Studies on the mode of sugar-phosphate product inhibition of brain hexokinase.

Hexokinase I (ATP:d-hexose 6-phosphotransferase, EC 2.7.1.1), a key regulatory glycolytic enzyme in certain tissues, is known to be markedly inhibited under physiological conditions. The action of the primary inhibitory effector, glucose-6-P, is reversed by inorganic orthophosphate (P_i). A molecular model for inhibition and deinhibition of hexokinase was recently proposed [Ellison, W. R., Lueck, J. D., and Fromm, H. J. (1975) J. Biol. Chem.250, 1864–1871]. One of the central assumptions of this model is that glucose-6-P is a normal product inhibitor of hexokinase. It has long been suggested that glucose-6-P is an allosteric inhibitor of hexokinase, whereas other sugar-phosphate products such as mannose-6-P are normal product inhibitors. In this report we investigated the kinetic mechanism of hexokinase action with mannose as substrate and mannose-6-P as an inhibitor. The data obtained show that there are no qualitative differences between glucose and mannose as substrates and glucose-6-P and mannose-6-P as inhibitors. Binding experiments indicate that glucose-6-P and mannose-6-P are competitive binding ligands with hexokinase I. Furthermore, the activation pattern observed with Pi and glucose-6-P inhibited hexokinase is also found with the mannose-6-P inhibited phosphotransferase. These findings suggest that the mechanism of inhibition of glucose-6-P and mannose-6-P represents a difference in degree rather than a difference in kind. An explanation of the results in terms of a stereochemical model is presented.     

Okadaic acid-induced transcriptional downregulation of type I collagen gene expression is mediated by protein phosphatase 2A

Summary In Saccharomyces cerevisiae, a small proportion of the glucose-6-P dehydrogenase activity is firmly associated with the mitochondrial fraction and is not removed by repeated washing or density-gradient centrifugation. However, the enzyme is released by sonic disruption. Mitochondrial glucose-6-P dehydrogenase that is released by sonication and partially purified has been found to be similar to cytosol glucose-6-P dehydrogenase with respect to electrophoretic mobility, isoelectric point, pH optimum, molecular size, and apparent K _m 's for NADP⁺ and glucose-6-P. These results indicate that a single species of glucose-6-P dehydrogenase is synthesized in S. cerevisiae and that the enzyme has more than one intracellular location. Mitochondrial glucose-6-P dehydrogenase may be a source of intramitochondrial NADPH and may function with hexokinase and transhydrogenase to provide a pathway for glucose oxidation that is coupled to the synthesis of mitochondrial ATP. A constant proportion of total glucose-6-P dehydrogenase activity remains compartmented in the mitochondrial fraction throughout the growth cycle.     

Effect of inorganic phosphate on the reverse reaction of bovine brain hexokinase

Kinetic studies were used to investigate the mode of brain hexokinase (EC 2.7.1.1, ATP:D-hexose 6-phosphotransferase) regulation by glucose 6-phosphate (glucose-6-P), ADP, and inorganic phosphate (Pi). A model for regulation of brain hexokinase by glucose-6-P and Pi had been proposed from initial-rate studies and binding experiments [Ellison, W. R., Lueck, J. D., & Fromm, H. J. (1975) J. Biol. Chem. 250, 1864-1871]. The results of the present investigation demonstrate that Pi is an activator of the brain hexokinase reaction when the reaction is studied in the nonphysiological direction. Evidence is presented which indicates that the back-reaction substrates and Pi can bind the enzyme simultaneously, and the suggestion is made that Pi binds to an allosteric site on the enzyme. These findings are in marked contrast to results obtained in the absence of ADP which convincingly demonstrate that glucose-6-P and Pi are mutually exclusive binding ligands for brain hexokinase. The kinetic data can be reconciled with the model for hexokinase regulation within the context of the well-established kinetic mechanism for brain hexokinase.     

