Pre-steady-state kinetic studies of Saccharomyces cerevisiae myristoylCoA:protein N-myristoyltransferase mutants identify residues involved in catalysis.

MyristoylCoA:protein N-myristoyltransferase (Nmt, EC 2.3.1.97), a member of the GCN5 acetyltransferase (GNAT) superfamily, is an essential eukaryotic enzyme that catalyzes covalent attachment of myristate (C14:0) to the N-terminal Gly of proteins involved in myriad cellular functions. The 2.5 A resolution structure of a ternary complex of Saccharomyces cerevisiae Nmt1p with a bound substrate peptide (GLYASKLA) and nonhydrolyzable myristoylCoA analogue [Farazi, T. A., et al. (2001) Biochemistry 40, 6335] was used as the basis for a series of mutagenesis experiments designed to define the enzyme's catalytic mechanism. The kinetic properties of an F170A/L171A Nmt mutant are consistent with the proposal that their main chain amides, located in a beta-bulge structure conserved among GNATs, function as an oxyanion hole to polarize the thioester carbonyl of bound myristoylCoA prior to subsequent nucleophilic attack. Removal of the two C-terminal residues (M454 and L455) produces a 300--400-fold reduction in the chemical transformation rate and converts the rate-limiting step from a step after the transformation to the transformation event itself. This finding is consistent with the main chain C-terminal carboxylate of L455 functioning as a catalytic base that abstracts a proton from the N-terminal Gly ammonium of the bound peptide to generate the nucleophilic amine. Mutating N169 and T205 in concert reduces the rate of the chemical transformation, supporting their role as components of an H-bonding network that facilitates attack of the Gly1 amine and stabilizes the tetrahedral intermediate.

Enhanced gluconeogenesis and increased energy storage as hallmarks of aging in Saccharomyces cerevisiae.

A relationship between life span and cellular glucose metabolism has been inferred from genetic manipulations and caloric restriction of model organisms. In this report, we have used the Snf1p glucose-sensing pathway of Saccharomyces cerevisiae to explore the genetic and biochemical linkages between glucose metabolism and aging. Snf1p is a serine/threonine kinase that regulates cellular responses to glucose deprivation. Loss of Snf4p, an activator of Snf1p, extends generational life span whereas loss of Sip2p, a presumed repressor of the kinase, causes an accelerated aging phenotype. An annotated data base of global age-associated changes in gene expression in isogenic wild-type, sip2Delta, and snf4Delta strains was generated from DNA microarray studies. The transcriptional responses suggested that gluconeogenesis and glucose storage increase as wild-type cells age, that this metabolic evolution is exaggerated in rapidly aging sip2Delta cells, and that it is attenuated in longer-lived snf4Delta cells. To test this hypothesis directly, we applied microanalytic biochemical methods to generation-matched cells from each strain and measured the activities of enzymes and concentrations of metabolites in the gluconeogenic, glycolytic, and glyoxylate pathways, as well as glycogen, ATP, and NAD(+). The sensitivity of the assays allowed comprehensive biochemical profiling to be performed using aliquots of the same cell populations employed for the transcriptional profiling. The results provided additional evidence that aging in S. cerevisiae is associated with a shift away from glycolysis and toward gluconeogenesis and energy storage. They also disclosed that this shift is forestalled by two manipulations that extend life span, caloric restriction and genetic attenuation of the normal age-associated increase in Snf1p activity. Together, these findings indicate that Snf1p activation is not only a marker of aging but also a candidate mediator, because a shift toward energy storage over expenditure could impact myriad aspects of cellular maintenance and repair.

Transient-state kinetic analysis of Saccharomyces cerevisiae myristoylCoA:protein N-myristoyltransferase reveals that a step after chemical transformation is rate limiting.

