Role of mitochondria-associated hexokinase II in cancer cell death induced by 3-bromopyruvate

It has long been observed that cancer cells rely more on glycolysis to generate ATP and actively use certain glycolytic metabolic intermediates for biosynthesis. Hexokinase II (HKII) is a key glycolytic enzyme that plays a role in the regulation of the mitochondria-initiated apoptotic cell death. As a potent inhibitor of hexokinase, 3-bromopyruvate (3-BrPA) is known to inhibit cancer cell energy metabolism and trigger cell death, supposedly through depletion of cellular ATP. The current study showed that 3-BrPA caused a covalent modification of HKII protein and directly triggered its dissociation from mitochondria, leading to a specific release of apoptosis-inducing factor (AIF) from the mitochondria to cytosol and eventual cell death. Co-immunoprecipitation revealed a physical interaction between HKII and AIF. Using a competitive peptide of HKII, we showed that the dissociation of hexokinase II from mitochondria alone could cause apoptotic cell death, especially in the mitochondria-deficient ρ⁰ cells that highly express HKII. Interestingly, the dissociation of HKII itself did not directly affect the mitochondrial membrane potential, ROS generation, and oxidative phosphorylation. Our study suggests that the physical association between HKII and AIF is important for the normal localization of AIF in the mitochondria, and disruption of this protein complex by 3-BrPA leads to their release from the mitochondria and eventual cell death.     

Metabolic dynamics in skeletal muscle during acute reduction in blood flow and oxygen supply to mitochondria: in-silico studies using a multi-scale, top-down integrated model

Control mechanisms of cellular metabolism and energetics in skeletal muscle that may become evident in response to physiological stresses such as reduction in blood flow and oxygen supply to mitochondria can be quantitatively understood using a multi-scale computational model. The analysis of dynamic responses from such a model can provide insights into mechanisms of metabolic regulation that may not be evident from experimental studies. For the purpose, a physiologically-based, multi-scale computational model of skeletal muscle cellular metabolism and energetics was developed to describe dynamic responses of key chemical species and reaction fluxes to muscle ischemia. The model, which incorporates key transport and metabolic processes and subcellular compartmentalization, is based on dynamic mass balances of 30 chemical species in both capillary blood and tissue cells (cytosol and mitochondria) domains. The reaction fluxes in cytosol and mitochondria are expressed in terms of a general phenomenological Michaelis-Menten equation involving the compartmentalized energy controller ratios ATP/ADP and NADH/NAD(+). The unknown transport and reaction parameters in the model are estimated simultaneously by minimizing the differences between available in vivo experimental data on muscle ischemia and corresponding model outputs in coupled with the resting linear flux balance constraints using a robust, nonlinear, constrained-based, reduced gradient optimization algorithm. With the optimal parameter values, the model is able to simulate dynamic responses to reduced blood flow and oxygen supply to mitochondria associated with muscle ischemia of several key metabolite concentrations and metabolic fluxes in the subcellular cytosolic and mitochondrial compartments, some that can be measured and others that can not be measured with the current experimental techniques. The model can be applied to test complex hypotheses involving dynamic regulation of cellular metabolism and energetics in skeletal muscle during physiological stresses such as ischemia, hypoxia, and exercise.     

High glucose concentrations inhibit glucose phosphorylation, but not glucose transport, in human endothelial cells.

Glucose uptake is autoregulated in a variety of cell types and it is thought that glucose transport is the major step that is subjected to control by sugar availability. Here, we examined the effect of high glucose concentrations on the rate of glucose uptake by human ECV-304 umbilical vein-derived endothelial cells. A rise in the glucose concentration in the medium led a dose-dependent decrease in the rate of 2-deoxyglucose uptake. The effect of high glucose was independent of protein synthesis and the time-course analysis indicated that it was relatively slow. The effect was not due to inhibition of glucose transport since neither the expression nor the subcellular distribution of the major glucose transporter GLUT1, nor the rate of 3-O-methylglucose uptake was affected. The total in vitro assayed hexokinase activity and the expression of hexokinase-I were similar in cells treated or not with high concentrations of glucose. In contrast, exposure of cells to a high glucose concentration caused a marked decrease in phosphorylated 2-deoxyglucose/free 2-deoxyglucose ratio. This suggests the existence of alterations in the rate of in vivo glucose phosphorylation in response to high glucose. In summary, we conclude that ECV304 human endothelial cells reduce glucose utilization in response to enhanced levels of glucose in the medium by inhibiting the rate of glucose phosphorylation, rather than by blocking glucose transport. This suggests a novel metabolic effect of high glucose on cellular glucose utilization.     