Further studies on the role of phospholipids in determining the characteristics of mitochondrial binding sites for type I hexokinase

Previous work has indicated that two types (A and B) of binding sites for hexokinase exist, but in different proportions, on brain mitochondria from various species. Hexokinase is readily solubilized from Type A sites by glucose 6-phosphate (Glc-6-P), while hexokinase bound to Type B sites remains bound even in the presence of Glc-6-P. Type A:Type B ratios are approximately 90:10, 60:40, 40:60, and 20:80 for brain mitochondria from rat, rabbit, bovine and human brain, respectively. The present study has indicated that MgCl2-dependent partitioning of mitochondrially bound hexokinase into a hydrophobic (Triton X-114) phase is generally correlated with the proportion of Type B sites. This partitioning behavior is sensitive to phospholipase C, implying that the factor(s) responsible for conferring hydrophobic character is(are) phospholipid(s). Substantial differences were also seen in the resistance of hexokinase, bound to brain mitochondria from various species, to solubilization by Triton X-100, Triton X-114, or digitonin. This resistance increased with proportion of Type B sites. Enrichment of bovine brain mitochondria in acidic phospholipids (phosphatidylserine or phosphatidylinositol), but not phosphatidylcholine or phosphatidylethanolamine, substantially increased solubilization of the enzyme after incubation at 37 degrees C. Collectively, the results imply that the Type A and Type B sites are located in membrane domains of different lipid composition, the Type A sites being in domains enriched in acidic phospholipids which lead to greater susceptibility to solubilisation by Glc-6-P.     

Magnetic resonance studies of the spatial arrangement of glucose-6-phosphate and chromium (III)-adenosine diphosphate at the catalytic site of hexokinase.

The interaction of CrADP, an exchange-inert paramagnetic analogue of Mg-ADP, with yeast hexokinase has been studied by measuring the effects of CrADP on the longitudinal nuclear relaxation rate (1/T1) of the protons of water and the protons and phosphorus atom of enzyme-bound glucose-6-P. The paramagnetic effect of CrADP on 1/T1 of water protons is enhanced upon complexation with the enzyme. Titrations measuring this paramagnetic effect at several enzyme concentrations in the presence of glucose-6-P yielded a characteristic enhancement factor for 1/T1 of water protons and the dissociation constant of CrADP from the ternary enzyme . ADPCr . glucose-6-P complex. The latter value (2 mM) is similar to that obtained from kinetic inhibition studies (Danenberg and Cleland [1975]. Biochemistry. 14:28). The presence of glucose-6-P increased the enhancement of the water relaxation rate by enzyme-bound CrADP, suggesting the formation of an enzyme . CrADP . glucose-6-P complex. The existence of such a complex was confirmed by the observation of a paramagnetic effect of enzyme-bound CrADP on the l/T1 of the 31P-nucleus and protons of enzyme-bound glucose-6-P. From the paramagnetic effects of enzyme-bound CrADP on the relaxation rates of the 31P-nucleus and the carbon-bound protons of glucose-6-P in the enzyme . ADPCr . glucose-6-P complex, using the correlation time of approximately 0.7 ns, determined from the magnetic field-dependence of 1/T1 of water protons over the range 24.3-360 MHz, a Cr3+ to phosphorus distance of 6.6 +/- 0.7 A and Cr3+ to alpha- and beta-anomeric proton distances of 8.9 and 9.7 A were calculated. These results imply the absence of a direct coordination of the phosphoryl group of glucose-6-P by the nucleotide-bound metal on hexokinase but indicate van der Waals contact between a phosphoryl oxygen of glucose-6-P and the hydration sphere of the nucleotide-bound metal. The distances are consistent with a model that assumes molecular contact between the phosphorus of glucose-6-P and a beta-phosphoryl oxygen of ADP suggesting an associative phosphoryl transfer. Because after phosphorylation of ADP, the metal ion is coordinated to the transferred phosphoryl group, the overall migration of the phosphoryl group during the phosphoryl transfer is approximately 3.6 A toward the nucleotide-bound metal. Little or no catalysis of phosphoryl transfer from glucose-6-P to alpha, beta-bidentate or beta-monodentate CrADP ( less than or equal to 0.05% of the rate found with MgADP) occurred in the presence of hexokinase, as monitored by glucose formation in a coupled assay system using glucose oxidase and peroxidase. The ability of beta, gamma-bidentate CrATP to act as a substrate (Danenberg and Cleland [1975].     

