely (i) glucose phosphate isomerase, phosphoglycerate kinase and lactate dehydrogenase, and (ii) triose phosphate isomerase, fructose diphosphate aldolase and pyruvate kinase. 8. Mechanisms for maintaining the observed relationships between the activities of the enzymes in the tissue are discussed.     

Experimental effect of bruneomycin on the energy metabolism of liver tissue]

The effect of an antitumor antibiotic bruneomycin on the energy metabolism in the liver tissue was studied. Four hours after the drug administration the consumption of glycogen and glucose in the liver tissue increased because of glycogenolysis activation, which was evident from increased activity of prosphorylase, phosphofructokinase, hexokinase and summation glycolytic activity. 24 and 48 hours after the antibiotic administration the balance of consumption and resynthesis of phosphate macroergs in the liver tissue impaired, which was evident from decreased levels of ATP, impairement of conjugation of the processes of oxidation due to impairement of permeability and structural integrity of the mitochondrial membranes. Further decrease in the glycogen liver levels was mainly due to suppressed resynthesis of glycogen because of destructive-necrotic processes. Simultaneously the processes of glycolytic splitting of glucose decreased which was evident from decreased activity of the enzymes and summation glycolytic activity.     

A histochemical study about the influence of lytic enzymes on plasma membrane enzyme activities in rat liver and kidney.

1. The effect of lipolytic, glycolytic and proteolytic enzymes on the activities of plasma membrane enzyme activities in rat liver and kidney has been investigated by a pretreatment of tissue sections with the lytic enzymes. 2. The action of the proteolytic enzymes causes a very strong decrease of leucyl-beta-naphthylamidase activity, whereas the activities of ATP-ase, 5'-nucleotidase and alkaline phosphatase show a lesser decrease. This indicates a different membrane anchorage of leucyl-beta-naphthylamidase as compared to that of the phosphatases. 3. Treatment with glycolytic enzymes results in a decrease of 5'-nucleotidase and ATP-ase activity, whereas liver alkaline phosphatase and leucyl-beta-naphthylamidase show an increase in activity. 4. Treatment with phospholipase C gives about the same results. The very strong decrease of 5'-nucleotidase activity indicates a great dependence on phospholipids.     

Comparative levels between enzymes of the glycolytic pathway from erythrocytes and somatic tissues of the chicken Gallus gallus domesticus

A comparative study on the glycolytic enzymes from chicken erythrocytes and somatic tissues has been carried out, the results being shown as active units per mg protein in supernatants of 1085, 12,100 and 106,000 g fractionated centrifugation. The profiles of the glycolytic enzymes have been analyzed in terms of their activity relative to hexokinase and as the ratios between pairs of enzymes bearing a product-substrate relationship. Chicken erythrocyte displays a very peculiar profile of glycolytic enzymes. It possesses a FruP2-activated pyruvate kinase of the L isoenzyme type, which does not seem to be the predominant isoenzyme together with the M type, the content in glycolytic enzymes being much lower than in the somatic tissues.     

Coordinate regulation of glycolysis by hypoxia in mammalian cells

The impact of hypoxic exposure on the activities of all 11 glycolytic enzymes was studied in cell culture into mammalian cells—mouse lung macrophages and L8 rat skeletal muscle cells. During hypoxic exposure, the measured activity of all glycolytic enzymes increased, establishing coordinate regulation. Three nonglycolytic cytoplasmic enzymes showed no change in activity under the same conditions, suggesting a specific mechanism. Hypoxia appears to increase the activities of all glycolytic enzymes whether rate-limiting or not, presumably increasing adenosine triphosphate availability despite decreased O₂ supply.     

Increased glucose metabolism and ATP level in brain tissue of Huntington's disease transgenic mice

