Brain Hexokinase Research Articles

Stimulation of Brain Hexokinase Gene Expression by Recombinant Brain Insulin-Like Growth Factor in C6 Glial Cells

Glycolysis is essential for cerebral energy generation. Hence, expression and regulation of gene-encoding brain hexokinase (HK I), the exclusive brain glucose phosphorylating enzyme, can be a critical step in this process. The present study demonstrates the ability of recombinant brain insulin-like growth factor (BIGF, a closely related member of insulin superfamily) to stimulate HK I gene expression in a concentration- and time-dependent manner in C6 glial cells. BIGF treatment (10 ng/ml) on quiescent C6 glial cells stimulates transcription and translation of HK I RNA to ∼2.5-fold within 4 h after the addition of growth factor. In contrast, insulin or epidermal growth factor could not mimic this effect. Coincubation of cycloheximide with BIGF abolished this stimulatory effect, indicating a requirement for prior protein synthesis for this effect. These results suggest that IGF may have a role in regulating hexokinase gene expression in brain and possibly of brain glucose metabolism.

Regulation of hexokinase I: crystal structure of recombinant human brain hexokinase complexed with glucose and phosphate

Hexokinase I, the pacemaker of glycolysis in brain tissue and red blood cells, is comprised of two similar domains fused into a single polypeptide chain. The C-terminal half of hexokinase I is catalytically active, whereas the N-terminal half is necessary for the relief of product inhibition by phosphate. A crystalline complex of recombinant human hexokinase I with glucose and phosphate (2.8 Å resolution) reveals a single binding site for phosphate and glucose at the N-terminal half of the enzyme. Glucose and phosphate stabilize the N-terminal half in a closed conformation. Unexpectedly, glucose binds weakly to the C-terminal half of the enzyme and does not by itself stabilize a closed conformation. Evidently a stable, closed C-terminal half requires either ATP or glucose 6-phosphate along with glucose. The crystal structure here, in conjunction with other studies in crystallography and directed mutation, puts the phosphate regulatory site at the N-terminal half, the site of potent product inhibition at the C-terminal half, and a secondary site for the weak interaction of glucose 6-phosphate at the N-terminal half of the enzyme. The relevance of crystal structures of hexokinase I to the properties of monomeric hexokinase I and oligomers of hexokinase I bound to the surface of mitochondria is discussed.

Identification of a Phosphate Regulatory Site and a Low Affinity Binding Site for Glucose 6-Phosphate in the N-terminal Half of Human Brain Hexokinase

Crystal structures of human hexokinase I reveal identical binding sites for phosphate and the 6-phosphoryl group of glucose 6-phosphate in proximity to Gly87, Ser88, Thr232, and Ser415, a binding site for the pyranose moiety of glucose 6-phosphate in proximity to Asp84, Asp413, and Ser449, and a single salt link involving Arg801 between the N- and C-terminal halves. Purified wild-type and mutant enzymes (Asp84 --> Ala, Gly87 --> Tyr, Ser88 --> Ala, Thr232 --> Ala, Asp413 --> Ala, Ser415 --> Ala, Ser449 --> Ala, and Arg801 --> Ala) were studied by kinetics and circular dichroism spectroscopy. All eight mutant hexokinases have kcat and Km values for substrates similar to those of wild-type hexokinase I. Inhibition of wild-type enzyme by 1,5-anhydroglucitol 6-phosphate is consistent with a high affinity binding site (Ki = 50 microM) and a second, low affinity binding site (Kii = 0.7 mM). The mutations of Asp84, Gly87, and Thr232 listed above eliminate inhibition because of the low affinity site, but none of the eight mutations influence Ki of the high affinity site. Relief of 1,5-anhydroglucitol 6-phosphate inhibition by phosphate for Asp84 --> Ala, Ser88 --> Ala, Ser415 --> Ala, Ser449 --> Ala and Arg801 --> Ala mutant enzymes is substantially less than that of wild-type hexokinase and completely absent in the Gly87 --> Tyr and Thr232 --> Ala mutants. The results support several conclusions. (i) The phosphate regulatory site is at the N-terminal domain as identified in crystal structures. (ii) The glucose 6-phosphate binding site at the N-terminal domain is a low affinity site and not the high affinity site associated with potent product inhibition. (iii) Arg801 participates in the regulatory mechanism of hexokinase I.

