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Proximal colon epithelial gene responses to diets containing increasing levels of dietary fermentable material (FM) from 2 different sources were measured to determine whether gene expression patterns were independent of the source of FM. Male Fischer 344 rats (10/group) were fed for 6 wk a control diet containing 10% (g/g) cellulose (0% FM); or a 2, 5, or 10% wheat bran (WB) diet (1, 2, 5% FM); or a 2, 5, or 8% fructooligosaccharides (FOS) diet (2, 5, 8% FM). WB and FOS were substituted for cellulose to give a final 10% nondigestible material content including FM. Gene responses were relative to expression in rats fed the control diet. The gene response patterns associated with feeding ~2% FM (5% WB and 2% FOS) were similar (~10 gene changes $ 1.6-fold; P # 0.01) and involved genes associated with transport (Scnn1g, Mt1a), transcription (Zbtb16, Egr1), immunity (Fkbp5), a gut hormone (Retn1b), and lipid metabolism (Scd2, Insig1). These changes were also similar to those associated with 5% FM but only in rats fed the 10% WB diet. In contrast, the 5% FOS diet (;5% FM) was associated with 68 gene expression changes $ 1.6-fold (P # 0.01). The diet with the highest level of fermentation (8% FOS, ;8% FM) was associated with 132 changes $ 1.6-fold (P # 0.01), including genes associated with transport, cellular proliferation, oncogene and tumor metastasis, the cell cycle, apoptosis, signal transduction, transcript regulation, immunity, gut hormones, and lipid metabolic processes. These results show that both the amount and source of FM determine proximal colon epithelial gene response patterns in rats. J. Nutr. 141: 790‐797, 2011.

Ctr1 (copper transporter 1) mediates high-affinity copper uptake. Ctr2 (copper transporter 2) shares sequence similarity with Ctr1, yet its function in mammalian cells is poorly understood. In African green monkey kidney COS-7 cells and rat tissues, Ctr2 migrated as a predominant band of approximately 70 kDa and was most abundantly expressed in placenta and heart. A transiently expressed hCtr2-GFP (human Ctr2-green fluorescent protein) fusion protein and the endogenous Ctr2 in COS-7 cells were mainly localized to the outer membrane of cytoplasmic vesicles, but were also detected at the plasma membrane. Biotinylation of Ctr2 with the membrane-impermeant reagent sulfo-NHS-SS-biotin [sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate] confirmed localization at the cell surface. Cells expressing hCtr2-GFP hyperaccumulated copper when incubated in medium supplemented with 10 microM CuSO(4), whereas cells depleted of endogenous Ctr2 by siRNAs (small interfering RNAs) accumulated lower levels of copper. hCtr2-GFP expression did not affect copper efflux, suggesting that hCtr2-GFP increased cellular copper concentrations by promoting uptake at the cell surface. Kinetic analyses showed that hCtr2-GFP stimulated saturable copper uptake with a K(m) of 11.0+/-2.5 microM and a K(0.5) of 6.9+/-0.7 microM when data were fitted to a rectangular hyperbola or Hill equation respectively. Competition experiments revealed that silver completely inhibited hCtr2-GFP-dependent copper uptake, whereas zinc, iron and manganese had no effect on uptake. Furthermore, increased copper concentrations in hCtr2-GFP-expressing cells were inversely correlated with copper chaperone for Cu/Zn superoxide dismutase protein expression. Collectively, these results suggest that Ctr2 promotes copper uptake at the plasma membrane and plays a role in regulating copper levels in COS-7 cells.

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.

A method is described that accurately and rapidly quantifies the free and total phosphorous content of a commercially available, purified, phytic acid preparation. This allows its use as a standard for phytic acid determinations in foods. The method involves a wet ashing step followed by phosphorous measurement with a 1-amino-2-naphthol-4-sulfonic acid-molybdate reagent in a microplate reader at 660 nm. The procedure can be performed in 3 h with as little as 50 mg sample.

The importance of fat oxidation and fatty acid synthesis were examined in rats fed approximately one half their ad libitum food intake for a period of 13 days followed by 7 days of ad libitum feeding (refed rats). This study was undertaken because previous reports demonstrated that refed rats rapidly accumulated body fat. Our results confirmed this observation: refed rats accrued body fat and body weight at rates that were approximately 3 times higher than controls. Evidence for a period of increased metabolic efficiency was demonstrated by measuring the net energy requirement for maintenance over the refeeding period: refed rats had a reduced metabolic rate during the period of energy restriction (approximately 30% lower than control) and this persisted up to 2 days after the reintroduction of ad libitum feeding. The major factor responsible for the rapid fat gain was a depressed rate of fatty acid oxidation. Calculations of protein and carbohydrate intake over the refeeding period showed that the simplest explanation for the decrease in fatty acid oxidation is fat sparing. This is possible because of the large increase in dietary carbohydrate and protein intake during the refeeding period when metabolic rates are still depressed. The increased carbohydrate and protein may adequately compensate for the increasing energy requirements of the ER rats over the refeeding period affording rats the luxury of storing the excess dietary fat energy.

The low-temperature metabolism of erythrocytes from the freeze-tolerant frog Rana sylvatica was investigated by (13)C and (31)P NMR spectroscopy. Erythrocytes readily took up high concentrations of the natural cryoprotectant, glucose, at both high (12 and 17 degrees C) and low (4 degrees C) temperatures but glucose was apparently not metabolized at 4 degrees C. Strong inhibition of glucose catabolism at low temperature would facilitate the maintenance of the very high concentrations of glucose (approximately 200 mM) that are accumulated to provide cryoprotection during freezing in wood frogs. Analysis of (13)C labeling of glycolytic intermediates at 4 degrees C showed mixing of label primarily in hexose (fructose) and hexose phosphate (glucose 6-phosphate, fructose 6-phosphate) pools but little label incorporation into triose phosphate intermediates. These data are consistent with a profound low-temperature-induced inhibition of phosphofructokinase (PFK). Investigations into potential PFK control mechanisms were undertaken. (31)P NMR analysis showed that the intracellular pH of erythrocytes increased from 7.0 to 7.3 as temperature decreased from 17 to 4 degrees C in a manner consistent with alphastat regulation. This change is exactly opposite to that expected if overall PFK activity was regulated by changes in cellular pH since PFK is less active at lower pH values in vitro. Other factors must, therefore, operate to regulate PFK at lower temperatures.

The effect of dietary fat on body composition, whole body lipogenesis, and enzyme activity was measured in rats over the first 16 weeks post-weaning. Rats were fed either a low fat (5% w/w fat) or high fat (20% w/w fat) diet for the first 4 weeks. After this time all rats were fed the low fat diet. The results showed no significant effect of diet on the rate of fat synthesis over the first 8 weeks of the experiment. However, the activities of the enzymes of fatty acid synthesis [glucose 6-phosphate dehydrogenase, malic enzyme, adenosine triphosphate-citrate lyase, acetyl-coenzyme A carboxylase (ACCX), fatty acid synthetase] were dependent on the age and dietary status of the animals. The exact pattern depended on the specific enzyme and the tissue source. No significant differences in pyruvate dehydrogenase (PDH) activity were observed. Mathematical analysis of the enzyme activities suggested that ACCX and PDH were the most likely sites of fat synthesis regulation. In addition, an examination of body composition and overall weight retention showed that the “weight increasing” effect of a high fat diet could be completely reversed by subsequent feeding of a low fat diet. However, the reversal required an additional 12 weeks. Interestingly, at this time the rats switched from a high fat to a low fat diet had a lower body weight and lower body fat content than rats fed a low fat diet throughout the course of the experiment.