Mitochondrial hexokinase and cardioprotection of the intact heart

The interaction of hexokinase with mitochondria has emerged as a powerful mechanism in protecting many cell types against cell death. However, the role of mitochondrial hexokinase (mitoHK) in cardiac ischemia-reperfusion injury has as of yet received little attention. In this review we examine whether increased binding of hexokinase to the mitochondrion is also an integral component of cardioprotective signalling. We discuss observations in cardiac mitochondrial activation that directed us to the hypothesis of hexokinase cellular redistribution with reversible, cardioprotective ischemia, summarize the data showing that many cardioprotective interventions, such as ischemic preconditioning, insulin, morphine and volatile anesthetics, increase mitochondrial hexokinase binding within the intact heart, and discuss similarities between mitochondrial hexokinase association and ischemic preconditioning. Although most data indicate that mitochondrial hexokinase may indeed be an integral part of cardioprotection, a definitive proof for a causal relation between the amount of mitoHK and cardiac ischemia-reperfusion injury in the intact heart is eagerly awaited. When such relationship is indeed observed, the association of hexokinase with mitochondria will offer an opportunity to develop new therapies to combat ischemic cardiac diseases.     

Altered metabolic states do not change the intracellular distribution of hexokinase in Zajdela hepatoma ascites cells.

The distribution of hexokinase between bound and soluble forms was studied by digitonin fractionation of Zajdela hepatoma ascites cells maintained under various metabolic conditions. Addition of glucose to Zajdela cells respiring on endogenous substrates induces an immediate inhibition of respiration by 50-60% ( Crabtree effect), and a production of acid due to glycolysis. Acid production decreases abruptly after 60s to 50% of the initial rate. The ATP/ADP ratio is not altered by the addition of glucose or by different rates of glycolysis. The uncoupling agent carbonyl cyanide m-chlorophenylhydrazone decreases the ATP/ADP ratio by 10-fold in cells respiring on endogenous substrate, but has little effect on cells oxidizing glucose. Rapid fractionation of the cells under these various metabolic conditions revealed no change in the distribution of hexokinase. Approx. 75% of hexokinase is bound in all cases, in contrast with lactate dehydrogenase, 95% of which was in the soluble form. Longer-term incubations (to 20 min) revealed only slight (10-15%) increases in soluble hexokinase in cells incubated with glucose. Various metabolic inhibitors had little additional affect on the subcellular distribution of hexokinase. Thus a rapid release of hexokinase from mitochondrial membrane is not a mechanism by which glycolysis is regulated in rapidly growing Zajdela hepatoma.     

Mitochondrial bound hexokinase activity as a preventive antioxidant defense: steady-state ADP formation as a regulatory mechanism of membrane potential and reactive oxygen species generation in mitochondria

Brain hexokinase is associated with the outer membrane of mitochondria, and its activity has been implicated in the regulation of ATP synthesis and apoptosis. Reactive oxygen species (ROS) are by-products of the electron transport chain in mitochondria. Here we show that the ADP produced by hexokinase activity in rat brain mitochondria (mt-hexokinase) controls both membrane potential (Deltapsi(m)) and ROS generation. Exposing control mitochondria to glucose increased the rate of oxygen consumption and reduced the rate of hydrogen peroxide generation. Mitochondrial associated hexokinase activity also regulated Deltapsi(m), because glucose stabilized low Deltapsi(m) values in state 3. Interestingly, the addition of glucose 6-phosphate significantly reduced the time of state 3 persistence, leading to an increase in the Deltapsi(m) and in H(2)O(2) generation. The glucose analogue 2-deoxyglucose completely impaired H(2)O(2) formation in state 3-state 4 transition. In sharp contrast, the mt-hexokinase-depleted mitochondria were, in all the above mentioned experiments, insensitive to glucose addition, indicating that the mt-hexokinase activity is pivotal in the homeostasis of the physiological functions of mitochondria. When mt-hexokinase-depleted mitochondria were incubated with exogenous yeast hexokinase, which is not able to bind to mitochondria, the rate of H(2)O(2) generation reached levels similar to those exhibited by control mitochondria only when an excess of 10-fold more enzyme activity was supplemented. Hyperglycemia induced in embryonic rat brain cortical neurons increased ROS production due to a rise in the intracellular glucose 6-phosphate levels, which were decreased by the inclusion of 2-deoxyglucose, N-acetyl cysteine, or carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Taken together, the results presented here indicate for the first time that mt-hexokinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.     

