The role of nicotinamide–adenine dinucleotide phosphate-dependent malate dehydrogenase and isocitrate dehydrogenase in the supply of reduced nicotinamide–adenine dinucleotide phosphate for steroidogenesis in the superovulated rat ovary

1. Superovulated rat ovary was found to contain high activities of NADP-malate dehydrogenase and NADP-isocitrate dehydrogenase. The activity of each enzyme was approximately four times that of glucose 6-phosphate dehydrogenase and equalled or exceeded the activities reported to be present in other mammalian tissues. Fractionation of a whole tissue homogenate of superovulated rat ovary indicated that both enzymes were exclusively cytoplasmic. The tissue was also found to contain pyruvate carboxylase (exclusively mitochondrial), NAD-malate dehydrogenase and aspartate aminotransferase (both mitochondrial and cytoplasmic) and ATP-citrate lyase (exclusively cytoplasmic). 2. The kinetic properties of glucose 6-phosphate dehydrogenase, NADP-malate dehydrogenase and NADP-isocitrate dehydrogenase were determined and compared with the whole-tissue concentrations of their substrates and NADPH; NADPH is a competitive inhibitor of all three enzymes. The concentrations of glucose 6-phosphate, malate and isocitrate in incubated tissue slices were raised at least tenfold by the addition of glucose to the incubation medium, from the values below to values above the respective K(m) values of the dehydrogenases. Glucose doubled the tissue concentration of NADPH. 3. Steroidogenesis from acetate is stimulated by glucose in slices of superovulated rat ovary incubated in vitro. It was found that this stimulatory effect of glucose can be mimicked by malate, isocitrate, lactate and pyruvate. 4. It is concluded that NADP-malate dehydrogenase or NADP-isocitrate dehydrogenase or both may play an important role in the formation of NADPH in the superovulated rat ovary. It is suggested that the stimulatory effect of glucose on steroidogenesis from acetate results from an increased rate of NADPH formation through one or both dehydrogenases, brought about by the increases in the concentrations of malate, isocitrate or both. Possible pathways involving the two enzymes are discussed.     

Polyacrylamide gel technique for the histochemical demonstration of soluble enzymes

Summary Soluble enzymes were immobilized and visualized by polyacrylamide gel slabs, impregnated with the incubation medium including auxiliairy enzymes. The method has several advantages over existing techniques which make use of gel films or a semipermeable membrane. The diffusion of tissue compounds is effectively limited, while auxiliary enzymes may be operative. Moreover the viscosity of the medium is temperature-independent so that the incubation temperature can be varied.To demonstrate the suitability of the method glycerol-3-phosphate dehydrogenase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, hexokinase, phosphoglucomutase and aldolase were visualized in human or rat skeletal muscle. Cytosolic and mitochondrial glycerol-3-phosphate dehydrogenase were both visualized in the absence of added NAD⁺ and menadione.For the visualization of ATP producing enzymes, like creatine kinase and pyruvate kinase, the method is not suitable.     

Polyacrylamide gel technique for the histochemical demonstration of soluble enzymes.

Soluble enzymes were immobilized and visualized by polyacrylamide gel slabs, impregnated with the incubation medium including auxiliary enzymes. The method has several advantages over existing techniques which make use of gel films or a semipermeable membrane. The diffusion of tissue compounds is effectively limited, while auxiliary enzymes may be operative. Moreover the viscosity of the medium is temperature-independent so that the incubation temperature can be varied. To demonstrate the suitability of the method glycerol-3-phosphate dehydrogenase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, hexokinase, phosphoglucomutase and aldolase were visulaized in human or rat skeletal muscle. Cytosolic and mitochondrial glycerol-3-phosphate dehydrogenase were both visualized in the absence of added NAD+ and menadione. For the visualization of ATP producint enzymes, like creatine kinase and pyruvate kinase, the method is not suitable.     

