Effect of NAD and ADP on glycolysis in the kidneys and liver of rats during ischemia

Experiments on albino rats have shown that kidney ischemia and its simulation by the anaerobic incubation of postmitochondrial kidney homogenate fraction without a substrate induce a considerable damage of the glycolytic system at the stage of the glucoso-6-phosphate transformation into fructoso-1.6-diphosphate and a less pronounced damage in the fructoso-1.6-diphosphate transformation into lactate. Administration of adenosine diphosphate (ADP) and nicotinamide adenine dinucleotide (NAD) to rats before kidney vessel occlusion or their addition to the postmitochondrial fraction before the anaerobic incubation without a substrate decreased a degree of the glycolytic system damage. The damage of the glycolytic system and protective action of NAD are also detected under simulation of liver ischemia. Possible mechanisms of the ischemic damage in the glycolytic liver and kidney tissue system are discussed.     

The Role of Phosphometabolites in Cell Proliferation, Energy Metabolism, and Tumor Therapy

A common characteristic of tumor cells is the constant overexpression of glycolytic and glutaminolytic enzymes. In tumor cells the hyperactive hexokinase and the partly inactive pyruvate kinase lead to an expansion of all phosphometabolites from glucose 6-phosphate to phosphoenolpyruvate. In addition to the glycolytic phosphometabolites, synthesis of their metabolic derivatives such as P-ribose-PP, NADH, NADPH, UTP, CTP, and UDP-N-acetyl glucosamine is also enhanced during cell proliferation. Another phosphometabolite derived from P-ribose-PP, AMP, inhibits cell proliferation. The accumulation of AMP inhibits both P-ribose-PP-synthetase and the increase in concentration of phosphometabolites derived from P-ribose-PP. In cells with low glycerol 3-phosphate and malate-aspartate shuttle capacities the inhibition of the lactate dehydrogenase by low NADH levels leads to an inhibition of glycolytic ATP production. Several tumor-therapeutic drugs reduce NAD and NADH levels, thereby inhibiting glycolytic energy production. The role of AMP, NADH, and NADPH levels in the success of chemotherapeutic treatment is discussed.     

Metabolite and enzyme contents of freeze-clamped liver of the marine fish Stenotomus chrysops

Hepatic metabolites and enzymes in the marine fish, scup or porgy (Stenotomus chrysops), were determined in freeze-clamped tissue taken either within a day of removing fish from their natural habitat or after scup were held in captivity for 6-8 months. The same determinations were made for liver from fed or 48 hr-starved rats (Mus norvegicus albinus). Compared with rat liver, both groups of fish had, per gram of liver, higher contents of AMP, inorganic phosphate, glucose, glucose-6-phosphate, malate, glutamate and NH4+. ATP was lower in fish liver, and ADP, lactate and pyruvate contents were similar in rats and fish. Fish held in captivity had significantly lower pyruvate, alpha-ketoglutarate, and cytosolic free NAD+/NADH and higher cytosolic free NADPH/NADP+. These decreases were similar to those seen when starved rats were compared with fed ones. In scup liver, glucose-6-phosphate dehydrogenase was 3-8 times, malic enzyme about 2 times, and alanine aminotransferase 2-4 times higher than those activities in rat liver. Those results and a higher cytosolic free NADPH/NADP+ are consistent with the liver being the major site of lipogenesis in fish.     

ATP synthesis kinetics and mitochondrial function in the postischemic myocardium as studied by 31P NMR

The effects of ischemia on mitochondrial function and the unidirectional rate of ATP synthesis (Pi----ATP rate) were studied using a Langendorff-perfused heart preparation and 31P NMR spectroscopy. There was significant postischemic depression of mechanical function assessed as the heart rate pressure product, and the myocardial oxygen consumption rate at a given rate pressure product was elevated. Experiments performed on glucose- and pyruvate-perfused hearts demonstrated the presence of a large contribution to the unidirectional Pi----ATP rate catalyzed by glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. This rate was much greater than the maximal glucose utilization rate in the myocardium, demonstrating that the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase reactions are near equilibrium both before and after ischemia. In the pyruvate-perfused postischemic hearts, the glycolytic contribution was eliminated and the net rate of ATP synthesis by oxidative phosphorylation was measurable. Despite the reduced mechanical function and increased myocardial oxygen consumption rate, the ratio of the net rate of ATP synthesis by oxidative phosphorylation to oxygen consumption rate (the P:O ratio) was not altered subsequent to ischemia (2.34 +/- 0.12 and 2.36 +/- 0.09 in normal and postischemic hearts, respectively). Therefore, mitochondrial uncoupling cannot be the cause of postischemic depression in mechanical function; instead, the data suggest the existence of ischemia-induced inefficiency in ATP utilization.     

