Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.1.1 (hexokinase)
5,274 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mitochondrially bound hexokinase (ATP-D-hexose-6-phosphotransferase; EC 2.7.1.1) was dissociatively extracted from normal rat brains and intracerebral and subcutaneous implants of the 36B-10 glioma. At least 70% of the total hexokinase enzyme activity in normal and glioma tissue was associated with the mitochondrial fraction. Purification of the crude tissue extracts by ion-exchange and affinity chromatography followed by analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a successive purification of the enzyme to homogeneity with a molecular size of 98 kilodaltons. Enzyme kinetics with glucose or 2-deoxyglucose (2-DG) as the substrate were measured spectrophotometrically by coupling the appropriate reactions to either NADPH or NAD+ formation. The Km of hexokinase with glucose as the substrate in the intracerebral glioma (0.138 mM) and subcutaneous glioma (0.183 mM) tissues was 2.1-2.7-fold higher than that observed in normal brain tissue (0.067 mM) (p less than 0.001). No significant differences were observed in the Km for hexokinase with 2-DG as the substrate in the glioma and normal brain tissue. The phosphorylation ratio for normal brain was 0.320 and was increased in the intracerebral glioma to 0.694 and in the subcutaneous glioma to 0.519. The ratios of deoxyglucose and glucose volumes of distribution in normal brain and intracerebral glioma tissues were 1.70 and 1.85, respectively. The lumped constants calculated directly from the phosphorylation ratios and the volumes of distribution of deoxyglucose and glucose were 0.517 in normal brain and 1.168 in intracerebral glioma. Our results indicate the lumped constant is increased 2.26-fold in intracerebral glioma compared with normal brain.
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PMID:Determination of the deoxyglucose and glucose phosphorylation ratio and the lumped constant in rat brain and a transplantable rat glioma. 272 62

Porcine hepatic glucokinase (ATP: D-hexose 6-phosphotransferase EC 2.7.1.1) has been purified by a modification of the procedure for its purification from rats. However, difficulties were encountered with endogenous proteases and the reliability of a source for porcine livers. The molecular weight has been determined to be 60,400 +/- 1,400 by sodium dodecyl sulfate, polyacrylamide gel electrophoresis. The enzyme has been characterized kinetically. The parameter values, S0.5 (glucose) and Hill coefficient (nH) are 2.4 mM and 1.9 respectively under sulfhydryl-reducing conditions. The enzyme undergoes the two sulfhydryl-related decays of its activity previously observed in the enzyme isolated from rat (Tippett PS, Neet KE: Arch Biochem Biophys 222:285-298, 1983). The enzyme is inhibited by palmitoyl-CoA, Ki (apparent) = 1.0 microM, nH = 1.8; this concentration of inhibitor is significantly below its critical micelle concentration. Physically and kinetically glucokinase isolated from pig is similar to the enzyme isolated from rat. The porcine system provides a second source for isolation and further characterization of this important and unusual enzyme.
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PMID:The regulatory kinetic properties of porcine hepatic glucokinase. 277 Jul 13

The post-mortem stability of some brain enzymes was studied. Over the time period under examination, the cytoplasmic enzymes investigated underwent a decisive decay, hexokinase being the most labile and acylphosphatase the most stable. On the other hand, structured activities such as Na+, K+-ATPase and Ca2+, Mg2+-ATPase showed an apparent transitory increase. The differences in post-mortem stability of soluble enzymes could be ascribed, at least in part, to their different susceptibility toward proteolytic activities, as suggested by the electrophoretic results.
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PMID:Post-mortem modifications of the specific activity of some brain enzymes. 283 61