Facultative metabolic rate depression is the common adaptive strategy underlying various animal mechanisms for surviving harsh environmental conditions. This strategy is common among molluscs, enabling animals to survive over days or even months in the absence of oxygen or undr extremely dry conditions. The large reductions in metabolic rate during estivation and anoxia can translate into considerable energy savings when dormant animals are compared to active animals. A complex metabolic coordination is required during the transition into the dormant state to maintain cellular homeostasis and involves both energy-consuming and energy-producing pathways. With regard to energy-producing pathways, several different mechanisms have been identified that participate in controlling flux. One such mechanism, enzyme phosphorylation, can have a wide-ranging effect. For example, phosphorylated enzymes exhibit altered substrate, activator, and inhibitor affinities. This effect may be magnified by changes in the concentrations of allosteric effectors, such as fructose 2,6-bisphosphate, that occur during hypometabolic states. Changes in fructose 2,6-bisphosphate are related to changes in enzyme phosphorylation through changes in the relative activity of phosphofructokinase-2. Alterations in glycolytic enzyme binding can also be brought about through changes in enzyme phosphorylation. The present review focuses on identifying hypometabolism-related changes in enzyme phosphorylation as well as characterizing the mechanisms involved in mediating these phosphorylation events.

This study was conducted to determine the time course of metabolic changes associated with a switch from a high-fat to a low-fat diet in rats. Adult rats, maintained on a high-fat diet (42% of energy from fat) for 4-5 weeks were switched to a low-fat diet (11% of energy from fat), and the activities of several liver enzymes were followed. Three different phases could be distinguished. The early phase, complete by 2 days after the switch in diets, included an increase in the activity of glucose 6-phosphate dehydrogenase (pentose phosphate pathway), an increase in pyruvate kinase and pyruvate dehydrogenase activities (terminal end of the glycolytic pathway) and an increase in ATP-citrate lyase and fatty acid synthetase (fatty acid synthesis pathway). The early phase also included a decrease in the activity of phosphoenolpyruvate carboxykinase (PEPCK, gluconeogenesis) and a lower branched-chain amino acid dehydrogenase activity (BCAADH, branched-chain amino acid degradation). The concentration of the allosteric phosphofructokinase regulator, fructose 2,6-bisphosphate (Fru-2,6-P2, glycolysis), decreased during the early phase. An intermediate phase could also be discerned between 3 and 10 days after the switch in diets. In this phase, the decreased Fru-2,6-P2 concentration and the decreased PEPCK and BCAADH activities observed in the early phase were reversed. The late phase occurred 10 days after the dietary switch and was characterized by an increase in the activities of glucokinase (glycolytic pathway) and glycogen phosphorylase (associated with glycogenolysis) and by a decrease in glutamate dehydrogenase, PEPCK and BCAADH activities. These measurements indicate that at least 20 days are required before metabolic changes associated with a switch in diet are complete.

The effect of cytoskeleton modulators on glycolytic enzyme binding was examined in the hepatopancreas of Otala lactea in an attempt to identify potential cellular binding sites. Binding was followed by measuring phosphofructokinase (PFK), aldolase (ALD), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and pyruvate kinase (PK) distribution between low speed pellets (12,000 xg), high speed pellets (100,000 xg) and high speed supernatants. Taxol (which stabilizes microtubules), colchicine (which destabilizes microtubules) and cytochalasin B and D (which destabilize F-actin filaments) were added to the homogenate prior to centrifugation. Addition of taxol increased the amount of PFK associated with the high speed pellet. Cytochalasin B and D reduced the binding of PFK and PK to the low speed pellet. ALD and GAPDH binding were unaffected by any treatment. Lowering the pH of the crude homogenate increased PFK binding to the low speed pellet by 33%. This effect could be reversed by addition of cytochalasin B and D suggesting that pH influences the PFK-F-actin interaction in vivo. The differential binding response of PFK, PK, ALD and GAPDH to added effectors suggests that, in the cell, PFK and PK are bound to different subcellular structural elements than are ALD and GAPDH.

Methane emissions represent a major environmental concern associated with manure management in the livestock industry. A more thorough understanding of how microbial communities function in manure storage tanks is a prerequisite for mitigating methane emissions. Identifying the microorganisms that are metabolically active is an important first step. Methanogenic archaea are major contributors to methanogenesis in stored swine manure, and we investigated active methanogenic populations by DNA stable isotope probing (DNA-SIP). Following a preincubation of manure samples under anoxic conditions to induce substrate starvation, [U- 13 C]acetate was added as a labeled substrate. Fingerprint analysis of density-fractionated DNA, using length-heterogeneity analysis of PCR-amplified mcrA genes (encoding the alpha subunit of methyl coenzyme M reductase), showed that the incorporation of 13 C into DNA was detectable at in situ acetate concentrations (∼7 g/liter). Fingerprints of DNA retrieved from heavy fractions of the 13 C treatment were primarily enriched in a 483-bp amplicon and, to a lesser extent, in a 481-bp amplicon. Analyses based on clone libraries of the mcrA and 16S rRNA genes revealed that both of these heavy DNA amplicons corresponded to Methanoculleus spp. Our results demonstrate that uncultivated methanogenic archaea related to Methanoculleus spp. were major contributors to acetate-C assimilation during the anoxic incubation of swine manure storage tank samples. Carbon assimilation and dissimilation rate estimations suggested that Methanoculleus spp. were also major contributors to methane emissions and that the hydrogenotrophic pathway predominated during methanogenesis.

Changes in the activities of alanine aminotransferase (Ala-AT), aspartate aminotransferase (Asp-AT), glutamate dehydrogenase (GDH), serine dehydratase (SDH), and branched chain amino acid dehydrogenase were monitored in weanling and adult male rats fed diets differing in fat content: 11% (low), 22% (medium), and 42% (high) of total energy from fat. The carbohydrate and fiber contents were adjusted to maintain the caloric density of the diets. The results showed that changing the total dietary fat and carbohydrate altered the activities of enzymes predominantly in liver and white muscle. This effect was dependent on the age of the animal. In adult animals, high fat diets decreased liver Ala-AT and SDH activity and increased GDH activity. Muscle Ala-AT activity was lowered in adults fed a high fat diet. In weanlings, high fat diets lowered liver Ala-AT and Asp-AT activities as well as muscle Ala-AT activity. Animals were fasted overnight to determine the effect of a high fat diet on fasting enzyme levels. In adults, fasting eliminated the decrease in muscle Ala-AT activity seen in high fatfed animals and decreased liver Ala-AT and SDH activity. In weanlings, fasting increased liver GDH activity and white muscle Ala-AT and Asp-AT activities. These results suggest a complex dependence of amino acid utilization that may depend on dietary regime and the age of the animal. (J. Nutr. Biochem. 6:414-421, 1995.)

The aim of the study was to find the optimal method (with respect to convenience and accuracy) for determining total protein in whole-body homogenates of rats. Three different protein extraction methods and five different protein concentration methods were assessed. The results were compared against a reference value measured by complete amino acid analysis after acid hydrolysis. The data demonstrated that extraction with 5% (w/v) sodium dodecylsulfate (SDS) in 0.5 N NaOH was far superior to that with water alone or to 6 N guanidine-HCl. A comparison of the Biuret, Bradford, and bicinchoninic acid methods on the SDS-NaOH-extracted samples showed that the Biuret method was optimal, giving a value that was 90% of the reference value with a small variation (2.4% of the mean). The Kjeldahl method gave the correct protein concentration only when a nitrogen factor of 5.51 +/- 0.03 (N = 5) was applied. The results suggest that extraction with SDS-NaOH followed by the Biuret procedure is a good method for measuring protein concentrations in whole body rat homogenates.

