Environmental Anoxia Research Articles

Low Glycolysis Is Neuroprotective during Anoxic Spreading Depolarization (SD) and Reoxygenation in Locusts.

Migratory locusts enter a reversible hypometabolic coma to survive environmental anoxia, wherein the cessation of CNS activity is driven by spreading depolarization (SD). While glycolysis is recognized as a crucial anaerobic energy source contributing to animal anoxia tolerance, its influence on the anoxic SD trajectory and recovery outcomes remains poorly understood. We investigated the effects of varying glycolytic capacity on adult female locust anoxic SD parameters, using glucose or the glycolytic inhibitors 2-deoxy-d-glucose (2DG) or monosodium iodoacetate (MIA). Surprisingly, 2DG treatment shared similarities with glucose yet had opposite effects compared with MIA. Specifically, although SD onset was not affected, both glucose and 2DG expedited the recovery of CNS electrical activity during reoxygenation, whereas MIA delayed it. Additionally, glucose and MIA, but not 2DG, increased tissue damage and neural cell death following anoxia-reoxygenation. Notably, glucose-induced injuries were associated with heightened CO2 output during the early phase of reoxygenation. Conversely, 2DG resulted in a bimodal response, initially dampening CO2 output and gradually increasing it throughout the recovery period. Given the discrepancies between effects of 2DG and MIA, the current results require cautious interpretations. Nonetheless, our findings present evidence that glycolysis is not a critical metabolic component in either anoxic SD onset or recovery and that heightened glycolysis during reoxygenation may exacerbate CNS injuries. Furthermore, we suggest that locust anoxic recovery is not solely dependent on energy availability, and the regulation of metabolic flux during early reoxygenation may constitute a strategy to mitigate damage.

Purification and Regulation of Pyruvate Kinase from the Foot Muscle of the Anoxia and Freeze Tolerant Marine Snail, Littorina littorea.

The intertidal marine snail, Littorina littorea, has evolved to survive bouts of anoxia and extracellular freezing brought about by changing tides and subsequent exposure to harsh environmental conditions. Survival in these anoxic conditions depends on the animals entering a state of metabolic rate depression in order to maintain an appropriate energy production-consumption balance during periods of limited oxygen availability. This study investigated the kinetic, physical, and regulatory properties of pyruvate kinase (PK), which catalyzes the final reaction of aerobic glycolysis, from foot muscle of L. littorea to determine if the enzyme is differentially regulated in response to anoxia and freezing exposure. PK purified from foot muscle of anoxic animals exhibited a lower affinity for its substrate phosphoenolpyruvate than PK from control and frozen animals. PK from anoxic animals was also more sensitive to a number of allosteric regulators, including alanine and aspartate, which are key anaerobic metabolites in L. littorea. Furthermore, PK purified from anoxic and frozen animals exhibited greater stability compared to the non-stressed control animals, determined through high-temperature incubation studies. Phosphorylation of threonine and tyrosine residues was also assessed and demonstrated that levels of threonine phosphorylation of PK from anoxic animals were significantly higher than those of PK from control and frozen animals, suggesting a potential mechanism for regulating PK activity. Taken together, these results suggest that PK plays a role in suppressing metabolic rate in these animals during environmental anoxia exposure.

Navigating oxygen deprivation: liver transcriptomic responses of the red eared slider turtle to environmental anoxia.

The best facultative anaerobes among vertebrates are members of the genera Trachemys (pond slider turtles) and Chrysemys (painted turtles), and are able to survive without oxygen for up to 12 to 18 weeks at ∼3 °C. In this study, we utilized RNAseq to profile the transcriptomic changes that take place in response to 20 hrs of anoxia at 5 °C in the liver of the red eared slide turtle (Trachemys scripta elegans). Sequencing reads were obtained from at least 18,169 different genes and represented a minimum 49x coverage of the C. picta bellii exome. A total of 3,105 genes showed statistically significant changes in gene expression between the two animal groups, of which 971 also exhibited a fold change equal to or greater than 50% of control normoxic values. This study also highlights a number of anoxia-responsive molecular pathways that are may be important to navigating anoxia survival. These pathways were enriched in mRNA found to significantly increase in response to anoxia and included molecular processes such as DNA damage repair and metabolic reprogramming. For example, our results indicate that the anoxic turtle may utilize succinate metabolism to yield a molecule of GTP in addition to the two molecules that results from lactate production, and agrees with other established models of anoxia tolerance. Collectively, our analysis provides a snapshot of the molecular landscape of the anoxic turtle and may provide hints into the how this animal is capable of surviving this extreme environmental stress.

