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)

The surface distribution of several proteins (porin, hexokinase, and two proteins associated with microtubules or actin filaments) on the outer membrane of brain mitochondria was analyzed by immunogold labelling of purified mitochondria in vitro. The results suggest the existence of specialized domains for the distribution of porin in the outer mitochondrial membrane. Similarities between the distribution of porin and the distribution of microtubule-associated proteins bound in vitro to mitochondria suggested that mitochondria and microtubules interact by binding microtubule-associated proteins to porin-containing domains of the outer membrane. This hypothesis was supported by biochemical studies on outer mitochondrial proteins involved in in vitro binding of cytoskeleton elements. In vitro interactions between mitochondria and microtubules or neurofilaments were analyzed by electron microscopy. These studies revealed cross-bridging between the outer membrane of mitochondria and the two cytoskeleton elements. Cross-bridging was influenced by ATP hydrolysis and by several proteins associated with the surface of mitochondria or with microtubules. In addition, unidentified proteins which were recognized by antibodies to all intermediate filaments subunits were associated either with the mitochondrial surface or with microtubules. This data suggest the participation of additional cytoplasmic proteins in the interactions between cytoskeleton elements and mitochondria.
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PMID:Interactions between brain mitochondria and cytoskeleton: evidence for specialized outer membrane domains involved in the association of cytoskeleton-associated proteins to mitochondria in situ and in vitro. 820 13

Hexokinase in mammalian brain is particulate and usually considered to be bound to the outer mitochondrial membrane. Investigation of rabbit brain mitochondria prepared either by differential centrifugation and discontinuous density gradient centrifugation has provided evidence that this particulate fraction also contains endoplasmic vesicles and synaptosomes. Solubilization of the bound hexokinase by different combinations of detergents and metabolites has proved the existence of different hexokinase binding sites. Electron microscopic examination of hexokinase location by immuno-gold labelling techniques confirmed that hexokinase is indeed predominantly bound to mitochondria but that a significant proportion is also bound to non-mitochondrial membranes. Attempts to quantify this distribution were unsuccessful since different figures were obtained using anti-hexokinase IgG affinity purified on immobilized native or denatured hexokinase. Binding studies of the purified rabbit brain mitochondrial hexokinase to rabbit liver mitochondria and microsomes confirmed that in addition to a binding site on mitochondria there is another binding site on microsomes. The N-terminal sequence of hexokinase has been shown to be important for mitochondria binding and also for microsome binding. These results suggest that the intracellular localization of hexokinase in rabbit brain is not exclusively mitochondrial and that the metabolic role of this enzyme should be reconsidered by including a binding site on the endoplasmic reticulum.
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PMID:Intracellular distribution of hexokinase in rabbit brain. 823 43

Hexokinases are comprised of two highly homologous approximately 50-kDa halves and are product-inhibited by glucose-6-P. Four amino acid residues, Ser603, Asp657, Glu708, and Glu742, located in the C-terminal half of the tumor mitochondrial enzyme have been shown to be essential for enzyme function (Arora, K. K., Filburn, C. R., and Pedersen, P. L. (1991) J. Biol. Chem. 266, 5359-5362). Here we have assessed also the role of the N-terminal half of the same enzyme. Site-directed mutagenesis of residues predicted to interact with glucose in the N-terminal half, i.e. Ser155, Asp209, and Glu260, to Ala, have no effect on hexokinase activity. In addition, inhibition by hexose mono- and bisphosphates is unchanged for each of the mutant enzymes. Significantly, the overexpressed N-terminal polypeptide is devoid of catalytic activity but does have the capacity to bind ATP-agarose and be released with ATP and glucose-6-P. In contrast, the overexpressed C-terminal polypeptide is catalytically active and shows the same product inhibition pattern as the complete 100-kDa parent enzyme. These results emphasize that the N-terminal half of tumor hexokinase is essential neither for catalysis nor product modulation. Rather, the N-terminal half may play another role, perhaps in modulation of the ATP/glucose-6-P-dependent binding of the enzyme to tumor mitochondria or by acting as a spacer between the outer mitochondrial membrane and the C-terminal catalytic unit.
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PMID:Structure/function relationships in hexokinase. Site-directed mutational analyses and characterization of overexpressed fragments implicate different functions for the N- and C-terminal halves of the enzyme. 834 2