Changes in [35S]methionine protein labeling patterns were examined by following incorporation into the acid precipitate protein fraction of land snails, Otala lactea (Müller) (Pulmonata, Helicidae). Labeled proteins were analyzed by SDS polyacrylamide gel electrophoresis and isoelectric focusing columns. Snails in four different physiological states were compared: active controls, short term aestivating snails (injected and allowed to enter aestivation), long term aestivating snails (aestivated for 14 days, injected, and maintained in the aestivating state), and snails aroused after aestivation (aestivated, injected, and aroused). Protein associated radioactivity was measured over a 7 day time course post injection. Autoradiographic analysis of SDS-polyacrylamide gels showed increases in the radioactivity of four proteins: 91 kDa (hepatopancreas, day 1 in long term aestivating animals), 50 kDa (hepatopancreas, day 2 in short term aestivating snails), 70 kDa and 30 kDa (foot, day 2 in short term aestivating animals). Hepatopancreas and foot from day 1 long term aestivating and day 2 short term aestivating animals were also analyzed by isoelectric focusing columns. Several pH-specific differences were apparent when controls and aestivating animals were analyzed. In particular a peak of radioactivity was observed at pH 5.05 in 1 d long term aestivating hepatopancreas and at pH 4.30 in 2d short term aestivating animals. Several differences were noted in foot with no specific pattern emerging. SDS-polyacrylamide gel electrophoresis analysis of the hepatopancreas peaks showed the appearance of several bands with increased radioactivity, including the 91 kDa and 50 kDa proteins described above. These results suggest that O. lactea aestivation specific proteins may be involved in the transition to a depressed metabolic state.

Isolated tissues from the land snail Otala lactea were used to examine the relationship between protein kinase activity and phosphorylation-induced changes associated with metabolic depression. Hepatopancreas and foot muscle were removed from active and estivating land snails and incubated in vitro under aerobic and anoxic conditions. Pyruvate kinase (PK), cAMP-dependent protein kinase (PKA), and protein kinase second messenger compounds (cyclic AMP and inositol 1,4,5-triphosphate) were measured after incubating the tissues for 4 hours. Pyruvate kinase from the hepatopancreas of active snails was phosphorylated during anoxic incubations as indicated by changes in the I50 value for L-alanine. However, measurements of PKA activity and of cellular cAMP concentrations suggested that PKA activity was lower in these incubated tissues. When foot muscle was used as the tissue source, incubation under anoxic conditions produced no changes in PK activity even though PKA activity was drastically reduced. Analysis of changes in inositol 1,4,5-triphosphate concentrations after tissue incubation showed that they were not consistent with changes in PK activity in either organ. These results suggest that PKA and Ca2+/phospholipid-dependent protein kinase C do not phosphorylate PK during anoxia in land snails. The differences between values measured in incubated tissues and those measured in vivo suggest that isolated O. lactea tissues are not a good in vitro model system for studying metabolic changes associated with depressed metabolism. © 1994 Wiley-Liss, Inc.

Protein labeling patterns with [32P]phosphorous and [35S]methionine plus [35S]cysteine were examined in normoxic and anoxic tissues of Otala lactea. Analysis of protein phosphorylation showed differences between normoxic and anoxic individuals in both the extent of labeling and the pattern of protein phosphorylation. These changes were confined to the hepatopancreas where anoxia caused overall protein phosphorylation to decrease by a factor of 3. Subcellular fractionation of the hepatopancreas showed that this overall decrease in protein phosphorylation was due to a lower level of protein phosphorylation in individual fractions. Thus, the level of protein phosphorylation during anoxia was 45% (plasma membrane fraction), 21% (mitochondrial fraction), 24% (microsomal fraction), and 22% (cytosolic fraction) of that measured in normoxic tissues. Isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis showed several changes in protein phosphorylation patterns. For example, the relative phosphorylation of cytosolic proteins of molecular mass 65, 49, 30, 25, 21, and 18 kDa was increased in normoxic individuals. Cytosol from anoxic snails showed a relative increase in the phosphorylation of proteins of molecular mass 68, 52, 44, 37, 18, and 16 kDa. No differences in the protein labeling patterns of the mitochondrial and microsomal fractions were apparent. In contrast, analysis of proteins labeled with [35S]methionine plus [35S]cysteine showed no differences when anoxic and normoxic snail tissues were compared.

Changes in de novo protein synthesis and protein phosphorylation were monitored during anoxia and recovery in the red-eared slider Trachemys (= Pseudemys) scripta elegans. Time courses of 35S-radiolabeled methionine incorporation into acid-precipitable material showed an increase up to 5 h postinjection and remained constant after this time. Comparison of the total and acid-precipitable 35S label incorporation into tissues from 20-h control, anoxic, and recovering animals showed differences between these groups: total radioactivity in brain was 2.9-fold lower in recovering turtles, whereas protein-associated radioactivity was 2.4-fold higher in anoxic liver, 2.3-fold lower in recovering skeletal muscle, and 3.7-fold lower in recovering brain tissue. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radiolabeled proteins showed the existence of a newly synthesized protein band (relative molecular mass = 72 kDa) that was apparent only in 20-h recovering liver and skeletal muscle. Use of 32P labeling to monitor changes in protein phosphorylation patterns during anoxia revealed 1.6-, 1.4-, and 1.5-fold increases in 32P incorporation in anoxic brain, heart, and liver, respectively. Changes in protein phosphorylation were localized to the plasma membrane and cytosolic fractions in brain and to the cytosolic fraction in liver.

Protein kinase C from the anoxia-tolerant turtle Pseudemys scripta elegans was investigated to determine its role in mediating changes in brain metabolism associated with anoxia. Measurements of protein kinase C distribution in cytosol and membrane-associated fractions of cerebrum and hindbrain were performed with warm (18 °C)- and cold (7 °C)-acclimated animals exposed to normoxic or anoxic conditions. In cerebrum, the percentage of bound protein kinase C decreased from 48.5% to 35.1% in warm-acclimated animals and from 45.0% to 25.6% in cold-acclimated animals. In the hind-brain, bound protein kinase C increased from 45.0% to 72.9% in warm-acclimated animals and from 40.3% to 68.8% in cold-acclimated animals. The presence of three distinct protein kinase C isozymes (Types I, II and III) was confirmed by hydroxylapatite chromatography. The distribution of isozymes between cytosolic and membrane-associated fractions in cerebrum was 24% I, 37% II and 39% III (cytosolic) and 32% I, 35% II and 34% III (membrane-associated). In the hindbrain, the protein kinase C isozyme distribution was 34% I, 40% II and 26% III (cytosolic) and 18% I, 47% II and 35% III (membrane-associated). Kinetic characterization of the three isozymes showed that Type I was 27% activated by Ca2+, whereas Types II and III were only 4% and 2% activated by Ca2+, respectively. Full activity for all enzymes was observed only in the presence of phosphatidylserine and diacylglycerol. No differences in the Km for ATP, the Ka for Ca2+ or the Ka for phosphatidylserine were observed.