Sieved Transport and Redistribution of Bioavailable Phosphorus from Watershed with Complex River Networks to Lake.

An innovative approach was developed to reveal phosphorus (P) transport and redistribution in large and complex river networks in the Lake Taihu basin by establishing the relations between sediment P spatial distribution and P sorption behavior on particles with different grain size, sorted by hydrodynamics. The sediment P fractionation composition changed greatly across the basin, where 69% consisted of acid-soluble fractions (HCl-P) in upstream rivers while 70% was in fractions associated with reducible metal hydroxides (BD-P) and amorphous hydroxides (NaOH25-P) in downstream rivers. Fine particles enriched in BD-P and NaOH25-P fractions tended to sorb liberated P during the resuspension process, and fine particles were more easily delivered downstream toward the lake, forming a sieved transport of P in the river networks. This will lead to a great potential for sediment P release when environmental anoxia develops in the sediments or high pH occurs in the sediment surface during intensive algal blooms in the shallow lake. Reduction of external P from point sources from urbanized areas is an important requirement at the basin scale; however, long-term efforts are needed to restore aquatic macrophytes in the lake, which would decrease P recycling rates at the water-sediment interface.

Postnatal loss of brainstem serotonin neurones compromises the ability of neonatal rats to survive episodic severe hypoxia

Pet-1(-/-) mice with a prenatal, genetically induced loss of 5-hydroxytryptamine (5-HT, serotonin) neurones are compromised in their ability to withstand episodic environmental anoxia via autoresuscitation. Given the prenatal role of 5-HT neurones in the development of neural networks, here we ask if a postnatal loss of 5-HT neurones also compromises autoresuscitation. We treated neonatal rat pups at postnatal day (P)2-3 with an intra-cisternal injection of 5,7-dihydroxytryptamine (5,7-DHT; ~40 μg; n = 8) to pharmacologically lesion the 5-HT system, or vehicle (control; n = 14). At P7-10 we exposed unanaesthetized treated and control pups to 15 episodes of environmental anoxia (97% N(2), 3% CO(2)). Medullary 5-HT content was reduced 80% by 5,7-DHT treatment (P < 0.001). Baseline ventilation (V(E)), metabolic rate (V(O(2))), ventilatory equivalent (V(E)/V(O(2))), heart rate (HR), heart rate variability (HRV) and arterial haemoglobin saturation (S(aO(2))) were no different in 5-HT-deficient pups compared to controls. However, only 25% of 5-HT-deficient pups survived all 15 episodes of environmental anoxia, compared to 79% of control littermates (P = 0.007). High mortality of 5,7-DHT-treated pups was associated with delayed onset of gasping (P < 0.001), delayed recovery of HR from hypoxic-induced bradycardia (P < 0.001), and delayed recovery of eupnoea from hypoxic-induced apnoea (P < 0.001). Treatment with 5,7-DHT affected neither the gasping pattern once initiated, nor HR, V(E)/V(O(2)) or S(aO(2)) during the intervening episodes of room air. A significant increase in HRV occurred in all animals with repeated exposure, and in 5-HT-deficient pups this increase occurred immediately prior to death. We conclude that a postnatal loss of brainstem 5-HT content compromises autoresuscitation in response to environmental anoxia. This report provides new evidence in rat pups that 5-HT neurones serve a physiological role in autoresuscitation. Our data may be relevant to understanding the aetiology of the sudden infant death syndrome (SIDS), in which there is medullary 5-HT deficiency and in some cases evidence of severe hypoxia and failed autoresuscitation.

Effects of environmental anoxia and re-oxygenation on mitochondrial function and metabolism of eastern oysters(Crassostrea virginica)

Effects of environmental anoxia and re-oxygenation on mitochondrial function and metabolism of eastern oysters(Crassostrea virginica)

Extreme Tolerance to Anoxia in the Lucinoma Aequizonata Symbiosis

Our study describes the extraordinary capability of the endosymbiont-bearing bivalve Lucinoma aequizonata to tolerate environmental anoxia. The clam survives without oxygen for 262 days (50% mortality). The total quantity of glycogen in a specimen does not decrease significantly after long-term anoxia (10.5 mo). Common glycogen-derived anaerobic products (opines, lactate, succinate, acetate, and propionate) are only produced in minor quantities. This indicates either severe metabolic depression or the utilization of alternative energy sources. We have found indications that the endosymbiotic bacteria might function as an important carbon source for the bivalve. Transmission electron microscopy studies showed that the symbionts are largely degraded after L. aequizonata was incubated anoxically for 10.5 mo. Polyphosphates detected in symbiont granules by energy dispersive X-ray spectrometry (EDX) might represent an alternative energy source for the clam's metabolism under this stress situation.