The Roles of Glycine Residues in the ATP Binding Site of Human Brain Hexokinase

Mutants of hexokinase I (Arg539 --> Lys, Thr661 --> Ala, Thr661 --> Val, Gly534 --> Ala, Gly679 --> Ala, and Gly862 --> Ala), located putatively in the vicinity of the ATP binding pocket, were constructed, purified to homogeneity, and studied by circular dichroism (CD) spectroscopy, fluorescence spectroscopy, and initial velocity kinetics. The wild-type and mutant enzymes have similar secondary structures on the basis of CD spectroscopy. The mutation Gly679 --> Ala had little effect on the kinetic properties of the enzyme. Compared with the wild-type enzyme, however, the Gly534 --> Ala mutant exhibited a 4000-fold decrease in kcat and the Gly862 --> Ala mutant showed an 11-fold increase in Km for ATP. Glucose 6-phosphate inhibition of the three glycine mutants is comparable to that of the wild-type enzyme. Inorganic phosphate is, however, less effective in relieving glucose 6-phosphate inhibition of the Gly862 --> Ala mutant, relative to the wild-type enzyme and entirely ineffective in relieving inhibition of the Gly534 --> Ala mutant. Although the fluorescence emission spectra showed some difference for the Gly862 --> Ala mutant relative to that of the wild-type enzyme, indicating an environmental alteration around tryptophan residues, no change was observed for the Gly534 --> Ala and Gly679 --> Ala mutants. Gly862 --> Ala and Gly534 --> Ala are the first instances of single residue mutations in hexokinase I that affect the binding affinity of ATP and abolish phosphate-induced relief of glucose 6-phosphate inhibition, respectively.

The mechanism of regulation of hexokinase: new insights from the crystal structure of recombinant human brain hexokinase complexed with glucose and glucose-6-phosphate.

Hexokinase I is the pacemaker of glycolysis in brain tissue. The type I isozyme exhibits unique regulatory properties in that physiological levels of phosphate relieve potent inhibition by the product, glucose-6-phosphate (Gluc-6-P). The 100 kDa polypeptide chain of hexokinase I consists of a C-terminal (catalytic) domain and an N-terminal (regulatory) domain. Structures of ligated hexokinase I should provide a basis for understanding mechanisms of catalysis and regulation at an atomic level. The complex of human hexokinase I with glucose and Gluc-6-P (determined to 2.8 A resolution) is a dimer with twofold molecular symmetry. The N- and C-terminal domains of one monomer interact with the C- and N-terminal domains, respectively, of the symmetry-related monomer. The two domains of a monomer are connected by a single alpha helix and each have the fold of yeast hexokinase. Salt links between a possible cation-binding loop of the N-terminal domain and a loop of the C-terminal domain may be important to regulation. Each domain binds single glucose and Gluc-6-P molecules in proximity to each other. The 6-phosphoryl group of bound Gluc-6-P at the C-terminal domain occupies the putative binding site for ATP, whereas the 6-phosphoryl group at the N-terminal domain may overlap the binding site for phosphate. The binding synergism of glucose and Gluc-6-P probably arises out of the mutual stabilization of a common (glucose-bound) conformation of hexokinase I. Conformational changes in the N-terminal domain in response to glucose, phosphate, and/or Gluc-6-P may influence the binding of ATP to the C-terminal domain.

Binding of rat brain hexokinase to recombinant yeast mitochondria: effect of environmental factors and the source of porin.

Heterologous binding of rat brain hexokinase to wild type, porinless, and recombinant yeast mitochondria expressing human porin was assessed, partially characterized, and compared to that in the homologous system (rat liver mitochondria). With porin-containing yeast mitochondria it is shown that (i) a significant, saturable association occurs; (ii) its extent and apparent affinity, correlated with the origin of porin, are enhanced in the presence of dextran; (iii) the binding requires Mg ions and apparently follows a complex cooperative mechanism. This heterologous association does not seem to differ fundamentally from that in the homologous system and represents a good basis for molecular studies in yeast. With porinless yeast mitochondria, binding occurs at much lower affinity, but to many more sites per mitochondrion. The results indicating a major but not exclusive role for porin in the binding are discussed in terms of (i) the mode and mechanism of binding, and (ii) the suitability of the rat hexokinase-yeast mitochondria couple for the study of heterogeneous catalysis in reconstituted cellular model systems.

