Soluble Hexokinase Research Articles

Effect of the antitumoral alkylating agent 3-bromopyruvate on mitochondrial respiration: role of mitochondrially bound hexokinase

The alkylating agent 3-Bromopyruvate (3-BrPA) has been used as an anti-tumoral drug due to its anti-proliferative property in hepatomas cells. This propriety is believed to disturb glycolysis and respiration, which leads to a decreased rate of ATP synthesis. In this study, we evaluated the effects of the alkylating agent 3-BrPA on the respiratory states and the metabolic steps of the mitochondria of mice liver, brain and in human hepatocarcinoma cell line HepG2. The mitochondrial membrane potential (ΔΨ(m)), O(2) consumption and dehydrogenase activities were rapidly dissipated/or inhibited by 3-BrPA in respiration medium containing ADP and succinate as respiratory substrate. 3-BrPA inhibition was reverted by reduced glutathione (GSH). Respiration induced by yeast soluble hexokinase (HK) was rapidly inhibited by 3-BrPA. Similar results were observed using mice brain mitochondria that present HK naturally bound to the outer mitochondrial membrane. When the adenine nucleotide transporter (ANT) was blocked by the carboxyatractiloside, the 3-BrPA effect was significantly delayed. In permeabilized human hepatoma HepG2 cells that present HK type II bound to mitochondria (mt-HK II), the inhibiting effect occurred faster when the endogenous HK activity was activated by 2-deoxyglucose (2-DOG). Inhibition of mt-HK II by glucose-6-phosphate retards the mitochondria to react with 3-BrPA. The HK activities recovered in HepG2 cells treated or not with 3-BrPA were practically the same. These results suggest that mitochondrially bound HK supporting the ADP/ATP exchange activity levels facilitates the 3-BrPA inhibition reaction in tumors mitochondria by a proton motive force-dependent dynamic equilibrium between sensitive and less sensitive SDH in the electron transport system.

Expression of several genes involved in sucrose/starch metabolism as affected by different strategies to induce phosphate deficiency in Arabidopsis

The effects of inorganic phosphate (Pi) deficiency on expression of genes encoding ADP-glucose pyrophosphorylase small and large subunits (ApS and ApL1, ApL2, ApL3 genes), UDP-glucose pyrophosphorylase (Ugp gene), sucrose synthase (Sus1), soluble and insoluble acid invertases (Inv and Invcw) and hexokinase (Hxk1 gene), all involved in carbohydrate metabolism, were investigated in Arabidopsis thaliana (L.) Heynh. We used soil-grown pho mutants affected in Pi status, as well as wild-type (wt) plants grown under Pi deficiency conditions in liquid medium, and leaves of wt plants fed with D-mannose. Generally, ApS, ApL1, Ugp and Inv genes were upregulated, although to a varied degree, under conditions of Pi-stress. The applied conditions had differential effects on expression of other genes studied. For instance, Sus1 was downregulated in pho1 (Pi-deficient) mutant, but was unaffected in wt plants grown in liquid medium under P-deficiency. Mannose had distinct concentration-dependent effects on expression of genes under study, possibly reflecting a dual role of mannose as a sink for Pi and as glucose analog. Feeding Pi (at up to 200 mM) to the detached leaves of wt plants strongly affected the expression of ApL1, ApL2, Sus1 and Inv genes, possibly due to an osmotic effect exerted by Pi. The data suggest that ADP-glucose and UDP-glucose pyrophosphorylases (enzymes indirectly involved in Pi recycling) as well as invertases (sucrose hydrolysis) are transcriptionally regulated by Pi-deficiency, which may play a role in homeostatic mechanisms that acclimate the plant to the Pi-stress conditions.

Hexokinases of Tobacco Leaves: Changes in the Cytosolic and Non-Cytosolic Isozyme Complexes Induced by Tobacco Mosaic Virus Infection