We have identified cDNAs representing three hexokinase mRNAs (Hk1-sa, Hk1-sb, Hk1-sc) by screening mouse spermatogenic cell cDNA libraries with a mouse hepatoma cell line hexokinase (Hk1) cDNA [Arora KK, Fanciulli M, Pederson PL. J Biol Chem 1990; 265:6481-6488]. Although all three cDNAs show 99% identity to the somatic Hk1 cDNA sequence throughout most of their coding region, they differ from this sequence at the 5' end. They contain a common spermatogenic cell-specific sequence and a sequence unique to each cDNA immediately 5' to the common domain. However, they lack the porin-binding domain (PBD) present in this region of Hk1, used for binding to a pore-forming protein in the outer mitochondrial membrane. These observations appear to support a model proposed by others for hexokinase gene evolution in mammals. In addition, we found that Hk1-sb has an internal sequence that is not present in Hk1, Hk1-sa, or Hk1-sc. Moreover, Hk1-sa and Hk1-sb transcripts are developmentally expressed in mouse spermatogenic cells. Hk1-sa mRNA is first expressed during meiosis and continues to be present in postmeiotic germ cells, while the more abundant Hk1-sb mRNA is detected only in postmeiotic germ cells. These and other findings suggest that enzymes encoded by Hk1-sa, Hk1-sb, and Hk1-sc are present only in spermatogenic cells.
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PMID:Unique hexokinase messenger ribonucleic acids lacking the porin-binding domain are developmentally expressed in mouse spermatogenic cells. 839 93

The voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane is a small abundant protein found in all eukaryotic kingdoms which forms a voltage-gated pore when incorporated into planar lipid bilayers. VDAC is also the site of binding of the metabolic enzymes hexokinase and glycerol kinase to the mitochondrion in what may be a significant metabolic regulatory interaction. Recently, there has been speculation that there may be multiple forms of VDAC in mammals which differ in their localization in the outer mitochondrial membrane and in their physiological function. In this report, we describe the identification and characterization of two human cDNAs encoding VDAC homologs (HVDAC1 and HVDAC2). To confirm VDAC function, each human protein has been expressed in yeast lacking the endogenous VDAC gene. Human proteins isolated from yeast mitochondria formed channels with the characteristics expected of VDAC when incorporated into planar lipid bilayers. In addition, expression of the human proteins in such strains can complement phenotypic defects associated with elimination of the endogenous yeast VDAC gene. Since VDAC is the site of binding of hexokinase to the outer mitochondrial membrane, the binding capacity of each VDAC isoform expressed in yeast mitochondria was assessed. When compared with the binding of hexokinase to mitochondria lacking VDAC, the results show that mitochondria expressing HVDAC1 are capable of specifically binding hexokinase, whereas mitochondria expressing HVDAC2 only bind hexokinase at background levels. The expression of each human cDNA has been assessed by Northern blot and polymerase chain reaction techniques. With one exception, each is expressed in all human cell lines and tissues examined.
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PMID:Cloning and functional expression in yeast of two human isoforms of the outer mitochondrial membrane channel, the voltage-dependent anion channel. 842 Sep 59