MyristoylCoA:protein N-myristoyltransferase is a member of the superfamily of GCN5-related N-acetyltransferases and catalyzes the covalent attachment of myristate to the N-terminal Gly residue of proteins with diverse functions. Saccharomyces cerevisiae Nmt1p is a monomeric protein with an ordered bi-bi reaction mechanism: myristoylCoA is bound prior to peptide substrate; after catalysis, CoA is released followed by myristoylpeptide. Analysis of the X-ray structure of Nmt1p with bound substrate analogues indicates that the active site contains an oxyanion hole and a catalytic base and that catalysis proceeds through the nucleophilic addition-elimination mechanism. To determine the rate-limiting step in the enzyme reaction, pre-steady-state kinetic analyses were performed using a new, sensitive nonradioactive assay that detects CoA. Multiple turnover quenched flow studies disclosed that a step after the chemical transformation limits the overall rate of the reaction. Multiple and single turnover analyses revealed that the rate for the chemical transformation step is 13.8+/-0.6 s(-1) while the slower steady-state phase is 0.10+/-0.01 s(-1). Stopped flow kinetic studies of substrate acquisition indicated that binding of myristoylCoA to the apo-enzyme occurs through at least a two-step process, with a fast phase rate of 3.2 x 10(8) M(-1) s(-1) and a slow phase rate of 23+/-2 s(-1) (defined at 5 degrees C). Binding of an octapeptide substrate, representing the N-terminal sequence of a known yeast N-myristoylprotein (Cnb1p), to a binary complex composed of Nmt1p and a nonhydrolyzable myristoylCoA analogue (S-(2-oxo)pentadecylCoA) has a second-order rate constant of 2.1+/-0.3 x 10(6) M(-1) s(-1) and a dissociation rate of 26+/-15 s(-1) (defined at 10 degrees C). These results are interpreted in light of the X-ray structures of this enzyme.

Sip2p and its partner snf1p kinase affect aging in S. cerevisiae.

For a number of organisms, the ability to withstand periods of nutrient deprivation correlates directly with lifespan. However, the underlying molecular mechanisms are poorly understood. We show that deletion of the N-myristoylprotein, Sip2p, reduces resistance to nutrient deprivation and shortens lifespan in Saccharomyces cerevisiae. This reduced lifespan is due to accelerated aging, as defined by loss of silencing from telomeres and mating loci, nucleolar fragmentation, and accumulation of extrachromosomal rDNA. Genetic studies indicate that sip2Delta produces its effect on aging by increasing the activity of Snf1p, a serine/threonine kinase involved in regulating global cellular responses to glucose starvation. Biochemical analyses reveal that as yeast age, hexokinase activity increases as does cellular ATP and NAD(+) content. The change in glucose metabolism represents a new correlate of aging in yeast and occurs to a greater degree, and at earlier generational ages in sip2Delta cells. Sip2p and Snf1p provide new molecular links between the regulation of cellular energy utilization and aging.

Effect of centrifugation at 2G for 14 days on metabolic enzymes of the tibialis anterior and soleus muscles.

BACKGROUND: The enzyme composition of different muscle types vary greatly, leading to different changes of enzyme level caused by exposure to various stimuli. METHODS: Male Wistar rats were centrifuged at 2G in a 12-ft radius centrifuge for 14 d. Tibialis anterior (TA) and soleus muscles from four centrifuge and four control rats were analyzed for three enzymes characteristic of fast twitch muscles (phosphofructokinase, glycerol-3-phosphate dehydrogenase, and pyruvate kinase), and four enzymes characteristic of slow twitch muscles (hexokinase, mitochondrial thiolase, B-hydroxyacyl CoA dehydrogenase, and citrate synthase). RESULTS: The centrifuged TA muscles lost 15% of their weight; the corresponding soleus muscles gained 4%. Calculated on the basis of dry weight, the fast twitch enzyme activities were reduced 3-15% in the TA muscles but increased 10-23% in the soleus muscles. The slow twitch enzymes were reduced 18-30% in TA muscles but were almost unchanged in the soleus muscles. When calculated on the basis of total muscle weight, all of the enzymes in TA muscles were significantly reduced by centrifugation. In contrast, in soleus muscles, on the basis of total muscle weight, centrifugation caused an average increase of 22% in the fast twitch enzymes but only marginal changes in the slow twitch enzymes.