Effects of environmental anoxia and different periods of reoxygenation on oxidative balance in gills of the estuarine crab Chasmagnathus granulata

We investigated the effects of anoxia (8 h) and different periods of reoxygenation (20 and 40 min) on the oxidative balance in anterior and posterior gills of the crab Chasmagnathus granulata. Enzyme activity of catalase and GST was increased in the gills of the animals submitted to anoxia, and SOD activity was decreased. These enzymes returned approximately to control levels during the anoxia recovery time. These results demonstrated enzyme activities change with variations in environmental oxygen levels. The posterior gills showed a higher antioxidant enzyme activity than anterior gills. In the gills, there were no changes in the non-enzymatic antioxidant system (TRAP) during anoxia. On the other hand, during anoxia recovery, an increase of TRAP in both gills was observed. Anoxia does not change lipid peroxidation (TBARS) in the gills. During anoxia recuperation, an increase in levels of TBARS was observed. Thus the results demonstrate that C. granulata has a similar strategy of preparation for oxidative stress as observed in other intertidal species, enabling the crabs to survive in an environment with extreme variations in physical and chemical characteristics, such as salt marshes.

Can the End Products of Anaerobic Metabolism, Tauropine and D-lactate, Be Used As Metabolic Stress Indicators During Transport of Live South African Abalone Haliotis Midae?

This study was undertaken to investigate whether the accumulation of end products of anaerobic metabolism can be used as an early indicator of deteriorating conditions during transport of live abalone Haliotis midae. A first series of experiments revealed that the enzyme tauropine dehydrogenase, responsible for the production of tauropine, is present in high activities (54 U g wet weight−1) in the shell adductor muscle, but D-lactate dehydrogenase, responsible for the production of D-lactate, is the predominantly active enzyme (10 U g wet weight−1) in foot muscle. The next series of experiments investigated the potential of anaerobic metabolism in the abalone by subjecting the gastropod to either functional anoxia (exercise metabolism) or 6 h of environmental anoxia (seawater gassed with nitrogen). Exercise, primarily powered by the shell adductor muscle, was mainly fueled by glycolysis resulting in the production of tauropine, whereas during 6 h of experimental anoxia, fermentation of glycogen led to the formation of mainly tauropine in the shell adductor muscle and mainly D-lactate in the foot muscle. The last experiment, investigating changes in these metabolites during simulated (abalone packed in oxygen-filled plastic bags resting on foam sponges soaked in seawater) transportation stress of up to 36 h at 7 and 10°C, clearly showed that tauropine accumulation in the shell adductor muscle and D-lactate accumulation in the foot muscle is time-dependent. Both metabolites are already produced during the first 6 h of simulated transportation (especially at 10°C), indicating that aerobic metabolism is impaired at an early stage of transportation. Hence, these metabolites can serve as indicators of the conditions abalone were subjected to during transport. Furthermore, abalone use the strategy of metabolic depression in this simulation experiment, as indicated by the decreased glycolytic flux in various tissues.

Oxygen, pHi and arrest of biosynthesis in brine shrimp embryos.