Degradation of glucose-metabolizing enzymes in the rat small intestine during starvation

1. The degradation rates and half-lives of hexokinase, 6-phosphogluconate dehydrogenase, lactate dehydrogenase, pyruvate kinase, glucose 6-phosphate dehydrogenase, phosphoglycerate kinase and aldolase were calculated from measurements of the decline in activities of these enzymes in rat small intestine during starvation. 2. The half-lives of the enzymes are: hexokinase, 5.7h; 6-phosphogluconate dehydrogenase, 7.6h; glucose 6-phosphate dehydrogenase, 6.0h; pyruvate kinase, 8.9h; lactate dehydrogenase, 8.7h; phosphoglycerate kinase, 8.7h; aldolase, 5.1h. 3. The significance of the results is discussed with respect to the regulation of enzyme concentrations in response to changes in diet.     

Glycolytic enzyme interactions with tubulin and microtubules

Interactions of the glycolytic enzymes glucose-6-phosphate isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, triose-phosphate isomerase, enolase, phosphoglycerate mutase, phosphoglycerate kinase, pyruvate kinase, lactate dehydrogenase type-M, and lactate dehydrogenase type-H with tubulin and microtubules were studied. Lactate dehydrogenase type-M, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and aldolase demonstrated the greatest amount of co-pelleting with microtubules. The presence of 7% poly(ethylene glycol) increased co-pelleting of the latter four enzymes and two other enzymes, glucose-6-phosphate isomerase, and phosphoglycerate kinase with microtubules. Interactions also were characterized by fluorescence anisotropy. Since the KD values of glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase for tubulin and microtubules were all found to be between 1 and 4 microM, which is in the range of enzyme concentration in cells, these enzymes are probably bound to microtubules in vivo. These observations indicate that interactions of cytosolic proteins, such as the glycolytic enzymes, with cytoskeletal components, such as microtubules, may play a structural role in the formation of the microtrabecular lattice.     

Phospholipids and the regulation of pyruvate dehydrogenase from rat adipocyte mitochondria

Aqueous dispersions of 4 out of 9 phospholipids added individually to the mitochondrial fraction from rat adipocytes altered the activity of pyruvate dehydrogenase in a dose-dependent manner from 1 to 300 microM. Phosphatidylserine increased and phosphatidylcholine, phosphatidylinositol and phosphatidylinositol-4-phosphate decreased enzyme activity. The stimulation of pyruvate dehydrogenase induced by phosphatidylserine may be reversed to below basal activity by phosphatidylinositol-4-phosphate and to basal activity by NaF, a pyruvate dehydrogenase phosphatase inhibitor. The inhibition of pyruvate dehydrogenase induced by phosphatidylinositol-4-phosphate may be restored to basal levels by the addition of calcium. These results suggest that phosphatidylserine activates pyruvate dehydrogenase activity through activation of the phosphatase, perhaps forming a phosphatidylserine-calcium complex. The inhibition by phosphatidylinositol-4-phosphate may be mediated by disruption of the enzyme complex. The phospholipids may play a physiological role in the regulation of pyruvate dehydrogenase activity.     

Regulation of glycolysis and fatty acid synthesis from glucose in sheep adipose tissue

1. The following were measured in adipose-tissue pieces, obtained from 7–9 month-old sheep, before or after the tissue pieces had been maintained in tissue culture for 24 h: the rates of synthesis from glucose of fatty acids, acylglycerol glycerol, pyruvate and lactate; the rate of glucose oxidation to CO₂; the rate of glucose oxidation via the pentose phosphate pathway; the activities of hexokinase, glucose 6-phosphate dehydrogenase, phosphofructokinase, pyruvate kinase, pyruvate dehydrogenase and ATP citrate lyase; the intra- and extra-cellular water content; the concentration of various metabolites and ATP, ADP and AMP. 2. The proportion of glucose carbon converted into the various products in sheep adipose tissue differs markedly from that observed in rat adipose tissue. 3. There was a general increase in the rate of glucose utilization by the adipose-tissue pieces after maintenance in tissue culture; largest changes were seen in the rates of glycolysis and fatty acid synthesis from glucose. These increases are paralleled by an increase in pyruvate kinase activity. There was no change in the activities of the other enzymes as measured, although the net flux through all the enzymes increased. 4. Incubation of fresh adipose-tissue pieces for 2–6h led to an increase in the affinity of pyruvate kinase for phosphoenolpyruvate. 5. The rate of pyruvate production by glycolysis was greater than the activity of pyruvate dehydrogenase of the tissue. 6. The results suggest that both pyruvate kinase and pyruvate dehydrogenase have important roles in restricting the utilization of glucose carbon for fatty acid synthesis in sheep adipose tissue.     