Purification and Properties of Buffalo (Bubalus bubalis) Erythrocyte Hexokinase

Buffalo erythrocytes contain one isozyme of hexokinase that apparently lacks microheterogeneity as shown by chromatographic properties. A single protein band was detected by means of Western blotting using an antibody raised in rabbits against homogeneous rat brain hexokinase I. The native protein has a molecular weight of 200,000 ± 2880 by gel filtration. Partial purification of erythrocyte hexokinase by a combination of several procedures, including affinity chromatography, which was previously applied successfully to the purification of other mammalian type I hexokinases, produced a partially purified enzyme that showed several contaminants after SDS-polyacrylamide gel electrophoresis. The affinity of buffalo erythrocyte hexokinase for glucose (Km = 0.012 ± 0.001 mM) is lower than most other mammal hexokinases type I. It phosphorylates other sugars, with considerably higher Km values. This isozyme is able to use MgATP but does not use MgGTP, MgCTP or MgUTP. We used inhibition patterns, obtained with products to elucidate enzyme sequential mechanisms. Our results are clearly in agreement with a random sequential mechanism and in disagreement with an ordered sequential mechanism with either glucose or ATP as the obligatory first substrates. The ADP inhibition was of mixed type with both ATP and glucose as substrates.

Brain Hexokinase I Expression and Function during Development. † 1391

Glucose, an essential substrate for brain growth, cellular maturation, and oxidative metabolism is transported across the blood-brain barrier, into neurons and glial cells. Intracellularly glucose is phosphorylated into glucose-6-phosphate by the hexokinase I (hxl) enzyme. In the adult brain, glucose phosphorylation comprises the rate-limiting step in the process of glucose uptake. To determine the ontogeny of this critical rate limiting step, we examined the brain hxl expression (Northern blots), concentration (Western blots), enzymatic activity (NADPNADPH conversion by spectrophotometric assay) and function (3H-2-deoxy-glucose uptake) in Balb-C mice at 1d(n=6), 14d (n=6), and 35d (n=6) postnatal ages. Hxl mRNA and protein concentrations declined 20-50% (p < 0.05) while activity increased 2-fold between the 1d and 14d or 35d brains. In contrast, a six-fold increase in3 H-2-deoxy-glucose uptake (p < 0.05) which quantitates glucose transport and phosphorylation was noted between the 1d and 14d old mice. Our previous investigation demonstrated a 3-fold increase in brain glucose transporter expression and levels (particularly neuronal Glut 3) between the 1d and 14d mouse brains (Ped Res 39:91A, 1996). We conclude that 1] brain Hxl enzyme activity and function peak by a post-translational mechanism at the 14d postnatal age, 2] the age-related increase in brain Hxl enzyme activity along with the previous observation of a parallel increase in brain glucose transporter concentrations substantiate the age-related increase in brain 2-deoxyglucose uptake with a peak at 14d postnatal age. We speculate that this age-dependent increase in the mechanisms mediating brain glucose uptake 1] may be initiated by the physiological surge in thyroid hormone levels and activity that occurs at 14d of age, and 2] is critical for fueling the process of rapid brain growth and cellular development that occurs at 14d postnatal age in preparation for acquiring the specialized function of neurotransmission.

Complexes between hexokinase, mitochondrial porin and adenylate translocator in brain: regulation of hexokinase, oxidative phosphorylation and permeability transition pore.

Complexes between hexokinase, mitochondrial porin and adenylate translocator in brain: regulation of hexokinase, oxidative phosphorylation and permeability transition pore.

Crystallization and preliminary X-ray analysis of human brain hexokinase

Human brain hexokinase type I, expressed in Escherichia coli, has been crystallized from polyethylene glycol 8000 in the presence of inorganic phosphate. The crystals are hexagonal needles of diameter 0.25 mm, diffracting to a resolution of 3.5 Å on a rotating-anode/area-detector system. The crystals belong to the space group P3 121/P3 221 with cell dimensions a = b = 171.5 A ̊ and c = 99.4 A ̊ . The specific volume of the crystal is 4.2 Å 3/Da, suggesting an asymmetric unit with a single 100 kDa molecule and a solvent content of 71% by volume or two molecules of hexokinase with a solvent content of 41%. The complex of hexokinase with glucose crystallizes under similar conditions, giving crystals of the same morphology.