Embryos of the brine shrimp Artemia franciscana are able to withstand bouts of environmental anoxia for several years by entering a quiescent state, during which time metabolism is greatly depressed. Within minutes of oxygen removal, intracellular pH (pHi) drops at least 1.0 unit. This acidification has been strongly implicated in the arrest of both catabolic and anabolic processes in the cytoplasm. A global arrest of cytoplasmic translation accompanies the transition into anoxia or into aerobic acidosis (artificial quiescence imposed by intracellular acidification with CO2 in the presence of oxygen). Similarly, protein synthesis in isolated mitochondria from these embryos is also reduced markedly in response to acidic pH (80% reduction) or anoxia (79% reduction). The constancy of mRNA levels during quiescence indicates that protein synthesis is likely to be controlled at the translational level. Mitochondrial matrix pH is 8.2 during protein synthesis assays performed at the extramitochondrial pH optimum of 7.5. When this proton gradient is abolished with the K+/H+ ionophore nigericin, the extramitochondrial pH optimum for protein synthesis displays an alkaline shift of approximately 0.7 pH unit. These data suggest the presence of proton-sensitive translational components within the mitochondrion. The oxygen dependency of mitochondrial protein synthesis is not explained simply by blockage of the electron transport chain or by the increased redox state. Whereas oxygen deprivation substantially depresses protein synthesis by 77% after 1 h, normoxic incubations with saturating concentrations of cyanide or antimycin A have only a modest effect (36% reduction, cyanide; 20%, antimycin A). This cyanide- and antimycin-insensitive, but hypoxia-sensitive, inhibitory signature for the arrest of protein synthesis suggests the presence of a molecular oxygen sensor within the mitochondrion.

Profiles of nuclear and mitochondrial encoded mRNAs in developing and quiescent embryos of Artemia franciscana.

Embryos of the brine shrimp Artemia franciscana are able to withstand long bouts of environmental anoxia by entering a quiescent state during which metabolism is greatly depressed. Recent evidence supports a global arrest of protein synthesis during quiescence. In this study we measured the amounts of mRNA for a mitochondrial-encoded subunit of cytochrome c oxidase (COX I) and for nuclear-encoded actin during aerobic development, anaerobiosis, and aerobic acidosis (artificial quiescence imposed by intracellular acidification under aerobic conditions). The levels of both COX I and actin transcripts increased significantly during aerobic development. COX I mRNA levels were tightly correlated with previous measures of COX catalytic activity, which suggests that COX synthesis could be regulated by message concentration during aerobic development. The ontogenetic increase for these mRNAs was blocked by anoxia and aerobic acidosis. Importantly, the levels of COX I and actin mRNA did not decline appreciably during the 6 h bouts of quiescence, even though protein synthesis is acutely arrested by these same treatments. Thus, the constancy of mRNA levels during quiescence indicate that reduced protein synthesis is not caused by message limitation, but rather, is likely controlled at the translational level. One advantage of this regulatory mechanism is the conservation of mRNA molecules during quiescence, which would potentially favor a quick resumption of translation as soon as oxygen is returned to the embryos. Finally, because anoxia and aerobic acidosis are both characterized by acidic intracellular pH, the reduction in pH may serve, directly or indirectly, as one signal regulating levels of mRNA in this embryo during quiescence.

Anoxic brain failure in an ectothermic vertebrate: release of amino acids and K+ in rainbow trout thalamus.

The release of excitatory amino acids such as glutamate contributes greatly to anoxic and/or ischemic brain damage in mammals. However, for anoxia-intolerant ectothermic vertebrates, there has been no information on how anoxia affects extracellular amino acid levels, or how such changes relate temporally to major ion movements. We have investigated the effects of environmental anoxia on extracellular amino acid and K+ concentrations in rainbow trout thalamus in vivo at 15 degrees C, using microdialysis and K(+)-selective microelectrodes. Systemic blood pressure was also monitored. In separate experiments, endogenous neurotransmitter release was provoked by perfusing the microdialysis probe with a high-K+ Ringer solution, thereby establishing which amino acids are released by depolarization. Anoxia exposure resulted in the release of several amino acids, including glutamate, aspartate, gamma-aminobutyric acid (GABA), glycine, and taurine. GABA release appeared to be delayed compared with that of glutamate, for example. The loss of ion homeostasis (starting after 23 min) preceded the release of amino acids (starting after > or = 45 min). The amino acid release had no apparent effect on the rate of increase in extracellular K+. Thus, if these events are interrelated, the loss of ion homeostasis is likely to trigger the amino acid release but not vice versa.

Metabolism of 1-14C-Acetate and 1-14C-Leucine by Anoxic Goldfish (Carasslus auratus,L.): Evidence for Anaerobic Lipid Synthesis

Goldfish (Carassius auratus), acclimated to 20°C and a Po₂ of 16 KPa, were subjected to environmental anoxia for a total period of 10.5 h. To determine the presence of anaerobic fatty acid synthesis, the animals were injected intraperitoneally with $1-^{14}C-acetate$ after 3.5 h of anoxia. The excretion of radiolabeled ethanol and CO₂ was determined in samples of the surrounding water. The results show that, during 7 h of anoxia, 55% of the injected $1-^{14}C-acetate$ was excreted as $^{14}C-ethanol$, and 2.5% was excreted as $^{14}CO_{2}$. The latter indicates TCAcycle activity. Amino acids are important carbon donors for normoxic fatty acid synthesis, and, because amino acid catabolism is likely to proceed during anoxia, we investigated the production of acetyl-units from $1-^{14}C-leucine$. During 7 h of anoxia, 1.8% of the injected $1-^{14}C-leucine$ was excreted as $^{14}CO_{2}$, which indicates that amino acid catabolism indeed proceeds during anoxia. At the end of the exposure, the animals were sac...

