Studies on the functional significance of mitochondrial bound hexokinase in rabbit reticulocytes

Mitochondria from rabbit reticulocytes contain about 50% of the total reticulocyte hexokinases. The proportion of mitochondrial hexokinases may be changed under different metabolic conditions. Mitochondrial bound and soluble hexokinases exhibit different kinetic properties (KMATP and glucose-6-phosphate inhibition). The respiratory rate of isolated reticulocyte mitochondria in the presence of glucose depends on the glucose-6-phosphate concentration, as the ADP generation by the endogenous hexokinases is strongly inhibited by glucose-6-phosphate. In the experimental system all intermediary states of mitochondrial respiration can be adjusted between the state of maximal activity (state 3 or active state) and the controlled or resting state (state 4) by different glucose-6-phosphate levels. The stationary levels of the extramitochondrial adenine nucleotides in this experimental system have been measured. The rate of mitochondrial respiration and ATP formation depends on the extramitochondrial ATP/ADP ratio. At ratios of about 10 and lower the mitochondria are in their maximum phosphorylation state, at higher ratios the mitochondrial ATP formation is controlled by the extramitochondrial ATP/ADP ratio. It is postulated that the close intercounnection between the mitochondrial hexokinase and the mitochondrial ATP forming system in reticulocytes is of funcitonal significance for mitochondrial-cytosolic interactions in rabbit reticulocytes and probably in other types of cells with mitochondrial hexokinases, too.     

Phosphofructokinase is responsible for the fructose 2,6-bisphosphate inhibition of hexokinase in tissue extracts

Mammalian and yeast hexokinases were reported to be reversibly inhibited by fructose 2,6-bisphosphate in the presence of cytosolic proteins (H. Niemeyer, C. Cerpa, and E. Rabajille (1987) Arch. Biochem. Biophys. 257, 17-26). Reinvestigation of this finding using a radioassay with [14C]glucose as substrate showed no effect of fructose 2,6-bisphosphate on hexokinase activity of rat liver cytosols. Detailed reexamination of the spectrophotometric assay resulted in the observation that the fructose 2,6-bisphosphate-dependent inhibition was a function of the cytosolic phosphoglucose isomerase and phosphofructokinase activities compared to the amount of glucose-6-phosphate dehydrogenase used as auxiliary enzyme. The diminution or loss of the fructose 2,6-bisphosphate-dependent inhibition produced in aged cytosols was restored by addition of crystalline muscle phosphofructokinase, as well as by decreasing the amount of glucose-6-phosphate dehydrogenase in the assay. When phosphoglucose isomerase, phosphofructokinase, and hexokinase activities were separated by DEAE-chromatography of liver cytosol, no fructose 2,6-bisphosphate-dependent inhibition of hexokinase was found in any single fraction of the chromatogram. However, combination of fractions containing both phosphoglucose isomerase and phosphofructokinase displayed the fructose 2,6-bisphosphate-dependent inhibition on either endogenous hexokinase or added yeast hexokinase. From these results we conclude that the activation of phosphofructokinase elicited by fructose 2,6-bisphosphate is responsible for the hexokinase inhibition observed in the coupled spectrophotometric assay.     

Glucose phosphorylation and dephosphorylation in chicken liver.

1. Glucokinase was absent from chicken liver and only the low Km hexokinases, inhibited by AMP, ADP but not ATP, were present. 2. The Km of chicken liver glucose-6-phosphatase for glucose-6-phosphate was reduced from 5.65 to 3.75 mM following starvation, and the enzyme was inhibited by glucose. 3. Starvation of chickens for 24 hr slightly lowered the hexokinase activity and doubled glucose-6-phosphatase activity; it did not change subcellular distribution of the enzymes. Oral glucose rapidly restored the activities to fed values. 4. It was concluded that glucose uptake into, and efflux from, chicken hepatocytes, was regulated by the activity and kinetic characteristics of glucose-6-phosphatase and by the glucose-6-phosphate concentration, and that the hexokinases had little regulatory function.     

Subcellular distribution and kinetic properties of cytosolic and non-cytosolic hexokinases in maize seedling roots: implications for hexose phosphorylation.