Glutamate and potassium stimulation of hippocampal slices metabolizing glucose or glucose and pyruvate.

Using 2-deoxyglucose phosphorylation as an index of glucose use and concentrations of selected intermediates to monitor metabolic pathways, responses of rat hippocampal slices to glutamate and K+ stimulation were examined. With glutamate, the glucose phosphorylation rate (GPR) increased, and the slices accumulated glutamate at a constant rate, for 10 min. The uptake rate at each glutamate level was matched, approximately, by the increase in GPR at that level, with 4 or 5 glutamate molecules accumulated for every glucose molecule phosphorylated. Phosphocreatine and ATP levels fell abruptly, and lactate rose, probably reflecting neuronal activity, found by others to be very brief in the presence of glutamate. K+ stimulation produced responses of phosphocreatine, ATP and lactate levels and of GPR similar to those due to glutamate. There were also prolonged changes in the levels of other metabolites: with both stimulants glucose 6-phosphate fell, and malate rose. The changes in malate may be the result of the participation of mitochondrial malate dehydrogenase in both citrate cycle and malate shuttle. Citrate and alpha-ketoglutarate rose only with K+. When pyruvate was added to the medium, resting GPR was reduced, but for both stimulants the relative increases in GPR with stimulation were the same as without pyruvate. The changes in metabolic intermediates in response to K+ were like those with glucose alone. But with glutamate, the rise in lactate was greatly diminished, and malate fell instead of rising. Glutamate interference with the transfer of both 3-carbon as well as 4- and 5-carbon intermediates from glia to neurons may explain these results. If so, this interference is greater with pyruvate supplementation than with glucose alone.

Attenuation of rat lung isograft reperfusion injury with a combination of anti-ICAM-1 and anti-beta2 integrin monoclonal antibodies.

Four different combinations of monoclonal antibodies against rat ICAM-1, CD-11a, and CD-18 were utilized to determine the relative importance of LFA-1, Mac-1, and ICAM-1 in a rat model of severe lung allograft reperfusion injury. Negative control animals were given phosphate buffered saline (the carrier solution for the antibodies), while positive control animals were rendered neutropenic by the administration of a polyclonal mouse IgG. Antibodies were given with the donor lung flush, prior to left lung graft reperfusion, or both. Isolated graft function was determined 24 hr after implantation by arterial blood gas (ABG), and after sacrifice the native and transplanted lungs underwent bronchoalveolar lavage for alveolar protein quantitation, cell count and differential, and myeloperoxidase assay. Additionally, whole lung homogenates were assayed for myeloperoxidase activity. We found that the combination of anti-ICAM-1 (1 mg/kg) added to the donor lung flush, and anti-CD11a, anti-CD18, and anti-ICAM-1 (2 mg/kg i.v. of each) given to the recipient prior to reperfusion, resulted in significantly improved lung graft pAO2 by ABG, and decreased alveolar protein, cell count, and myeloperoxidase activity compared with control animals. Improvement was less than that seen in the neutropenic recipients, however. We conclude that LFA-1, Mac-1, and ICAM-1 are all important adhesion molecules in lung allograft reperfusion injury--yet even with antibody blockade of all three there are additional mechanisms allowing for neutrophil influx into the lungs.

Changes in ATP, phosphocreatine, and 16 metabolites in muscle stimulated for up to 96 hours.