The kinetic properties and regulation of pyruvate dehydrogenase (PDH) activity during estivation and recovery were examined in foot muscle and hepatopancreas of the terrestrial snail, Otala lactea (Muller) (Pulmonata, Helicidae). During estivation, the percentage of PDH in the active a form (PDHa) decreased from a control value of 98% to an estivating value of approximately 60% in both foot and hepatopancreas but the time course for changes in PDHa activity was different for the two tissues. Foot PDHa steadily decreased from the initiation of estivation until 20 h; after this time, the levels of PDHa showed no further change. Hepatopancreas PDHa levels did not decrease until 20 h after the initiation of estivation, after which time they rapidly dropped to reach a new, constant, estivating level by 30 h. Recovery curves for both tissues showed that PDHa had reached control levels 1 h after the reintroduction offood and water. Kinetic parameters of PDHa were also examined. The Michaelis constant value ($K_{...

Biochemical mechanisms underlying anaerobiosis were assessed in two Mediterranean bivalve species, Scapharca inaequivalvis and Venus gallina, with widely differing tolerances for oxygen lack. These species displayed LT50 values for anoxic survival at 17–18°C of 17 and 4 d, respectively. Succinate and alanine were the major products of 24 h anaerobic metabolism in both species but only S. inaequivalvis further metabolized succinate to propionate. Both species reduced metabolic rate while anoxic but metabolic arrest was more pronounced in S. inaequivalvis. Calculated ATP turnover rate (MATP) during exposure to N2-bubbled seawater was only 4.51% of the aerobic rate in S. inaequivalvis but was 12.68% in V. gallina. To counteract a greater load of acid end products, V. gallina foot showed a significantly greater buffering capacity, 23.38±0.20 slykes, compared to 19.6±0.79 slykes in S. inaequivalvis. The two species also differed distinctly in the enzymatic regulation of anaerobiosis. In V. gallina anoxia exposure caused only a small change in PFK kinetic parameters (a decrease in Ka AMP) and had no effect on glycogen phosphorylase. By contrast, S. inaequivalvis foot showed a strong modification of enzyme properties in anoxia. The percentage of glycogen phosphorylase in the a form dropped significantly only in S. inaequivalvis. Other changes included alterations in the properties of PFK leading to a less active enzyme form in anoxia. Compared to the aerobic enzyme form, PFK from anoxic foot showed a reduced affinity for fructose-6-P (Km increased 2.4-fold), greater inhibition by ATP (I50 decreased 6.8-fold), and an increase in sensitivity to AMP activation (Ka decreased by 50%). These enzyme changes appear to be key to a glycolytic rate depression during anaerobiosis in S. inaequivalvis foot muscle.

The role of pH and protein kinase second messengers in triggering or potentiating anoxia-linked changes in enzyme binding to particulate matter were evaluated using in vitro incubations of isolated ventricle strips of Busycon canaliculatum (L.) (Prosobranchia, Melongenidae). Incubating whelks under anoxic conditions for 4h reduced the percentage of phosphofructokinase (PFK), aldolase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and pyruvate kinase (PK) activity associated with cellular particulate matter. Triose phosphate isomerase, phosphoglycerate kinase, phosphoglyceromutase and enolase showed no changes in enzyme binding when whelks were subjected to anoxic stress in vivo. Incubating isolated ventricle strips in vitro under anoxic conditions simulated the changes seen in vivo in whole, anoxic whelks with respect to the percentage of PFK and PK bound during anoxic stress; both whole-animal studies and isolated tissue studies showed reduced PFK and PK binding after 4 h of anoxic incubation. Tissue pH could be artificially changed by incubating isolated ventricle strips in sea water buffered to a desired pH. This permitted an investigation of the effect of intracellular pH on PFK and PK binding in situ. PFK and PK responded to altered intracellular pH with increased enzyme binding at lower intracellular pH values and decreased enzyme binding at higher intracellular pH values. These binding patterns were exactly the opposite of those observed during anoxia; during anoxia stress, both intracellular pH and the percentage of PFK and PK associated with particulate matter decreased. Addition of the second messenger compounds dibutyryl cyclic AMP, dibutyryl cyclic GMP or phorbol 12-myristate 13-acetate plus the calcium ionophore A23187 had no effect on the percentage of activity bound to subcellular structures measured under either normoxic or anoxic conditions. This study suggests that enzyme binding in vivo is not regulated by changes in intracellular pH or concentrations of protein kinase second messenger compounds during anoxia.

Assocaiations between glycolytic enzymes and subscellular structures have been interpreted as presenting a novel mechanism of glycolytic control; reversible enzymes binding to subcellular structural components is believed to regulate enzymes activity in vivo through the formation of a multi-enzyme complex. However, three lines of evidence suggest that eyzyme binding to cellular structures is not involved in the control of glycolsis, (i) Calculations of the distribution of glycolytic enzymes under the physiological cellular conditions of higher ionic strenght and higher enzyme concentrations indicate that a large multi-enzyme complex would not exist, (ii) In many eases, binding to subcellular structures is accompanied by changes in enzyme kinetic parameters brought about by allosteric modification, but these changes often inhibit enzyme activity, (iii) In the case where formation of binary enzyme/enzyme complexes activates enzymes, the overall increase in flux through the enzyme reaction is negible.

A cystosolic protein kinase that phosphorylates pyruvate kinase (PK) in vitro has been identified in crude homogenates of heart, radular retractor, and foot muscle from the anoxia-tolerant marine whelk Busycon canaliculatum. Protein kinase action was measured by following changes in PK kinetic parameters: phosphorylated PK has a higher K m value for phosphoenolpyruvate and a lower I50 value for l-alanine. The crude protein kinase readily phosphorylated PK in a Mg2+-and ATP-dependent manner in the absence of any added effector. This activity was not affected by the addition of either cAMP (a stimulator of protein kinase A) or Ca2+ plus phorbol 12-myristate 13-acetate (stimulators of protein kinase C) to the incubation medium. Addition of cGMP to the homogenate, however, increased the rate of PK phosphorylation giving a 3–4-fold increase in the rate of change in PK kinetic parameters that was readily apparent after 5h. Complete time-courses of changes in PK kinetic parameters in the presence and absence of cGMP showed that cGMP increased the rate, but not the final extent, of PK phosphorylation. These results indicate that PK inactivation by enzyme phosphorylation in response to anoxia in whelk tissues may be mediated by a cyclic GMP stimulated protein kinase in response to changing levels of cGMP. This conclusion was further supported by data indicating that the total activity of protein kinase was the same in both anoxic and aerobic animals, and that the total PK phosphatase activity was also constant. Changes in PK phosphorylation during anoxia are not, therefore, the result of changes in the total amount of protein kinase or phosphatase.