The binding of glycerol kinase to the outer membrane of rat liver mitochondria: its importance in metabolic regulation

Glycerol kinase was found to associate with the hexokinase binding protein. The binding of glycerol kinase has a high specificity as illustrated by the fact that the magnitude of binding was reduced by glycerophosphate and antibodies against the hexokinase binding protein. A possible function of glycerol kinase binding to the mitochondria with respect to metabolic regulation is proposed for the following reasons: (i) Glycerol kinase seems to bind to the same binding protein as hexokinase. (ii) Both kinases were observed to be reversibly bound to the mitochondria in different metabolic situations, i.e., 10% of total cellular activity from both kinases is bound in starved rats whereas no activity of glycerol kinase and 30% of hexokinase become bound in fed rats. (iii) The kinetic properties of the associated glycerol kinase change in an analogous manner to those known for structure-bound hexokinase. (iv) With the binding of glycerol kinase to the mitochondria, it is possible to propose a metabolic pathway for glycerol oxidation to dihydroxyacetone phosphate by a combined action involving the enzyme, glycerol phosphate oxidase, and oxidative phosphorylation.     

Systematic Analysis of Small RNAs Associated with Human Mitochondria by Deep Sequencing: Detailed Analysis of Mitochondrial Associated miRNA

Mitochondria are one of the central regulators of many cellular processes beyond its well established role in energy metabolism. The inter-organellar crosstalk is critical for the optimal function of mitochondria. Many nuclear encoded proteins and RNA are imported to mitochondria. The translocation of small RNA (sRNA) including miRNA to mitochondria and other sub-cellular organelle is still not clear. We characterized here sRNA including miRNA associated with human mitochondria by cellular fractionation and deep sequencing approach. Mitochondria were purified from HEK293 and HeLa cells for RNA isolation. The sRNA library was generated and sequenced using Illumina system. The analysis showed the presence of unique population of sRNA associated with mitochondria including miRNA. Putative novel miRNAs were characterized from unannotated sRNA sequences. The study showed the association of 428 known, 196 putative novel miRNAs to mitochondria of HEK293 and 327 known, 13 putative novel miRNAs to mitochondria of HeLa cells. The alignment of sRNA to mitochondrial genome was also studied. The targets were analyzed using DAVID to classify them in unique networks using GO and KEGG tools. Analysis of identified targets showed that miRNA associated with mitochondria regulates critical cellular processes like RNA turnover, apoptosis, cell cycle and nucleotide metabolism. The six miRNAs (counts >1000) associated with mitochondria of both HEK293 and HeLa were validated by RT-qPCR. To our knowledge, this is the first systematic study demonstrating the associations of sRNA including miRNA with mitochondria that may regulate site-specific turnover of target mRNA important for mitochondrial related functions.     

Mitochondria-associated hexokinases play a role in the control of programmed cell death in Nicotiana benthamiana

Recent findings suggest a pivotal role for mitochondria-associated hexokinase in the regulation of apoptosis in animal cells. In this study, virus-induced gene silencing (VIGS) of a hexokinase-encoding Hxk1 caused necrotic lesions on leaves, abnormal leaf morphology, and retarded plant growth in Nicotiana benthamiana. Hxk1 was associated with the mitochondria, and this association required the N-terminal membrane anchor. VIGS of Hxk1 reduced the cellular glucose-phosphorylating activity to approximately 31% of control levels without changing the fructose-phosphorylating activity and did not alter hexose phosphate content severely. The affected cells showed programmed cell death (PCD) morphological markers, including nuclear condensation and DNA fragmentation. Similar to animal cell apoptosis, cytochrome c was released into the cytosol and caspase-9- and caspase-3-like proteolytic activities were strongly induced. Furthermore, based on flow cytometry, Arabidopsis thaliana plants overexpressing Arabidopsis HXK1 and HXK2, both of which are predominantly associated with mitochondria, exhibited enhanced resistance to H(2)O(2)- and alpha-picolinic acid-induced PCD. Finally, the addition of recombinant Hxk1 to mitochondria-enriched fractions prevented H(2)O(2)/clotrimazole-induced cytochrome c release and loss of mitochondrial membrane potential. Together, these results show that hexokinase critically regulates the execution of PCD in plant cells, suggesting a link between glucose metabolism and apoptosis.     

