Uptake of Yolk Very Low Density Lipoprotein by Chicken and Quail Embryos Is Not Mediated by a Homologue of the Oocyte Vitellogenesis Receptor

In chicken (Gallus domesticus) embryos, a limited amount of yolk engulfment occurs via coated invaginations at the yolk sac membrane apical surface. Because the presence of these so-called “coated pits” is associated with receptor-mediated endocytosis, the purpose of the present study was to demonstrate the existence on the yolk sac membrane of receptor sites for the interaction with very low density lipoprotein (VLDL), the major component of egg yolk. Ligand blotting experiments revealed the presence of a VLDL-binding protein (M_r ∼95 kDa) in yolk sac membranes of both chicken and Japanese quail (Coturnix coturnix japonica) embryos 8 days of age and older. However, these VLDL-binding proteins were present in very low abundance relative to that of another apolipoprotein B receptor that is found in the plasma membrane of chicken and quail oocytes (the so-called oocyte vitellogenesis receptor [OVR]; M_r 95 kDa). Furthermore, no signals were detected when chicken and quail yolk sac membrane proteins were probed with a rabbit polyclonal antibody raised against the 14 C-terminal amino acids of the chicken OVR. It was concluded that chicken and quail yolk sac membrane VLDL-binding proteins were structurally different from the chicken OVR and that receptor-mediated endocytosis plays a minor role in the uptake of yolk VLDL by developing avian embryos.     

Hexose kinases from Rhodotorula glutinis. Identification and properties of an hexokinase and a glucokinase.

An hexokinase (EC 2.7.1.1) and a glucokinase (EC 2.7.1.2) from the red yeast Rhodotorula glutinis are described. Both enzymes have been separated and some of their properties studied. The two enzymes share many properties, the K_mfor glucose is 0.1 mm for both enzymes and the K_m values for ATP are 0.5 mm and 0.6 mm respectively for hexokinase and glucokinase. The hexokinase shows a K_m of 2 mm for fructose and 0.1 mm for mannose; the glucokinase has a K_m for mannose of 0.2 mm. Both enzymes are constitutive, show competitive inhibition by N-acetylglucosamine and xylose, have weak affinity for glucosamine and exhibit a broad pH optimum. The molecular weights determined by gel filtration are 110,000 for glucokinase and 96,000 for hexokinase. The maximal activity of both hexose kinases nearly accounts for glucose utilization by Rh. glutinis.     

Incorporation of ZP1 into perivitelline membrane after in vivo treatment with exogenous ZP1 in Japanese quail (Coturnix japonica)

In birds, the egg envelope surrounding the oocyte prior to ovulation is called the perivitelline membrane and it plays important roles in fertilization. In a previous study we demonstrated that one of the components of the perivitelline membrane, ZP3, which is secreted from the ovarian granulosa cells, specifically interacts with ZP1, another constituent that is synthesized in the liver of Japanese quail. In the present study, we investigated whether ZP1 injected exogenously into the blood possesses the ability to reconstruct the perivitelline membrane of Japanese quail. When ZP1 purified from the serum of laying quail was injected into other female birds, the signal of this exogenous ZP1 was detected in the perivitelline membrane. In addition, we revealed, by means of ligand blot analysis, that serum ZP1 interacts with both ZP1 and ZP3 of the perivitelline membrane. By contrast, when ZP1 derived from the perivitelline membrane was administered, it failed to become incorporated into the perivitelline membrane. Interestingly, serum ZP1 recovered from other Galliformes, including chicken and guinea fowl, could be incorporated into the quail perivitelline membrane, but the degree of interaction between quail ZP3 and ZP1 of the vitelline membrane of laid eggs from chicken and guinea fowl appeared to be weak. These results demonstrate that exogenous ZP1 purified from the serum, but not ZP1 from the perivitelline membrane, can become incorporated into the perivitelline membrane upon injection into other types of female birds. To our knowledge, this is the first demonstration that the egg envelope component, when exogenously administered to animals, can reconstruct the egg envelope in vivo.     