Initial rate and isotope exchange studies of rat skeletal muscle hexokinase

The kinetic mechanism of rat skeletal muscle hexokinase (hexokinase II) was investigated in light of a proposal by Cornish-Bowden and his co-workers (Gregoriou, M., Trayer, I. P., and Cornish-Bowden, A. (1983) Eur. J. Biochem. 134, 283-288). These investigators reported that the kinetic mechanism is ordered, with glucose adding before ATP and ADP dissociating from hexokinase before glucose-6-P. In addition, these workers suggest that glucose-6-P and ATP add to allosteric sites on hexokinase. We investigated the mechanism of action of hexokinase II by studying initial rate kinetics in the nonphysiological direction and by isotope exchange at chemical equilibrium. The former experiments were carried out in the absence of inhibitors and then with AMP, which is a competitive inhibitor of ADP, and with glucose 1,6-bisphosphate, a competitive inhibitor of glucose-6-P. The findings from these experiments suggest that the kinetic mechanism is rapid equilibrium Random Bi Bi. Isotope exchange at equilibrium studies also supports the random nature of the muscle hexokinase reaction; however, they also suggest that the mechanism is partially ordered, i.e. there is a preferred pathway associated with the branched mechanism. Approximately two-thirds of the flux through the hexokinase reaction involves the glucose on first glucose-6-P off last branch of the Random Bi Bi mechanism. These results imply that the kinetic mechanism is steady state Random Bi Bi. There is some evidence to suggest that glucose-6-P binds to an allosteric site on muscle hexokinase, but none to suppose that ATP binds allosterically. Analysis of the mechanism of Gregoriou et al. suggests that it is at variance with the findings of this report as well as with data available from other laboratories.     

Studies on the kinetics and mechanism of orthophosphate activation of bovine brain hexokinase

The binding of glucose to bovine brain hexokinase, isozyme I, exhibited one binding site per 100,000 molecular weight. Glucose-6-P binding was examined in the absence and presence of ATP. ATP and glucose-6-P were shown to compete for the same binding site on the enzyme. A model was proposed to account for these findings and the previously reported data that glucose-6-P and P_i exhibit mutually exclusive, non-cooperative binding to the enzyme. The model shows that brain hexokinase exists in two rapidly interconvertible states, either with or without P_i and that glucose-6-P binding to the phosphate associated enzyme form is relatively very poor. This proposal has been tested kinetically and the data appear to support the suggested model.     

Studies on the interactions between phospholipids and membrane-bound enzymes in microsomes. Effects of phospholipases C on the glucose-6-phosphatase system of rat liver microsomes

The role of phospholipids in the glucose-6-phosphatase system, including glucose-6-P phosphohydrolase and glucose-6-P translocase, was studied in rat liver microsomes by using phospholipases C and detergents. In the time course experiments on detergent exposure, the maximal activation of glucose-6-P phosphohydrolase varied according to the nature of the detergent used. On treatment of microsomes with phospholipase C of C. perfringens, the activity of glucose-6-P phosphohydrolase without detergent (i.e. without rupture of translocase activity) was gradually decreased with the progressive hydrolysis of phosphatidylcholine and phosphatidylethanolamine on the microsomal membrane, and was restored by incubation of these microsomes with egg yolk phospholipids. The extent of decrease in this phosphohydrolase activity in the detergent-exposed microsomes (with rupture of translocase activity) also varied depending on the detergent used (Triton X-114 or taurocholate). When 66% of the phosphatidylinositol on the membrane was hydrolyzed by phosphatidylinositol-specific phospholipase C of B. thuringiensis, the inhibition of glucose-6-P phosphohydrolase activity without detergent was very small. Although the inhibition of enzyme activity with detergent was apparently greater than that without detergent, the enzyme activity was stimulated by the breakdown of phosphatidylinositol when the enzyme activity was measured at lower concentration (0.5 mM) of substrate, glucose-6-P. The latency of mannose-6-P phosphohydrolase, a plausible index of microsomal integrity, remained above 70% after the hydrolysis of phosphatidylcholine, phosphatidylethanolamine, or phosphatidylinositol. The results show that the glucose-6-phosphatase system requires microsomal phospholipids for its integrity, suggesting that there exists a close relation between phosphatidylinositol and glucose-6-P translocase.     

The role of adenylate kinase in the regulation of the rate and effectiveness of energy transfer from mitochondria to hexokinase in vitro

The effect of adenylate kinase activity on the rate and efficiency of energy transport from mitochondria to hexokinase was studied in a system containing isolated rabbit heart mitochondria, hexokinase and adenylate kinase at low concentrations of adenine nucleotides. Oxygen consumption by mitochondria and glucose-6-phosphate synthesis by hexokinase were recorded. It was found that with adenylate kinase being active both in mitochondria and in the washing solution, the rate and efficiency of glucose-6-phosphate synthesis considerably increases. The effects of adenylate kinase activity are fully abolished by diadenosine pentaphosphate, an inhibitor of adenylate kinase. The experimental results based on the use of adenylate kinase demonstrate the possibility of increasing the rate and efficiency of energy transfer between two spatially uncoupled biochemical processes in vitro with the aid of an enzymatic system.     