Inhibition studies of glucose-6-phosphate dehydrogenase from tetracycline-producing Streptomyces aureofaciens

NAD-linked activity of glucose-6-phosphate dehydrogenase from both low-producing and high-producing strains of Streptomyces aureofaciens was inhibited by ATP, ADP, AMP and Pi. The inhibition constants indicate that ADP was the most potent inhibitor. The NADP-linked activity remained unaffected even at relatively high concentrations of these inhibitors. All inhibitions of the NAD-linked activity were competitive with respect to NAD and noncompetitive with respect to glucose-6-phosphate. The results represent a possible new regulatory mechanism of glucose-6-phosphate dehydrogenase from a streptomycete and emphasize its involvement in the regulation of the biosynthesis of tetracyclines.     

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

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

Impaired glucose homeostasis during postimplantation pregnancy in the mouse following acute exposure to ethanol, with particular reference to the uterus and embryo.

Dramatic changes in the levels of plasma glucose and lactate and liver glycogen were observed in mice, given an intraperitoneal injection of ethanol (3.5 g/kg body weight) on Day 9 of pregnancy, during the period of time (6 h) required to clear the drug from the circulatory system. These alterations were accompanied by significant changes in the rates of accumulation of some glycolytic and citric acid cycle intermediates in the uterus, including glucose-6-phosphate, fructose-6-phosphate, lactate, citrate, alpha-ketoglutarate, and succinate. Although the changes in some metabolic parameters were very transient, not all metabolites returned to control values by the time that the drug had been cleared from the maternal system. Alcohol also impaired the capacity of Day 9 mouse embryos to metabolize [14C]glucose under culture conditions in vitro and significantly increased the amount of the aldohexose accumulating in the fetal membrane fluid when administered on Day 14 of pregnancy. However, ethanol neither influenced the ratio of NADH to NAD+ in the uterus nor changed the glycolytic and respiratory activity of the uterine endometrium when coincubated with the tissue in vitro. The results indicate that glucose homeostasis is impaired in both the embryo and the maternal system of mice acutely exposed to alcohol during the teratogenically sensitive period of postimplantation pregnancy and support the thesis that this phenomenon may present an important mechanism underlying the embryo-toxic effects of alcohol consumed under "binge" drinking conditions during pregnancy. However, the results also suggest that the effects registered at the uterine level most likely involve stress reactions and acetate rather than primary actions of the drug on the organ.     

Control of the shift from homolactic acid to mixed-acid fermentation in Lactococcus lactis: predominant role of the NADH/NAD+ ratio.

During batch growth of Lactococcus lactis subsp. lactis NCDO 2118 on various sugars, the shift from homolactic to mixed-acid metabolism was directly dependent on the sugar consumption rate. This orientation of pyruvate metabolism was related to the flux-controlling activity of glyceraldehyde-3-phosphate dehydrogenase under conditions of high glycolytic flux on glucose due to the NADH/NAD+ ratio. The flux limitation at the level of glyceraldehyde-3-phosphate dehydrogenase led to an increase in the pool concentrations of both glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate and inhibition of pyruvate formate lyase activity. Under such conditions, metabolism was homolactic. Lactose and to a lesser extent galactose supported less rapid growth, with a diminished flux through glycolysis, and a lower NADH/NAD+ ratio. Under such conditions, the major pathway bottleneck was most probably at the level of sugar transport rather than glyceraldehyde-3-phosphate dehydrogenase. Consequently, the pool concentrations of phosphorylated glycolytic intermediates upstream of glyceraldehyde-3-phosphate dehydrogenase decreased. However, the intracellular concentration of fructose-1,6-bisphosphate remained sufficiently high to ensure full activation of lactate dehydrogenase and had no in vivo role in controlling pyruvate metabolism, contrary to the generally accepted opinion. Regulation of pyruvate formate lyase activity by triose phosphates was relaxed, and mixed-acid fermentation occurred (no significant production of lactate on lactose) due mostly to the strong inhibition of lactate dehydrogenase by the in vivo NADH/NAD+ ratio.     