Hexose phosphorylation by hexokinases plays an important role in glycolysis, biosynthesis and control of sugar-modulated genes. Several cytosolic hexokinase and fructokinase isoforms have been characterized and organelle-bound hexokinases have also been detected in higher plants. In this study a hexokinase activity is described that is inhibited by ADP (K(i)=30 microM) and mannoheptulose (K(i) congruent with 300 microM) in non-cytosolic fractions (mitochondria, Golgi apparatus and microsomes) obtained from preparations of seedling roots of maize (Zea mays L.). The catalytic efficiency (Vmax/Km) for both ATP and glucose in all non-cytosolic hexokinase fractions is more than one order of magnitude higher than that of cytosolic hexokinase and fructokinases. Low (30%) or no ADP and mannoheptulose inhibition is observed with hexokinase and fructokinase activities derived from the cytosolic compartment obtained after ion exchange and affinity chromatography. The soluble fructokinase (FK) shows fructose cooperativity (Hill n>2). The Vmax/Km ratio is about 3-fold higher for ATP than for other NTPs and no difference for hexose phosphorylation efficiencies is found between cytosolic hexokinase and fructokinase isoforms (FK1, FK2) with ATP as substrate. The K(i) for fructose inhibition is 2 mM for FK1 and 25 mM for FK2. The data indicate that low energy-charge and glucose analogues preferentially inhibit the membrane-bound hexokinases possibly involved in sugar-sensing, but not the cytosolic hexokinases and fructokinases.     

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.     

Glucose repression in Saccharomyces cerevisiae is directly associated with hexose phosphorylation by hexokinases PI and PII

Genetic and biochemical analyses showed that hexokinase PII is mainly responsible for glucose repression in Saccharomyces cerevisiae, indicating a regulatory domain mediating glucose repression. Hexokinase PI/PII hybrids were constructed to identify the supposed regulatory domain and the repression behavior was observed in the respective transformants. The hybrid constructs allowed the identification of a domain (amino acid residues 102-246) associated with the fructose/glucose phosphorylation ratio. This ratio is characteristic of each isoenzyme, therefore this domain probably corresponds to the catalytic domain of hexokinases PI and PII. Glucose repression was associated with the C-terminal part of hexokinase PII, but only these constructs had high catalytic activity whereas opposite constructs were less active. Reduction of hexokinase PII activity by promoter deletion was inversely followed by a decrease in the glucose repression of invertase and maltase. These results did not support the hypothesis that a specific regulatory domain of hexokinase PII exists which is independent of the hexokinase PII catalytic domain. Gene disruptions of hexokinases further decreased repression when hexokinase PI was removed in addition to hexokinase PII. This proved that hexokinase PI also has some function in glucose repression. Stable hexokinase PI overproducers were nearly as effective for glucose repression as hexokinase PII. This showed that hexokinase PI is also capable of mediating glucose repression. All these results demonstrated that catalytically active hexokinases are indispensable for glucose repression. To rule out any further glycolytic reactions necessary for glucose repression, phosphoglucoisomerase activity was gradually reduced. Cells with residual phosphoglucoisomerase activities of less than 10% showed reduced growth on glucose. Even 1% residual activity was sufficient for normal glucose repression, which proved that additional glycolytic reactions are not necessary for glucose repression. To verify the role of hexokinases in glucose repression, the third glucose-phosphorylating enzyme, glucokinase, was stably overexpressed in a hexokinase PI/PII double-null mutant. No strong effect on glucose repression was observed, even in strains with 2.6 U/mg glucose-phosphorylating activity, which is threefold increased compared to wild-type cells. This result indicated that glucose repression is only associated with the activity of hexokinases PI and PII and not with that of glucokinase.     

Inhibition of brain hexokinase by a multisubstrate analog results from binding to a discrete regulatory site

P1-(adenosine-5')-P3-(glucose-6)-triphosphate (Ap3glucose) is a linear uncompetitive inhibitor vs glucose and a linear mixed inhibitor vs ATP of brain hexokinase, an inhibition pattern inconsistent with binding of Ap3glucose to the catalytic site when either the rapid equilibrium random or ordered sequential mechanism, which have been proposed for this enzyme, is considered. It is concluded that inhibition results from binding to a discrete regulatory site. The apparent ability of the regulatory site to accommodate both hexose and nucleotide moieties is consistent with suggestions by previous investigators that the regulatory site on mammalian hexokinases may have evolved from what was originally a catalytic site.     