Rabbit tibialis anterior muscles were stimulated continuously at 10 Hz for periods ranging from 2 min to 96 h and were analyzed for energy reserves and metabolic intermediates. Glycogen, ATP and phosphocreatine fell rapidly during the first 5 min of stimulation. Glycogen continued to fall to very low levels, whereas ATP and phosphocreatine rose, reaching 70% of control by 1 h, despite ongoing stimulation. After 2 h, glycogen also increased, regaining control levels in 4 days. Glucose rose to 4.5 times control in 30 min and still exceeded 2.5 times control at 24 h. In the first 2 min, glycolytic intermediates, glucose 6-phosphate (G-6-P), fructose 1,6-bisphosphate, lactate, and pyruvate more than doubled and then returned to control levels or below. Malate and 3-glycerophosphate rose 600 and 200%, respectively. Both of these compounds participate in shuttling reducing equivalents from cytoplasm into mitochondria. Citrate and alpha-ketoglutarate underwent much more modest changes. Glucose 1,6-bisphosphate (G-1,6-P2) fell to one-third of control by 2 h and then rose dramatically at 4 h. At 4 days it was still twice control. The 6-phosphogluconate (6PG) doubled at 2 min, then rose to 12 times control at 2 h, fell somewhat, and peaked at 16 times control at 24 h. Aspartate and alanine both exhibited a biphasic rise in concentration, whereas glutamate fell to 30% in 15 min and rose slowly after 4 h. The rise in glucose was interpreted to be the consequence of rapid glycogenolysis together with inhibition of hexokinase by G-1,6-P2 and elevated G-6-P. Paradoxically, glycogen resynthesis apparently occurred when the glycogen synthase stimulator, G-6-P, was very low, and the glycolysis stimulator, G-1,6-P2, was high. Although G-1,6-P2 is an inhibitor of 6PG dehydrogenase, the timing of the changes in G-1,6-P2 and 6PG levels suggests that the accumulation of 6PG was initiated by some other influence.

Alterations of intraembryonic metabolites in preimplantation mouse embryos exposed to elevated concentrations of glucose: a metabolic explanation for the developmental retardation seen in preimplantation embryos from diabetic animals.

Preimplantation mouse embryos exposed to hyperglycemia, whether in vivo or in vitro, experience delayed development from the 2-cell to blastocyst stage. By comparing metabolites from embryos exposed to high vs. normal glucose conditions, a metabolic explanation for the delayed growth pattern was sought. Fertilized 1-cell embryos obtained from superovulated B5 x CBA F1 mice were cultured for 96 h in medium containing 2.8 mM glucose (C) or in medium with added glucose to give 10 mM, 30 mM, or 52 mM glucose (HG). After incubation, each embryo was quick-frozen and freeze-dried. Metabolites were assayed by the ultramicrofluorometric technique and enzymatic cycling to obtain measurable levels in single embryos. Embryos cultured in HG exhibited 7-fold higher intracellular glucose levels than those cultured in C (C: 2.25 +/- 0.6 vs. HG: 16.61 +/- 2.4 mmol/kg wet weight; p < 0.001; C, n = 9; HG, n = 16). This accumulation of glucose was dose-related and stage-dependent. Citrate (C: 1.07 +/- 0.14 vs. HG: 1.98 +/- 0.12; p < 0.001), sorbitol (C: 0.41 +/- 0.06 vs. HG: 0.57 +/- 0.03; p < 0.01), malate (C: 0.81 +/- 0.13 vs. HG: 1.72 +/- 0.17; p < 0.001), and fructose (C: 2.1 +/- 0.3 vs. HG: 5.3 +/- 0.6; p < 0.001) were all significantly higher in HG. Also, these metabolites were highest in the most delayed embryos. Glycogen and 6-phosphogluconate levels were not significantly different. In conclusion, intraembryonic levels of glucose, and polyol pathway and Krebs cycle metabolites are elevated and correspond to the degree of developmental delay. These findings suggest that a metabolic abnormality may be responsible for retarded development experienced by embryos exposed to high glucose.

Administration of prostaglandin E1 after lung transplantation improves early graft function.