The mechanisms controlling glycolytic rate were examined in foot muscle of the terrestrial snail Otala lactea (Müller) (Pulmonata, Helicidae), during short and long periods of estivation and anoxia. Binding associations between glycolytic enzymes and the particulate fraction of the cell were assessed in both states. The percentage of enzyme activity bound to particulate matter decreased significantly over the short term (4 days estivation and 14·5 h anoxia); significant changes were seen for hexokinase (HK), phosphofructokinase (PFK), aldolase and lactate dehydrogenase (LDH) in estivation and, for these enzymes plus triosephosphate isomerase and pyruvate kinase (PK), in anoxia. Over the longer term in estivation (22 days) and anoxia (45 h), enzyme binding returned to control values. Tissue content of fructose-2,6-bisphosphate, a potent phosphofructokinase activator, decreased under all experimental conditions. Total glycogen phosphorylase activity decreased during short-term anoxia (14·5 h) and during long-term estivation (22 days), but the percentage of the active a form decreased significantly during anoxia only. Significant changes in the maximal activities of several enzymes were observed during both estivation and anoxia. Decreases in the maximal activity of HK, PFK, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase (PGK) and LDH were observed during long-term estivation. Increases in PGK and PK maximal activity in short-term anoxia and aldolase and PGK in long-term anoxia were also observed. These results suggest that changes in glycolytic enzyme binding may be part of an immediate mechanism used to cause a rapid decrease in glycolytic flux and initiate glycolytic rate depression, which also includes a reduction of fructose-2,6-bisphosphate content and decreased glycogen phosphorylase activity. In the long term, however, control of snail glycolytic rate is reorganized, so that enzyme binding associations revert to the control values. In the long term, then, control is mediated by lower fructose-2,6-bisphosphate concentrations and, during estivation, also by a decrease in maximal enzyme activities.

The effects of low temperature and anoxia were determined on phosphofructokinase (PFK) purified from white skeletal muscle of the freshwater turtle, Pseudemys scripta. These effects were assayed by comparing PFK kinetic constants measured at a high (20 degrees C) and low (6 degrees C) temperature using enzyme obtained from animals held under normoxic and anoxic conditions. When assayed at 20 degrees C, PFK from anoxic animals had a lower Ka for phosphate, a lower Ka for AMP and showed no inhibition with increasing concentrations of ATP (up to 10 mM) when compared to enzyme from normoxic animals. At 6 degrees C, anoxic enzyme had a higher Km for fructose 6-phosphate and a higher I50 value for citrate with respect to normoxic enzyme. Decreasing temperature also had a differential effect on PFK kinetic parameters depending on the source of the enzyme. When normoxic enzymes were compared at 20 and 6 degrees C, the enzyme measured at 6 degrees C showed a lower Km for ATP and a lower Ka for AMP. Comparison of anoxic enzymes at these two temperatures showed that anoxic PFK at 6 degrees C had a higher Ka for phosphate, a higher Ka for AMP, and a larger Hill coefficient. A comparison of maximal velocities at varying temperature showed that normoxic enzyme (Q10 = 2.22) was more temperature sensitive than the anoxic enzyme (Q10 = 1.80). It is possible to interconvert the normoxic and anoxic forms of PFK by incubating normoxic enzyme with the active subunit of protein kinase, suggesting that the kinetic changes observed during anoxia resulted from enzyme phosphorylation. These data are discussed with respect to the mechanisms underlying white muscle function during diving and hibernation in red-eared turtles.

Phospholipid Signaling Protocols Edited by Ian M. Bird. Totowa, Humana, 1998, $79.50 (xii +380 pages), ISBN 0-896-03491-7.

Free and membrane-associated fractions of protein kinase C (PKC) from rainbow trout (Onchorynchus mykiss) brain tissue were separated by hydroxylapatite chromatography and characterized kinetically. In both resting fish and in fish swum to exhaustion, approximately 40% of the total PKC activity was bound to membranes. Quantification of the three distinct hydroxylapatite chromatography peaks (PKC types gamma, beta and alpha) in cytosolic and membrane fractions revealed different isozyme distributions. The cytosolic fraction contained 21% PKC type gamma, 52% PKC type beta and 27% PKC type alpha. The membrane-associated fraction contained 23% PKC type gamma, 28% PKC type beta and 49% PKC type alpha. Kinetic characterization of the three isozymes showed that PKC type gamma was almost completely activated by Ca2+ alone whereas PKC type beta and PKC type alpha were 40% and 60% activated by Ca2+. Full activity for all enzymes was observed only in the presence of phosphatidylserine and diacylglycerol. Differences in the kinetic constants for the three isozymes were also apparent. PKC type gamma had a much lower affinity for Histone III-S when compared with PKC types beta and alpha (100 micrograms/ml as compared with 1.7 and 5.7 micrograms/ml). PKC type gamma also had a lower affinity for calcium (0.22 microM) when compared with PKC type beta (0.08 microM) and PKC type alpha (0.05 microM). PKC type alpha had a lower affinity for phosphatidylserine (8.6 micrograms/ml) when compared with PKC type gamma (0.37 microgram/ml) and PKC type beta (0.89 microgram/ml).

A double-blind, placebo-controlled, randomized crossover clinical trial involving 30 healthy adults was conducted to examine the impact of fructans on both the gut microbiota and immune system. Along with their regular diet, trial participants consumed either an oligofructose-enriched inulin supplement as the test diet or food grade maltodextrin as the control diet for a 28 day period, with a minimum 2 week washout period before cross-over. The trial supplements were coded as “A” or “B”, and will remain coded until the completion of all analyses. Serum immunoglobulin concentrations were quantified by Luminex multiplexing technology. IgM was significantly higher after 28 days of supplement A consumption (821.3 ng/mL ± 138.3) than after supplement B consumption (485.8 ng/mL ± 66.5) ( P = 0.0118). IgA was also significantly higher ( P = 0.0092) after supplement A consumption (1967.3 ng/mL ± 374.4) than after supplement B consumption (911.7 ng/mL ± 138.2). Active transforming growth factor-beta (TGF- β ) concentration was measured in plasma and serum cytokine concentrations were quantified by Enzyme-linked immunosorbent assay (ELISA). Levels of active TGF- β were 5.4 pg/mL ± 2.1 after consumption of supplement B, in comparison to 3.4 pg/mL ± 1.5 after supplement A consumption, although this difference was not statistically significant. IL-10 concentrations were significantly lower ( P = 0.0172) after supplement B consumption (41.2 pg/mL ± 29.0) than after supplement A consumption (79.8 pg/mL ± 42.1). Granulocyte colony-stimulating factor (G-CSF) levels were also significantly lower ( P = 0.0265) after supplement B consumption (5.4 pg/mL ± 2.5) than levels after supplement A consumption (14.4 pg/mL ± 3.6). No differences were observed between subjects consuming supplement A and B in Interleukin-8 (IL-8) and IL-1Ra concentrations. Analysis of cytokine profiles will provide insight into the extent of the potential effects of dietary fructans on the immune system mediated through the gut microbiota.

Gut microbiota-mediated effects on the immune system can influence cytokine production in gut-associated lymphoid tissues (GALT). The extent to which gender-associated differences in gut microbiota composition influence mucosal cytokine patterns is as yet undetermined, as is the impact such differences may have on the ability of fermentable material to modulate immune activity through effects on the gut microbiota. To explore these questions, we analyzed the effects of a fructo-oligosaccharide (FOS)-supplemented Purina chow diet on mucosal cytokine profiles, focusing on IL-6 (pro-inflammatory), IL-4 (TH 2 ) and IL-10 (regulatory T reg ). To compare gender-associated effects of FOS at the gut microbial and intestinal epithelial level, we measured fecal short chain fatty acid profiles (by gas chromatography) and mucin gene MUC2 expression (determined by quantitative real-time PCR). No major FOS-induced changes in cytokine profiles (vs. control) were observed in the ileum of male or female rats, although gender-specific differences in cytokine levels were observed. Cecal IL-6 (7732.49 ± 863 pg/g) was higher in FOS-fed male rats compared to FOS-fed female rats (5057.9 ± 536.5 pg/g). Cecal IL-10 was significantly lower in male rats fed the control diet (5301.2 ± 241.6 pg/g) relative to female rats fed the same diet (8164 ± 843.6 pg/g). However, cecal IL-10 was elevated in FOS-fed male rats (6484.3 ± 1269.7 pg/g) to levels similar to those of FOS-fed female rats (6662.4 ± 335.6 pg/g), indicating a potential differential effect of dietary FOS on this regulatory cytokine in males and females. Fecal butyric acid concentrations in FOS-fed male rats (35.1 ± 7.1 mmol/g) were significantly higher than those in FOS-fed female rats (13.6 ± 2.6 mmol/g), suggesting a gender-associated difference in the effects of FOS at the gut microbial level. Preliminary results indicate higher cecal MUC2 expression in male rats relative to female rats. These findings illustrate that fermentation-associated luminal changes may affect GALT immune activity in a gender-specific fashion.