Genetic and biochemical evidence for hexokinase PII as a key enzyme involved in carbon catabolite repression in yeast

Summary Mutants with reduced hexokinase activity previously isolated as resistant to carbon catabolite repression of invertase and maltase (Zimmermann and Scheel, 1977) were allele tested with mutant strains of Lobo and Maitra (1977) which had defects in one or several of the genes coding for glucokinase and the two unspecific hexokinases. It could be demonstrated, that the mutation abolishing carbon catabolite repression had occurred in a gene allelic to the structural gene of hexokinase PII. Moreover, the defective mutant allele for hexokinase PII isolated by Lobo and Maitra (1977) was also defective in carbon catabolite repression. Neither glucokinase nor hexokinase PI showed any effect on this regulatory system. Biochemical analysis in crude extracts also showed altered kinetic properties of hexokinases in the hex1 mutants. The results directly support the hypothesis previously put forward, that one of the hexokinases is not only active as a catalytic, but also as a regulatory protein.     

Inhibition of hexokinase activity by a fructose 2,6-bisphosphate-dependent cytosolic protein from liver

Mammalian and yeast hexokinases were found to be reversibly inhibited by fructose 2,6-bisphosphate, an effect requiring the presence of a cytosolic protein factor. Experimental evidence suggests that this factor (inhibitor) is a regulatory protein, the interactions of which with hexokinases are modulated by fructose 2,6-bisphosphate. The Vmax of hexokinase D was decreased, and no changes on other kinetic parameters were observed. The inhibitor was present in fresh liver cytosol filtered through Sephadex G-25 and was partially isolated by negative absorption on DEAE-cellulose followed by ammonium sulfate fractionation. The inhibitor was also present in brain and kidney, but not in muscle. A molecular mass of 200,000 was determined by gel filtration. The inhibition was dependent on the concentrations of both the inhibitory protein and fructose 2,6-bisphosphate. No delay in fructose 2,6-bisphosphate inhibition was observed. Several other hexose phosphates were tested and were not effective. In the presence of amounts of inhibitor sufficient to produce complete inhibition of hexokinase D, the concentration of fructose 2,6-bisphosphate required to produce 50% inhibition was about 0.5 microM. The inhibitor was unstable and was stabilized by the presence of fructose 2,6-bisphosphate.     

D-glucose anomeric preference of hexokinases from animals and yeast

The D-glucose anomeric preference of hexokinases partially purified from animals (rat, mouse, and chicken) and baker's yeast (Saccharomyces cerevisiae) were investigated by the assay system with glucose-6-phosphate dehydrogenase as a coupling enzyme. With low Km hexokinases in animal tissues and cells, the ratios of Vmax for the beta-anomer to Vmax for the alpha-anomer (V beta/V alpha) were within a range from 1.3 to 1.5. In yeast, the V beta/V alpha value was 1.1 for hexokinase A, 0.8 for hexokinase B, and 1.4 for glucokinase. The possible explanation for D-glucose anomeric preference of hexokinase is discussed.     

Stabilization of hexokinases I and II of ELD cells by binding to mitochondria

Significance of the binding of hexokinase to mitochondria was examined with respect to stabilization of the enzyme by the binding. Stability during the incubation of the mitochondria-bound forms of hexokinases I and II, both prepared from Ehrlich-Lettre ascites hyperdiploid tumor cells (ELD cells), were compared with that of the corresponding free forms. During the incubation at pH 7.4 and 37 degrees C up to 60 min, hexokinase activities decreased gradually, and the decrease in the activity of the free form was much more marked than that of the bound form for both hexokinases. Hexokinase II was much less stable than I, and the activity of the free form of the former was almost lost by the incubation for 15 min. But, more than a half of the original activity of hexokinase II was retained even after 60 min of the incubation when the enzyme was bound to mitochondria. Addition of 50 mM glucose increased the stability of hexokinase II, but the stabilizing effect was less marked for hexokinase I. On the other hand, addition of 28 mg/ml of bovine serum albumin markedly stabilized hexokinase I to almost the same extent as was observed with mitochondria. On the contrary, the serum albumin had little stabilizing effect on hexokinase II. These findings indicate that the binding to mitochondria stabilizes the hexokinases of ELD cells, though the stability is different by nature between hexokinases I and II.     