Early graft dysfunction continues to be a major clinical problem after lung transplantation. The objective of this experiment was to determine whether continuous administration of prostaglandin E1 (PGE1) after lung transplantation has a beneficial effect on early graft function. Left lung allotransplantation was performed in 10 size-matched mongrel dogs (weight, 24.4 to 31.4 kg). Lung preservation consisted of a bolus injection of PGE1 (250 micrograms) into the pulmonary artery, followed by a pulmonary artery flush with 50 mL/kg of 4 degrees C modified Euro-Collins solution. The lungs were then stored at 1 degree C for 12 hours. Left lung transplantation was performed using standard technique. The right pulmonary artery and right bronchus were ligated prior to chest closure. Animals were placed in the supine position and ventilated for 6 hours with 100% oxygen at a rate of 20 breaths/min, a tidal volume of 550 mL, and a positive end-expiratory pressure of 5 cm H2O. Animals were randomly allocated to one of two groups. Group I animals (n = 6) received continuous PGE1 infusion from the onset of implantation. The dose was gradually increased and fixed when mean systemic pressure showed a 10% decrease (mean PGE1 dose, 31.7 +/- 6.9 ng.kg-1.min-1). Group II animals (n = 4) received no PGE1. After the 6-hour assessment period, arterial oxygen tension and alveolar-arterial oxygen pressure difference were preserved in group I compared with group II (group I versus group II: arterial oxygen tension, 255.8 +/- 37.6 mm Hg versus 64.7 +/- 7.9 mm Hg [p < 0.05]; alveolar-arterial oxygen pressure difference, 411.1 +/- 70.5 mm Hg versus 597.5 +/- 1.3 mm Hg [p < 0.05]).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of tetrodotoxin-induced neural inactivation on single muscle fiber metabolic enzymes.

Selected enzymes were measured in mixed-fiber bundles and individual fibers from rat plantaris (PL) and soleus (Sol) muscles that had undergone either 2 wk of tetrodotoxin (TTX) inactivation of the sciatic nerve, a sham operation, or were contralateral to the TTX limb. TTX disuse caused severe wasting of PL (46%) and Sol (26%) muscles and of single fibers (50% and 40%, respectively). TTX PL and Sol also had reduced (50%) glycogen content. In TTX, PL, and Sol macro samples and single fibers, the activities (mol.h-1.kg dry wt-1) of hexokinase, glycogen phosphorylase, and lactate dehydrogenase were higher, lower, and unchanged, respectively, compared with controls. Single-fiber data showed that these changes occurred in all fibers. In TTX PL macro samples, activities of glycerol-3-phosphate dehydrogenase (GPDH), pyruvate kinase (PK), malate dehydrogenase (MDH), citrate synthase (CS), beta-hydroxyacyl-CoA dehydrogenase (BOAC), and thiolase were, or tended to be, lower. Single-fiber data showed a disappearance of high-oxidative moderate glycolytic fibers (i.e., usually fast-twitch oxidative in control) and the appearance of more fibers with a metabolic enzyme profile approaching that of control slow-oxidative fibers. In TTX Sol macro samples, GPDH and PK tended to be higher, and thiolase, BOAC, CS, and MDH lower. Single-fiber data corroborated these findings and suggested the appearance of fast fibers with downregulated oxidative enzyme profiles. Our results suggest that neuromuscular activity is a major, but not the sole, determinant of the size and metabolic heterogeneity that exists in muscle cells.

An unusual active hexose transport system in human and mouse preimplantation embryos.

In a metabolic study of human and mouse preimplantation embryos (preembryos), we measured glucose uptake and phosphorylation with nonradioactive 2-deoxyglucose (DG) as tracer. Initial experiments indicated an active hexose transport capacity, a property thought to be restricted in mammals to intestinal villi and kidney tubules [Baly, D. L. & Horuk, R. (1988) Biochim. Biophys. Acta 947, 571-590]. Significant findings are as follows: (i) During a 60-min incubation with a low level of DG, mouse blastocyst DG rose to levels up to 30 times that of the medium. (The intestinal active system does not transport DG [Crane, R. K. (1960) Physiol. Rev. 40, 789-825].) (ii) Active preembryo transport was not blocked (as it would have been in the intestine) by phlorizin [Alvarado, F. & Crane, R. K. (1962) Biochem. Biophys. Acta 56, 170-172 and Sacktor, B. (1989) Kidney Int. 36, 342-350] or by replacement of Na+ with choline+ or K+ [Crane (1960) and Sacktor (1989)]. (iii) Transport of DG was blocked by cytochalasin B (which is not true for the intestinal transporter). We conclude that a distinct active hexose transporter and at least one facilitated transporter are present in preembryos, perhaps appearing in tandem on different membranes during formation of the increasingly complex preembryo structure.