Radioiodination of chicken luteinizing hormone without affecting receptor binding potency

By improving the currently used lactoperoxidase method, we were able to obtain radioiodinated chicken luteinizing hormone (LH) that shows high specific binding and low nonspecific binding to a crude plasma membrane fraction of testicular cells of the domestic fowl and the Japanese quail, and to the ovarian granulosa cells of the Japanese quail. The change we made from the original method consisted of 1) using chicken LH for radioiodination that was not only highly purified but also retained a high receptor binding potency; 2) controlling the level of incorporation of radioiodine into chicken LH molecules by employing a short reaction time and low temperature; and 3) fractionating radioiodinated chicken LH further by gel filtration using high-performance liquid chromatography. Specific radioactivity of the final 125I-labeled chicken LH preparation was 14 microCi/micrograms. When specific binding was 12-16%, nonspecific binding was as low as 2-4% in the gonadal receptors. 125I-Labeled chicken LH was displaced by chicken LH and ovine LH but not by chicken follicle-stimulating hormone. The equilibrium association constant of quail testicular receptor was 3.6 x 10(9) M-1. We concluded that chicken LH radioiodinated by the present method is useful for studies of avian LH receptors.     

Purification and properties of pyruvate kinase type M2 from rat lung

(1) Pyruvate kinase type M₂ from rat lung has been purified 840-fold with an overall yield of 20%. The enzyme gave a single band upon SDS-electrophoresis and isoelectrofocusing and had a specific activity of 1340 U/mg protein. The homotetramer of M_r = 224 000 and an isoelectric point of pH 5.8 had an amino acid composition closely resembling that of other pyruvate kinase isoenzymes type M₂, excepts that of the chicken liver. The enzyme was crystallized. (2) The enzyme has its pH optimum at pH 6.5. The K_0.5 value for phosphoenolpyruvate is 0.26 mM (n_H = 1.81) which decreases in the presence of 0.2 mM fructose 1,6-bisphosphate to 0.056 mM (n_H = 1.06). 1 μM fructose 1,6-bisphosphate activates the enzyme at 0.1 mM phosphoenolpyruvate half-maximally. The K_m value for ADP at 1 mM phosphoenolpyruvate is 0.4 mM. The K_m value for other nucleoside diphosphates increases in the order ADP<GDP<IDP<UDP. (3) No evidence for an interconversion of pyruvate kinase type M₂ from rat or chicken lung was found. The enzyme was neither a substrate for the cAMP-dependent protein kinase from rabbit muscle nor for the cAMP-independent protein kinase from chicken liver. Since pyruvate kinase type M₂ from chicken liver is inactivated by phosphorylation catalyzed by a cAMP-independent protein kinase (Eigenbrodt, E., Abdel-Fattah Mostafa, M. and Schoner, W. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358, 1047–1055) we suggest that the interconvertible form of pyruvate kinase type M₂ may represent a separate form of the pyruvate kinase type M₂ family.     

Allelic inhibition at the autosomally inherited gene locus for liver alcohol dehydrogenase in chicken-quail hybrids

At the gene locus for liver alcohol dehydrogenase (ADH) of the Japanese quail, three alleles which give electrophoretic variants, A, B, and C, exist. This enzyme is autosomally inherited. Allelic polymorphism was not observed in the chicken, but the wild-type ADH of the chicken can readily be distinguished from A, B, and C of the quail by starch gel electrophoresis. In the development of both species, ADH activity reached a near adult level at about the nineteenth day (a few days after hatching in the quail and a few days before hatching in the chicken). Chicken-quail hybrids at the day of hatching (nineteenth day) revealed the presence of maternally derived quail ADH only, and their ADH activities were about half that of both parental species. Those hybrids which received either A or C allele from the mother quail showed three bands of ADH at the third day after hatching. The chicken and quail alleles began to function in synchronous harmony. One 3-day-old and two adult hybrids which received B allele from the quail, however, still revealed complete absence of the paternally derived chicken ADH.This work was supported in part by a grant (CA-05138) from the National Cancer Institute, U.S. Public Health Service, and in part by a research fund established in honor of General James H. Doolittle. Contribution No. 20-67, Department of Biology, City of Hope Medical Center.Dr. Eduardo Castro-Sierra is a fellow of the Institute for Advanced Learning of the City of Hope Medical Center.     

Subcellular localization of hexose kinases in pea stems: mitochondrial hexokinase

The subcellular localization of hexose phosphorylating activity in extracts of pea stems has been studied by differential centrifugation and sucrose density gradient centrifugation. The hexokinase (EC 2.7.1.1) was associated with the mitochondria, whereas fructokinase (EC 2.7.1.4) was in the cytosolic fraction. Some properties of the mitochondrial hexokinase were studied. The enzyme had a high affinity for glucose (K_m 76 micromolar) and mannose (K_m 71 micromolar) and a relatively low affinity for fructose (K_m 15.7 millimolar). The K_m for MgATP was 180 micromolar. The addition of salts stimulated the activity of the hexokinase. Al³⁺ was a strong inhibitor at pH 7 but not at the optimum pH (8.2). The enzyme was not readily solubilized but, in experiments with intact mitochondria, was susceptible to proteolysis. A location on the outer mitochondrial membrane is suggested for the hexokinase of pea stems.     