Huntington's disease (HD) is a progressive neurodegenerative disorder characterized by multifarious dysfunctional alterations including mitochondrial impairment. In the present study, the formation of inclusions caused by the mutation of huntingtin protein and its relationship with changes in energy metabolism and with pathological alterations were investigated both in transgenic and 3-nitropropionic acid-treated mouse models for HD. The HD and normal mice were characterized clinically; the affected brain regions were identified by immunohistochemistry and used for biochemical analysis of the ATP-producing systems in the cytosolic and the mitochondrial compartments. In both HD models, the activities of some glycolytic enzymes were somewhat higher. By contrast, the activity of glyceraldehyde-3-phosphate dehydrogenase was much lower in the affected region of the brain compared to that of the control. Paradoxically, at the system level, glucose conversion into lactate was enhanced in cytosolic extracts from the HD brain tissue, and the level of ATP was higher in the tissue itself. The paradox could be resolved by taking all the observed changes in glycolytic enzymes into account, ensuing an experiment-based detailed mathematical model of the glycolytic pathway. The mathematical modelling using the experimentally determined kinetic parameters of the individual enzymes and the well-established rate equations predicted the measured flux and concentrations in the case of the control. The same mathematical model with the experimentally determined altered V(max) values of the enzymes did account for an increase of glycolytic flux in the HD sample, although the extent of the increase was not predicted quantitatively. This suggested a somewhat altered regulation of this major metabolic pathway in HD tissue. We then used the mathematical model to develop a hypothesis for a new regulatory interaction that might account for the observed changes; in HD, glyceraldehyde-3-phosphate dehydrogenase may be in closer proximity (perhaps because of the binding of glyceraldehyde-3-phosphate dehydrogenase to huntingtin) with aldolase and engage in channelling for glyceraldehyde-3-phosphate. By contrast to most of the speculation in the literature, our results suggest that the neuronal damage in HD tissue may be associated with increased energy metabolism at the tissue level leading to modified levels of various intermediary metabolites with pathological consequences.     

Glycolytic enzymes associate dynamically with mitochondria in response to respiratory demand and support substrate channeling

In Arabidopsis thaliana, enzymes of glycolysis are present on the surface of mitochondria and free in the cytosol. The functional significance of this dual localization has now been established by demonstrating that the extent of mitochondrial association is dependent on respiration rate in both Arabidopsis cells and potato (Solanum tuberosum) tubers. Thus, inhibition of respiration with KCN led to a proportional decrease in the degree of association, whereas stimulation of respiration by uncoupling, tissue ageing, or overexpression of invertase led to increased mitochondrial association. In all treatments, the total activity of the glycolytic enzymes in the cell was unaltered, indicating that the existing pools of each enzyme repartitioned between the cytosol and the mitochondria. Isotope dilution experiments on isolated mitochondria, using (13)C nuclear magnetic resonance spectroscopy to monitor the impact of unlabeled glycolytic intermediates on the production of downstream intermediates derived from (13)C-labeled precursors, provided direct evidence for the occurrence of variable levels of substrate channeling. Pull-down experiments suggest that interaction with the outer mitochondrial membrane protein, VDAC, anchors glycolytic enzymes to the mitochondrial surface. It appears that glycolytic enzymes associate dynamically with mitochondria to support respiration and that substrate channeling restricts the use of intermediates by competing metabolic pathways.     

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.     

Evidence for a functional ATP-citrate lyase:NADP-malate dehydrogenase pathway in bovine adipose tissue: Enzyme and metabolite levels

Activities of key lipogenic and glycolytic enzymes were determined in extracts of crude homogenates to elucidate the rate-limiting step(s) for lipogenesis from lactate and glucose in bovine subcutaneous adipose tissue. The enzymes ATP-citrate lyase, NADP-malate dehydrogenase, and pyruvate carboxylase were shown to have enough activity to account for the rates of in vitro lipogenesis from 10 mm lactate with or without 2 mm glucose. Glucose utilization for fatty acid synthesis appears to be limited by the low activities of key glycolytic enzymes, especially hexokinase. Attempts were also made to estimate enzyme activities in bovine subcutaneous adipose tissue being incubated in vitro by relating primary substrate levels to kinetic characteristics for the enzymes. ATP-citrate lyase was estimated to be operating at levels equivalent to the rates of lactate incorporation into fatty acids in the absence or presence of 2 mm glucose in the incubation media. Additionally, metabolite levels were measured in rapidly frozen samples of bovine subcutaneous adipose tissue to estimate the relative importance of key lipogenic enzymes in vivo. At the citrate and malate levels measured in vivo, ATP-citrate lyase would be operating at levels that approximate those estimated in vitro.     