Changes in alveolar oxygen and carbon dioxide concentration and oxygen consumption during lung preservation. The maintenance of aerobic metabolism during lung preservation.

The lung is the only organ to which oxygen may be supplied after its blood supply is stopped. Before this study, we were not certain whether lung cells were able to maintain aerobic metabolism with the oxygen in the alveoli during preservation. Excised rabbit lungs were used to measure changes in the concentration of oxygen and carbon dioxide in the airway and changes in glucose, glucose-6-phosphate, lactate, adenosine triphosphate, and phosphocreatine levels in the lung tissue during preservation under different conditions. Twenty-seven lungs were flushed with low-potassium dextran electrolyte solution, inflated with room air, and preserved at 1 degree C (n = 8), 10 degrees C (n = 8), or 22 degrees (n = 11) for 4, 12, or 24 hours. Eight additional lungs were inflated with 100% nitrogen and preserved at 10 degrees C for 4 (n = 4) or 24 (n = 4) hours. Oxygen levels decreased and carbon dioxide levels increased in the airway of the lungs that were inflated with room air at rates dependent on the preservation temperature. The increase of carbon dioxide in the lungs that were inflated with 100% nitrogen was very small. When the oxygen was not available in the alveoli, lactate accumulated, and adenosine triphosphate and phosphocreatine decreased in the lung tissue. We concluded that lung cells are able to maintain aerobic metabolism with the oxygen in the alveoli during preservation and that the maintenance of aerobic metabolism may be essential to maintain the optimum viability of preserved lung tissue.

A refinement of the Akabayashi-Saito-Kato modification of the enzymatic methods for 2-deoxyglucose and 2-deoxyglucose 6-phosphate.

Akabayashi et al. made a valuable modification of the enzymatic methods from our laboratory for measuring 2-deoxyglucose and 2-deoxyglucose 6-phosphate. Their modified procedure eliminates glucose and glucose 6-phosphate by conversion to fructose-1,6-bisphosphate, thereby saving two analytical steps. However, the present report describes a limitation of this new elimination procedure which is due to its unexpected reversibility, and provides an easy way to circumvent this limitation, namely heating to destroy the reagent enzymes before proceeding. The final result is a more flexible analytical scheme that is capable of measuring 2-deoxyglucose and its phosphate down to extremely low levels in the presence of up to thousandfold higher glucose concentrations. The completeness of glucose elimination eliminates both the problem of contamination of available glucose-6-phosphate dehydrogenases with 6-phosphogluconate dehydrogenase, and also the effect of the presence in this same enzyme of a trace of glucose dehydrogenase activity, which is an apparent side reaction.

Evaluation of lung metabolism during successful twenty-four-hour canine lung preservation.

We used a canine left lung allotransplantation model to evaluate 24-hour lung preservation with two different electrolyte solutions, low-potassium dextran and low-potassium dextran with 1% glucose. To investigate changes in the energy status during preservation, we analyzed the lungs for adenosine triphosphate, phosphocreatine, and several metabolites of the glycolysis pathway and the citric acid cycle: glucose, glucose-6-phosphate, lactate, citrate, and malate. We also devised and evaluated a pulmonary cooling jacket to prevent rewarming of the lung during implantation. The lungs were divided into four groups. Groups I (n = 10) and II (n = 6) were flushed with low-potassium dextran and groups III (n = 6) and IV (n = 6) were flushed with low-potassium dextran solution with 1% glucose. The cooling jacket was used for groups II and IV only. After 24-hour preservation at 10 degrees C, the left lungs were implanted into the recipient animals. Function of the transplanted left lung was assessed during temporary (10 minutes) occlusion of the contralateral pulmonary artery while both lungs were ventilated with 100% oxygen. This assessment was performed at 1 hour and at 3, 8, and 22 days after transplantation. Immediately after transplantation the arterial oxygen tension was 279 +/- 70 mm Hg in group I, 376 +/- 56 mm Hg in group II, 523 +/- 41 mm Hg in group III, and 518 +/- 50 mm Hg in group IV. The arterial oxygen tension in groups III and IV were significantly greater than in group I (p < 0.05). Of the lungs preserved with low-potassium dextran solution with 1% glucose solution, 11 of 12 (92%) showed excellent lung function (arterial oxygen tension > 300 mm Hg) at 3 days; only 10 of 16 lungs preserved with low-potassium dextran achieved this level of function. Glucose, glucose-6-phosphate, lactate, citrate and malate levels decreased significantly during 24-hour preservation with low-potassium dextran solution; they were stable with low-potassium dextran solution with 1% glucose. Adenosine triphosphate and phosphocreatine were stable for 24 hours with both low-potassium dextran and low-potassium dextran solution with 1% glucose. The cooling jacket provided uniform cooling of the lung parenchyma during implantation, and significant increase in temperature was observed in its absence, with topical cooling by cold saline solution.(ABSTRACT TRUNCATED AT 400 WORDS)