Type I brain hexokinase: axonal transport and membrane associations within central nervous system presynaptic terminals.

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

Limitation of glycolysis by hexokinase in rat brain synaptosomes during intense ion pumping

Incubation of rat brain synaptosomes under conditions of either increased energy utilization (addition of Na + channel opener, veratridine, or ionophores, monensin and nigericin) or inhibition of oxidative phosphorylation (addition of rotenone), or a combination thereof, decreased [ATP], increased [ADP] and stimulated glycolysis. The rates of lactate generation were linear over a 15-min interval in the presence of rotenone alone but decreased in the other two conditions. During the first 5 min, the amount of lactate formed with veratridine, monensin or nigericin was as high or higher than with rotenone, but it was lower in the last 10 min. With a combination of one of the stimulators of ion movements and rotenone the rate of glycolysis was always markedly lower than with each compound added singly. The stimulated rates of lactate formation correlated positively with the synaptosomal content of [ATP]. After 15 min, [ATP] was 0.9–1.0 nmol/mg with rotenone, 0.5–0.9 nmol/mg with veratridine (or ionophores), and <0.3 nmol/mg with a combination of the two. Under the conditions used, calcium did not affect glycolytic activity directly. The Lineweaver-Burk plot of the rate of lactate formation against [ATP] yielded a straight line with a K m for ATP of about 0.1 mM, which is very similar to the K m for this nucleotide of brain hexokinase bound to mitochondria. In C6 cells glycolytic rate measured with a combination of an ionophore and rotenone was higher than with each of these compounds added singly while [ATP] never declined below about 9 nmol/mg prot. It is concluded that in synaptosomes, the high rate of energy utilization required for intense ion movements decreases [ATP] to a level that limits hexokinase activity kinetically. This may contribute to a reduction in the rate of glycolysis and hence energy production in brain hypoxia and ischemia.

Hexokinase present in human sperm is not tyrosine phosphorylated but its antibodies affect fertilizing capacity.

The present study was conducted to investigate the presence of hexokinase in human sperm cell, its subcellular localization, its modulation and role in capacitation/acrosome reaction, and tyrosine kinase activity, if any. These studies were conducted using antibodies (Ab) raised against rat brain hexokinase (type I isozyme) that have significant cross-reaction with various human tissue hexokinases and neutralize the catalytic activity of the enzyme. Hexokinase Ab reacted with acrosomal, mid-piece and tail regions of methanol-fixed (approximately 70-80%) and live (approximately 35-52%) sperm in the indirect immunofluorescence technique (IFT) and the immunobead binding technique (IBT), respectively. Hexokinase Ab specifically recognized a band of 116 kDa on the Western blot of detergent-solubilized human sperm preparation that was different from the 95-kDa phosphotyrosine protein. Hexokinase Ab caused a significant (P < 0.01 to < 0.001) and concentration-dependent inhibition of human sperm penetration of zona-free hamster oocytes in the sperm penetration assay (SPA). These data indicate that the hexokinase of 116-kDa molecular weight is present in acrosomal, mid-piece, and tail regions of human sperm. The sperm hexokinase is a glycoprotein that is different from the 95-kDa phosphotyrosine protein and is not phosphorylated at tyrosine residues; however, its antibodies cause agglutination and a concentration-dependent inhibition of fertilizing capacity of human sperm.

ATP-binding site of human brain hexokinase as studied by molecular modeling and site-directed mutagenesis.