Changes in the cytosotic (soluble) and the non-cytosolic (particulate) isozyme composition of hexokinases and in their properties were studied by ion exchange chromatography on DEAE cellulose after the subcellular fractionation both in the healthy and the tobacco mosaic virus (TMV) infected tobacco leaves. Three main isozyme complexes were obtained: one particulate fraction (the particulate hexokinase phosphorylating both glucose and fructose, EC 2.7.1.1), and two soluble fractions (the soluble hexokinase phosphorylating both the glucose and the fructose, and the soluble fructokinase, which phosphorylates primarily fructose, EC 2.7.1.4). The total fructokinase activities were nearly twice higher than the total glucokinase activities (188.6 % of glucokinase activity in healthy plants and 181.3 % in infected plants). The total particulate glucokinase activity was increased to 120.6 % and the fructokinase to 118.9 % in TMV infected tissue when compared with healthy control. The similar pattern of activity was observed for soluble hexokinase isozymes - the sum of soluble glucokinase activity was increased to 175.4 % and of fructokinase activity to 131.2 % in TMV infected tissue. The isozymes isolated both from the healthy control and TMV-infected leaves had the similar elution profiles, displayed Michaelis-Menten kinetics, showed the identical profiles of pH optima and were Mg2+ dependent with the highest enzyme activity at equimolar Mg2+ and ATP concentration.

Mutations that confer resistance to 2-deoxyglucose reduce the specific activity of hexokinase from Myxococcus xanthus.

The glucose analog 2-deoxyglucose (2dGlc) inhibits the growth and multicellular development of Myxococcus xanthus. Mutants of M. xanthus resistant to 2dGlc, designated hex mutants, arise at a low spontaneous frequency. Expression of the Escherichia coli glk (glucokinase) gene in M. xanthus hex mutants restores 2dGlc sensitivity, suggesting that these mutants arise upon the loss of a soluble hexokinase function that phosphorylates 2dGlc to form the toxic intermediate, 2-deoxyglucose-6-phosphate. Enzyme assays of M. xanthus extracts reveal a soluble hexokinase (ATP:D-hexose-6-phosphotransferase; EC 2.7.1.1) activity but no phosphotransferase system activities. The hex mutants have lower levels of hexokinase activities than the wild type, and the levels of hexokinase activity exhibited by the hex mutants are inversely correlated with the ability of 2dGlc to inhibit their growth and sporulation. Both 2dGlc and N-acetylglucosamine act as inhibitors of glucose turnover by the M. xanthus hexokinase in vitro, consistent with the finding that glucose and N-acetylglucosamine can antagonize the toxic effects of 2dGlc in vivo.

Hexokinases of tobacco leaves: influence of plant age on particulate and soluble isozyme composition

Changes in hexokinase particulate and soluble isozyme composition and activities in leaves of 65- and 115-d-old tobacco plants were determined by ion exchange chromatography on DEAE cellulose. During plant ageing, the activities of glucose and of fructose phosphorylating isozymes of particulate hexokinase decreased to 9.9 and 9.2 % of initial value, respectively. The activity of soluble hexokinase decreased to a lesser extent: that of glucose phosphorylating isozyme to 49.8 % and of fructose phosphorylating isozyme to 37.8 %. The activity of soluble fructokinase isozyme dropped to 34.8 %. Thus also the ratio of particulate and soluble isozymes was dependent on the age of leaf tissue.

Enhanced catalytic activity of hexokinase by work-induced mitochondrial binding in fast-twitch muscle of rat

Using a teased muscle fiber preparation, we determined the activity of mitochondrially bound hexokinase in rat fast-twitch muscle under control conditions and after low-frequency stimulation periods for up to 2 h. As compared to soluble hexokinase, mitochondrial binding led to stimulation of glucose 6-phosphate production. Low-frequency stimulation greatly enhanced glucose 6-phosphate formation which was 100% and 250% elevated after 1 and 2 h, respectively. These observations point to a mechanism which rapidly increases the catalytic activity of hexokinase through binding to the mitochondrial surface. © 1997 Federation of European Biochemical Societies.

Structural determinants that make hexokinase susceptible to ubiquitin-and ATP- dependent proteolysis