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.     

Liver metabolism in cold hypoxia: a comparison of energy metabolism and glycolysis in cold-sensitive and cold-resistant mammals

The effects of cold hypoxia were examined during a time-course at 2 °C on levels of glycolytic metabolites: glycogen, glucose, glucose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, phosphoenolpyruvate, pyruvate, lactate and energetics (ATP, ADP, AMP) of livers from rats and columbian ground squirrels. Responses of adenylate pools reflected the energy imbalance created during cold hypoxia in both rat and ground squirrel liver within minutes of organ isolation. In rat, ATP levels and energy charge values for freshly isolated livers were 2.54 mol·g^-1 and 0.70, respectively. Within 5 min of cold hypoxia, ATP levels had dropped well below control values and by 8 h storage, ATP, AMP, and energy charge values were 0.21 mol·g^-1, 2.01 mol·g^-1, and 0.17, respectively. In columbian ground squirrels the patterns of rapid ATP depletion and AMP accumulation were similar to those found in rat. In rat liver, enzymatic regulatory control of glycolysis appeared to be extremely sensitive to the decline in cellular energy levels. After 8 h cold hypoxia levels of fructose-6-phosphate decreased and fructose-1,6-bisphosphate increased, thus reflecting an activation of glycolysis at the regulatory step catalysed by phospho-fructokinase fructose-1,6-bisphosphatase. Despite an initial increase in flux through glycolysis over the first 2 min (lactate levels increased 3.7 mol·g^-1), further flux through the pathway was not permitted even though glycolysis was activated at the phosphofructokinase/fructose-1,6-bisphosphatase locus at 8 h, since supplies of phosphorylated substrate glucose-1-phosphate or glucose-6-phosphate remained low throughout the duration of the 24-h period. Conversely, livers of Columbian ground squirrels exhibited no activation or inactivation of two key glycolytic regulatory loci, phosphofructokinase/fructose-1,6-bisphosphatase and pyruvate kinase/phosphoenolpyruvate carboxykinase and pyruvate carboxylase. Although previous studies have shown similar allosteric sensitivities to adenylates to rat liver phospho-fructokinase, there was no evidence of an activation of the pathway as a result of decreasing high energy adenylate, ATP or increasing AMP levels. The lack of any apparent regulatory control of glycosis during cold hypoxia may be related to hibernator-specific metabolic adaptations that are key to the survival of hypothermia during natural bouts of hibernation.Abbreviations DHAP dihydroxyacetonephosphate - EC energy charge - F1,6P₂ fructose-1,6-bisphosphate - F2,6P₂ fructose-2,6-bisphosphate - F6P fructose-6-phosphate - FBP fructose-1,6-bisphosphatase - G1P glucose-1-phosphate - G6P glucose-6-phosphate - GAP glyceraldehyde-3-phosphate - GAPDH glyceraldehyde-3-phosphate dehydrogenase - L/R lactobionate/raffinose-based solution - MR metabolic rate - PDH pyruvate dehydrogenase - PEP phosphoenolpyruvate - PEPCK & PC phosphoenolpyruvate carboxykinase and pyruvate carboxylase - PFK phosphofructokinase; PK, pyruvate kinase - Q ₁₀ the effect of a 10 °C drop in temperature on reaction rates (generally, Q ₁₀=2–3) - TA total adenylates - UW solution University of Wisconsin solution (L/R-based)     

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.     

Glucose-6-phosphate dehydrogenase from a tetracycline producing strain of Streptomyces aureofaciens: some properties and regulatory aspects of the enzyme

Glucose-6-phosphate dehydrogenase from Streptomyces aureofaciens exhibited activity with both NAD and NADP, the maximum reaction rate being 1.6 times higher for NAD-linked activity than for the NADP-linked one. The KM values for NAD-linked activity were 2.5 mM for glucose-6-phosphate and 0.27 mM for NAD, and for NADP-linked activity 0.8 mM for glucose-6-phosphate and 0.08 mM for NADP. NAD- and NADP-linked activities were inhibited by both NADH and NADPH. (2'-phospho-)adenosinediphospho-ribose inhibited only NAD-linked activity. The inhibition was competitive with respect to NAD and noncompetitive with respect to glucose-6-phosphate.     