BRAIN MITOCHONDRIA : II. The Relationship of Brain Mitochondria to Glycolysis

A mitochondrial fraction prepared from calf brain cortex possessed negligible glycolytic activity in the absence of the enzymes of the high speed supernatant fraction. When mitochondria were added to a supernatant system supplemented with optimal amounts of crystalline hexokinase, a 20 per cent stimulation of glycolysis was observed. The supernatant fraction produced minimal amounts of lactate in the absence of exogenous hexokinase; the addition of mitochondria doubled the lactate production. The substitution of glycolytic intermediates for glucose as substrates as well as the addition of exogenous glycolytic enzymes to the supernatant fraction or supernatant fraction plus mitochondria indicated that the mitochondria contributed mainly hexokinase and phosphofructokinase. By direct assay of all of the enzymes of the glycolytic pathway, only hexokinase and phosphofructokinase were shown to be concentrated in the mitochondrial fraction. All other glycolytic enzymes were found to exhibit higher total and specific activities in the supernatant fraction.     

Identification of proteins interacting with lactate dehydrogenase in claw muscle of the porcelain crab Petrolisthes cinctipes

Biochemical adaptation of enzymes involves conservation of activity, stability and affinity across a wide range of intracellular and environmental conditions. Enzyme adaptation by alteration of primary structure is well known, but the roles of protein–protein interactions in enzyme adaptation are less well understood. Interspecific differences in thermal stability of lactate dehydrogenase (LDH) in porcelain crabs (genus Petrolisthes) are related to intrinsic differences among LDH molecules and by interactions with other stabilizing proteins. Here, we identified proteins that interact with LDH in porcelain crab claw muscle tissue using co-immunoprecipitation, and showed LDH exists in high molecular weight complexes using size exclusion chromatography and Western blot analyses. Co-immunoprecipitated proteins were separated using 2D SDS PAGE and analyzed by LC/ESI using peptide MS/MS. Peptide MS/MS ions were compared to an EST database for Petrolisthes cinctipes to identify proteins. Identified proteins included cytoskeletal elements, glycolytic enzymes, a phosphagen kinase, and the respiratory protein hemocyanin. Our results support the hypothesis that LDH interacts with glycolytic enzymes in a metabolon structured by cytoskeletal elements that may also include the enzyme for transfer of the adenylate charge in glycolytically produced ATP. Those interactions may play specific roles in biochemical adaptation of glycolytic enzymes.     

Reduced maximum capacity of glycolysis in brown adipose tissue of genetically obese, diabetic (db/db) mice and its restoration following treatment with a thermogenic beta-adrenoceptor agonist

The maximal activities of the key glycolytic enzymes hexokinase and 6-phosphofructokinase, were reduced in brown adipose tissue in db/db mice compared to their lean littermates. Treatment of db/db mice with the thermogenic beta-adrenoceptor agonist, BRL 26830, restored normoglycaemia. The only significant increase in activity of hexokinase and 6-phosphofructokinase in the BRL 26830-treated db/db mice occurred in brown adipose tissue where the total tissue activity increased 10- and 11-fold respectively. These changes together with increased 2-deoxyglucose uptake in vivo suggest that brown adipose tissue can play a quantitatively important role in the removal of glucose from the blood.     

Studies on glycolysis in vitro: role of glucose phosphorylation and phosphofructokinase activity on total velocity

An in vitro glycolysis system has been developed to study the regulation of glycolysis on kinetic structure basis, in order to determine the extent of regulatory effects on the whole system of individual enzymes according to their kinetic data, in rat liver and muscle. Hexokinase or glucose-6-phosphate addition to the system with glucose as substrate increases lactate production rate by 2.5 in liver and by 10 in muscle, which suggest glucose phosphorylation step is a limiting step in this system. Fructose 2,6-bisphosphate addition to the system increases lactate production rate in liver only when glucose is the substrate, but not with glucose-6-phosphate as substrate. There is a linear relationship between glycolytic activity, as lactate produced per min and protein quantity, which suggests that this system can also be used to assay glycolytic activity in tissue extracts. Specific glycolytic activity found, as mumol of L-lactate produced per min, per protein mg was 0.1 for muscle and 0.01 for liver.     