Intracellular protein degradation is highly selective, although the regulation of the mechanism(s) involved this phenomenon have not been fully elucidated. Protein ubiquitination has been implicated a variety of biological processes including the marking of protein for degradation. Hershko et al. [ l ] found that a free NHz-terminal group is essential for protein ligation to ubiquitin and subsequently others [2,3] reported that also the nature of the NH,-terminal residue has a strong influence. However other structural determinants appear to be important protein recognition by the ubiquitin system [4,5]. We have previously shown that purified rabbit hexokinase (HK) type I, the same HK isozyme present also mammalian brain is conjugated to ubiquitin and then degraded by a rabbit reticulocyte fraction I1 when is soluble 161 but not when is the mitochondrial bound form [7]. In fact we reported that the mitochondrii bound hexokinase is stable for several hours the same proteolytic system both the presence or absence of ATP. El, & and E,, the enzymes of the ubiquitin conjugating system, are able to incorporate 'Ior biotin-labelled ubiquitin, an ATP dependent manner, soluble HK as well as other mitochondria1 proteins and furthermore the mitochondria by themselves have a marked ATPdependent ability to conjugate lzsI-ubiquitin [8]. However the results of Western blotting experiments, using a specific anti-hexokinase type I antibody, confmed that there is a time-dependent decrease the HK immunoreactive material the presence of ATP when the soluble enzyme was tested contrast, the bound hexokinase was absolutely stable both the presence or absence of ATP (Fig. 1). Thus, mitochondrial bound hexokinase is neither ubiquitinated nor degraded. These results suggest that the intracellular distribution of a protein is an important feature which determines its susceptibility to ubiquitin dependent degradation. The different behaviour of the enzyme when is the soluble or the bound form may be explained different ways: one possibility is that the ubiquitin conjugating system is not able to ligate ubiquitin to hexokinase when it is bound to mitochondria due to the masking of the binding sites. The second explanation is that the soluble enzyme can undergo post-translational modifications, including partial NH2-terminal proteolysis producing in vivo truncate forms of hexokinase type I (Fig. 2) [9]. To differentiate between these possibilities and especially to investigate the role of the last phenomenon on ATPand ubiquitin-dependent proteolysis we have cloned and expressed bacterial cells the full-length human hexokinase type I, a truncate form lacking the first 11 amino acids and a mini-hexokinase corresponding to the COOH-terminal half of the enzyme (which contains the catalytic site). The expression E. coli cells of the different forms of recombinant HKs was obtained by the PET expression system (Novagen). Briefly, the cDNAs coding for the full-length HK, the HK lacking the first 11 aa and the COOH-terminal domain were all derived from the hhHEX-15 clone, purchased from ATCC, Maryland, (which includes nucleotides 383598, encoding a complete 917 amino acid protein) and then ligated with a PET expression plasmid. The recombinant PET expression vectors were then transformed into the E. coli cells BL21(DE3) and cultures of the transformants were induced with IPTG to express the recombinant HKs. The bacterial cells after induction were harvested and sonicated as reported (101 and the recombinant catalitycally active hexokinase proteins were purified to homogeneity as described [ 111. All these purified recombinant HKs were fmally tested for their stability a cell-free, ATPand ubiquitindependent pruteolytic system.

Rapid Changes in Carbohydrate Metabolism in Muscle Induced by Triiodothyronine; the Role of Glucose 1,6-Bisphosphate

All of the past research on glucose utilization by muscles focused on the slow action of thyroid hormones. Here we show that experimental hyperthyroidism, which was induced in rats by a single intramuscular injection of 3,3′,5-triiodothyronine (T3) at high concentrations, resulted in rapid changes (within minutes) in carbohydrate metabolism in tibialis anterior muscle, There was an increase in lactate content, in the allosteric activity of soluble phosphofructokinase (the rate-limiting enzyme in glycolysis), and in its product fructose 1,6-bisphosphate, 5 min following the injection of T3, suggesting stimulation of glycolysis. The allosteric activity of mitochondrial-bound, and, to a lesser extent, of soluble hexokinase, was also enhanced. However, the intracellular distribution of the enzymes was unchanged by the hormone. The allosteric stimulation of hexokinase may be attributed to the decrease in glucose 1,6-bisphosphate, which is a potent inhibitor of hexokinase. The level of glucose 6-phosphate, another known inhibitor of hexokinase, was not changed by the hormone. The activation of phosphofructokinase following T3 injection may be attributed to the decrease in ATP, an allosteric inhibitor of the enzyme, and the increase in the levels of Pi and fructose 1,6-bisphosphate, allosteric activators of the enzyme. Glycogen content was also significantly decreased in muscle 5 min following the injection of T3. These results suggest that in hyperthyroidism, muscle reacts rapidly to the excess of thyroid hormones by stimulation of glycogenolysis, glucose phosphorylation, and glycolysis, to provide ATP, which may serve as a compensatory mechanism to ATP depletion.