Activity of glucose-6-phosphate metabolism enzymes in the livers of rats with experimental valekson poisoning]

The activity of hexokinase, glucose-6-phosphatase and glucose-6-phosphoric dehydrogenase was studied in the liver of rats after one hour, one and five days after a single oral administration of organic phosphorus insecticide valekson. It was determined that administration of the preparation led to an increase of activity in the homogenate and solubilization of glucose-6-phosphatase, activation of glucose-6-phosphoric dehydrogenase and inhibition of hexokinase. The changes were maximum one hour after the administration of the compound. The results show that a decrease of the intensity of glucose-6-phosphate formation and metabolism is one of the pathogenetic factors in the development of valekson-induced intoxication.     

Hexokinase activity alters sugar-nucleotide formation in maize root homogenates

Two pools of hexokinase activities differing in sensitivity to ADP inhibition were characterised in maize roots. In order to evaluate how glucose utilisation could be affected by these hexokinases, glucose-6-P and NDP-5'-sugar levels were measured after a D-[U-14C]glucose pulse in root extracts in the presence of 0 or 1 mM ADP. Analysis of radio-labelled activated sugars by paper chromatography revealed that: (1) without ADP, nearly 20% of the 14C appeared in NDP-5'-sugars; (2) 0.1 mM ADP inhibited 14C-NDP-5'-sugar formation by 85%; and (3) with 1 mM ADP, 14C-NDP-5'-sugars were undetectable, but substantial (14%) 14C accumulated as glucose-6-P. Mannoheptulose, a hexokinase inhibitor, blocked the NDP-5'-sugar formation, but did not modify the amount of 14C-glucose-6-P in root extracts either with or without ADP. The analysis of the hexokinase activities with 0.8 mM glucose in maize root extracts showed that: (1) mitochondrial hexokinase activity was totally inhibited by 30 mM mannoheptulose; and (2) the cytosolic hexokinase was inhibited by only 30%. These data suggest that NDP-5'-sugar synthesis is sensitive to ADP fluctuations and that mannoheptulose affects preferentially the mitochondrial-bound hexokinase, but the cytosolic form is less sensitive. We propose that the mitochondrial hexokinase is the main energy charge sensor in this pathway in maize.     

Kinetic properties of hexokinase under near-physiological conditions. Relation to metabolic arrest in Artemia embryos during anoxia

Previous analyses of glycolytic metabolites in Artemia embryos indicate that an acute inhibition of glucose phosphorylation occurs during pHi-mediated metabolic arrest under anoxia. We describe here kinetic features of hexokinase purified from brine shrimp embryos in an attempt to explain the molecular basis for this inhibition. At saturating concentrations of cosubstrate, ADP is an uncompetitive inhibitor toward glucose and a partial noncompetitive inhibitor toward ATP (Kis = 0.86 mM, Kii = 1.0 mM, Kid = 1.9 mM). With cosubstrates at subsaturating concentrations, the uncompetitive inhibition versus glucose becomes noncompetitive, while inhibition versus ATP remains partial noncompetitive. The partial noncompetitive inhibition of ADP versus ATP is characterized by a hyperbolic intercept replot. These product inhibition patterns are consistent with a random mechanism of enzyme action that follows the preferred order of glucose binding first and glucose-6-P dissociating last. We propose that inhibition by glucose-6-P (Kis = 65 microM) occurs primarily by competing with ATP at the active site, resulting in the formation of the dead-end complex, enzyme-glucose-glucose-6-P. Versus glucose, inhibition by glucose-6-P is uncompetitive at pH 8.0 and noncompetitive at pH 6.8. Over a physiologically relevant pH range of 8.0 to 6.8 alterations in Km and Ki values do not account for the reduction in glucose phosphorylation, and no evidence suggests that Artemia hexokinase activity is modulated by reversible binding to intracellular structures. Total aluminum in the embryos is 4.01 +/- 0.36 micrograms/g dry weight, or, based upon tissue hydration, 72 microM. This concentration of aluminum dramatically reduces enzyme activity at pH values less than 7.2, even in the presence of physiological metal ion chelators (citrate, phosphate). When pH, aluminum, citrate, phosphate, substrates, and products were maintained at cellular levels measured under anoxia, we can account for a 90% inhibition of hexokinase relative to activity under control (aerobic) conditions.     