In response to environmental stress (low water, low oxygen) snails sharply suppress their metabolic rate, a process that is coordinated at the molecular level by reversible protein phosphorylation of key enzymes and functional proteins. Factors affecting protein kinase activity are, therefore, critical to metabolic suppression. Changes in the concentration of protein kinase second messenger compounds were followed over the first 24 h of aestivation and anoxia exposure in the terrestrial snail Otala lactea (Muller) (Pulmonata, Helicidae). The results showed declining concentrations of cyclic AMP over the first 24 h of anoxia exposure and aestivation in foot. Cyclic AMP concentrations in hepatopancreas transiently decreased with the lowest concentration observed at 4 h in both anoxic and aestivating animals. A transient increase in foot muscle cyclic GMP concentrations was apparent 4 h after the start of aestivation whereas a slow, steady increase was seen in anoxic foot muscle. Foot muscle 1,4,5-inositol triphosphate (IP3) concentrations decreased transiently during anoxia exposure and aestivation. Hepatopancreas IP3 concentrations were significantly lower in 24 h anoxic snails and foot IP3 concentrations were significantly lower in 24 h aestivating snails. Kinetic characterization of purified PKA catalytic subunit was also performed. Snail PKA catalytic subunit had an absolute requirement for Mg2+ ion but was inhibited at Mg2+ concentrations above 0.5 mM. Increasing concentrations of neutral salts and phosphate also inhibited activity although the inhibition by phosphate appeared to be specific since the inhibition constant (I50 = 39 mM) was much lower than that of the neutral salts (I50 approximately 240 mM). The enzyme exhibited a broad pH optimum between pH 6.5-8.5. Arrhenius plots gave an activation energy of 13.3 kcal/mol corresponding to a Q10 value of 2.3. The relationship between these results and temporal control of enzyme phosphorylation is discussed.

It is commonly believed that certain reactions in a metabolic sequence may be at or close to equilibrium because of the large excess of catalytic capacity compared to the flux through these enzyme loci. Simple algebraic manipulations can show that the equilibrium and steady state conditions are mutually exclusive. However, solution of the complete reaction schemes for model "equilibrium" reactions shows that they can remain far from equilibrium even though the ratio of enzyme flux to steady state flux through the overall pathway is high. These calculations show that a reaction's proximity to equilibrium depends on the overall flux through the enzyme locus as well as on the kinetic parameters of the other enzymes in the pathway. Thus, combinations of kinetic parameters may exist that allow certain reactions to approach equilibrium but these conditions are not universal.Key words: equilibria, theoretical kinetics, metabolic control.

Protein phosphorylation patterns were investigated in whole tissues and subcellular fractions of active and aestivatingOtala lactea (Muller) (Pulmonata, Helicidae). Measurement of overall protein phosphorylation showed that incorporation of32P increased until the second day after injection and remained constant for the remaining 4 days of the time course. Comparison of tissues from aestivating and active snails on day 3 showed a decreased protein phosphorylation in aestivating snails (44% of active). No differences in total and protein-associated radioactivity for foot, mantle or haemolymph were observed. Subcellular fractionation of the hepatopancreas localized the changes to plasma membrane, microsomal, and cytosolic fractions: values for aestivating animals were reduced to 71, 37 and 58% of the corresponding active values. Separation of the individual subcellular fractions on isoelectric focusing columns revealed differences in the phosphate incorporation patterns. Plasma membrane from aestivating animal hepatopancreas had a lower overall level of incorporation and fewer radioactive peaks in the pH 7–10 region than did the plasma membrane fraction from active animals. SDS-PAGE analysis of plasma membrane fractions from active and aestivating snails showed a relative decrease in phosphorylation between 60–80 kDa and 30–40 kDa. IEF analysis of cytosolic proteins from aestivating snail hepatopancreas also showed peaks of radioactivity that were apparently shifted by 0.3 pH units toward higher pI values. Increased phosphate incorporation was observed at a peak that corresponded to the pI value for pyruvate kinase in aestivating snails but definite assignment of peaks was not possible. SDS-PAGE analysis of cytosolic proteins showed an aestivation-related decrease in relative protein phosphorylation between 30–35 kDa and 40–45 kDa. A relative increase in phosphorylation during aestivation was observed for proteins between 16–22 kDa. Overall, the data indicate that snails dramatically alter their protein phosphorylation pattern in hepatopancreas during aestivation. (Mol Cell Biochem143: 7–13, 1995)

Summary We have discussed six different types of mechanisms that are used to adapt enzyme function to allow metabolism to respond to changing physiological demands or environmental stresses. Although the present discussion has been largely limited to enzymes of glycolysis, the principles are equally applicable to virtually all aspects of metabolic function. In particular, control via reversible protein phosphorylation is proving to be widespread for the coordination of diverse cellular enzymes and proteins ( Boyer and Krebs, 1987 ). For example, reversible phosphorylation is now well known to extend to the control of membrane ion-channel proteins, regulating both their incorporation into membranes and their activity once inserted ( Reuter, 1987 ). Such coordinated control over a wide variety of cellular processes is proving to be critical to metabolic rate depression. Hibernating mammals, for example, retain control over membrane potential difference as body temperature falls to ambient; the same species in the summer-adapted state, however, are just as susceptible to the destructive effects of hypothermia on membrane ion regulation as are humans. The lessons to be learned from comparative biochemistry are many. The combinations of metabolic controls that can be applied for the adaptation of enzyme function are nearly limitless and mechanisms can be found to overcome almost any environmental constraint or meet any physiological demand. Increasingly, we are applying the information gained from comparative studies and nowhere is this more true that in the development of organ preservation technology. Both the mechanisms of metabolic rate depression and of freeze tolerance that are found in animals are directly applicable to the development of mechanisms for organ transplant technology and some of the most effective therapies presently used (e.g., glycerol as a cryoprotectant; inhibitors of membrane ion transport to reduce energy demands) effectively mimic the strategies used in the natural situation.

Publisher Summary This chapter analyzes the mechanism of glycolytic rate and glycolytic control. In the classical view, glycolytic flux is controlled primarily through allosteric regulation of four enzymes: glycogen phosphorylase (GP), hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK). Glycogen phosphorylase, HK, and PFK are found at the start of the glycolytic sequence, whereas PK is one of the terminal glycolytic enzymes. In fish, all four enzymes show several features common to many regulatory enzymes: they catalyze high energy steps that are (essentially) irreversible, they catalyze reactions that involve a high energy intermediate, and they are multi-subunit enzymes. They are also allosterically regulated by several different compounds, which serve to link enzyme activity to the energy state of the cell. The complexity of controls governing glycolytic enzyme activity becomes apparent when the widely different metabolic states that fish can endure are considered. The chapter examines the evidence for and against the formation of a functional glycolytic complex. It defines the glycolytic complex as a multi-enzyme structure containing all the enzymes necessary to metabolize glucose to pyruvate.