Hexose-6-kinases in germinating honey locust cotyledons: Substrate specificity of d-fructo-6-kinase

Extracts of the cotyledons of germinated honey locust ( Gleditsia triacanthos) seeds, which contain galactomannan as a reserve polysaccharide in the endosperm, were fractionated by chromatography and the fractions examined for the presence of a specific manno-6-kinase which could phosphorylate the d-mannose released by hydrolysis of galactomannan. One particulate hexokinase (the major hexose-6-kinase fraction) and two soluble hexokinase fractions (the minor portion), as well as a soluble fructo-6-kinase fraction, were initially separated. From chromatography, electrophoresis and kinetic studies, no evidence for a specific manno-kinase was obtained. This and the level and kinetic behaviour of the particulate hexokinase implicated it as the enzyme catalysing the phosphorylation of released d-mannose. The fructo-kinase activity was further separated into three fractions. Kinetic studies on one of these with native and synthetic substrates indicated that the structural requirements for the monosaccharide substrate were a β- d-anomeric 2-OH in the furanose ring, a 4-OH trans to the d-5-CH 2OH and a -CH 2OH substituent on C 2 ( trans to the 5-CH 2OH) which could be modified. The orientation of the hydroxyl on C-3 had only a limited effect.

Various vectorial behaviours of a spatially structured substrate cycle.

The dynamic properties of the glucose/glucose-6-phosphate cycle are studied under conditions where the phosphatase and kinase interconverting enzymes are spatially distributed. A semi-artificial membrane made of compacted plant cell walls bearing active phosphatase in its natural state, separates two compartments, one of these compartments containing soluble hexokinase. Depending only upon the two enzyme activity levels and the initial distribution of the substrates, numerous asymmetrical and vectorial behaviours can be observed, such as facilitated glucose 6-phosphate diffusion, active transport of either glucose or glucose 6-phosphate and sequential (alternative) transport between glucose and glucose 6-phosphate. A diffusion-partition reaction coupling can account for these oriented mass transfers. The possibility of such a coupling in this model system is clearly dictated by the global analog of the Curie principle. These results may provide new insight on (a) the still obscure role played by the cell-wall phosphatase activities, particularly their involvement in the transport of exogenous phosphomonoesters, and (b) the actual in vivo operation of substrate and protein cycles in view of the heterogeneity and anisotropy of the cellular milieu.

Hexokinase kinetics in human skeletal muscle after hyperinsulinaemia, hyperglycaemia and hyperepinephrinaemia

The effects of 120 min of euglycaemic hyperinsulinaemia (UH, approximately 5 mM; 40 mU m-2 min-1), UH plus adrenaline infusion (0.05 microgram kg-1 min-1), and hyperglycaemic normoinsulinaemia (26 mM) on hexokinase kinetics in human skeletal muscle were examined. Biopsies were obtained from the quadriceps femoris muscle before and after each clamp. Total muscle hexokinase activity (HKt) (measured on a 2500 g supernatant) at a saturating level of the substrate glucose (1 mM) averaged 13 mmol kg dry wt-1 min-1 in the basal state and did not change significantly under any condition. Soluble hexokinase activity (HKs) (16,000 g supernatant) accounted for approximately 65% of HKt in the basal state, and this percentage was not significantly affected by any condition, suggesting that there was no major transfer of HK between cytosol and mitochondria. The activity of HKt and HKs was inhibited by glucose 1,6-bisphosphate (G-1,6-P2) in a concentration dependent manner in the basal state, and the sensitivity to G1,6-P2 inhibition was not altered by any condition. The activity of HKt and HKs in the presence of a subsaturating level of glucose (0.1 mM) accounted for approximately 70% of the activity at 1 mM glucose, and this percentage was not altered by any condition. These data suggest that under the present conditions alterations in the rates of whole body glucose disposal cannot be associated with alterations in HK distribution between cellular compartments nor its measured kinetics properties.

The soluble but not mitochondrially bound hexokinase is a substrate for the ATP- and ubiquitin-dependent proteolytic system