Glucose metabolism and proliferation in glia: role of astrocytic gap junctions

Astrocytes play a well-established role in brain metabolism, being a key element in the capture of energetic compounds from the circulation and in their delivery to active neurons. Their metabolic status is affected in many pathological situations, such as gliomas, which are the most common brain tumors. This proliferative dysfunction is associated with changes in gap junctional communication, a property strongly developed in normal astrocytes studied both in vitro and in vivo. Here, we summarize and discuss the findings that have lead to the identification of a link between gap junctions, glucose uptake, and proliferation. Indeed, the inhibition of gap junctional communication is associated with an increase in glucose uptake due to a rapid change in the localization of both GLUT-1 and type I hexokinase. This effect persists due to the up-regulation of GLUT-1 and type I hexokinase and to the induction of GLUT-3 and type II hexokinase. In addition, cyclins D1 and D3 have been found to act as sensors of the inhibition of gap junctions and have been proposed to play the role of mediators in the mitogenic effect observed. Conversely, in C6 glioma cells, characterized by a low level of intercellular communication, an increase in gap junctional communication reduces glucose uptake by releasing type I and type II hexokinases from the mitochondria and decreases the exacerbated rate of proliferation due to the up-regulation of the Cdk inhibitors p21 and p27. Identification of the molecular actors involved in these pathways should allow the determination of potential therapeutic targets that could lead to the testing of alternative strategies to prevent, or at least slow down, the proliferation of glioma cells.     

Vimentin supports mitochondrial morphology and organization

Vimentin is one of the intermediate filaments that functions in structural support, signal transduction and organelle positioning of a cell. In the present study, we report the contribution of vimentin in mitochondrial morphology and organization. Using subcellular fractionation, immunoprecipitation and fluorescence microscopy analyses, we found that vimentin was associated with mitochondria. Knockdown of vimentin resulted in mitochondrial fragmentation, swelling and disorganization. We further demonstrated that the vimentin cytoskeleton co-localized and interacted with mitochondria to a greater extent than other cytoskeletal components known to support mitochondria. Our results also suggest that vimentin could participate in the mitochondrial association of microtubules. As mitochondrial morphologies determine mitochondrial function, our findings revealed a potentially important relationship between the vimentin-based intermediate filaments and the regulation of mitochondria.     

Hexokinase-mitochondria interaction mediated by Akt is required to inhibit apoptosis in the presence or absence of Bax and Bak

The serine/threonine kinase Akt inhibits mitochondrial cytochrome c release and apoptosis induced by a variety of proapoptotic stimuli. The antiapoptotic activity of Akt is coupled, at least in part, to its effects on cellular metabolism. Here, we provide genetic evidence that Akt is required to maintain hexokinase association with mitochondria. Targeted disruption of this association impairs the ability of growth factors and Akt to inhibit cytochrome c release and apoptosis. Targeted disruption of mitochondria-hexokinase (HK) interaction or exposure to proapoptotic stimuli that promote rapid dissociation of hexokinase from mitochondria potently induce cytochrome c release and apoptosis, even in the absence of Bax and Bak. These effects are inhibited by activated Akt, but not by Bcl-2, implying that changes in outer mitochondrial membrane (OMM) permeability leading to apoptosis can occur in the absence of Bax and Bak and that Akt inhibits these changes through maintenance of hexokinase association with mitochondria.     

Why does the brain (not) have glycogen?

In the present paper we formulate the hypothesis that brain glycogen is a critical determinant in the modulation of carbohydrate supply at the cellular level. Specifically, we propose that mobilization of astrocytic glycogen after an increase in AMP levels during enhanced neuronal activity controls the concentration of glucose phosphates in astrocytes. This would result in modulation of glucose phosphorylation by hexokinase and upstream cell glucose uptake. This mechanism would favor glucose channeling to activated neurons, supplementing the already rich neuron-astrocyte metabolic and functional partnership with important implications for the energy compounds used to sustain neuronal activity. The hypothesis is based on recent modeling evidence suggesting that rapid glycogen breakdown can profoundly alter the short-term kinetics of glucose delivery to neurons and astrocytes. It is also based on review of the literature relevant to glycogen metabolism during physiological brain activity, with an emphasis on the metabolic pathways identifying both the origin and the fate of this glucose reserve.     