Carbohydrate metabolism in the rat peritoneal macrophages

Rat peritoneal macrophages derive energy differently from other tissues. Resting rat peritoneal macrophages have been taken for the present investigation. Lactate produced by extracellular glycolysis in the peritoneal lavage fluid, is readily converted into pyruvate by resting peritoneal macrophages and is oxidised in mitochondria. Glycolytic enzymes other than phosphoglucoisomerase and lactate dehydrogenase could not be substantially demonstrated. Glucose-6-phosphate dehydrogenase was detected. The presence of glucose-6-phosphate dehydrogenase along with phosphoglucoisomerase indicates the operation of the hexose monophosphate shunt as a pathway supplementary to glycolysis. Resting rat peritoneal macrophages thus appear to utilize extracellular lactate as their main energy source instead of glucose, bypass glycolysis and have active hexose monophosphate shunt.     

Aerobic glycolysis: a study of human articular cartilage

Cartilage generally is one of those tissues that exhibit aerobic glycolysis. In a previous study on rat epiphyseal cartilage it had been suggested that this phenomenon is related to potentially excessive production of pyruvate and acetyl coenzyme A, the latter derived from fatty acid oxidation and inhibiting pyruvate dehydrogenase activity. The present study has shown that, in human articular cartilage, the contribution from fatty acid oxidation is too small to account for this phenomenon although the total potential production of pyruvate could still be in excess of the requirements for acetyl coenzyme A for the Krebs' cycle. Of greater relevance may be the apparent correlations that have been found between the activities of lactate and glyceraldehyde 3-phosphate dehydrogenases (r = 0 X 82: 0.01 greater than p greater than 0.001) and between those of lactate and glucose 6-phosphate dehydrogenases (r = 0.92; p less than 0.001).     

Assaying for pyruvate,orthophosphate dikinase activity: Necessary precautions with phosphoenolpyruvate carboxylase as coupling enzyme

Phosphoenolpyruvate carboxylase (EC 4.1.1.31), used as a coupling enzyme in the assay of the pyruvate, orthophosphate dikinase (EC 2.7.9.1) forward reaction, is a serious limiting factor for the overall rate when added at a level of 0.2–0.3 unit/ml of assay medium. Nonlimiting assay conditions are obtained by either increasing the level of the coupling enzyme to 3 units/ml or adding 6mM glucose-6-phosphate as an activator/stabilizer of phosphoenolpyruvate carboxylase.Abbreviations G-6-P glucose-6-phosphate - LDH lactate dehydrogenase - MDH malate dehydrogenase - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase - PVP polyvinylpyrrolidone - PPDK pyruvate, orthophosphate dikinase - U unit of enzyme activity (mol/min)     

Fluorometric procedures for measuring triglyceride concentrations in small amounts of tissue and plasma

It has been previously shown that triglycerides can be specifically hydrolyzed by lipase from Rhizopus arrhizus in the presence of hog liver esterase and sodium dodecyl sulfate. The glycerol produced can then be measured by sequential reactions with glycerokinase, pyruvate kinase, and lactate dehydrogenase: glycerol and ATP are converted to glycerol-3-phosphate and ADP by glycerokinase; the ADP reacts with phosphoenolpyruvate and pyruvate kinase to yield pyruvate; the pyruvate is converted to lactate with lactate dehydrogenase, and the cofactor NAD+ is simultaneously reduced to NADH. This report describes procedures by which either the disappearance of NADH or the appearance of NAD+ was determined fluorometrically, with 10- to 100-fold greater sensitivity than by spectrophotometry. In addition, enzymatic cycling of NAD+ was used to increase the sensitivity of the assay over 1000-fold, and thereby provided accurate measurement of less than 1 ng of triglyceride. Results obtained from the three fluorometric methods were highly correlated with an automated periodate oxidation method using serum samples and lipid extracts of muscle tissue.     