The participation of glucose-1,6-diphosphate in the regulation of hexokinase and phosphoglucomutase activities in brains of young and adult rats

1. The level of glucose-1,6-diphosphate (Glc-1,6-P2), the powerful regulator of carbohydrate metabolism, was found to be strikingly decreased in brains of adult rats (5 months of age) as compared to young (10-14 days of age). 2. This age-related decrease in Glc-1,6-P2, the potent inhibitor of hexokinase and activator of phosphoglucomutase, was accompanied by a correlated increase in the activity of hexokinase and a reduction in phosphoglucomutase. 3. Evidence is provided showing that Glc-1,6-P2 participates in the regulation of these enzymes' activities with age. 4. The age-related changes in Glc-1,6-P2 and in the enzymes' activities in brain were opposite to those which we previously found in skeletal muscle. 5. These results suggest that Glc-1,6-P2 is involved in the regulation of carbohydrate metabolism during growth in both brain and muscle, as well as in the interrelationship between these two tissues.     

Ligand-induced conformations of rat brain hexokinase: effects of glucose-6-phosphate and inorganic phosphate

Binding of glucose-6-P induces conformational change in rat brain hexokinase (ATP:d-hexose 6-phosphotransferase, EC 2.7.1.1) as indicated by decreased susceptibility to digestion by chymotrypsin and an increased sedimentation coefficient on sucrose density gradients. These effects are competitively reversed by P_i, as are solubilization (of the mitochondrial form of hexokinase) and inhibition by glucose-6-P. Thus, the observed conformational changes are likely to be directly related to the effect of these ligands on catalytic activity and the interaction of the hexokinase with the mitochondrial membrane.Both glucose-6-P and P_i stabilize the enzyme against heat inactivation; this effect, as well as the effect of glucose-6-P on inactivation by chymotrypsin, have been used to estimate the dissociation constants for the complexes of hexokinase with glucose-6-P and P_i; the values are 7–8 μm, and 0.25 mm, respectively.These observations are consistent with a model in which brain hexokinase may exist in two distinct conformations, rapidly and reversibly interconvertible. The effect of glucose-6-P and P_i are explained by highly preferential binding to one or the other of these conformations.     

Mitochondrial hexokinase from differentiated and undifferentiated HT29 colon cancer cells: effect of some metabolites on the bound/soluble equilibrium

1. Solubilization of mitochondrial bound hexokinase (HK), which represents 75-80% of the total enzyme activity in the cells, was investigated in freshly isolated mitochondria from undifferentiated (Glc+) or differentiated (Glc-) HT29 adenocarcinoma cells. In both models, the bound HK is almost completely released in vitro by 100 microM glucose 6-P (G 6-P). 2. Free ATP (5 mM) or palmitate (800 microM) produce a partial solubilization of bound HK, more markedly in the case of Glc- mitochondria. 3. Glucose or glucose 1-P are found unable to solubilize bound HK. Glucose 1,6-P2, 2-deoxyglucose 6-P or glucosamine 6-P can solubilize the enzyme but are less efficient than G 6-P. 4. Mg2+ and Pi are found to counteract the glucose 6-P induced solubilization of HK in both types of mitochondria. Taking into account the intracellular concentrations of these ions, this could in part explain why, in HT29 cells, HK is predominantly bound to the mitochondria.     

Hysteretic behavior of the hepatic microsomal glucose-6-phosphatase system.

Carbamyl-P:glucose and PPi:glucose phosphotransferase, but not inorganic pyrophosphatase, activities of the hepatic microsomal glucose-6-phosphatase system demonstrate a time-dependent lag in product production with 1 mM phosphate substrate. Glucose-6-P phosphohydrolase shows a similar behavior with [glucose-6-P] less than or equal to 0.10 mM, but inorganic pyrophosphatase activity does not even at the 0.05 or 0.02 mM level. The hysteretic behavior is abolished when the structural integrity of the microsomes is destroyed by detergent treatment. Calculations indicate that an intramicrosomal glucose-6-P concentration of between 20 and 40 microM must be achieved, whether in response to exogenously added glucose-6-P or via intramicrosomal synthesis by carbamyl-P:glucose or PPi:glucose phosphotransferase activity, before the maximally active form of the enzyme system is achieved. It is suggested that translocase T1, the transport component of the glucose-6-phosphatase system specific for glucose-6-P, is the target for activation by these critical intramicrosomal concentrations of glucose-6-P.     