The interaction of ATP with the active site of hexokinase is unknown since the crystal structure of the hexokinase-ATP complex is unavailable. It was found that the ATP binding site of brain hexokinase is homologous to that of actin, heat shock protein hsc70, and glycerol kinase. On the basis of these similarities, the ATP molecule was positioned in the catalytic domain of human brain hexokinase, which was modeled from the X-ray structure of yeast hexokinase. Site-directed mutagenesis was performed to test the function of residues presumably involved in interaction with the tripolyphosphoryl moiety of ATP. Asp532, which is though to be involved in binding the Mg2+ ion of the MgATP2- complex, was mutated to Lys and Glu. The kcat values decreased 1000- and 200-fold, respectively, for the two mutants. Another residue, Thr680 was proposed to interact with the gamma-phosphoryl group of ATP through hydrogen bonds and was mutated to Val and Ser. The kcat value of the Thr680Val mutant decreased 2000-fold, whereas the kcat value of the Thr680Ser decreased only 2.5-fold, implying the importance of the hydroxyl group. The Km and dissociation constant values for either ATP or glucose of all the above mutants showed little or no change relative to the wild-type enzyme. The Ki values for the glucose 6-phosphate analogue 1,5-anhydroglucitol 6-phosphate, were the same as that of the wild-type enzyme, and the inhibition was reversed by inorganic phosphate (Pi) for all four mutants. The circular dichroism spectra of the mutants were the same as that of the wild-type enzyme. The results from the site-directed mutagenesis demonstrate that the presumed interactions of investigated residues with ATP are important for the stabilization of the transition state.

Application of a Double Isotopic Labeling Method to a Study of the Interaction of Mitochondrially Bound Rat Brain Hexokinase with Intramitochondrial Compartments of ATP Generated by Oxidative Phosphorylation

γ-Labeled ATP was produced by rat brain mitochondria utilizing [32P]Pias substrate for oxidative phosphorylation. The32P/14C ratio of Glc-6-P produced by the endogenous mitochondrially bound hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) using [U-14C]Glc as substrate was determined as a function of time after initiation of oxidative phosphorylation. This same ratio was determined for Glc-6-P formed by added yeast hexokinase using extramitochondrial ATP as substrate. The specific activity of ATP formed by oxidative phosphorylation was manipulated either by initiating the reaction with labeledPiand subsequently adding excess unlabeledPior by initiating the reaction with unlabeledPiand introducing the labeled substrate at a later time. The32P/14C ratio of Glc-6-P formed by yeast hexokinase, reflecting the specific activity of ATP in the extramitochondrial space, was rapidly responsive to such manipulations, but the corresponding changes in the32P/14C ratio of Glc-6-P produced by the endogenous hexokinase were markedly different. The results are consistent with the view that mitochondrially bound hexokinase does not utilize extramitochondrial ATP as substrate but rather is functionally coupled to a discrete intramitochondrial compartment of ATP produced by oxidative phosphorylation.

Active Site Residues of Human Brain Hexokinase as Studied by Site-specific Mutagenesis

The truncated gene of hexokinase, mini-hexokinase, starting with methionine 455 and ending at the C terminus was expressed in Escherichia coli. Mini-hexokinase lost its ability to ameliorate inhibition of glucose-6-P-inhibited mini-hexokinase in the presence of phosphate (P(i)). We suggest that the P(i) site either resides in the N-terminal half of hexokinase I or requires the N-terminal portion of the enzyme. Site-directed mutagenesis was performed to obtain two mutants of mini-hexokinase: C606S and C628S. Both are thought to be associated with the active site of hexokinase I. These mutants exhibited a 3-fold increase in Km for glucose but no change in either the Km for ATP or the kcat. The circular dichroism (CD) spectra showed no differences among the wild-type or mutant enzymes. These results suggest that Cys606 and Cys628 are not involved in glucose binding directly. The putative ATP-binding site of full-length human brain hexokinase may involve Arg539 and Gly679, and these residues were mutated to Ile. For the mutant R539I, the kcat value decreased 114-fold relative to wild-type hexokinase, whereas the Km values for ATP and glucose changed only slightly. No change was observed in the Ki value for 1,5-anhydroglucitol 6-phosphate. CD spectra showed only a slight change in secondary structure. For the mutant G679I, overexpressed hexokinase is insoluble. We suggest that Arg539 is important for catalysis because it stabilizes the transition state product ADP-hexokinase. Gly679 is probably important for proper folding of the protein.