Physiologically, a postprandial glucose rise induces metabolic signal sequences that use several steps in common in both the pancreas and peripheral tissues but result in different events due to specialized tissue functions. Glucose transport performed by tissue-specific glucose transporters is, in general, not rate limiting. The next step is phosphorylation of glucose by cell-specific hexokinases. In the beta-cell, glucokinase (or hexokinase IV) is activated upon binding to a pore protein in the outer mitochondrial membrane at contact sites between outer and inner membranes. The same mechanism applies for hexokinase II in skeletal muscle and adipose tissue. The activation of hexokinases depends on a contact site-specific structure of the pore, which is voltage-dependent and influenced by the electric potential of the inner mitochondrial membrane. Mitochondria lacking a membrane potential because of defects in the respiratory chain would thus not be able to increase the glucose-phosphorylating enzyme activity over basal state. Binding and activation of hexokinases to mitochondrial contact sites lead to an acceleration of the formation of both ADP and glucose-6-phosphate (G-6-P). ADP directly enters the mitochondrion and stimulates mitochondrial oxidative phosphorylation. G-6-P is an important intermediate of energy metabolism at the switch position between glycolysis, glycogen synthesis, and the pentose-phosphate shunt. Initiated by blood glucose elevation, mitochondrial oxidative phosphorylation is accelerated in a concerted action coupling glycolysis to mitochondrial metabolism at three different points: first, through NADH transfer to the respiratory chain complex I via the malate/aspartate shuttle; second, by providing FADH2 to complex II through the glycerol-phosphate/dihydroxy-acetone-phosphate cycle; and third, by the action of hexo(gluco)kinases providing ADP for complex V, the ATP synthetase. As cytosolic and mitochondrial isozymes of creatine kinase (CK) are observed in insulinoma cells, the phosphocreatine (CrP) shuttle, working in brain and muscle, may also be involved in signaling glucose-induced insulin secretion in beta-cells. An interplay between the plasma membrane-bound CK and the mitochondrial CK could provide a mechanism to increase ATP locally at the KATP channels, coordinated to the activity of mitochondrial CrP production. Closure of the KATP channels by ATP would lead to an increase of cytosolic and, even more, mitochondrial calcium and finally to insulin secretion. Thus in beta-cells, glucose, via bound glucokinase, stimulates mitochondrial CrP synthesis. The same signaling sequence is used in the opposite direction in muscle during exercise when high ATP turnover increases the creatine level that stimulates mitochondrial ATP synthesis and glucose phosphorylation via hexokinase. Furthermore, this cytosolic/mitochondrial cross-talk is also involved in activation of muscle glycogen synthesis by glucose. The activity of mitochondrially bound hexokinase provides G-6-P and stimulates UTP production through mitochondrial nucleoside diphosphate kinase. Pathophysiologically, there are at least two genetically different forms of diabetes linked to energy metabolism: the first example is one form of maturity-onset diabetes of the young (MODY2), an autosomal dominant disorder caused by point mutations of the glucokinase gene; the second example is several forms of mitochondrial diabetes caused by point and length mutations of the mitochondrial DNA (mtDNA) that encodes several subunits of the respiratory chain complexes. Because the mtDNA is vulnerable and accumulates point and length mutations during aging, it is likely to contribute to the manifestation of some forms of NIDDM.(ABSTRACT TRUNCATED)
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PMID:Mitochondria and diabetes. Genetic, biochemical, and clinical implications of the cellular energy circuit. 854 53

Several enzymes in the glycolytic pathway are reported to have spermatogenic cell-specific isozymes. We reported recently the cloning of cDNAs representing three unique type 1 hexokinase mRNAs (mHk1-sa, mHk1-sb, and mHk1-sc) present only in mouse spermatogenic cells and the patterns of expression of these mRNAs (Mori et al., 1993: Biol Reprod 49:191-203). The mRNAs contain a spermatogenic cell-specific sequence, but lack the sequence for the porin-binding domain that somatic cell hexokinases use to bind to a pore-forming protein in the outer mitochondrial membrane. We now report the cloning of cDNAs representing three unique human type 1 hexokinase mRNAs (hHK1-ta, hHK1-tb, and hHK1-tc) expressed in testis, but not detected by Northern analysis in other human tissues. These mRNAs also contain a testis-specific sequence not present in somatic cell type 1 hexokinase, but lack the sequence for the porin-binding domain. The hHK1-tb and hHK1-tc mRNAs each contain an additional unique sequence. The testis-specific sequence of the human mRNAs is similar to the spermatogenic cell-specific sequence of the mouse mRNAs. Furthermore, Northern analysis of RNA from mouse, hamster, guinea pig, rabbit, ram, human, and rat demonstrated expression of type 1 hexokinase mRNAs lacking the porin-binding domain in the testes of these mammals. These results suggest that hexokinase may have unique structural or functional features in spermatogenic cells and support a model proposed by others for hexokinase gene evolution in mammals.
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PMID:Testis-specific expression of mRNAs for a unique human type 1 hexokinase lacking the porin-binding domain. 872 88