Lipogenesis in rat and guinea-pig isolated epididymal fat-cells

Fat-cells were prepared from rat and guinea-pig epididymal adipose tissue and compared on the basis of the intracellular distributions and activities of enzymes and with respect to their utilization of various U-(14)C-labelled substrates for lipogenesis. 1. Compared with the rat, guinea-pig extramitochondrial enzyme activities differed in that aconitate hydratase, alanine aminotransferase, ATP-citrate lyase, lactate dehydrogenase, NAD-malate dehydrogenase, NADP-malate dehydrogenase and phosphoenolpyruvate carboxykinase activities were appreciably lower, whereas aspartate aminotransferase, glucose 6-phosphate dehydrogenase, NADP-isocitrate dehydrogenase and 6-phosphogluconate dehydrogenase activities were appreciably higher. Mitochondrial activities of citrate synthase, NADP-isocitrate dehydrogenase and pyruvate carboxylase were appreciably lower, whereas mitochondrial activities of aspartate aminotransferase, glutamate dehydrogenase, NAD-malate dehydrogenase and phosphoenolpyruvate carboxykinase were higher in the guinea pig compared with the rat. 2. In general guinea-pig fat-cells incorporated acetate and lactate into fatty acids more readily than rat fat-cells, whereas rat fat-cells incorporated glucose and pyruvate more readily than guinea-pig fat-cells. 3. Acetate stimulated the incorporation of glucose into fatty acids in rat fat-cells, but had no appreciable effect upon this process in guinea-pig fat-cells. Acetate greatly decreased the incorporation of lactate into fatty acids in cells from both species. 4. Lactate/pyruvate ratios produced by incubation of guinea-pig cells with glucose+insulin were very low compared with those found with rat cells under the same conditions. 5. With glucose (+insulin) or with glucose+acetate (+insulin) as substrates guinea-pig cells produced enough NADPH by the hexose monophosphate pathway to satisfy the NADPH requirements of lipogenesis. In rat fat-cells under the same conditions, hexose monophosphate-pathway NADPH provision was not sufficient to meet the requirements of lipogenesis. 6. These results are discussed, particularly in relationship to the disposition of cytosolic reducing equivalents in the cells.     

The regulation of triglyceride synthesis and fatty acid synthesis in rat epididymal adipose tissue. Effects of insulin, adrenaline and some metabolites in vitro

1. Adipose tissues from rats fed a balanced diet were incubated in the presence of glucose (20mm) with the following additions: insulin, anti-insulin serum, insulin+acetate, insulin+pyruvate, insulin+lactate, insulin+phenazine methosulphate, insulin+oleate+albumin, insulin+adrenaline+albumin, insulin+6-N-2'-O-dibutyryl 3':5'-cyclic AMP+albumin. 2. Measurements were made of the whole tissue concentrations of adenine nucleotides, hexose phosphates, triose phosphates, glycerol 1-phosphate, 3 phosphoglycerate, 6-phosphogluconate, long-chain fatty acyl-CoA, acid-soluble CoA, citrate, isocitrate, malate and 2-oxoglutarate, and of the release into the incubation medium of lactate, pyruvate and glycerol after 1h of incubation. 3. Fluxes of [(14)C]glucose carbon through the major pathways of glucose metabolism were calculated from the yields of (14)C in various products after 2h of incubation. Fluxes of [(14)C]acetate, [(14)C]pyruvate or [(14)C]lactate carbon in the presence of glucose were also determined. 4. Measurements were also made of the whole-tissue concentrations of metabolites in tissues taken directly from Nembutal-anaesthetized rats. 5. Whole tissue mass-action ratios for phosphofructokinase, phosphoglucose isomerase and the combined (aldolasextriose phosphate isomerase) reaction were similar in vivo and in vitro. The reactants of phosphofructokinase appeared to be far from mass-action equilibrium. In vitro, the reactants of hexokinase also appeared to be far from mass-action equilibrium. 6. Correlation of observed changes in glycolytic flux with changes in fructose 6-phosphate concentration suggested that phosphofructokinase may show regulatory behaviour. The enzyme appeared to be activated in the presence of oleate or adrenaline and to be inhibited in the presence of lactate or pyruvate. 7. Evidence is presented that the reactants of lactate dehydrogenase and glycerol 1-phosphate dehydrogenase may be near to mass-action equilibrium in the cytoplasm. 8. No satisfactory correlations could be drawn between the whole-tissue concentrations of long-chain fatty acyl-CoA, citrate and glycerol 1-phosphate and the observed rates of triglyceride and fatty acid synthesis. Under the conditions employed, the concentration of glycerol 1-phosphate appeared to depend mainly on the cytoplasmic [NAD(+)]/[NADH] ratios. 9. Calculated hexose monophosphate pathway flux rates roughly correlated with fatty acid synthesis rates and with whole tissue [6-phosphogluconate]/[glucose 6-phosphate] ratios. The relative rates of production of NADPH for fatty acid synthesis by the hexose monophosphate pathway and by the ;malic enzyme' are discussed. It is suggested that all NADH produced in the cytoplasm may be used in that compartment for reductive synthesis of fatty acids, lactate or glycerol 1-phosphate.     