Enzyme levels in chick embryo heart and brain from 1 to 21 days of development

Enzyme activity levels were measured in chick embryo brain and heart during development, beginning with medullary plate and cardiogenic mesentoderm.To study heart and brain during the period of morphogenesis (1–4 days) a method for freezedrying whole chick embryos was developed. In three divisions of brain—diencephalon, telencephalon, and hindbrain-hexokinase, glyceraldehyde-3-P dehydrogenase, and 6-P-gluconic dehydrogenase maintained approximately constant levels of activity during this period. Brain glucose-6-P dehydrogenase levels fell somewhat, but contrary to earlier reports showed no wide fluctuations. In heart, glucose-6-P dehydrogenase activity fell to one-half between 1 and 4 days, 6-P-gluconic dehydrogenase activity remained constant, while hexokinase activity doubled in atrium from 1 to 2 days, and tripled in ventricle from 1 to 4 days.From 6 to 21 days of development, homogenates of hearts and brains were used. Hexokinase activity in brain increased four-fold during this period, while in heart the specific activity did not change. Glyceraldehyde-3-P dehydrogenase activity showed no change in either organ. NAD-dependent isocitric dehydrogenase increased in both heart and brain, fourfold in brain, nearly twofold in heart. α-Ketoglutaric dehydrogenase increased 50% in brain and 250% in heart.The increasing levels of citric acid cycle enzymes probably reflect an increasing energy demand in both organs during the last 2 weeks before hatching. Since adult brain depends primarily upon glucose for energy, it seems reasonable that the hexokinase activity continued to increase. Adult heart, however, obtains its energy from substrates other than glucose, which may account for the fact that during the last 2 weeks no change in heart hexokinase activity was seen.     

The role of hexokinase as a possible modulator of Ca2+ movements in isolated rat brain mitochondria

The present study shows that in brain mitochondria the active calcium uptake and the sodium-dependent calcium efflux are modulated by the porin-bound enzyme hexokinase. The release of the enzyme, promoted by glucose-6-phosphate (G-6-P), under conditions which do not affect mitochondrial functions, is accompanied by a decrease of the rates of fluxes of the cation. This phenomenon is discussed and correlated with the formation of microcompartments between the inner and outer mitochondrial membranes, where the hexokinase-porin complex may constitute a regulating gate system for calcium.     

Age-dependent changes in glucose 1,6-bisphosphate levels and in the activities of glucose 1,6-bisphosphatase, and particulate hexokinase and 6-phosphogluconate dehydrogenase in rat skin

The levels of glucose 1,6-bisphosphate (Glc-1,6-P2), the powerful regulator of carbohydrate metabolism, changed in rat skin during growth: Glc-1,6-P2 increased during the first week of age, and thereafter was dramatically reduced during maturation. The activity of glucose 1,6-bisphosphatase, the enzyme that degradates Glc-1,6-P2, changed with age in an invert manner as compared to the changes in Glc-1,6-P2. These findings suggest that the age dependent changes in this enzyme's activity may account for the changes in intracellular Glc-1,6-P2 concentration. The age-related changes in Glc-1,6-P2 were accompanied by concomitant changes in the activities of particulate (mitochondrial) hexokinase and 6-phosphogluconate dehydrogenase, the two enzymes known to be inhibited by Glc-1,6-P2. The activities of both these enzymes in the soluble fraction were not changed with age. The particulate enzymes were more susceptible to inhibition by Glc-1,6-P2 than the soluble activities, which may explain why only the particulate, but not the soluble activities, correlated with the age-dependent changes in tissue Glc-1,6-P2. These results suggest that the changes in particulate hexokinase and 6-phosphogluconate dehydrogenase resulted from changes in intracellular concentration of Glc-1,6-P2. The marked reduction in Glc-1,6-P2 during maturation, accompanied by activation of mitochondrial hexokinase and 6-phosphogluconate dehydrogenase, may reflect an enhancement in skin metabolism during growth.