Type I Brain Hexokinase: Axonal Transport and Membrane Associations Within Central Nervous System Presynaptic Terminals

Abstract: While studying the delivery of cytoplasmic proteins to the presynaptic terminals of CNS neurons, we discovered unique characteristics of one protein (p118) conveyed in slow component b (SCb) of axonal transport, the large group of proteins representing the cytoplasmic matrix. Alone among the SCb group, p118 coisolated with the synaptic junctional complex on biochemical fractionation of the radiolabeled synaptic regions. Purification and amino acid sequencing of this protein revealed it is most likely the guinea pig form of type I (brain) hexokinase (ATP: d -hexose 6-phosphotransferase, EC 2.7.1.1). Further biochemical treatments were consistent with this identity. The majority of type I brain hexokinase has been thought to be associated primarily with membranes, in particular the mitochondrial outer membrane. We found that the majority of type I hexokinase is transported toward the terminals at a rate at least 10 times slower than that exhibited by the maximal or average rate of mitochondria. This suggests that, in the axon, the enzyme exhibits transient or dynamic interactions with mitochondria that are moving more rapidly. It is not clear whether hexokinase binds exclusively to mitochondria, or also exhibits association with nonmitochondrial membranes. The unexpected enrichment of hexokinase during synaptic junctional complex purification may result from its strong association with the presynaptic membrane portion of the synapse.     

The role of the mitochondrial outer membrane in energy metabolism of tumor cells

The outer mitochondrial membrane contains a pore structure which is composed of a 30,000 Da protein, porin. The pore has an internal diameter of 2 nm and exhibits a molecular-sieving exclusion limit between 3000 and 6000 Da. These pores, therefore, provide the exit/entrance port for metabolites moving between mitochondria and the cytosol. Hexokinase binds to porin on the outer surface of mitochondria. The location of hexokinase has evoked a number of theories in which bound hexokinase is given a central role in regulating glycolysis, and, perhaps, the metabolic communication between oxidative and glycolytic metabolism. This is of particular importance in rapidly growing tumor cells in which the aerobic production of lactate and hexokinase activity are highly induced. In the present paper, we summarize the suggested roles of the outer membrane and bound hexokinase in regulation glycolysis of tumor cells. Experiments attempting to elucidate the role of hexokinase binding in the regulation of tumor cell metabolism are presented.     

Actin-based cellular framework for glucose signaling by Arabidopsis hexokinase1

Glucose functions in plants both as a metabolic resource as well as a hormone that regulates expression of many genes. Arabidopsis hexokinase1 (HXK1) is the best understood plant glucose sensor/transducer, yet we are only now appreciating the cellular complexity of its signaling functions. We have recently shown that one of the earliest detectable responses to plant glucose treatments are extensive alterations of cellular F-actin. Interestingly, AtHXK1 is predominantly located on mitochondria, yet also can interact with actin. A normal functioning actin cytoskeleton is required for HXK1 to act as an effector in glucose signaling assays. We have suggested that HXK1 might alter F-actin dynamics and thereby influence the formation and/or stabilization of cytoskeleton-bound polysomes. In this Addendum, we have extended our initial observations on the subcellular targeting of HXK1 and its interaction with F-actin. We then further consider the cellular context in which HXK1 might regulate gene expression.Key words: Arabidopsis, F-actin, glucose signaling, hexokinase, hTalin, mitochondria, polysomes, protoplasts, transient expression assay, fluorescence microscopy     

Glucose metabolism in the rat small intestine: the effect of glucose analogues on hexokinase activity (Short Communication)

The effect of intubation of starved rats with solutions of glucose, 3-O-methylglucose, mannose, 2-deoxyglucose and sorbitol on the subcellular location of hexokinase in the mucosa of the small intestine was studied. Glucose, 3-O-methylglucose and mannose caused the soluble hexokinase activity to rise to a value similar to that found in fed animals, whereas sorbitol and 2-deoxyglucose caused smaller increases.     