Mode of action of alpha-chlorohydrin as a male anti-fertility agent. Inhibition of the metabolism of ram spermatozoa by alpha-chlorohydrin and location of block in glycolysis

1. The effect of alpha-chlorohydrin on the metabolism of glycolytic and tricarboxylate-cycle substrates by ram spermatozoa was investigated. The utilization and oxidation of fructose and triose phosphate were much more sensitive to inhibition by alpha-chlorohydrin (0.1-1.0mm) than lactate or pyruvate. Inhibition of glycolysis by alpha-chlorohydrin is concluded to be between triose phosphate and pyruvate formation. Oxidation of glycerol was not as severely inhibited as that of the triose phosphate. This unexpected finding can be explained in terms of competition between glycerol and alpha-chlorohydrin. A second, much less sensitive site, of alpha-chlorohydrin inhibition appears to be associated with production of acetyl-CoA from exogenous and endogenous fatty acids. 2. Measurement of the glycolytic intermediates after incubation of spermatozoal suspensions with 15mm-fructose in the presence of 3mm-alpha-chlorohydrin showed a ;block' in the conversion of glyceraldehyde 3-phosphate into 3-phosphoglycerate. alpha-Chlorohydrin also caused conversion of most of the ATP in spermatozoa into AMP. After incubation with 3mm-alpha-chlorohydrin, glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase activities were decreased by approx. 90% and 80% respectively, and in some experiments aldolase was also inhibited. Other glycolytic enzymes were not affected by a low concentration (0.3mm) of alpha-chlorohydrin. Loss of motility of spermatozoa paralleled the decrease in glyceraldehyde 3-phosphate dehydrogenase activity. alpha-Chlorohydrin, however, did not inhibit glyceraldehyde 3-phosphate dehydrogenase or triose phosphate isomerase in sonicated enzyme preparations when added to the assay cuvette. 3. Measurement of intermediates and glycolytic enzymes in ejaculated spermatozoa before, during and after injection of rams with alpha-chlorohydrin (25mg/kg body wt.) confirmed a severe block in glycolysis in vivo at the site of triose phosphate conversion into 3-phosphoglycerate within 24h of the first injection. Glyceraldehyde 3-phosphate dehydrogenase activity was no longer detectable and both aldolase and triose phosphate isomerase were severely inhibited. Spermatozoal ATP decreased by 92% at this time, being quantitatively converted into AMP. At 1 month after injection of alpha-chlorohydrin glycolytic intermediate concentrations returned to normal in the spermatozoa but ATP was still only 38% of the pre-injection concentration. Motility of spermatozoa was, however, as good as during the pre-injection period. The activity of the inhibited enzymes also returned to normal during the recovery period and 26 days after injection were close to pre-injection values. 4. An unknown metabolic product of alpha-chlorohydrin is suggested to inhibit glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase of spermatozoa. This results in a lower ATP content, motility and fertility of the spermatozoa. Glycidol was shown not to be an active intermediate of alpha-chlorohydrin in vitro.     

In vitro effect of extracellular AMP on MCF-7 breast cancer cells: inhibition of glycolysis and cell proliferation.

MCF-7 human breast cancer cells propagated in vitro were treated with adenosine derivatives added to the culture medium. The effects on cell proliferation, glycolysis, and glutaminolysis were investigated. Of all adenosine derivatives tested, AMP was the most efficient inhibitor of cell proliferation. In AMP-treated cells, DNA synthesis decreased, whereas RNA and protein syntheses rose normally with time. In terms of carbohydrate metabolism, lactate production from glucose was drastically reduced; therefore, most of lactate produced must have been derived from glutamine. Increases in the enzyme activities involved in glutamate degradation and in the malate-aspartate shuttle were observed. In contrast, actual glycolytic flux rates declined, whereas key glycolytic enzyme activities increased. Metabolites such as fructose 1,6-bisphosphate and pyruvate accumulated in AMP-arrested cells. Based on the lowered NAD level in the AMP-treated cells, lactate dehydrogenase, but not malate dehydrogenase, was impaired; thereby the whole of glycolysis was inhibited. In compensation, glutamine catabolism was increased. NAD concentrations fell drastically because of the known inhibition of P-ribose-PP synthesis through heightened intracellular AMP levels. A hypothetical metabolic scheme to explain these results and to show how extracellular AMP may influence carbohydrate metabolism and cell proliferation is presented.     

Renal cortical intermediary metabolism in the recovery phase of postischemic acute renal failure in the dog.