Impact of anoxia and hydrogen sulphide on the metabolism of Arctica islandica L. (Bivalvia)

The effects of environmental anoxia and anoxic sulphide exposure on metabolism are measured in tissues of the clam, Arctica islandica. Under anoxia the total activity of glycogen phosphorylase and the percentage of the enzyme in the active a form are significantly reduced. Alterations in pyruvate kinase kinetics produce slightly increased V max values, strongly increased S 0.5 PEP, slightly increased S 0.5 ADP in the muscular tissues and mantle, and strongly reduced I 50 for alanine (up to 90-fold increased sensitivity). Anoxia also stimulates a reduction of fructose-2,6-bisphosphate levels, an effector of phosphofructokinase, in all tissues tested. These effects are consistent with enzyme modifications induced by phosphorylation to produce a restricted activity. Anoxic sulphide exposure produced similar effects on the glycogenolytic enzyme glycogen phosphorylase (GP), as does anoxia alone. In the course of the experiments, mitochondrial energy metabolism is not affected by sulphide. The accumulation of the anaerobic indicator metabolite succinate is almost identical in adductor and foot under both stresses. The data suggest a similar coordinated metabolic rate control under environmental anoxia and anoxic sulphide exposure, i.e. H 2S has no distinctly different effects on the parameters tested. This study provides evidence that while being burrowed in anoxic sediments, the clams are able to withstand the detrimental effects of sulphide for a substantial period of time.

Conversion of phosphoenolpyruvate in venus gallina and scapharca inaequivalvis at anoxia stress—I. time dependency

1. 1. The origin of carbon incorporated in succinate in the first 12 hr of environmental anoxia was studied in two bivalve species of the Northern Adriatic Sea. 2. 2. Posterior adductor muscles were excised after various periods of exposure to anoxic seawater and incubated under nitrogen in the presence of metabolic inhibitors (aminooxyacetate, iodoacetate, 3-mercaptopicolinate) and UL- 14C substrates, in order to examine the redirection of glycolytic flux from pyruvate kinase to phosphoenolpyruvate carboxykinase. 3. 3. Changes in the levels of key metabolites and the distribution of radiolabeled glucose and aspartate indicate that PEP-carboxykinase did not participate in anaerobic metabolism of these species for at least the initial 12 hr of anaerobiosis.

Sulfide and cyanide induced mortality and anaerobic metabolism in the arcid blood clam Scapharca inaequiv alvis

1. The survival and metabolic adjustments of the blood clam S. inaequivalvis have been determined at environmental anoxia and tissue anoxia induced by sulfide and cyanide. 2. Times to 50% mortality were established in clams placed in oxygenated seawater with and without dissolved sulfide or free cyanide or deoxygenated seawater with and without dissolved sulfide. 3. Anaerobic metabolism was studied in live animals and in red blood cells incubated in vitro. Tissue anoxia due to sulfide and cyanide caused greater changes in the levels of aspartate and the pyruvate derivatives, compared to environmental anoxia.

Normoxic and anoxic heat output of the freshwater bivalves Pisidium and Sphaerium : I. Rhythms of spontaneous quiescence and behaviour.