The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by alpha-Cyano-4-hydroxycinnamate and related compounds.

1. Effects of alpha-cyano-4-hydroxycinnamate and alpha-cyanocinnamate on a number of enzymes involved in pyruvate metabolism have been investigated. Little or no inhibition was observed of any enzyme at concentrations that inhibit completely mitochondrial pyruvate transport. At much higher concentrations (1 mM) some inhibition of pyruvate carboxylase was apparent. 2. Alpha-Cyano-4-hydroxycinnamate (1-100 muM) specifically inhibited pyruvate oxidation by mitochondria isolated from rat heart, brain, kidney and from blowfly flight muscle; oxidation of other substrates in the presence or absence of ADP was not affected. Similar concentrations of the compound also inhibited the carboxylation of pyruvate by rat liver mitochondria and the activation by pyruvate of pyruvate dehydrogenase in fat-cell mitochondria. These findings imply that pyruvate dehydrogenase, pyruvate dehydrogenase kinase and pyruvate carboxylase are exposed to mitochondrial matrix concentrations of pyruvate rather than to cytoplasmic concentrations. 3. Studies with whole-cell preparations incubated in vitro indicate that alpha-cyano-4-hydroxycinnamate or alpha-cyanocinnamate (at concentrations below 200 muM) can be used to specifically inhibit mitochondrial pyruvate transport within cells and thus alter the metabolic emphasis of the preparation. In epididymal fat-pads, fatty acid synthesis from glucose and fructose, but not from acetate, was markedly inhibited. No changes in tissue ATP concentrations were observed. The effects on fatty acid synthesis were reversible. In kidney-cortex slices, gluconeogenesis from pyruvate and lactate but not from succinate was inhibited. In the rat heart perfused with medium containing glucose and insulin, addition of alpha-cyanocinnamate (200 muM) greatly increased the output and tissue concentrations of lactate plus pyruvate but decreased the lactate/pyruvate ratio. 4. The inhibition by cyanocinnamate derivatives of pyruvate transport across the cell membrane of human erythrocytes requires much higher concentrations of the derivatives than the inhibition of transport across the mitochondrial membrane. Alpha-Cyano-4-hydroxycinnamate appears to enter erythrocytes on the cell-membrane pyruvate carrier. Entry is not observed in the presence of albumin, which may explain the small effects when these compounds are injected into whole animals.     

Intracellular localization of enzymes in white-adipose-tissue fat-cells and permeability properties of fat-cell mitochondria. Transfer of acetyl units and reducing power between mitochondria and cytoplasm