Renal metabolism has been studied in eight dogs before and 48 hr after a 60-min period of renal ischemia induced by clamping the left renal artery with the simultaneous removal of the right kidney, and in 12 sham-operated animals. The study involved the measurement of renal uptake and production of lactate, glutamine, glutamate, alanine, ammonium, and oxygen, and the measurement of the tissue concentrations of ATP, glutamine, lactate, alpha-ketoglutarate, aspartate, and alanine in the renal cortex. Two days after a temporary renal ischemia, the remaining kidney showed a 22% decrease in glomerular filtration rate (GFR) and a 25% decrease in renal plasma flow. Fractional sodium and potassium excretions were similar to those of control dogs. Renal production or extraction of glutamine, glutamate, alanine, ammonium, and oxygen (all expressed by 100 ml of GFR) was not significantly different in basal conditions or 2 days after ischemia, but lactate extraction was reduced in postischemic kidneys (-101 +/- 29 vs -204 +/- 38 mumol/100 ml GFR in control dogs). The cortical concentrations of glutamine and glutamate were lower in postischemic than in control kidneys. No differences were found in cortical concentration of alpha-ketoglutarate, aspartate, lactate, pyruvate, or ATP, but total nucleotides and inorganic phosphate were decreased in postischemic kidneys. It is concluded that in the recovery phase of the ischemia, a decreased lactate uptake is the main metabolic change, and total ATP production is adapted to the decrease of GFR and sodium reabsorption.     

Heteromerous interactions among glycolytic enzymes and of glycolytic enzymes with F-actin: effects of poly(ethylene glycol)

Interactions of glucose-6-phosphate isomerase (D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9), aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13), glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12), triose-phosphate isomerase (D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1), phosphoglycerate mutase (D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1), phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.3), enolase (2-phospho-D-glycerate hydro-lyase, EC 4.2.1.11), pyruvate kinase (ATP:Pyruvate O2-phosphotransferase, EC 2.7.1.40) and lactate dehydrogenase [S)-lactate:NAD+ oxidoreductase, EC 1.1.1.27) with F-actin, among the glycolytic enzymes listed above, and with phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) were studied in the presence of poly(ethylene glycol). Both purified rabbit muscle enzymes and rabbit muscle myogen, a high-speed supernatant fraction containing the glycolytic enzymes, were used to study enzyme-F-actin interactions. Following ultracentrifugation, F-actin and poly(ethylene glycol) tended to increase and KCl to decrease the pelleting of enzymes. In general, the greater part of the pelleting occurred in the presence of both F-actin and poly(ethylene glycol) and the absence of KCl. Enzymes that pelleted more in myogen preparations than as individual purified enzymes in the presence of poly(ethylene glycol) and the absence of F-actin were tested for specific enzyme-enzyme associations, several of which were observed. Such interactions support the view that the internal cell structure is composed of proteins that interact with one another to form the microtrabecular lattice.     

Glucose stimulation of heart phosphorylase phosphatase activity in vitro and in vivo

Summary To determine the mechanism of the glucose stimulation, glucose or glucose-6-phospate was added to dilute heart extracts in the presence or absence of AMP. The intracellular glucose, tissue glucose-6-phosphate, and tissue AMP concentrations were also determined in 24-h starved animals given glucose; 24-h starved animals given insulin as well as diabetic starved and diabetic starved insulin-treated animals were also studied.The A_0.5 for glucose stimulation of cardiac phosphorylase phosphatase activity was approximately 1 .2 mM. The A_0.5 for glucose-6-phosphate was approximately 0.02 mM. The glucose-6-phosphate concentration in all animals exceeded the Ao.5 by 10-fold. However, the intracellular glucose concentration in the glucose-treated, insulin-treated, diabetic, and diabetic insulin-treated rats was in the range of the A_0.5 for stimulation of phosphorylase phosphatase activity. AMP completely inhibited phosphorylase phosphatase activity at a concentration of 0.2 mM. Physiological concentrations of glucose and glucose-6-phosphate partially reversed this inhibition. Administration of glucose or insulin resulted in an increase in intracellular glucose concentration, an increase in tissue glucose-6-phosphate and a decrease in tissue AMP concentrations. These data suggest that glucose may be a physiological regulator of phosphorylase phosphatase in heart muscle as it is in liver.Recipient ofaMedical InvestigatorshipAward from theVeterans Administration.     

Combined glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase in catecholamine-stimulated guinea-pig cardiac muscle. Comparison with mass-action ratio of creatine kinase.