CYTOCHEMICAL LOCALIZATION OF TWO GLYCOLYTIC DEHYDROGENASES IN WHITE SKELETAL MUSCLE

The cytochemical localization, by conventional methods, of lactate and glyceraldehyde-3-phosphate dehydrogenases is limited, firstly, by the solubility of these enzymes in aqueous media and, secondly, by the dependence of the final electron flow from reduced nicotinamide-adenine dinucleotide (NADH) to the tetrazolium on tissue diaphorase activity: localization is therefore that of the diaphorase, which in rabbit adductor magnus is mitochondrial. NADH has been found to have great affinity to bind in the sarcoplasmic reticulum, and, therefore, if it is generated freely in the incubation media containing 2,2',5,5'-tetra-p-nitrophenyl-3,3'-(3,3'-dimethoxy-4,4'-phenylene)-ditetrazolium chloride (TNBT) and N-methyl phenazonium methyl sulfate (PMS), it can bind there and cause a false staining. Since such a production of NADH can readily occur in the incubation media for glycolytic dehydrogenases due to diffusion of these soluble enzymes from tissue sections, the prevention of enzyme solubilization is extremely important. Fixation in formaldehyde prevented such enzyme diffusion, while at the same time sufficient activity persisted to allow for adequate staining. The incubation media contained PMS, so that the staining system was largely independent of tissue diaphorase activity. Application of these methods to adductor magnus of rabbit revealed by light microscopy, for both enzymes, a fine network which was shown by electron microscopy to represent staining of the sarcoplasmic reticulum. Mitochondria also reacted. These findings add further support for the notion that the sarcoplasmic reticulum is probably involved in glycolytic activity.     

The tentative identification in Escherichia coli of a multienzyme complex with glycolytic activity.

Penicillin spheroplasts of Escherichia coli were ruptured osmotically, by freezing and thawing, or mechanically. Differential centrifugation sedimented 20-30% of the glycolytic enzymes without increasing their specific activities. There was, however, evidence of distinct groups of sedimenting enzymes; growth on different carbon sources could influence the distribution. Sucrose gradient studies gave no evidence of enzyme association but provided estimations of the molecular weight of each enzyme which were close to those subsequently observed on gel filtration. Using the determined molecular weight and a literature value for specific activity, the measured activity ratio of the enzymes was compared with that expected from an equimolar mixture. All values agreed within a factor of five, except for hexokinase. The relative roles of hexokinase and phosphotransferase in E. coli are briefly considered. An equimolar multienzyme aggregate of all the enzymes of glycolysis would have a molecular weight of about 1.6 X 10(6). Chromatography on a Biogel column yielded one fraction, corresponding to a molecular weight of 1.6 X 10(6), which contained a proportion of all the glycolytic enzyme studied; the remaining portion of each enzyme activity was eluted from the column at the position expected from its individual molecular weight. The fraction of mol. wt 1 600 000 was tested for complete glycolysis pathway activity and found not to be different from a reconcentrated mixture of the separated enzymes. Both the eluted and the reconstructed systems showed unexpected activity changes at different protein concentrations. The specific radioactivity of pyruvate formed by these systems from [14C]glucose 6-phosphate was reduced by the presence of unlabelled 3-phosphoglycerate, but by less than would have been expected had the latter been able to participate fully in glycolytic activity. This result indicates that these preparations were capable of selectivity compartmenting glycolytic intermediates. Electron microscope investigation of both systems showed large numbers of regular 30 nm diameter particles which, on disruption, appeared to be composed of smaller units: it is possible that these particles may have been aggregates containing glycolytic enzymes. The possible advantages of a glycolytic multienzyme complex are briefly discussed.     

Decrease in phosphofructokinase activity during blood preservation and the effect of intracellular ATP

The activities of 15 enzymes, including all the glycolytic enzymes, were observed during the preservation of human red cells at 4°C. After 8 weeks, phosphofructokinase (PFK) activity had decreased most. The decrease was prevented by the addition of adenine and inosine to the preservation medium, but not at all by inosine alone and only slightly by adenine alone. This decrease paralleled that of the decrease of intracellular ATP. PFK in the hemolysate was inactivated rapidly, but the inactivation was effectively prevented by ATP. The decrease in glycolytic activity during preservation was concluded to be a result of the loss of PFK activity, and this in turn was due to the decrease of ATP.     

Enzymes of glycolysis are functionally associated with the mitochondrion in Arabidopsis cells