Differences in the responses of brain cytosolic and mitochondrial hexokinases to three essential divalent metal ions

Rat brain cytosolic and mitochondrial hexokinase activities were undetectable without added divalent cations. Mg2+ activated cytosolic (K0.5 of Mg2+ = 343 +/- 13 microM) and mitochondrial (K0.5 of Mg2+ = 183 +/- 8 microM) hexokinase in a concentration-related manner. The corresponding values for Mn2+ were 702 +/- 99 and 413 +/- 21 microM respectively. Ca2+, however, activated both forms of hexokinase poorly. In the presence of Mg2+, both Mn2+ and Cu2+ were more potent inhibitors of cytosolic hexokinase than mitochondrial hexokinase, whereas the inhibition of Cd2+ and Ca2+ did not show such selectivity. These results demonstrate that brain mitochondrial and cytosolic hexokinases differ significantly in their responses to divalent cations.     

A novel type of chloroplast stromal hexokinase is the major glucose-phosphorylating enzyme in the moss Physcomitrella patens

Hexokinase catalyzes the first step in the metabolism of glucose but has also been proposed to be involved in sugar sensing and signaling both in yeast and in plants. We have cloned a hexokinase gene, PpHXK1, in the moss Physcomitrella patens where gene function can be studied directly by gene targeting. PpHxk1 is a novel type of chloroplast stromal hexokinase that differs from previously studied membrane-bound plant hexokinases. Enzyme assays on a knock-out mutant revealed that PpHxk1 is the major glucose-phosphorylating enzyme in Physcomitrella, accounting for 80% of the total activity in protonemal tissue. The mutant is deficient in the response to glucose, which in wild type moss induces the formation of caulonemal filaments that protrude from the edge of the colony. Growth on glucose in the dark is strongly reduced in the mutant. Sequence data suggest that most plants including Physcomitrella and Arabidopsis have both chloroplast-imported hexokinases similar to PpHxk1 and traditional membrane-bound hexokinases. We propose that the two types of plant hexokinases have distinct physiological roles.     

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 Mr 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 (Mr 118,000), preparative isoelectric focusing (pI 5.9), 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.     

Contrasting molecular population genetics of four hexokinases in Drosophila melanogaster, D. simulans and D. yakuba

As part of a larger study contrasting patterns of variation in regulatory and nonregulatory enzymes of the central metabolic pathways we have examined the molecular variation in four uncharacterized hexokinase genes unique to muscle, fat body, and testis in Drosophila melanogaster, D. simulans, and D. yakuba. Earlier isoenzyme studies had designated these genes as Hex-A, Hex-C, and Hex-t. There are two tightly linked testes-specific genes designated here as Hex-t1 and Hex-t2. Substantial and concordant differences across species are seen in levels of both amino acid and silent polymorphism. The flight muscle form Hex-A is the most conserved followed by the fat body hexokinase Hex-C and testis-specific hexokinases Hex-t1 and Hex-t2. While constraints acting at the amino acid level are expected, the silent polymorphisms follow this pattern as well. All genes are in regions of normal recombination, therefore hitchhiking and background selection are not likely causes of interlocus differences. In D. melanogaster latitudinal clines are seen for amino acid polymorphisms at the Hex-C and Hex-t2 loci. There is evidence for accelerated amino acid substitution in Hex-t1 that has lost residues known to be associated with glucose and glucose-6-phosphate binding. D. simulans shows substantial linkage phase structuring that suggests historical population subdivision.     

Cloning and biochemical characterization of a novel mouse ADP-dependent glucokinase

Glycolysis, the catabolism of glucose to pyruvate, is an iconic central metabolic pathway and often used as a paradigm for explaining the general principles of the regulation/control of cellular metabolism. The ubiquitous mammalian ATP-dependent hexokinases I-III and hexokinase IV, also termed glucokinase, initiate the process by phosphorylating glucose to glucose-6-phosphate. Despite glycolysis having been studied extensively for over 70 years and the last new mammalian ATP-dependent hexokinase isotype having been described in the 1960s, we report here the biochemical characterization of a recombinant ADP-dependent glucokinase cloned from a full-length Mus musculus cDNA, identified by sequence analysis. The recombinant enzyme is quite specific for glucose, is monomeric, has an apparent Km for glucose and ADP of 96 and 280 microM, respectively, and is inhibited by both high concentrations of glucose and AMP. The metabolic role of this enzyme in cells would be dependent on the relative level of its activity to those of the ATP-dependent hexokinases. The greatest advantage of an ADP-GK would clearly be during ischemia/hypoxia, clinically relevant conditions in multiple major disease states, by decreasing the priming cost for the phosphorylation of glucose, saving ATP.     