1. A method is described for extracting separately mitochondrial and extramitochondrial enzymes from fat-cells prepared by collagenase digestion from rat epididymal fat-pads. The following distribution of enzymes has been observed (with the total activities of the enzymes as units/mg of fat-cell DNA at 25 degrees C given in parenthesis). Exclusively mitochondrial enzymes: glutamate dehydrogenase (1.8), NAD-isocitrate dehydrogenase (0.5), citrate synthase (5.2), pyruvate carboxylase (3.0); exclusively extramitochondrial enzymes: glucose 6-phosphate dehydrogenase (5.8), 6-phosphogluconate dehydrogenase (5.2), NADP-malate dehydrogenase (11.0), ATP-citrate lyase (5.1); enzymes present in both mitochondrial and extramitochondrial compartments: NADP-isocitrate dehydrogenase (3.7), NAD-malate dehydrogenase (330), aconitate hydratase (1.1), carnitine acetyltransferase (0.4), acetyl-CoA synthetase (1.0), aspartate aminotransferase (1.7), alanine aminotransferase (6.1). The mean DNA content of eight preparations of fat-cells was 109mug/g dry weight of cells. 2. Mitochondria showing respiratory control ratios of 3-6 with pyruvate, about 3 with succinate and P/O ratios of approaching 3 and 2 respectively have been isolated from fat-cells. From studies of rates of oxygen uptake and of swelling in iso-osmotic solutions of ammonium salts, it is concluded that fat-cell mitochondria are permeable to the monocarboxylic acids, pyruvate and acetate; that in the presence of phosphate they are permeable to malate and succinate and to a lesser extent oxaloacetate but not fumarate; and that in the presence of both malate and phosphate they are permeable to citrate, isocitrate and 2-oxoglutarate. In addition, isolated fat-cell mitochondria have been found to oxidize acetyl l-carnitine and, slowly, l-glycerol 3-phosphate. 3. It is concluded that the major means of transport of acetyl units into the cytoplasm for fatty acid synthesis is as citrate. Extensive transport as glutamate, 2-oxoglutarate and isocitrate, as acetate and as acetyl l-carnitine appears to be ruled out by the low activities of mitochondrial aconitate hydratase, mitochondrial acetyl-CoA hydrolyase and carnitine acetyltransferase respectively. Pathways whereby oxaloacetate generated in the cytoplasm during fatty acid synthesis by ATP-citrate lyase may be returned to mitochondria for further citrate synthesis are discussed. 4. It is also concluded that fat-cells contain pathways that will allow the excess of reducing power formed in the cytoplasm when adipose tissue is incubated in glucose and insulin to be transferred to mitochondria as l-glycerol 3-phosphate or malate. When adipose tissue is incubated in pyruvate alone, reducing power for fatty acid, l-glycerol 3-phosphate and lactate formation may be transferred to the cytoplasm as citrate and malate.     

The role of citrate on the oxidative metabolism of submerged cultures of Claviceps purpurea 129

In submerged cultures of Claviceps purpurea citrate utilization was stopped when the pool phosphate concentration decreased to 0.44–0.55 mmol per g of mycelial protein. The absence of citrate in the medium resulted in a 50% decrease of biomass, cessation of the alkaloid (agro-, elymo-, chanoclavine) synthesis, formation of a central large vacuole, cell enlargement and medium acidification by lactate. Citrate synthase and malate dehydrogenase gradually disappeared. Glucose-6-phosphate dehydrogenase was not detectable. The activities of glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase increased by one or two orders of magnitude higher than in control culture (with 80 mM citrate). Substrate competition on the pyruvate level is discussed.     

Carbohydrate metabolism in erythrocytes of copper deficient rats

Dietary copper deficiency is known to adversely affect the circulatory system of fructose-fed rats. Part of the problem may lie in the effect of copper deficiency on intermediary metabolism. To test this, weanling male Long-Evans rats were fed for 4 or 8 weeks on sucrose-based diets containing low or adequate copper content. Copper deficient rats had significantly lower plasma and tissue copper as well as lower plasma copper, zinc-superoxide dismutase activity. Copper deficient rats also had a significantly higher heart:body weight ratio when compared to pair-fed controls. Direct measurement of glycolysis and pentose phosphate pathway flux in erythrocytes using (13)C NMR showed no differences in carbon flux from glucose or fructose to pyruvate but a significantly higher flux through the lactate dehydrogenase locus in copper deficient rats (approximately 1.3 times, average of glucose and glucose + fructose measurements). Copper-deficient animals had significantly higher erythrocyte concentrations of glucose, fructose, glyceraldehyde 3-phosphate and NAD(+). Liver metabolite levels were also affected by copper deficiency being elevated in glycogen and fructose 1-phosphate content. The results show small changes in carbohydrate metabolism of copper deficient rats.     