Schistosoma mansoni Hexokinase: CDNA Cloning and Immunogenicity Studies

DNA encoding a Schistosoma mansoni hexokinase (SHEX) was amplified from cDNA by the polymerase chain reaction using opposing oligonucleotide primers designed to hybridize with two short segments of hexokinase coding sequences that an well-conserved through evolution. The resulting DNA fragment was then used as a probe to identify a full-length hexokinase cDNA clone. SHEX cDNA encodes a 50-kDa protein that is approximately 46% homologous to rat hexokinase, 40% to rat glucokinase, and 34% to yeast hexokinase A. SHEX coding DNA was expressed within Escherichia coli cells and the 50-kDa recombinant product (rSHEX) was partially purified. Mice repeatedly immunized with rSHEX produced antibodies which recognize rSHEX but this offered no significant protection against subsequent cercarial challenge. On Western blots, rSHEX is weakly recognized by antisera against rat brain hexokinase but not by sera from three strains of mice experimentally infected with S. mansoni parasites or from numerous human schistosomiasis patients. Thus, unlike other reported S. mansoni glycolytic enzymes, hexokinase appears to be poorly immunogenic during schistosome infection and of limited potential as a vaccine candidate.

Mitochondrial Hexokinase in Brain: Coexistence of Forms Differing in Sensitivity to Solubilization by Glucose-6-Phosphate on the Same Mitochondrial

Hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) is associated with mitochondria from brain of various species. The fraction of the bound activity that can be released in soluble form after incubation of the mitochondria with glucose-6-phosphate (Glc-6-P) has been shown to vary markedly among species (F. Kabir and J. E. Wilson, Arch. Biochem. Biophys. 300, 641-650, 1993). A method has been developed by which mitochondria bearing significant amounts of bound hexokinase can be selectively immunoprecipitated by Stasymphylococcus aureus cells coated with anti-Type I hexokinase antibodies. Treatment of mitochondria from guinea pig, bovine, and human brain with Glc-6-P solubilized asympproximately 60, 40, and 20% of the bound hexokinase activity, respectively, but had no effect on the extent to which the mitochondria could be immunoprecipitated. This is consistent with the view that the residual hexokinase, resistant to solubilization with Glc-6-P, coexists on the same mitochondria bearing the Glc-6-P-sensitive form, i.e., removal of the latter does not prevent immunoprecipitation mediated by the Glc-6-P-resistant form. Mitochondrial porin has previously been shown to be involved in binding of hexokinase to mitochondria. Four porin species, having a common molecular weight but differing in isoelectric point, were detected, in the same relative amounts, in mitochondria from bovine and rat brain. The extent to which Glc-6-P solubilizes hexokinase from rat and bovine brain mitochondria is ≍90 and ≍40%, respectively. Thus the marked difference in sensitivity to solubilization with Glc-6-P cannot be attributed to differences in the relative amounts of different porin species present in rat and bovine mitochondria.

P95, the major phosphotyrosine-containing protein in mouse spermatozoa, is a hexokinase with unique properties.

Mouse sperm contain a major phosphotyrosine-containing protein of M(r) 95,000 (nonreducing conditions) which has been implicated as a sperm membrane receptor for the egg zona pellucida glycoprotein, ZP3 (Leyton, L., and Saling, P. (1989) Cell 57, 1123-1130; Leyton, L., LeGuen, P., Bunch, D., and Saling, P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11692-11695). This protein was purified and subjected to limited tryptic digestion and subsequent amino acid analysis. Three sequenced peptides revealed 100% amino acid identity to a mouse hepatoma hexokinase (Arora, K. K., Fanciulli, M., and Pederson, P. L. (1990) J. Biol. Chem. 265, 6481-6488). The purified protein, which migrated at M(r) 116,000 under reducing conditions (p95/116), reacted with an antiserum to the purified rat brain hexokinase, type 1, and comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the purified rat brain enzyme under both nonreducing and reducing conditions. Unlike p95/116, the rat brain enzyme was not a phosphotyrosine-containing protein. The p95/116 protein could be immunoprecipitated with the hexokinase antiserum or an O-phosphotyrosine antibody. Limited tryptic digestion of the purified p95/116 and the rat brain enzyme generated subsets of identical peptides which reacted with the hexokinase antiserum. However, p95/116 also contained phosphotyrosine-containing peptides that were not present in the rat brain hexokinase. When different mouse tissues were probed with the hexokinase antiserum all tissues, with the exception of liver, contained immunoreactive protein. In contrast, only sperm and testis possessed a phosphotyrosine-containing form of hexokinase. These data suggest that the germ cell component of the testis possesses a unique tyrosine-phosphorylated form of hexokinase.

Brain Hexokinase and Intramitochondrial Compartments of ATP: Fact and Artifact

Brain Hexokinase and Intramitochondrial Compartments of ATP: Fact and Artifact