The effect of changing concentrations of glycolytic intermediates on the binding of phosphofructokinase, aldolase and pyruvate kinase to cellular particulate matter was investigated. Concentrations of glycolytic intermediates were altered by adding 2 mM iodoacetic acid (IAA) to an incubation medium containing tissues isolated from the channelled whelk Busycon canaliculatum. Iodoacetic acid inhibited glyceraldehyde 3-phosphate dehydrogenase activity causing a 100-400 fold increase in the concentration of fructose 1,6-bisphosphate as well as 3-20 fold increases in glucose 6-phosphate, fructose 6-phosphate, and dihydroxyacetone phosphate levels depending on the experimental protocol. Cellular pH values were not statistically different in the presence of IAA. Measurement of enzyme binding to particulate matter showed that the binding of phosphofructokinase, aldolase and pyruvate kinase was unaffected by iodoacetic acid under any experimental condition. These results show that changes in the tissue concentrations of enzyme substrates and products do not regulate enzyme binding to particulate matter in the cell.

A computer program that allows the preparation of buffers containing known concentrations of metal-ligand complexes at defined pH values and temperatures is described. Ligands are defined as compounds that bind metals and may include AMP, ADP, ATP, GMP, GDP, GTP, EGTA, EDTA, BAPTA, phosphate, sulfate, chloride, monocarboxylic acids, dicarboxylic acids, organophosphates, and/or citric acid. Metals may include sodium, potassium, magnesium, calcium, and/or manganese. The program uses association constants corrected for temperature and ionic strength so that solutions between 0 and 40 degrees C and between pH values of 4 and 10 can be defined. The program can perform the following: (i) calculate the concentration of all metal-ligand complexes when total metal and total ligand concentrations are known, (ii) calculate the concentration of metal ion required to make a solution of known free metal ion concentration when total ligand concentrations are known, (iii) calculate the concentration of ligand required to make a solution of known free metal ion concentration when total metal concentrations are known, and (iv) calculate the total concentrations of metal and ligand required to make a buffer of known metal-ligand concentration. Options i-iii are useful for making buffers of defined free metal ion concentrations; option iv is useful for making buffers of defined metal-nucleotide concentrations.

The mechanisms of glycolytic rate control during hibernation in the ground squirrel Spermophilus lateralis were investigated in four tissues: heart, liver, kidney, and leg muscle. Overall glycogen phosphorylase activity decreased significantly in liver and kidney to give 50% or 75% of the activity found in the corresponding euthermic organs, respectively. The concentration of fructose-2,6-bisphosphate (F-2,6-P2) decreased significantly in heart and leg muscle during hibernation to 50% and 80% of euthermic tissue concentrations, respectively, but remained constant in liver and kidney. The overall activity of pyruvate dehydrogenase (PDH) in heart and kidney from hibernators was only 4% of the corresponding euthermic values. Measurements of phosphofructokinase (PFK) and pyruvate kinase (PK) kinetic parameters in euthermic and hibernating animals showed that heart and skeletal muscle had typical rabbit skeletal M-type PFK and M1-type PK. Liver and kidney PFK were similar to the L-type enzyme from rabbit liver, whereas liver and kidney PK were similar to the M2 isozyme found primarily in rabbit kidney. The kinetic parameters of PFK and PK from euthermic vs hibernating animals were not statistically different. These data indicate that tissue-specific phosphorylation of glycogen phosphorylase and PDH, as well as changes in the concentration of F-2,6-P2 may be part of a general mechanism to coordinate glycolytic rate reduction in hibernating S. lateralis.

An investigation of the direct transfer of metabolites from rabbit muscle L-lactate dehydrogenase (LDH, EC 1.1.1.27) to glycerol-3-phosphate dehydrogenase (GPDH, EC 1.1.1.8) revealed discrepancies between theoretical predictions and experimental results. Measurements of the GPDH reaction rate at a fixed NADH concentration and in the presence of increasing LDH concentrations gave experimental results similar to those previously obtained by Srivastava, Smolen, Betts, Fukushima, Spivey & Bernhard [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6464-6468]. However, a mathematical solution of the direct-transfer-mechanism equations as described by Srivastava et al. (1989) showed that the direct-transfer model did not adequately describe the experimental behaviour of the reaction rate at increasing LDH concentrations. In addition, experiments designed to measure the formation of an LDH4.NADH.GPDH2 complex, predicted by the direct-transfer model, indicated that no significant formation of tertiary complex occurred. An examination of other kinetic models, developed to describe the LDH/GPDH/NADH system better, revealed that the experimental results may be best explained by assuming that free NADH, and not E1.NADH, is the sole substrate for GPDH. These results suggest that direct transfer of NADH between rabbit muscle LDH and GPDH does not occur in vitro.

The cellular distribution of free and bound glycolytic enzymes in vivo was estimated by means of a model based on previously determined association constants for individual binding interactions and in vivo protein concentrations. The calculations revealed that a significant proportion of the enzymes would be either associated with F-actin, or bound in binary enzyme-enzyme complexes in vivo . An analysis of the relative concentration, and relative activity, of F-actin-bound enzymes suggested that a complete glycolytic complex, composed of all enzymatic steps from phosphofructokinase (PFK) to lactate dehydrogenase (LDH) does not exist. This was indicated by a very low concentration of F-actin-associated phosphoglycerate kinase (PGK) and by a very low activity of F-actin bound aldolase and PGK; this model showed that aldolase and PGK would be absent from any F-actin bound complex. An analysis of soluble enzyme-enzyme associations indicated that formation of binary enzyme complexes may lead to an increased overall flux through glyceraldehyde 3-phosphate dehydrogenase and LDH, but would serve to decrease flux through PFK and aldolase. A 1·4-fold activation of PFK, which occurs when the soluble enzyme binds to F-actin, suggested that reversible binding of PFK to F-actin may represent a novel cellular mechanism for controlling glycolytic flux during periods of increased metabolic demand by controlling the key regulatory enzyme of glycolysis.

A protein kinase which phosphorylates pyruvate kinase (PK) in vitro was purified and characterized from the foot muscle of the anoxia-tolerant gastropod mollusc Busycon canaliculatum. Purification involved four steps: poly(ethylene glycol) fractionation, affinity chromatography on Blue agarose, ion-exchange chromatography on phosphocellulose and preparative isoelectric focusing (pI = 5.5). The activity was monitored by following changes in pyruvate kinase I50 values for L-alanine which have previously been linked to changes in the degree of enzyme phosphorylation. The correlation between enzyme phosphorylation and changes in the L-alanine inhibition constant was also directly demonstrated in the present paper by radioactively labelling PK with [tau-32P]ATP. The final purified protein kinase solution gave a single band on SDS-gel electrophoresis with a molecular weight of 37,000 +/- 2000. Kinetic analysis of the purified protein kinase (PK-kinase) showed a pH optimum of 7.0, an absolute requirement for magnesium ions (Km = 1.29 mM), a relatively high affinity for MgATP (Km = 57 microM), and inhibition by increasing salt concentrations (I50 = 55 mM KCl). The protein kinase activity was not affected by either spermine, heparin, cAMP, cGMP or concentrations of CaCl2 less than 10 mM. The enzyme did not phosphorylate either phosphofructokinase or glycogen phosphorylase, two enzymes that are also phosphorylated during anoxia in whelks. The purified enzyme is different from the catalytic subunit of cAMP-dependent protein kinase as shown by the inability of cAMP to stimulate the protein kinase at all stages of the preparation; cAMP did not activate either crude enzyme, the 7% poly(ethylene glycol) supernatant, or any of the column eluant peak fractions when measured by changes in pyruvate kinase kinetic parameters.