Mitochondria fulfill a wide range of metabolic functions in addition to the synthesis of ATP and contain a diverse array of proteins to perform these functions. Here, we present the unexpected discovery of the presence of the enzymes of glycolysis in a mitochondrial fraction of Arabidopsis cells. Proteomic analyses of this mitochondrial fraction revealed the presence of 7 of the 10 enzymes that constitute the glycolytic pathway. Four of these enzymes (glyceraldehyde-3-P dehydrogenase, aldolase, phosphoglycerate mutase, and enolase) were also identified in an intermembrane space/outer mitochondrial membrane fraction. Enzyme activity assays confirmed that the entire glycolytic pathway was present in preparations of isolated Arabidopsis mitochondria, and the sensitivity of these activities to protease treatments indicated that the glycolytic enzymes are present on the outside of the mitochondrion. The association of glycolytic enzymes with mitochondria was confirmed in vivo by the expression of enolase- and aldolase-yellow fluorescent protein fusions in Arabidopsis protoplasts. The yellow fluorescent protein fluorescence signal showed that these two fusion proteins are present throughout the cytosol but are also concentrated in punctate regions that colocalized with the mitochondrion-specific probe Mitotracker Red. Furthermore, when supplied with appropriate cofactors, isolated, intact mitochondria were capable of the metabolism of (13)C-glucose to (13)C-labeled intermediates of the trichloroacetic acid cycle, suggesting that the complete glycolytic sequence is present and active in this subcellular fraction. On the basis of these data, we propose that the entire glycolytic pathway is associated with plant mitochondria by attachment to the cytosolic face of the outer mitochondrial membrane and that this microcompartmentation of glycolysis allows pyruvate to be provided directly to the mitochondrion, where it is used as a respiratory substrate.     

Glycosis in the eggs of the echiuroid, Urechis unicinctus and the oyster, Crassostrea gigas. Rate-limiting steps and activation at fertilization.

The content of glycolytic intermediates and of adenine nucleotides was measured in eggs of the echiuroid, Urechis unicinctus and the oyster, Crassostrea gigas, before and after fertilization. On the whole, the profile of the change in each glycolytic intermediate in Urechis eggs upon fertilization was found to be essentially similar to that in oyster eggs. Calculation of the mass action ratio for each glycolytic step from the amounts of glycolytic intermediates determined suggests that there are at least three limiting enzymes in the glycolysis system in unfertilized and fertilized eggs of each species examined. Phosphorylase (EC 2.4.1.1), phosphofructokinase (EC 2.7.1.11), and pyruvate kinase (EC 2.7.1.40) may be rate-limiting enzymes for the glycolysis system in Urechis eggs as well as in oyster eggs. These enzymes are thought to be activated upon fertilization, though even the reactions of the enzymes in fertilized eggs do not reach a state of equilibrium. In eggs of Urechis and oyster, phosphorylase is the first enzyme to be activated following fertilization. In Urechis eggs, pyruvate kinase is activated after the instant increase in the phosphorylase activity upon fertilization, followed by phosphofructokinase activation. In oyster eggs, however, pyruvate kinase and phosphofructokinase seem to be stimulated simultaneously, subsequent to phosphorylase activation upon fertilization. The mechanism controlling phosphorylase and pyruvate kinase activity is unknown, but the phosphofructokinase activity in both species may be regulated by the intracellular concentration of adenine nucleotides, since the enzyme activity is enhanced along with a decline in the phosphate potential in the eggs of both Urechis and of oyster.     

Content and synthesis of glycolytic enzymes and creatine kinase in skeletal muscles and normal and dystrophic chickens

A number of workers have reported that avian muscular dystrophy causes alterations in the levels of certain enzyme activities in "fast-twitch" muscle fibers but has little effect on enzyme activities in "slow-twitch" muscle fibers. In the present work, the effects of this disease on the content and relative rates of synthesis of a number of glycolytic enzymes and the skeletal muscle-specific MM isoenzyme of creatine kinase in chicken muscles was investigated. It was shown that (i) the approximate 50% reductions in steady-state concentrations of three glycolytic enzymes (aldolase, enolase, and glyceraldehyde-3-P dehydrogenase) in dystrophic breast (fast-twitch) muscle result predominantly from decreases in relative rates of synthesis, rather than accelerations in relative rates of degradation, of these proteins in the diseased tissue; (ii) in contrast to the situation with the glycolytic enzymes, muscular dystrophy has only minor effects (25% or less) on the content and relative rate of synthesis of MM creatine kinase in breast muscle fibers; (iii) the muscular dystrophy-associated alterations in content and synthesis of the glycolytic enzymes in breast muscle fibers become apparent only during postembryonic maturation of this tissue; and (iv) as expected, muscular dystrophy has no significant effect on the content or relative rates of synthesis of glycolytic enzymes in slow-twitch lateral adductor muscles of the chicken. These results are discussed in terms of the apparent similarities between the effects of muscular dystrophy and surgical denervation on the protein synthetic programs expressed by mature fast-twitch muscle fibers.