Intracellular protein degradation is highly selective, however, the mechanism(s) underlying this selectivity are not fully understood. We bave previously shown that purified rabbit hexokinase type I, an enzyme present in mammalian brain both in soluble and mitochondrial bound form, is conjugate to ubiquitin and then degraded by a rabbit reticulocyte fraction II. In the present study we report that the mitochondrial bound hexokinase is stable for several hours in the same proteolytic system both in the presence or absence of ATP. E 1, E 2 and E 3 the enzymes of the ubiquitin conjugating system, are able to incorporate 125I- or biotin-labelled ubiquitin in an ATP-dependent manner in soluble hexokinase as well as in a number of mitochondrial proteins. Furthermore, the mitochondria by themselves have a pronounced ATP-dependent ability to conjugate 125I-ubiquitin. However, Western blotting experiments, using a specific antibody against hexokinase, or against ubiquitin, showed that the mitochondrial bound enzyme is neither ubiquitinated nor degraded. This result has been confirmed by purification of bound hexokinase from the brain mitochondrial fraction or following the incubation of intact mitochondria with ATP, 125I-ubiquitin and E 1, E 2 and E 3. Thus, mitochondrial bound hexokinase is not recognized by the ubiquitin conjugating system while the soluble enzyme is conjugate to ubiquitin and then degraded. Furthermore, the soluble hexokinase from rabbit brain was isolated by immunoaffinity chromatography and shown to be recognized by an anti-ubiquitin antibody. These results suggest that the intracellular distribution of protein is an important feature of a protein which determines its susceptibility to ubiquitin-dependent degradation.

Erythrocyte membrane-bound acetylcholinesterase in vitamin E deficient rabbits

1. The specific activities of erythrocyte membrane-bound acetylcholinesterase (EC 3.1.1.7.) and soluble hexokinase (EC 2.7.1.1.) in vitamin E deficient and vitamin E sufficient rabbits were investigated. 2. Acetylcholinesterase specific activities values of 43.4 in deficient and 57.4 in sufficient vitamin E rabbits were obtained. Hexokinase specific activity was not modified, and values of 3.31 in deficient and 3.6 in controls were found. 3. No peroxidation process was detected by us on vitamin E deficient diets. 4. These observations would suggest that the membrane stabilizing effect of vitamin E may be accomplished by a mode of action not necessarily related to its ability to prevent lipid peroxidation.

Effects of kainate on glucose metabolising enzymes in the brain

Hexokinase and glucose-6-phosphate dehydrogenase activities were studied in brain regions after intraventricular injection of kainic acid. Hexokinase activity was decreased by 10–15% in various regions while glucose-6-phosphate dehydrogenase activity remained unaltered. Soluble hexokinase activity, which remained the smaller fraction of total hexokinase activity, showed slightly more dramatic decreases of 15–35% compared to normal activities in brain regions. This decrease of hexokinase activity in the cytosolic compartment could partly account for the kainate-induced decreases seen in glucose metabolism.

Treatment of frostbite with the calmodulin antagonists thioridazine and trifluoperazine

1. 1. Thioridazine and trifluoperazine, which have been previously found in this laboratory to be the most effective calmodulin antagonists in treatment of burns, are shown here to be also effective in the treatment of frostbite. 2. 2. Electron microscopic studies have revealed a complete reversal of both the vascular and skin tissue damage induced by frostbite. 3. 3. The reversal of the vascular damage was also demonstrated by the ability of these compounds to abolish the increase in hemoglobin content in the skin. 4. 4. The reversal of the skin tissue damage was also revealed by the ability of these compounds to raise the decreased ATP level and the reduced activities of 6-phosphogluconate dehydrogenase and mitochondrial and soluble hexokinase in skin, induced by frostbite, to normal control levels.

Therapeutic and prophylactic treatment of skin burns with several calmodulin antagonists

1. 1. Several calmodulin antagonists abolished the decrease in ATP level and in the activities of 6-phosphogluconate dehydrogenase and mitochondrial and soluble hexokinase, induced by burns in the rat skin. 2. 2. These antagonists had also a protective action on the blood capillaries and erythrocyte membrane, as judged by the electron microscopic appearance, as well as the abolishment of hemoglobin increase and burn edema. 3. 3. Of all the compounds investigated here, the most effective were trifluoperazine and thioridazine, which are also known as the more potent calmodulin antagonists. 4. 4. The present experiments suggest that calmodulin antagonists may be effective drugs in treatment of burns, having both therapeutic and prophylactic action.

Insulin effect on [ 14C]-valine incorporation and its relation to hexokinase activity in developing brain

Using minced brain cortex from fetal and postnatal rats, we studied the incorporation of [ 14C]-valine into protein in the presence of insulin. We also assayed the “particle bound” and soluble hexokinase in these tissues. Insulin significantly stimulated the incorporation of [ 14C]-valine into brain proteins from fetal stage upto 2 days of life. After this period the insulin effect was minimal, with no effect by day 5. The “particle bound” (40,000g pellet) brain hexokinase, on the other hand, remained low till about 2 days of life and then increased to almost adult level by 5 days. Our results show that there is an inverse relation between this anabolic effect of insulin and the “particle bound” hexokinase activity in the cortex of developing rat brain.