Isolated mouse liver mitochondria are devoid of glucokinase

Glucokinase is a hexokinase isoform with low affinity for glucose that has previously been identified as a cytosolic enzyme. A recent report claims that glucokinase physically associates with liver mitochondria to form a multi-protein complex that may be physiologically important in apoptotic signaling [N.N. Danial, C.F. Gramm, L. Scorrano, C.Y. Zhang, S. Krauss, A.M. Ranger, S.R. Datta, M.E. Greenberg, L.J. Licklider, B.B. Lowell, S.P. Gygi, S.J. Korsmeyer, Nature 424 (2003) 952-956]. Here, we re-examined the association of glucokinase with isolated mouse liver mitochondria. When glucokinase activity was measured by coupled enzyme assay, robust activity was present in whole liver homogenates and their 9500 g supernatants (cytosol), but activity in the purified mitochondrial fraction was below detection (<0.2% of homogenate). Furthermore, addition of 45 mM glucose in the presence of ATP did not increase mitochondrial respiration, indicating the absence of ADP formation by glucokinase or any other hexokinase isoform. Immunoblots of liver homogenates and cytosol revealed strong glucokinase bands, but no immunoreactivity was detected in mitochondria. In conclusion, mouse liver mitochondria lack measurable glucokinase. Thus, functional linkage of glucokinase to mitochondrial metabolism and apoptotic signaling is unlikely to be mediated by the physical association of glucokinase with mitochondria.     

Hexokinase isoenzymes of RIN-m5F insulinoma cells. Expression of glucokinase gene in insulin-producing cells.

We have analysed the pattern of expression of the hexokinase isoenzyme group in RIN-m5F insulinoma cells. Three hexokinase forms were resolved by DEAE-cellulose chromatography. The most abundant isoenzyme co-eluted with hexokinase type II from rat adipose tissue and displayed a Km for glucose of 0.15 mM, similar to the adipose-tissue enzyme. Hexokinase type II was in large part associated with a particulate subcellular fraction in RIN-m5F cells. The two other hexokinases separated by ion-exchange chromatography were an enzyme similar to hexokinase type I from brain and glucokinase (or hexokinase type IV). The latter isoenzyme was identified as the liver-type glucokinase by the following properties: co-elution with hepatic glucokinase from DEAE-cellulose and DEAE-Sephadex; sigmoid saturation kinetics with glucose with half-maximal velocity at 5.6 mM and Hill coefficient (h) of 1.54; suppression of enzyme activity by antibodies raised against rat liver glucokinase; apparent Mr of 56,500 and pI of 5.6, as shown by immunoblotting after one- and two-dimensional gel electrophoresis; peptide map identical with that of hepatic glucokinase after proteolysis with chymotrypsin and papain. These data indicate that the gene coding for hepatic glucokinase is expressed in RIN-m5F cells, a finding consistent with indirect evidence for the presence of glucokinase in the beta-cell of the islet of Langerhans. On the other hand, the overall pattern of hexokinases is distinctly different in RIN-m5F cells and islets of Langerhans, since hexokinase type II appears to be lacking in islets. Alteration in hexokinase expression after tumoral transformation has been reported in other systems.     

Kinetic enhancement of bound hexokinase activity by mitochondrial respiration

These studies were designed to examine the functional relationship between respiring rat liver mitochondria and bound hexokinase. Kinetic studies were peformed varying either exogenously supplied ATP or ATP synthesized endogenously by respiring mitochondria and varied concentrations of ADP. Michaelis-Menten constants and maximum velocities were determined at two, five, and ten minutes after initiating the reactions. The K_m's and Vmax's were invariant with respect to added ATP, but the apparent K_m's varied considerably when endogenous substrate was utilized. At two minutes, the K_m for endogenous ATP was 25% of the K_m for provided ATP, but, by ten minutes, it had reached 70%. The Vmax's varied far less markedly. This is a clear demonstration of preferential utilization of mitochondrial ATP by bound hexokinase.     