In vitro incubation of isolated hexokinase isozyme I or isolated dimer of mitochondrial creatine kinase with the outer mitochondrial membrane pore led to high molecular weight complexes of enzyme oligomers. Similar complexes of hexokinase and mitochondrial creatine kinase could be extracted by 0.5% Triton X-100 from homogenates of rat brain. Hexokinase and creatine kinase complexes could be separated by subsequent chromatography on DEAE anion exchanger. The molecular weight, as determined by gel-permeation chromatography, was approximately 400 kDa for both complexes. The Mr suggested tetramers of hexokinase (monomer 100 kDa) and creatine kinase (active enzyme is a dimer of 80 kDa). The composition of the complexes was further characterised by specific antibodies. Besides either hexokinase or creatine kinase molecules the complexes contained porin and adenylate translocator. It was possible to incorporate the complexes into artificial bilayer membranes and to measure conductance in 1 M KCI. The incorporating channels had a high conductance of 6 nS that was asymmetrically voltage dependent. The complexes were also reconstituted in phospholipid vesicles that were loaded with ATP. Complex containing vesicles retained ATP while vesicles reconstituted with pure porin were leaky. The internal ATP could be used by creatine kinase and hexokinase in the complex to phosphorylate external creatine or glucose. This process was inhibited by atractyloside. The hexokinase complex containing vesicles were furthermore loaded with malate or ATP that was gradually released by addition of Ca2+ between 100 and 600 microM. The liberation of malate or ATP by Ca2+ could be inhibited by N-methylVal-4-cyclosporin, suggesting that the porin translocator complex constitutes the permeability transition pore. The results show the physiological existence of kinase porin translocator complexes at the mitochondrial surface. It is assumed that such complexes between inner and outer membrane components are the molecular basis of contact sites observed by electron microscopy. Kinase complex formation may serve three regulatory functions, firstly regulation of the kinase activity, secondly stimulation of oxidative phosphorylation and thirdly regulation of the permeability transition pore.
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PMID:Complexes between kinases, mitochondrial porin and adenylate translocator in rat brain resemble the permeability transition pore. 891 85

Binding of the Type I isozyme of mammalian hexokinase to mitochondria is mediated by the porin present in the outer mitochondrial membrane. Type I hexokinase from rat brain is avidly bound by rat liver mitochondria while, under the same conditions, there is no significant binding to mitochondria from S. cerevisiae. Previously published work demonstrates the lack of significant interaction of yeast hexokinase with mitochondria from either liver or yeast. Thus, structural features required for the interaction of porin and hexokinase must have emerged during evolution of the mammalian forms of these proteins. If these structural features serve no functional role other than facilitating this interaction of hexokinase with mitochondria, it seems likely that they evolved in synchrony since operation of selective pressures on the hexokinase-mitochondrial interaction would require the simultaneous presence of hexokinase and porin capable of at least minimal interaction, and be responsive to changes in either partner that affected this interaction. Recent studies have indicated that a second type of binding site, which may or may not involve porin, is present on mammalian mitochondria. There are also reports of hexokinase binding to mitochondria in plant tissues, but the nature of the binding site remains undefined.
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PMID:Homologous and heterologous interactions between hexokinase and mitochondrial porin: evolutionary implications. 906 7

When rat pancreatic islets are incubated in 5.5 or 16.7 mmol/l glucose for 3 h, an increased sensitivity is observed in islets pre-exposed to high glucose, as indicated by a shift to the left of the glucose dose-response curve (EC50 7.1 +/- 0.9 and 11.5 +/- 1.2 in high- and low-glucose-exposed islets, respectively; n = 5, P < 0.05). To investigate the mechanism(s) responsible for this effect, we measured hexokinase and glucokinase activity both in the cytosolic fraction and in a mitochondrion-enriched fraction, since binding to the outer mitochondrial membrane has been reported to result in an increased enzyme activity. In islets cultured at 16.7 mmol/l glucose, the cytosolic hexokinase activity was similar to control islets, but mitochondrial enzyme activity was significantly increased (124 +/- 7 vs. 51 +/- 9 nmol x microg(-1) x 90 min(-1), P < 0.01). As a consequence, the cytosolic:mitochondrial fraction ratio was altered in comparison with control islets. In contrast, glucokinase activity in the two groups of islets was similar in the cytosolic fraction and undetectable in the mitochondrial fraction. Hexokinase I quantitation by Western blot confirmed the enzyme translocation from the free cytosolic to the mitochondria-bound form in islets cultured at 16.7 mmol/l glucose. Glucose-induced alterations were reversible after 1 h exposure to 5.5 mmol/l glucose. Moreover, in islets exposed to 16.7 mmol/l glucose, inhibition of hexokinase binding to mitochondria by the addition of 20 nmol/l dicyclohexylcarbodiimide resulted in no increase of glucose sensitivity (EC50 10.9 +/- 0.4, n = 3, similar to that of control islets). These data indicate that after chronic exposure to high glucose, the beta-cell becomes more sensitive to glucose before eventually getting desensitized. This increased sensitivity is associated with (and may be due to) an increased hexokinase activity secondary to a subcellular shift of the enzyme from the free cytosolic to the mitochondria-bound, more active form.
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PMID:Hexokinase shift to mitochondria is associated with an increased sensitivity to glucose in rat pancreatic islets. 920 Jun 49


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