In fasting Pisidium amnicum and Sphaerium corneum, regular periods of behavioural and metabolic quiescence were shown to occur in the normoxic, constant environment of the flow-through chamber of a heat-flow microcalorimeter. The metabolic rate was suppressed to 7.5% of normal at 10° C and to 8.5-9.7% at 20° C for periods exceeding the period of active metabolism by a factor of 3.5 at 10° C and 8.3 at 20° C. The rate of heat output during normoxic quiescence was equal to that during environmental anoxia, suggesting spontaneous achievement of body anoxia by complete shell closure. The mass-specific integrated heat output during closure periods was independent of size. Parallel observations on clam behaviour suggested that metabolic quiescence coincided with shell closure, and bursts of heat flow with active ventilation. Shell closure was accompanied by pronounced bradycardia, down to 20% of the active rate. In a constant environment, the rhythmic quiescence is regulated by shell closure which is probably triggered by lack of food. Regular quiescence of fasting bivalves may conserve energy reserves considerably, the amount depending on the possible excretion rate of the end products, and the post-quiescence recovery costs, which were not measured. Heat output during the active period was close to the average metabolic rate found earlier for Sphaeriidae. However, all the values determined so far are likely to be underestimates of the natural metabolism because the effects of digestion and growth are not included.

Effects of environmental anoxia on concentrations of free amino acids and kinetic properties of glutamate dehydrogenase in three body parts of Phascolosoma arcuatum (Sipuncula)

The body wall (BW), introvert cum retractor muscles (ICRM) and digestive tract (DT) of the sipunculid worm Phascolosoma arcuatum had different biochemical adaptations to anoxia with reference to amino acid metabolism. Although aspartate degradation occurred in all three body parts of the anoxic worm, alanine and glutamate accumulated only in the ICRM and DT, and not the BW. Hence, in the BW, the amino group of aspartate was not transferred to alanine during anoxia, but was possibly released as ammonia through glutamate. The kinetic properties of glutamate dehydrogenase (GDH) from the BW and ICRM were altered during anoxia to enhance the glutamate deaminating activity. Although alanine and glutamate were accumulated in the ICRM of the anoxic P. arcuatum, the decrease in aspartate content was unable to account for the increase. Results indicate that it was in the DT of the anoxic worm that glutamate was likely to act as an intermediate to shuttle the amino group between aspartate and alanine. The kinetic properties of the GDH from this body part of P. arcuatum were unaltered by anoxia.

Biochemical adaptations of the mudskipper Boleophthalmus boddaerti to a lack of oxygen

Specimens of the mudskipper Boleophthalmus boddaerti, collected along the estuarine canal at Pasir Ris, Singapore between April 1987 and December 1989, were examined in the laboratory. After being exposed to environmental hypoxia for 6 h, no accumulation of lactate, alanine or succinate was observed in the muscle tissue of B. boddaerti. In addition, the blood lactate content and muscle creatine phosphate (CrP), adenosine triphosphate (ATP) and glycogen contents were not significantly different from those of the control. During normoxic recovery, oxygen debt repayment was only a small fraction of the oxygen deficit incurred during the 6 h of hypoxic exposure. Therefore, it would appear that B. boddaerti coped with environmental hypoxia by undergoing metabolic rate reduction. After anoxic exposure, the CrP, ATP and adenosine diphosphate (ADP) contents in the muscle tissue of B. boddaerti were significantly lower than the corresponding control values. Lactate accumulated in the blood and muscle tissue of the anoxic fish though the muscle energy charge and glycogen content remained constant. Hence, B. boddaerti was able to cope with such a period of anoxia without increasing its glycolytic rate. Succinate, which was not detected in the muscle tissue of the control and hypoxia-exposed fish, accumulated in significant quantities in the muscle tissue of B. boddaerti exposed to environmental anoxia and functional hypoxia. In the case of functional hypoxia, there was a significant decrease in the muscle glycogen content, and the muscle lactate content increased 17.3-fold, indicating that glycolysis was activated to provide the source of energy during muscular exercise.

Subcellular enzyme binding and the regulation of glycolysis in anoxic turtle brain.

The association of glycolytic enzymes with the particulate fraction of the cell was assessed in the brain of the freshwater turtle, Pseudemys scripta elegans, using three different methodologies. Each method showed that a large percentage of each of eight enzymes was bound in brain. The effect of environmental anoxia (5 or 20 h submergence in N2-bubbled water at 7 degrees C) on the distribution of enzymes between free and bound states was analyzed. All three techniques showed a significant increase in the percentages of brain aldolase and glyceraldehyde-3-phosphate dehydrogenase bound during anoxia and no change in lactate dehydrogenase or creatine kinase binding. Two methodologies also showed an increase in the percent bound during anoxia for hexokinase, phosphofructokinase, and phosphoglycerate kinase. An increased association of glycolytic enzymes with structural elements of the cell during anoxia may physically position the glycolytic pathway to facilitate coupling between this ATP-generating pathway and ATP-utilizing processes, such as membrane ion pumps.