Effect of oleate on the apparent Km of monoamine oxidase and the amount of membrane-bound hexokinase in isolated rat hepatocytes: Further evidence for the controlling role of the surface charge in hexokinase binding

Brief incubation of isolated rat hepatocytes in the presence of the oleate-bovine serum albumin complex resulted in a release to the cytosol of a portion of hexokinase (EC 2.7.1.1) normally bound to intracellular membranes. This was correlated with an increase of the negative surface potential of the outer mitochondrial membrane, as measured in situ by determining changes of K_m of monoamine oxidase (EC 1.4.3.4). It is suggested that non-esterified fatty acids produce a partial release of bound hexokinase in the liver cell by changing the surface charge of intracellular membranes.     

FISH on avian lampbrush chromosomes produces higher resolution gene mapping

Giant lampbrush chromosomes, which are characteristic of the diplotene stage of prophase I during avian oogenesis, represent a very promising system for precise physical gene mapping. We applied 35 chicken BAC and 4 PAC clones to both mitotic metaphase chromosomes and meiotic lampbrush chromosomes of chicken (Gallus gallus domesticus) and Japanese quail (Coturnix coturnix japonica). Fluorescence in situ hybridization (FISH) mapping on lampbrush chromosomes allowed us to distinguish closely located probes and revealed gene order more precisely. Our data extended the data earlier obtained using FISH to chicken and quail metaphase chromosomes 1–6 and Z. Extremely low levels of inter- and intra-chromosomal rearrangements in the chicken and Japanese quail were demonstrated again. Moreover, we did not confirm the presence of a pericentric inversion in Japanese quail chromosome 4 as compared to chicken chromosome 4. Twelve BAC clones specific for chicken chromosome 4p and 4q showed the same order in quail as in chicken when FISH was performed on lampbrush chromosomes. The centromeres of chicken and quail chromosomes 4 seem to have formed independently after centric fusion of ancestral chromosome 4 and a microchromosome.     

Noradrenaline and glycogen content and the activity of several enzymes of carbohydrate metabolism in normal, embryonic, and partly denervated livers and in hepatomas of the rat]

The noradrenaline and glycogen contents as well as hexokinase, glucokinase and glucose-6-phosphatase activities were determined in normal, embryonic and partially denervated (bilateral dissection of the Nervus splanchnicus or Nervus vagus) rat liver and in two transplantable hepatomas. In embryonic liver and hepatomas a strong decrease or complete loss of noradrenaline and glycogen levels and glucokinase and glucose-6-phosphatase activities is demonstrable as compared to the livers of adult animals, while the hexokinase activity is enhanced. Following bilateral splanchnicotomy the glycogen content and hexokinase activity are enhanced; the glucose-6-phosphatase activity is reduced, and the liver does not contain any noradrenaline. Bilateral vagotomy causes decrease of the glycogen content, of the hexokinase and glucokinase activities and an enhancement of glucose-6-phosphatase activity. The results lend support to the idea of antagonistic action of the sympathetic and parasympathetic nervous systems upon several partial reactions of carbohydrate metabolism of liver. In addition, it can be assumed that the alterations of the carbohydrate metabolism demonstrable in hepatomas as compared to normal liver are not solely attributable to disturbance or breakdown of the nervous regulation.     

Maintenance of glucokinase activity in primary hepatocyte cultures

When primary cultures of hepatocytes are maintained for 2 weeks from the time of perfusion, the activity of the enzyme glucokinase decreases rapidly, so that the activity can no longer be detected after the fourth day in culture. Concomitantly, there occurs an increase in the activity of hexokinases, the low-K_M isozymes, which predominate in fetal liver. We have made several modifications of the culture medium in an attempt to prevent the decrease in glucokinase activity. When the medium was supplemented with a mixture of insulin, thyroxine, glucagon, dexamethasone, testosterone, and estradiol, the activity of the enzyme in the hepatocytes was present at approximately 15% of in vivo levels after 2 weeks in culture. When this hormone mixture was present during the first 4 hrs of culture and when the hepatocytes were allowed to attach to the collagen support and were maintained thereafter in medium supplemented with fetal bovine serum, insulin, and dexamethasone, the activity of glucokinase increased after an initial decrease for 3 days and was maintained thereafter at levels comparable to those observed in vivo. This effect of the hormone mixture was found to be the result of the presence of glucagon in the mixture, since the presence of glucagon with no other hormones added, except insulin, during the attachment period produced the same pattern of increased glucokinase activity. Immunoprecipitation of glucokinase from the hepatocytes, using monospecific antibody, indicated that the increase in enzyme activity was the result of increased glucokinase enzyme protein and not an increased synthesis of the other hexokinase isozymes. These studies demonstrate the specific hormonal requirements for the maintenance of glucokinase levels in primary hepatocyte culture at those seen in vivo and lends support to the hypothesis that fetal gene expression in primary hepatocyte cultures is selectively regulated rather than being a general effect with a common regulatory mechanism.     