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.     

Hexokinase isoenzymes from the Novikoff hepatoma. Purification, kinetic and structural characterization, with emphasis on hexokinase C.

The purification to homogeneity of hexokinases B and C from the cytosol of rat Novikoff hepatoma was achieved by a protocol using an initial chromatography on Blue 2-agarose to separate the isoenzymes from each other. After that step each hexokinase was subjected to chromatography on DEAE-cellulose, hydroxyapatite and Sephacryl S-300, followed by re-chromatography on hydroxyapatite. The final preparations of hexokinases B and C had specific activities of 86 and 23.5 units/mg of protein respectively, and gave single bands on electrophoresis under non-denaturing conditions or in SDS/polyacrylamide gels. Mr values of about 100,000 were found for both isoenzymes either by Sephacryl S-300 chromatography or by SDS/polyacrylamide-gel electrophoresis. Values of apparent Km for glucose and ATP of pure hexokinase B were similar to those reported for the enzyme from other sources. The apparent Km value for glucose of hexokinase C was 0.025 mM. Marked inhibition of hexokinase C by glucose concentrations above 0.2 mM was found. The effect was partially relieved by ATP concentrations above 1 mM and was independent of pH. Glucose 6-phosphate was inhibitory, but the Ki value (0.18 mM) is higher than those reported for other animal hexokinases. The amino acid composition of hexokinase C was found to be similar to those reported for hexokinases B and D. Also, an immune serum directed against hexokinase A was able, at low dilutions, to bind hexokinases B and C. An immune serum directed against hexokinase C was able, at low dilutions, to bind hexokinase B and also, but weakly, hexokinase A.     

Pig red blood cell hexokinase: Regulatory characteristics and possible physiological role

The regulatory properties of pig erythrocyte hexokinase III have been studied. Among mammalian erythrocyte hexokinases, the pig enzyme shows the highest affinity for glucose and a positive cooperative effect with n_H = 1.5 at all the MgATP concentrations studied (for 0.5 to 5 mm). Glucose at high concentrations is also an inhibitor of hexokinase III. Similarly, the apparent affinity constant for MgATP is independent of glucose concentration. Uncomplexed ATP and Mg are both competitive inhibitors with respect to MgATP. Glucose 6-phosphate, known as a stronger inhibitor of all mammalian erythrocyte hexokinases, is a poor inhibitor for the pig enzyme (K_i = 120 μm). Furthermore, this inhibition is not relieved by orthophosphate as with other mammalian red blood cell hexokinases. A variety of red blood cell-phosphorylated compounds were tested and found to be inhibitors of pig hexokinase III. Of these, glucose 1,6-diphosphate and 2,3-diphosphoglycerate displayed inhibition constants in the range of their intracellular concentrations. In an attempt to investigate the role of hexokinase type III in pig erythrocytes some metabolic properties of this cell have been studied. The adult pig erythrocyte is able to utilize 0.27 μmol of glucose/h/ml red blood cells (RBC) compared with values of 0.56–2.85 μmol/h/ml RBC for the other mammalian species. This reduced capacity to metabolize glucose results from a relatively poor ability of the cell membrane to transport glucose. In fact, all the glycolytic enzymes were present and a low intracellular glucose concentration was measured (0.5 mm against a plasma level of 5 mm). Furthermore, transport and utilization were concentration-dependent processes. Inosine, proposed as the major energy substrate of the pig erythrocyte, at physiological concentrations is not as efficient as glucose in maintaining reduced glutathione levels under oxidative stress. Furthermore, newborn pig erythrocytes (fully permeable to glucose) possess hexokinase type II as the predominant glucose-phosphorylating activity. This fact and the information derived from the study of the regulatory characteristics of hexokinase III and from metabolic studies on intact pig erythrocytes permit the hypothesis that the presence of this peculiar hexokinase isozyme (type III) enables the adult pig erythrocyte to metabolize low but appreciable amounts of glucose.