Key enzymes of carbohydrate metabolism as targets of the 11.5-kDa Zn(2+)-binding protein (parathymosin)

The 11.5-kDa Zn(2+)-binding protein (ZnBP) was covalently linked to Sepharose. Affinity chromatography with a cytosolic subfraction from liver resulted in purification of a predominant 38-kDa protein. In comparable experiments with brain cytosol a 39-kDa protein was enriched. The ZnBP-protein interactions were zinc-specific. Both proteins were identified as fructose-1,6-bisphosphate aldolase. Experiments with crude cytosol showed zinc-specific interaction of additional enzymes involved in carbohydrate metabolism. From liver cytosol greater than 90% of the following enzymes were specifically retained: aldolase, phosphofructokinase-1, hexokinase/glucokinase, glucose-6-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and fructose-1,6-bisphosphatase. Glucose-6-phosphate isomerase, phosphoglycerate kinase, enolase, lactate dehydrogenase, and most of triosephosphate isomerase remained unbound. From L-type pyruvate kinase only the phosphorylated form seems to interact with ZnBP. Using brain cytosol hexokinase, phosphofructokinase-1, and aldolase were completely bound to the affinity column, whereas glucose-6-phosphate isomerase, phosphoglycerate kinase, enolase, lactate dehydrogenase, pyruvate kinase, and most of triose-phosphate isomerase remained unbound. The behavior of glucose-6-phosphate dehydrogenase and glycerol-3-phosphate dehydrogenase from this tissue could not be followed. A possible function of ZnBP in supramolecular organization of carbohydrate metabolism is proposed.     

Distribution of some enzymes concerning carbohydrate metabolism in sea urchin eggs

As an aid to the elucidation of the mechanism of activation of glycolysis upon fertilization, the activity and the distribution of the enzymes concerned were measured in unfertilized and fertilized eggs of Hemicentrotus pulcherrimus and Pseudocentrotus depressus. The enzymes investigated were phosphorylase, exo-1,4-α-glucosidase, hexokinase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, 6-phosphofructokinase, hexosediphosphatase, fructose-bisphosphate aldolase, pyruvate kinase, and lactate dehydrogenase.Phosphorylase and pyruvate kinase were the enzymes which were activated upon fertilization. Glucose-6-phosphate dehydrogenase and a part of aldolase changed their distribution from the particulate to the soluble fraction upon fertilization. Advantages of enzyme activation over changes in enzyme distribution upon fertilization were discussed as a mechanism for the fertilization-induced activation of glycolysis.     

Evidence for a higher glycolytic than oxidative metabolic activity in white matter of rat brain

Different values exist for glucose metabolism in white matter; it appears higher when measured as accumulation of 2-deoxyglucose than when measured as formation of glutamate from isotopically labeled glucose, possibly because the two methods reflect glycolytic and tricarboxylic acid (TCA) cycle activities, respectively. We compared glycolytic and TCA cycle activity in rat white structures (corpus callosum, fimbria, and optic nerve) to activities in parietal cortex, which has a tight glycolytic-oxidative coupling. White structures had an uptake of [(3)H]2-deoxyglucose in vivo and activities of hexokinase, glucose-6-phosphate isomerase, and lactate dehydrogenase that were 40-50% of values in parietal cortex. In contrast, formation of aspartate from [U-(14)C]glucose in awake rats (which reflects the passage of (14)C through the whole TCA cycle) and activities of pyruvate dehydrogenase, citrate synthase, alpha-ketoglutarate dehydrogenase, and fumarase in white structures were 10-23% of cortical values, optic nerve showing the lowest values. The data suggest a higher glycolytic than oxidative metabolism in white matter, possibly leading to surplus formation of pyruvate or lactate. Phosphoglucomutase activity, which interconverts glucose-6-phosphate and glucose-1-phosphate, was similar in white structures and parietal cortex ( approximately 3 nmol/mg tissue/min), in spite of the lower glucose uptake in the former, suggesting that a larger fraction of glucose is converted into glucose-1-phosphate in white than in gray matter. However, the white matter glycogen synthase level was only 20-40% of that in cortex, suggesting that not all glucose-1-phosphate is destined for glycogen formation.