31P NMR and 13C NMR studies of recombinant Saccharomyces cerevisiae with altered glucose phosphorylation activities

Manipulation of cellular metabolism to maximize the yield and rate of formation of desired products may be achieved through genetic modification. Batch fermentations utilizing glucose as a carbon source were performed for three recombinant strains of Saccharomyces cerevisiae in which the glucose phosphorylation step was altered by mutation and genetic engineering. The host strain (hxk1 hxk2 glk) is unable to grow on glucose or fructose; the three plasmids investigated expressed hexokinase PI, hexokinase PII, or glucokinase, respectively, enabling more rapid glucose and fructose phosphorylation in vivo than that provided by wild-type yeast.Intracellular metabolic state variables were determined by ³¹P NMR measurements of in vivo fermentations under nongrowth conditions for high cell density suspensions. Glucose consumption, ethanol and glycerol production, and polysaccharide formation were determined by ¹³C NMR measurements under the same experimental conditions as used in the ³¹P NMR measurements. The trends observed in ethanol yields for the strains under growth conditions were mimicked in the nongrowth NMR conditions.Only the strain with hexokinase PI had higher rates of glucose consumption and ethanol production in comparison to healthy diploid strains in the literature. The hexokinase PII strain drastically underutilized its glucose-phosphorylating capacity. A regulation difference in the use of magnesium-free ATP for this strain could be a possible explanation. Differences in ATP levels and cytoplasmic pH values among the strains were observed that could not have been foreseen. However, cytoplasmic pH values do not account for the differences observed among in vivo and in vitro glucose phosphorylation activities of the three recombinant strains.     

Studies on proinsulin and proglucagon biosynthesis and conversion at the subcellular level: I. Fractionation procedure and characterization of the subcellular fractions

Anglerfish islets were homogenized in 0.25 M sucrose and separated into seven separate subcellular fractions by differential and discontinuous density gradient centrifugation. The objective was to isolate microsomes and secretory granules in a highly purified state. The fractions were characterized by electron microscopy and chemical analyses. Each fraction was assayed for its content of protein, RNA, DNA, immunoreactive insulin (IRI), and immunoreactive glucagon (IRG). Ultrastructural examination showed that two of the seven subcellular fractions contain primarily mitochondria, and that two others consist almost exclusively of secretory granules. A fifth fraction contains rough and smooth microsomal vesicles. The remaining two fractions are the cell supernate and the nuclei and cell debris. The content of DNA and RNA in all fractions is consistent with the observed ultrastructure. More than 82 percent of the total cellular IRI and 89(percent) of the total cellular IRG are found in the fractions of secretory granules. The combined fractions of secretory granules and microsomes consistently yield >93 percent of the total IRG. These results indicate that the fractionation procedure employed yields fractions of microsomes and secretory granules that contain nearly all the immunoassayable insulin and glucagons found in whole islet tissue. These fractions are thus considered suitable for study of proinsulin and proglucagon biosynthesis and their metabolic conversion at the subcellular level.     

2-Deoxyglucose and cytochalasin D modulate aldolase mobility in living 3T3 cells

Approximately 23% of the glycolytic enzyme aldolase in the perinuclear region of Swiss 3T3 cells is immobile as measured by FRAP. Previous studies suggest that the immobile fraction may be associated with the actin cytoskeleton (Pagliaro, L. and D. L. Taylor. 1988. J. Cell Biol. 107:981-991), and it has been proposed that the association of some glycolytic enzymes with the cytoskeleton could have functional significance, perhaps involving a fundamental relationship between glycolysis, cytoplasmic organization, and cell motility. We have tested the effect of a key glycolytic inhibitor and an actin cytoskeletal modulator on the mobility of aldolase in living cells directly, using fluorescent analog cytochemistry and FRAP. We report here that the competitive hexokinase inhibitor 2-deoxyglucose releases the bound fraction of aldolase in 3T3 cells within 10 min, and that this process is reversible upon washout of the inhibitor. A similar result is produced with the actin-binding agent, cytochalasin D. These results are consistent with models in which glycolytic enzymes are not exclusively diffusion-limited, soluble proteins, but may exist partially in the solid phase of cytoplasm. Such organization has significant implications for both the modulation of cytoplasmic structure and for cellular metabolism.