Glucose metabolism assessed with 2-deoxyglucose and the effect of glutamate in subdivisions of rat hippocampal slices.

A new approach to the study of glucose phosphorylation in brain slices is described. It is based on timed incubation with nonradioactive 2-deoxyglucose (DG), after which the tissue levels of DG and 2-deoxyglucose-6-phosphate (DG6P) are measured separately with sensitive enzymatic methods applied to specific small subregions. The smallest samples had dry weights of approximately 0.5 microgram. Direct measurements in different regions of hippocampal slices showed that within 6 min after exposure to DG, the ratios of DG to glucose in the tissue were almost the same as in the incubation medium, which simplifies the calculation of glucose phosphorylation rates and increases their reliability. Data are given for ATP, phosphocreatine, sucrose space, and K+ in specific subregions of the slices. DG6P accumulation proceeded at a constant rate for at least 10 min, even when stimulated by 10 mM glutamate in the medium. The calculated control rate of glucose phosphorylation was 2 mmol/kg (dry weight)/min. In the presence of 10 mM glutamate it was twice as great. The response to 10 mM glutamate of different regions of the slice was not uniform, ranging from 164% of control values in the molecular layer of CA1 to 256% in the stratum radiatum of CA1. There was a profound fall in phosphocreatine levels (75%) in response to 10 mM glutamate despite a 2.4-fold increase in glucose phosphorylation. Even in the presence of 1 mM glutamate, the increase in glucose phosphorylation (50%) was not great enough to prevent a significant drop in phosphocreatine content.

Effects of microgravity and tail suspension on enzymes of individual soleus and tibialis anterior fibers.

Selected enzymes of energy metabolism were measured in random individual fibers of soleus and tibialis anterior (TA) muscles from rats exposed for 2 wk to spaceflight (F) aboard COSMOS 2044 or tail suspension (T) and from synchronous controls. Average size of soleus fibers (dry weight per unit length) was reduced 37% in F and T fibers; there was little change in TA fibers. Enzyme changes were more pronounced in soleus than in TA fibers. Three enzymes characteristic of fast-twitch muscles, pyruvate kinase, glycerol-3-phosphate dehydrogenase, and 1-phosphofructokinase, were elevated in F and T soleus fibers, but changes in phosphofructokinase were not statistically significant. 3-Ketoacid-CoA transferase, characteristic of slow-twitch muscles, did not change significantly in either F or T fibers. Hexokinase, usually moderately higher in slow- than in fast-twitch muscles, increased markedly in both F and T fibers. In TA fibers analyzed for hexokinase, malate dehydrogenase, phosphohexoisomerase, and pyruvate kinase, only hexokinase and malate dehydrogenase showed significant changes. Hexokinase increased 83% in one of two T muscles. Enzyme data for TA fibers typed by myosin adenosinetriphosphatase were more informative: phosphofructokinase, phosphorylase, and glycerol-3-phosphate dehydrogenase were increased in type IIb fibers of either F or T muscles or both. Malate dehydrogenase was not changed in fibers of any type in either F or T muscle.