Presentation des connaissances acquises concernant la theorie des systemes enzymatiques couples, a l'aide des modeles mathematiques elabores depuis plusieurs annees ou plus recemment

Rabbit heart mitochondria synthesize comparably more monoacyl-than diacylglycerol 3-phosphate as compared to heart microsomes. Since the fatty acid specificity of the biosynthesis by cardiac enzymes differs from that by hepatic enzymes, the positional specificity of monoacylglycerol 3-phosphate formation by mitochondria was investigated by using a method utilizing lysosomal phosphatidate phosphohydrolase and borate-impregnated thin layer chromatography. Contrary to the results of studies with liver particulates, ca. one-third of palmitate was found at position 2 of the glycerol moiety and the remainder at position 1, whereas one-third of oleate was bound at position 1. Only a slight, asymmetric distribution was observed with linoleate as the acyl donor. Thus, our study indicates that preferential acylation of individual fatty acids during the formation of monoacylglycerol 3-phosphate is not as distinct in the heart mitochondria as in the liver mitochondria and microsomes.

The glycolytic enzymes glycogen phosphorylase, phosphofructokinase (PFK), and pyruvate kinase (PK) were assessed in liver, heart, red muscle, and white muscle of aerobic and 5-h anoxic turtles (Pseudemys scripta) for changes in total activity and kinetic parameters. Anoxia induced statistically significant changes in these glycolytic enzymes in each of the four organs assayed. Compared with normoxic controls, anoxic liver showed a 3.3-fold increase in glycogen phosphorylase activity, a 1.5-fold increase in the PFK I50 value for citrate (concentration that inhibits initial activity by 50%), a 1.5-fold increase in the PFK Michaelis constant (Km) value for fructose 6-phosphate (P), and an increased maximal activity of PK. Anoxic heart muscle showed a 2.6-fold decrease in glycogen phosphorylase activity and, for PFK, a 1.7-fold decrease in the Km value for ATP and a twofold increase in the I50 value for citrate. In anoxic white muscle, PFK showed a fivefold lower Km value for fructose-6-P and a threefold lower activator concentration producing half-maximal activation (A50) for potassium phosphate than the aerobic enzyme form. Changes in anoxic white muscle PK included a twofold increase in the Km value for ADP and a 1.7-fold decrease in the I50 value for alanine. In red muscle, anoxia affected only the Km value for ATP, which was 50% higher than the value for the aerobic enzyme form. Fructose 2,6-diphosphate (P2) levels also decreased in heart muscle and increased in red and white muscle during anoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel purification of phosphofructokinase has been achieved in a two step process using ion-exchange affinity chromatography and a transition-state analogue affinity column matrix. The procedure can be performed in one day, and gives a 25% yield of the starting material. The transition-state analogue chromatography is carried out using an ADP-agarose column in the presence of fructose 6-phosphate, magnesium ions and nitrate ions. In the presence of nitrate ion plus substrate, phosphofructokinase binds immobilized ADP while other proteins pass through the column. Previous studies with creatine kinase have shown that the nitrate ion mimics the planar phosphate in the transition state resulting in a complex which is stable under the relatively high ionic strength of the column buffer. This permits the elution of phosphofructokinase in a single peak of high specific activity. This column typically results in a 20-30 fold increase in specific activity with only a small loss of activity.

The effect of exercise on the binding of glycolytic enzymes to subcellular structures was examined in rainbow trout (Salmo gardneri). Both “burst” and “endurance” type exercise produced an increase of approximately 50% in the percentage of phosphofructokinase (PFK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), aldolase, and 3-phosphoglycerate kinase associated with particulate matter in white skeletal muscle. In contrast, cardiac muscle showed no change in enzyme binding when trout were exercised, suggesting that the effects seen in white muscle are related to an increased anaerobic glycolytic flux in this tissue. Modulation of binding by altered pH or calcium ion concentration was tested: a decrease in pH increased PFK binding to subcellular particles, whereas 2 mM CaCl2 decreased GAPDH binding. These results are discussed with respect to the formation of a glycolytic complex during exercise in trout white muscle.

The roles of hormones, second messengers and pH in triggering or potentiating biochemical responses to anaerobiosis were evaluated using in vitro incubations of isolated muscle tissues (foot, radular retractor, ventricle) from the marine whelk Busycon canaliculatum (L.). Incubating tissues in vitro under anoxic conditions stimulated changes in muscle fructose-2,6-bisphosphate levels and pyruvate kinase kinetics (Km values for phosphoenolpyruvate, values for L-alanine) that were virtually equivalent to those that occur in vivo. Additions of hormones (epinephrine, norepinephrine, octopamine, serotonin, glucagon, insulin) or inhibitors of prostaglandin synthesis (dexamethasone, aspirin) had no effect on these metabolic responses to anoxia. The second messenger compounds, dibutyryl cyclic AMP and Ca2+ + ionophore A23187 + phorbol myristate acetate, produced isolated and tissue-specific responses in muscles incubated under aerobic conditions, but the magnitude and pattern of these responses differed from those seen in anoxia. Second messengers also had no effect on the development of biochemical responses in anoxic muscles. Tissue pH was artificially altered in order to evaluate the role of pH change (acidification occurs during anoxia in vivo) in the control of metabolic responses to anoxia. In all cases, the changes in the kinetic properties of pyruvate kinase (PK) correlated with the state of oxygenation of the tissue and not with the measured tissue pH value; higher tissue pH did not prevent anoxia-induced phosphorylation of PK and lower tissue pH did not alter the kinetic patterns of the aerobic enzyme. Overall, the study indicates that cells and tissues of the whelk respond individually to anoxia and that coordination of the action of protein kinases during anoxia is not mediated by pH or by common second-messenger mechanisms.

The stoichiometry and dissociation constant for the binding of homogeneous chicken heart mitochondrial creatine kinase (MiMi-CK) to mitoplasts was examined under a variety of conditions. Salts and substrates release MiMi-CK from mitoplasts in a manner that suggests an ionic interaction. The binding of MiMi-CK to mitoplasts is competitively inhibited by Adriamycin, suggesting that they compete for the same binding site. Fluorescence measurements also show that Adriamycin binds to MiMi-CK so that the effect of Adriamycin on the binding of MiMi-CK to mitoplasts is not simple. Titrating mitoplasts with homogeneous MiMi-CK at different pH values shows a pH-dependent equilibrium involving a group(s) on either the membrane or the enzyme with a pKa = 6. Extrapolating these titrations to infinite MiMi-CK concentration gives 14.6 IU bound/ nmol cytochrome aa3 corresponding to 1.12 mol MiMi-CK/mol cytochrome aa3. Chicken heart mitochondria contain, after isolation, 2.86 ± 0.42 IU/nmol cytochrome aa3. Titrating respiring mitoplasts with carboxyatractyloside gives at saturation 3.3 mol ADP/ ATP translocase/mol cytochrome aa3. Therefore, chicken heart mitoplasts can maximally bind about 1 mol of MiMi-CK per 3 mol translocase; in normal chicken heart mitochondria about 1 mol of MiMi-CK is present per 13 mol translocase.