Isoenzyme electrophoretic patterns (zymodemes) are increasingly used to distinguish between pathogenic and non-pathogenic strains of Entamoeba histolytica. Isolates of E. histolytica from asymptomatic and symptomatic cases have been shown to differ in the electrophoretic mobility of their hexokinase and phosphoglucomutase isoenzymes. The hexokinase isoenzymes from a non-pathogenic strain and from a pathogenic strain of E. histolytica were purified by fast protein liquid chromatography in several steps, which included a separation by size, chromatofocusing, and anion exchange chromatography. The isoenzymes differed in their isoelectric points, which ranged from pH 4.8-5.4, but had very similar kinetic properties and almost identical apparent molecular weights (48,000) in sodium dodecyl sulfate polyacrylamide gels, as well as on gel filtration columns. Comparison of tryptic peptide analysis of each of the isoenzymes indicated considerable homology between the non-pathogenic and pathogenic forms. Antibodies produced against each of the two pathogenic hexokinase isoenzymes inhibited their enzymatic activity. The antibodies also inhibited the activity of the isoenzymes of the non-pathogenic strain. Our findings suggest that the isoenzymes have structural similarities, and that the pathogenic ones differ from the non-pathogenic ones in their electromobility due to post-translational modifications.
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PMID:Isolation and partial characterization of the hexokinase isoenzymes from pathogenic and non-pathogenic strains of Entamoeba histolytica. 289 Jan 4

Inhibition of glutamate transport is a potential indirect cause of excitotoxic damage by glutamate in the CNS. The mercuric ion, the form in which metallic mercury vapor is believed to exert its neurotoxic action, is a known inhibitor of amino acid transport. This study examines the specificity with which HgCl2 inhibits glutamate transport in mouse cerebral astrocytes by means of comparative measurements of 2-deoxyglucose uptake. Uptake of 2-deoxyglucose is an index of glucose utilization that reflects the function of Na+,K+-ATPase and hexokinase, and is sensitive to Na+ entry. The kinetic parameters, ionic dependence, and substrate specificity of glutamate transport in these astrocyte cultures were consistent with the commonly occurring system designated X-AG. Acute exposure to 0.5 microM HgCl2 inhibited by 50% the initial rate of glutamate transport but did not affect 2-deoxyglucose uptake. Glutamate transport was not detectably inhibited by Al2+, Pb2+, Co2+, Sr2+, Cd2+, or Zn2+ (10 microM as chlorides). The inhibitory action of 0.5 microM HgCl2 on glutamate transport was rapidly reversible. The action of 1-2 microM HgCl2 was progressive when exposures were extended to 1-3 h, and was more slowly reversible. These results suggest that Hg2+ can impair glial glutamate transport reversibly at exposure levels that do not compromise some other vital cell functions.
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PMID:Specificity and reversibility of the inhibition by HgCl2 of glutamate transport in astrocyte cultures. 289 9

In rapidly growing, highly glycolytic hepatoma cells as much as 65% of the total cell hexokinase is bound to the outer mitochondrial membrane [Parry, D.M., & Pedersen, P.L. (1983) J. Biol. Chem. 258, 10904-10912]. In this paper, we describe the purification to apparent homogeneity of a mitochondrial pore-forming protein from the highly glycolytic AS-30D rat hepatoma cell line. The purified protein shows a single 35 000-dalton band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an amino acid composition slightly more hydrophobic than that of the rat liver pore protein (also known as VDAC or mitochondrial porin), and a channel-forming activity of 136 channels min-1 (microgram of protein)-1. In addition to displaying the properties characteristic of VDAC (single-channel conductance, voltage dependence, and preference for anions), we observe that the AS-30D VDAC protein is one of only three mitochondrial proteins that bind [14C]dicyclohexylcarbodiimide (DCCD) at relatively low dosages (2 nmol of DCCD/mg of mitochondrial protein). Significantly, treatment of intact mitochondria isolated from either rat liver or the AS-30D hepatoma with DCCD results in an almost complete inhibition of their ability to binding hexokinase. Fifty percent inhibition of binding occurs at less than 2 nmol of DCCD/mg of mitochondrial protein. In contrast to DCCD, water-soluble carbodiimides are without effect on hexokinase binding. These results suggest that the pore-forming protein of tumor mitochondria forms at least part of the hexokinase receptor complex. In addition, they indicate that a carboxyl residue located within a hydrophobic region of the receptor complex may play a critical role in hexokinase binding.
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PMID:Hexokinase receptor complex in hepatoma mitochondria: evidence from N,N'-dicyclohexylcarbodiimide-labeling studies for the involvement of the pore-forming protein VDAC. 300 16