Sepsis does not impair tricarboxylic acid cycle in the heart.

Sepsis has been reported to cause mitochondrial dysfunction and inhibition of key enzymes that regulate the tricarboxylic acid (TCA) cycle. We investigated the effect of sepsis on high-energy phosphates, glycolytic and TCA cycle intermediates, and specific amino acids that are involved in regulating the size of the TCA cycle pool during changes in metabolic state of the heart. Sepsis was induced in 12 female rats by the cecal ligation and perforation technique under halothane anesthesia; seven control rats underwent cecal manipulation without ligation. At 36-42 h postsurgery, the rats were reanesthetized, the chest was opened, and the hearts were freeze-clamped. Perchloric acid extracts of the hearts were analyzed with fluorometric enzymatic methods and 31P nuclear magnetic resonance spectroscopy. There were no significant differences in the levels of the TCA cycle intermediates or high-energy phosphates between the septic and control rats. The major metabolic changes were the 28% decrease in alanine and the 31% decrease in glutamate in the septic hearts compared with control (P less than 0.05 and P less than 0.005, respectively). Phosphocholine, a component of membrane phospholipids, was increased by 91% in the septic hearts (P less than 0.01). We conclude that sepsis does not impair the TCA cycle or induce significant cellular ischemia in the heart. The increase in phosphocholine may represent significant cellular membrane disruption during sepsis.

Changes in intracellular pH caused by high K in normal and acidified frog muscle. Relation to metabolic changes.

We examined the effect of depolarization on intracellular pH (pHi) of normal (pHi approximately 7.37) and acidified (pHi 5.90-6.70) frog semitendinosus muscle using microelectrodes. A small bundle was superfused with a Na(+)-free buffered solution (10 mM HEPES, 100% O2, pH 7.35) containing either 2.5 or 25 mM K+. An NH4Cl prepulse was used to lower pHi. At normal pHi, depolarization usually produced a slight (0.04) alkalinization, followed by a fall in pHi of approximately 0.2. In contrast, in all 25 acidified bundles pHi rose by 0.1-0.7. The rise was greater the lower the initial pHi. It could be imitated by caffeine and blocked by tetracaine and thus was, most likely, initiated by release of calcium. We ascribed the alkalinization to hydrolysis of phosphocreatine (PCr); 2,4-dinitrofluorobenzene abolished it. Biochemical analysis on fibers at the peak of alkalinization showed PCr to be reduced by one-half, while PCr in normal fibers that had been depolarized for the same period (4-6 min) showed no change. We postulated that low pHi slows glycolysis with its associated ATP formation by reducing glycogenolysis and particularly by reducing conversion of fructose-6-phosphate to fructose-1,6-diphosphate through inhibition of phosphofructokinase (PFK), an enzyme which is known to be highly pH sensitive. Thus PCr hydrolysis would be required to replace much of the hydrolyzed ATP. This postulated effect on PFK is in agreement with the finding that glucose-6-phosphate (in near-equilibrium with fructose-6-phosphate) was increased nearly fivefold in the depolarized acid fibers, but not in the depolarized normal fibers. However, fructose-1,6-diphosphate also increased significantly; 3-phosphoglycerate was not affected. This suggests an additional acid-induced bottleneck between the latter two substrates. We measured the intrinsic buffering power, beta, of frog semitendinosus muscle with small pulses of NH4Cl. It was found to vary with pHi according to beta = 144.6 - 17.2 (pHi).

Measurement of 2-deoxyglucose and 2-deoxyglucose 6-phosphate in tissues.

The enzymatic methods previously described for 2-deoxyglucose (DG) and 2-deoxyglucose 6-phosphate have been refined and adapted to measurements of brain samples ranging from 50 mg wet weight to less than a microgram dry weight. Procedures for preparing such samples for assay are described. Analytical properties of the enzymes employed are given together with means for overcoming their possible short comings. Emphasis is placed on information useful for employing DG to assess rapid changes in glucose metabolism.