Modulation of human glucokinase intrinsic activity by SH reagents mirrors post-translational regulation of enzyme activity

The low-affinity glucose phosphorylating enzyme glucokinase plays a key role in the process of glucose recognition in pancreatic B-cells. To evaluate mechanisms of intrinsic regulation of enzyme activity human pancreatic B-cell and liver glucokinase and for comparison rat liver glucokinase were expressed in E. coli bacteria. A one-step purification procedure through metal chelate affinity chromatography revealed 58 kDa proteins with high specific activities in the range of 50 U/mg protein and K_m values around 8 mM for the substrate d-glucose with a preference for the α-anomer. There were no tissue specific differences, no species differences in the electrophoretic mobility, and no differences of the kinetic properties of these well conserved enzymes. The deletion of the 15 tissue-specific NH₂-terminal amino acids of the human glucokinase resulted in a catalytically active enzyme whose kinetic properties were not significantly different from those of the wild-type enzymes. The human and rat glucokinase isoforms were non-competitively inhibited by the sulfhydryl group reagents alloxan and ninhydrin with K_i values in the range of 1 μM. The inhibition of glucokinase enzyme activity was reversed by dithiothreitol with an EC₅₀ value of 9 μM for alloxan and of 60 μM for ninhydrin. d-Glucose provided protection against alloxan-induced inhibition of human and rat glucokinase isoenzymes with half-maximal effective concentrations between 11 and 16 mM. The enzyme inhibition by alloxan was accompanied by a change in the electrophoretic mobility with a second lower molecular 49 kDa glucokinase band which can be interpreted as a compact glucokinase molecule locked by disulfide bonds. Quantification of free sulfhydryl groups revealed an average number of 3.6 free sulfhydryl groups per enzyme molecule for the native human glucokinase isoforms. Alloxan decreased the average number of free sulfhydryl groups to 1.9 per enzyme molecule indicating that more than one SH side group is oxidized by this compound. The extraordinary sensitivity of the SH side groups of the glucokinase may be a possible mechanism of enzyme regulation by interconversion of stable (active) and unstable (inactive) conformations of the enzyme. In pancreatic B-cells the glucose-dependent increase of reduced pyridine nucleotides may stabilize the enzyme in the 58 kDa form and provide optimal conditions for glucose recognition and glucose-induced insulin secretion.     

Complete nucleotide sequence of the Coturnix chinensis (blue-breasted quail) mitochondrial genome and a phylogenetic analysis with related species

Coturnix chinensis (blue-breasted quail) has been classically grouped in Galliformes Phasianidae Coturnix, based on morphologic features and biochemical evidence. Since the blue-breasted quail has the smallest body size among the species of Galliformes, in addition to a short generation time and an excellent reproductive performance, it is a possible model fowl for breeding and physiological studies of the Coturnix japonica (Japanese quail) and Gallus gallus domesticus (chicken), which are classified in the same family as blue-breasted quail. However, since its phylogenetic position in the family Phasianidae has not been determined conclusively, the sequence of the entire blue-breasted quail mitochondria (mt) genome was obtained to provide genetic information for phylogenetic analysis in the present study. The blue-breasted quail mtDNA was found to be a circular DNA of 16,687 base pairs (bp) with the same genomic structure as the mtDNAs of Japanese quail and chicken, though it is smaller than Japanese quail and chicken mtDNAs by 10 bp and 88 bp, respectively. The sequence identity of all mitochondrial genes, including those for 12S and 16S ribosomal RNAs, between blue-breasted quail and Japanese quail ranged from 84.5% to 93.5%; between blue-breasted quail and chicken, sequence identity ranged from 78.0% to 89.6%. In order to obtain information on the phylogenetic position of blue-breasted quail in Galliformes Phasianidae, the 2,184 bp sequence comprising NADH dehydrogenase subunit 2 and cytochrome b genes available for eight species in Galliformes [Japanese quail, chicken, Gallus varius (green junglefowl), Bambusicola thoracica (Chinese bamboo partridge), Pavo cristatus (Indian peafowl), Perdix perdix (gray partridge), Phasianus colchicus (ring-neck pheasant), and Tympanchus phasianellus (sharp-tailed grouse)] together with that of Aythya americana (redhead) were examined using a maximum likelihood (ML) method. The ML analyses on the first/second codon positions, the third codon positions, and amino acid sequence consistently demonstrated that blue-breasted quail and Japanese quail are in the same phylogenetic cluster.     