The steady-state reactant levels of triose-phosphate isomerase and the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system were examined in guinea-pig cardiac muscle. Key glycolytic intermediates, including glyceraldehyde 3-phosphate were directly measured and compared with those of creatine kinase. Non-working Langendorff hearts as well as isolated working hearts were perfused with 5 mM glucose (plus insulin) under normoxia conditions to maintain lactate dehydrogenase near-equilibrium. The cytosolic phosphorylation potential ([ATP]/([ADP].[Pi])) was derived from creatine kinase and the free [NAD+]/([NADH].[H+]) ratio from lactate dehydrogenase. In Langendorff hearts glycolysis was varied from near-zero flux (hyperkalemic cardiac arrest) to higher than normal flux (normal and maximum catecholamine stimulation). The triose-phosphate isomerase was near-equilibrium only in control or potassium-arrested Langendorff hearts as well as in postischemic 'stunned' hearts. However, when glycolytic flux increased due to norepinephrine or due to physiological pressure-volume work the enzyme was displaced from equilibrium. The alternative phosphorylation ratio [ATP]'/([ADP]).[Pi]) was derived from the magnesium-dependent glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system assigning free magnesium different values in the physiological range (0.1-2.0 mM). As predicted, [ATP]/([ADP].[Pi]) and [ATP]'/([ADP]'.[Pi]') were in excellent agreement when glycolysis was virtually halted by hyperkalemic arrest (flux approximately 0.2 mumol C3.min-1.g dry mass-1). However, the equality between the two phosphorylation ratios was not abolished upon resumption of spontaneous beating and also not during adrenergic stimulation (flux approximately 5-14 mumol C3.min-1.g dry mass-1). In contrast, when flux increased due to transition from no-work to physiological pressure-volume work (rate increase from approximately 3 to 11 mumol C3.min-1.g dry mass-1), the two ratios were markedly different indicating disequilibrium of the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase. Only during adrenergic stimulation or postischemic myocardial 'stunning', not due to hydraulic work load per se, glyceraldehyde-3-phosphate levels increased from about 4 microM to greater than or equal to 16 microM. Thus the guinea-pig cardiac glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system can realize the potential for near-equilibrium catalysis at significant flux provided glyceraldehyde-3-phosphate levels rise, e.g., due to 'stunning' or adrenergic hormones.     

Functional and morphological changes in the kidneys in acute embologenic arterial occlusion of the extremities

The results of biochemical, radioisotope and morphological investigations of dog kidneys in experimental acute occlusion of hind limb arteries during ischemia and in postischemic periods are reviewed. Morphological and functional changes in the kidneys occur in ischemia. Blood flow recovery in the extremities aggravates these changes leading in 12-hour ischemia to acute renal failure.     

Inhibition of Lactate Production in Rat Brain Extracts and Synaptosomes by 3-[4-(Reduced 3-Pyridine Aldehyde-Adenine Dinucleotide)]-Pyruvate

In basic solutions, pyruvate enolizes and reacts (through its 3-carbon) with the 4-carbon of the nicotinamide ring of NAD+, yielding an NAD-pyruvate adduct in which the nicotinamide ring is in the reduced form. This adduct is a strong inhibitor of lactate dehydrogenase, presumably because it binds simultaneously to the NADH and pyruvate sites. The potency of the inhibition, however, is muted by the adduct's tendency to cyclize to a lactam. We prepared solutions of the pyruvate adduct of NAD+ and of NAD+ analogues in which the -C(O)NH2 of NAD+ was replaced with -C(S)NH2, -C(O)CH3, and -C(O)H. Of the four, only the last analogue, 3-[4-(reduced 3-pyridine aldehyde-adenine dinucleotide)]-pyruvate (RAP) cannot cyclize and it was found to be the most potent inhibitor of beef heart and rat brain lactate dehydrogenases. The inhibitor binds very tightly to the NADH site (Ki approximately 1 nM for the A form). Even at high concentrations (20 microM), RAP had little or no effect on rat brain glyceraldehyde-3-phosphate, pyruvate, alpha-ketoglutarate, isocitrate, soluble and mitochondrial malate, and glutamate dehydrogenases. The glycolytic enzymes, hexokinase and phosphofructokinase, were similarly unaffected. RAP strongly inhibited lactate production from glucose in rat brain extracts but was less effective in inhibiting lactate production from glucose in synaptosomes.