In rabbit reticulocytes, the hexokinase (EC 2.7.1.1)-specific activity is 4-5 times that of corresponding mature red cells. Immunoprecipitation of hexokinase by a polyclonal antibody made in vitro shows that this maturation-dependent hexokinase decay is not due to accumulation of inactive enzyme molecules but to degradation of hexokinase. A cell-free system derived from rabbit reticulocytes, but not mature erythrocytes, was found to catalyze the decay of hexokinae activity and the degradation of 125I-labeled enzyme. This degradation is ATP-dependent and requires both ubiquitin and a proteolytic fraction retained by DEAE-cellulose. Maximum ATP-dependent degradation was obtained at pH 7.5 in the presence of MgATP. MgGTP could replace MgATP with a relative stimulation of 0.90. 125I-Hexokinase incubated with reticulocyte extract in the presence of ATP forms high molecular weight aggregates that reach a steady-state concentration in 1 h, whereas the degradation of the enzyme is linear up to 8 h, suggesting that the formation of protein aggregates precedes enzyme catabolism. These aggregates are stable upon boiling in 2% sodium dodecyl sulfate, 3% mercaptoethanol and probably represent an intermediate step in the enzyme degradation with hexokinase and other proteins covalently conjugate to ubiquitin. That hexokinase could be conjugated to ubiquitin was shown by the formation of 125I-ubiquitin-hexokinase complexes in the presence of ATP and the enzymes of the ubiquitin-protein ligase system. Thus, the decay of hexokinase during reticulocyte maturation is ATP- and ubiquitin-dependent and suggests a new physiological role for the energy-dependent degradation system of reticulocytes.
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PMID:Rabbit red blood cell hexokinase. Decay mechanism during reticulocyte maturation. 301 48

6-Phosphofructokinase (PFK) plays a central role in the regulation of glycolysis in both normal and neoplastic cells. Since PFK also mediates the Pasteur effect, it coordinates the two modes of energy production in most cell systems, i.e., glycolysis and respiration. The energy production in the cancer cell is characterized by a predominance of aerobic glycolysis (the Warburg effect) and a diminution or lack of the Pasteur effect. Previous studies from this laboratory have demonstrated that PFK in humans and in the rat exists in multiple tetrameric isozymic forms consisting of three unique subunits under separate genetic controls, M, L, and P types. These isozymes are distinguishable from one another by ion-exchange chromatography and subunit-specific antibodies. Various organs exhibit unique isozyme distribution patterns which essentially reflect the preferred mode of carbohydrate metabolism utilized, i.e., glycolysis or gluconeogenesis or both. In order to investigate whether the high aerobic glycolysis of the cancer cell can be explained on the basis of a lack of the regulatory function of PFK due to an altered isozyme distribution pattern, we compared the activity and isozymic profile of the enzyme from malignant cells of human leukemias, lymphomas, virus-transformed cell lines, and established malignant cell lines of lymphoid, myeloid, erythroid, and fibroblastic origin and their normal counterparts. The myeloid and erythroid cell lines were also investigated after in vitro differentiation induced by dimethyl sulfoxide, sodium butyrate, hemin, etc. Our results show that, as is the case with hexokinase and pyruvate kinase, the other two rate-limiting enzymes of glycolysis, PFK shows both quantitative increases and isozymic alterations secondary to altered gene expression during neoplastic transformation, both in vivo and in vitro. In contradistinction to the isozymic alteration in hexokinase and pyruvate kinase, where highly regulated liver-type isozymes decrease or disappear and are replaced by the nonregulated ones, in the case of PFK, the highly regulated liver-type isozyme not only persists but actually increases, followed by an increase in the platelet-type isozyme. These isozymic alterations closely parallel the quantitative increases in total PFK activity, which in turn is closely related to the rate of replication of cancer cells and hence an increase in metabolism. Thus, human PFK is both a transformation- and a progression-linked discriminant of malignancy (For definitions of these terms, see Weber et al., N. Engl. J. Med., 296: 486-493, 1977.).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Alterations in the activity and isozymic profile of human phosphofructokinase during malignant transformation in vivo and in vitro: transformation- and progression-linked discriminants of malignancy. 315 73