Purification, properties, and evidence for two subtypes of human placenta hexokinase type I

In human placenta 85% of total hexokinase activity (EC 2.7.1.1) was found in a soluble form. Of this, 70% is hexokinase type I while the remaining 30% is hexokinase type II. All the bound hexokinase is type I. Soluble hexokinase I was purified 11,000-fold by a combination of ion-exchange chromatography, affinity chromatography, and dye-ligand chromatography. The specific activity was 190 units/mg protein with a 75% yield. The enzyme shows only one band in nondenaturing polyacrylamide gel electrophoresis that stains for protein and enzymatic activity; however, two components (with M r 112,000 and 103,000) were constantly seen in sodium dodecyl sulfate-gel electrophoresis. Many attempts were made to separate these two proteins under native conditions; however, only one peak of activity was obtained when the enzyme was submitted to gel filtration ( M r 118,000), preparative isoelectric focusing (p I5.9), anion-exchange chromatography, hydroxylapatite chromatography, and affinity chromatography on immobilized dyes and immobilized glucosamine. The high and low molecular weight hexokinases show the same isoelectric point under denaturing conditions as determined by two-dimensional gel electrophoresis. Each hexokinase subtype was obtained by preparative sodium dodecyl sulfate electrophoresis followed by electroelution. Monospecific antibodies raised in rabbits against electroeluted high and low molecular weight hexokinases were not able to recognize the native enzymes but each of them detected both hexokinases on immunoblots. Amino acid compositions and peptide mapping by limited proteolysis of the high and low molecular weight hexokinases were also performed and suggested a strong homology between these two subtypes of human hexokinase I.

Continuous monitoring of adenosine 5'-triphosphate in the microenvironment of immobilized enzymes by firefly luciferase.

The study of enzymes sequestered in artificial or biological systems is generally conducted by indirect methodology with macroscopic measurements of reactants in the bulk medium. This paper describes a new approach with firefly luciferase to monitor ATP concentration directly in the microenvironment of enzymes producing or consuming ATP. Upon addition of ATP to immobilized firefly luciferase, the onset of light production is slower than that observed with the soluble enzyme, due to a slower diffusion of ATP to the immobilized enzyme. With immobilized pyruvate kinase, a relative accumulation of ATP inside the beads is demonstrated, as measured with coimmobilized firefly luciferase. The accumulation of product (ATP) is enhanced when the bead suspension is not stirred. This ATP in the beads is relatively inaccessible to soluble hexokinase added to the bulk medium. Similarly, a rapid ATP depletion in the microenvironment of immobilized hexokinase is demonstrated. This microscopic event is kinetically distinguishable from the slower macroscopic depletion of substrate in the bulk medium. The rate of depletion in the microenvironment depends on the local activity of the immobilized enzyme but not on the total amount of enzyme in suspension, as does the macroscopic phenomenon. The theoretical principles for the interaction of diffusion and catalysis in these systems are briefly summarized and discussed. These results are relevant to various molecular mechanisms proposed for membrane-bound enzyme action and regulation, derived from macroscopic kinetic measurements assuming a negligible diffusion control.

Regulation of glucokinase activity in the domestic fowl

The factors which regulate soluble and particulate glucokinase and hexokinase activity in the liver of domestic chickens has been investigated. Pretreatment with oral administration (via tube feeding) of glucose plus injection of insulin resulted in a significant increase in the activity of soluble (p less than 0.01) and particulate (p less than 0.01) glucokinase activity whereas fasting for 48 hours reduced glucokinase and hexokinase activity (p less than 0.01) in the particulate fraction only. Treatment of fed chickens for 2 weeks with thyroxine (50 micrograms: i.m. daily) plus triiodothyronine (50 micrograms) resulted in a marginal decrease (NS) in soluble glucokinase activity but significantly increased soluble hexokinase (p less than 0.05) activity. Thyroidectomized animals showed a decline in both soluble glucokinase (p less than 0.01) and hexokinase (p less than 0.025) activity. There was no effect of thyroid hormone manipulation on particulate glucokinase activity although there was a significant reduction in particulate hexokinase activity (p less than 0.05) in thyroidectomized birds. These data establish a physiological role for the glucokinase enzyme activity in avian carbohydrate metabolism and suggest that in contrast with the mammal, the particulate fraction is the more physiologically important enzyme.