A comparative cytogenetic study of chromosome homology between chicken and Japanese quail

In order to construct a chicken (Gallus gallus) cytogenetic map, we isolated 134 genomic DNA clones as new cytogenetic markers from a chicken cosmid DNA library, and mapped these clones to chicken chromosomes by fluorescence in situ hybridization. Forty-five and 89 out of 134 clones were localized to macrochromosomes and microchromosomes, respectively. The 45 clones, which localized to chicken macrochromosomes (Chromosomes 1-8 and the Z chromosome) were used for comparative mapping of Japanese quail (Coturnix japonica). The chromosome locations of the DNA clones and their gene orders in Japanese quail were quite similar to those of chicken, while Japanese quail differed from chicken in chromosomes 1, 2, 4 and 8. We specified the breakpoints of pericentric inversions in chromosomes 1 and 2 by adding mapping data of 13 functional genes using chicken cDNA clones. The presence of a pericentric inversion was also confirmed in chromosome 8. We speculate that more than two rearrangements are contained in the centromeric region of chromosome 4. All 30 clones that mapped to chicken microchromosomes also localized to Japanese quail microchromosomes, suggesting that chromosome homology is highly conserved between chicken and Japanese quail and that few chromosome rearrangements occurred in the evolution of the two species.     

Chicken microsatellite primers are not efficient markers for Japanese quail

Domestic fowl or chicken (Gallus gallus) and Japanese quail (Coturnix japonica) belong to the family Phasianidae. The exchange of marker information between chicken and quail is an important step towards the construction of a high-resolution comparative genetic map in Phasianidae, which includes several poultry species of agricultural importance. We tested chicken microsatellite markers to see if they would be suitable as genetic linkage markers in Japanese quail. Twenty-six per cent (31/120) of chicken primers amplified individual loci in Japanese quail and 65% (20/31) of the amplified loci were found to be polymorphic. Eleven of the polymorphic loci were excluded as uninformative because of the lack of amplification in some individuals or high frequency of nonspecific amplification. The sequence information of the remaining nine loci revealed six of them to contain microsatellites that were nearly identical with those of the orthologous regions in chicken. For these six loci, allele frequencies were estimated in 50 unrelated quails. Although the very few chicken markers that do work well in quail could be used as anchor points for a comparative mapping, most chicken markers are not useful for studies in quail. Therefore, more effort should be committed to developing quail-specific markers rather than attempting to adapt chicken markers for work in quail.     

Glucose phosphorylation in tumor cells. Cloning, sequencing, and overexpression in active form of a full-length cDNA encoding a mitochondrial bindable form of hexokinase

In rapidly growing tumor cells exhibiting high glucose catabolic rates, the enzyme hexokinase is markedly elevated and bound in large amounts (50-80% of the total cell activity) to the outer mitochondrial membrane (Arora, K.K., and Pedersen, P.L. (1988) J. Biol. Chem. 263, 17422-17428; Parry, D.M., and Pedersen, P.L. (1983) J. Biol. Chem. 258, 10904-10912). In extending these studies, we have isolated a cDNA clone of hexokinase from a lambda gt11 library of the highly glycolytic, c37 mouse hepatoma cell line. This clone, comprising 4,198 base pairs, contains a single open reading frame of 2,754 nucleotides which encode a 918-amino acid hexokinase with a mass of 102,272 daltons. This enzyme exhibits, respectively, 68 and 32 amino acid differences, including several charge differences, from the recently sequenced human kidney and rat brain enzymes. The putative glucose and ATP binding domains present in the latter two enzymes and in rat liver glucokinase are conserved in the tumor enzyme. At its N-terminal region, tumor hexokinase has a 12-amino acid hydrophobic stretch which is present in the rat brain enzyme but absent in the rat liver glucokinase, a cytoplasmic enzyme. The mature tumor hexokinase protein has been overexpressed in active form in Escherichia coli and purified 9-fold. The overexpressed enzyme binds to rat liver mitochondria in the presence of MgCl2. This is the first report describing the cloning and sequencing of a tumor hexokinase, and the first report documenting the overexpression of any hexokinase type in E. coli. Questions pertinent to the enzyme's mechanism, regulation, binding to mitochondria, and its marked elevation in tumor cells can now be addressed.