Glucose 6-phosphate as well as several other hexose mono- and diphosphates were found by kinetic studies to be competitive inhibitors of human hexokinase I (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) versus MgATP. Limited proteolysis by trypsin does not destroy the hexokinase activity but produces as well-defined peptide map when the digested enzyme is electrophoresed in the presence of sodium dodecyl sulfate. MgATP at subsaturating concentration protects hexokinase from trypsin digestion, while phosphorylated sugars, Mg2+, glucose and inorganic phosphate have no effect. Addition of glucose 6-phosphate to the MgATP-hexokinase complex at a concentration 100-times higher than its Ki was not able to reverse the MgATP-induced conformation of hexokinase, suggesting that the binding of glucose 6-phosphate and MgATP are not mutually exclusive. Similar evidence was also obtained by studies of the induced modifications of ultraviolet spectra of hexokinase by the binding of MgATP, glucose 6-phosphate and both compounds. Among a library of monoclonal antibodies produced against rat brain hexokinase I and that recognize human placenta hexokinase I, one (4A6) was found to be able to modify the Ki of glucose 6-phosphate (from 25 to 140 microM) for human hexokinase I. The same antibody also weakens the inhibition by all the other hexoses phosphate studied without affecting the apparent Km for MgATP (from 0.6 to 0.75 mM) or for glucose. These data support the view for the binding of glucose 6-phosphate at a regulatory site on the enzyme.
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PMID:The interaction of phosphorylated sugars with human hexokinase I. 325 34

Recent studies from this laboratory have demonstrated that a form of hexokinase characteristic of rapidly growing, highly glycolytic tumor cells is bound to an outer mitochondrial membrane receptor complex containing a Mr 35,000 pore protein (D. M. Parry and P. L. Pedersen, J. Biol. Chem., 258: 10904-10912, 1983; R. A. Nakashima, et al., Biochemistry, 25: 1015-1021, 1986). In new studies reported here the specificity of this receptor complex for binding hexokinase is defined, and a purification scheme is described which leads to a homogeneous and bindable form of the tumor hexokinase. In the AS-30D hepatoma, hexokinase activity is elevated more than 100-fold relative to liver tissue. The relative increase in hexokinase activity is 8 times greater than that of any other glycolytic enzyme. Hexokinase is the only glycolytic enzyme of AS-30D cells to exhibit a mitochondrial/cytoplasmic specific activity ratio greater than 1, showing a 3.5-fold elevation in the mitochondrial fraction. Purification of hexokinase is accomplished by preferential solubilization of the mitochondrial bound enzyme with glucose-6-phosphate, followed by high-performance liquid chromatography on gel permeation and anion exchange columns. The final fraction has a specific activity of 144 units per mg of protein, with a Km for glucose of 0.13 mM and for ATP of 1.4 mM. The purified tumor enzyme migrates as a single species upon sodium dodecyl sulfate: polyacrylamide gel electrophoresis with an apparent molecular weight of 98,000. Significantly, the purified tumor enzyme retains its activity for mitochondrial binding. Additional results derived from chromatographic, polyclonal antibody, and amino acid analysis studies indicate that the predominant rat hepatoma hexokinase species is related most closely to isozymic form(s) of the enzyme commonly referred to as type II, and least related to the liver type IV isozyme (glucokinase).
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PMID:Purification and characterization of a bindable form of mitochondrial bound hexokinase from the highly glycolytic AS-30D rat hepatoma cell line. 333 84


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