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 subcellular location of hexokinase was investigated in rat kidney. Both soluble and particulate locations are indicated by differential centrifugation. The particulate form is predominant, representing about 80% of the total activity. None of the activity is latent. Density gradient centrifugation followed by marker enzyme analysis reveals the presence of two populations of mitochondria with distinct densities. Hexokinase is associated primarily with the mitochondrial population having the lower density. Association of hexokinase with brush border, plasma membrane, lysosomes, and endoplasmic reticulum is considered unlikely on the basis of density gradient centrifugation and enzyme analysis. About 95% of the hexokinase activity associated with the mitochondrial fraction can be released in soluble form by repeated incubations with glucose 6-phosphate. An incubation time of about 4 min at 30 degrees C is required to achieve a maximal solubilizing effect. Release is accomplished without disrupting the mitochondrial compartments. Hexokinase is released also by treatment of the mitochondrial fraction with increasing concentrations of digitonin. This technique disrupts and differentially releases the mitochondrial compartments. As observed with liver, but in contrast to that observed with tumor (Parry, D. M., and Pedersen, P. L. (1983) J. Biol. Chem. 258, 10904-10912), the release of hexokinase from the mitochondrial fraction of kidney does not correlate with the release of enzymes known to mark the mitochondrial membranes or compartments. These studies provide the first critical evidence about the subcellular location of hexokinase in kidney. They show that in this tissue hexokinase is associated primarily with low density mitochondria, a finding that adds credibility to the existence of this discrete population of mitochondria in vivo. Significantly, this association of hexokinase with kidney mitochondria appears unique in that its release on submitochondrial fractionation does not correlate with the release of known mitochondrial marker enzymes. These results are directly relevant to those cells in the kidney which utilize glucose as an energy source. It is suggested that the enhanced glycolytic capacity of these cells may be due, at least in part, to an association of hexokinase with low density mitochondria.
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PMID:Intracellular localization of rat kidney hexokinase. Evidence for an association with low density mitochondria. 674 30

Schizosaccharomyces pombe was treated with either cycloheximide or anisomycin at levels sufficient to inhibit > 95% of protein synthesis for periods upon to 3 h, equivalent to one cell cycle. Treatment for as little as 1 h caused significant loss of the Golgi apparatus by both immunofluorescence and electron microscopy. The loss was quantitated by stereology on electron micrographs. Nearly 90% of the stacked Golgi was lost over a 3 h period. No other intracellular membrane compartment seemed to be affected. Measurement of enzyme activities confirmed these observations. The activity of a resident of the Golgi apparatus, alpha-1,2 galactosyltransferase, was reduced over this time, whereas the endoplasmic reticulum marker, BiP, and the cytoplasmic enzyme, hexokinase, were unaffected. The morphological changes associated with cycloheximide addition were reversed on its removal, though there was a lag before cells recommenced growth or secretion of the enzyme, acid phosphatase.
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PMID:Inhibition of protein synthesis disrupts the Golgi apparatus in the fission yeast, Schizosaccharomyces pombe. 820 44

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

We investigated the kinetics of 2-deoxy-D-glucose (DG) uptake and metabolism in Caco-2 cells, because this human cell line may represent a valid enterocyte model to assess the dynamics between sugar transport and metabolism and hence to obtain insights into the factors involved during the intracellular phase of glucose absorption. When studied in 14-day-old monolayers, DG uptake is characterized by a lag phase with a time course matching the decrease in intracellular glucose concentrations, and no intracellular glucose 6-phosphate (G-6-P) can be detected at any time during incubation. After 1 h of preincubation of Caco-2 cells in substrate-free transport medium, however, steady-state DG uptake matches 2-deoxy-D-glucose 6-phosphate (DG-6-P) accumulation with undetectable levels of free DG. This complex behavior in DG uptake is linked to high hexokinase activity in Caco-2 cells, and the enzyme has a Michaelis-Menten constant K(m) for glucose that is typical of hexokinase type II (0.120 +/- 0.003 mM). Caco-2 cells also contain low-level glucose-6-phosphatase (G-6-Pase) activity, which may account for the leveling off in DG uptake, and the kinetics of DG transport may be attributed to the existence of a predominant pathway with a K(m) of 1.7 +/- 0.2 mM. Finally, analysis of the growth-related expression of DG transport and hexokinase activity clearly shows that DG uptake is lowest in postconfluent cells when hexokinase is at its highest levels. We thus conclude that 1) transport is the rate-limiting step during DG accumulation, 2) G-6-P is a potent inhibitor of hexokinase activity compared with DG-6-P, so that enzyme inhibition may have physiological relevance in diverting glucose from metabolism during its active reabsorption in the small intestine, and 3) low levels of G-6-Pase activity seem to exclude this enzyme, and hence the endoplasmic reticulum, as important factors during the intracellular phase of glucose transport.
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PMID:2-Deoxyglucose transport and metabolism in Caco-2 cells. 877 13

The uptake and release properties of Ca2+ by several subcellular fractions of the bovine adrenal medulla were investigated. Investigation by the 45Ca2+ tracer method showed that permeabilized cells and the fractions of mitochondria (MT) and microsomes (MC) caused ATP-dependent Ca2+ uptake in a Ca2+ concentration-dependent manner (pCa 8-4), whereas permeabilized cells and the fractions of secretory granules (SG) were able to accumulate a significant amount of Ca2+ even in the absence of ATP, which was completed by the addition of hexokinase and glucose. In these organelle fractions, Ca2+ uptake in the presence of ATP at pCa 7 and pCa 5.8 was well-correlated with the activity of the NADPH cytochrome c reductase (marker enzyme for the endoplasmic reticulum) and cytochrome c oxidase (marker enzyme for mitochondria), respectively. As detected by Fura-2 ratiometry, both inositol 1,4,5-trisphosphate (IP3) and caffeine caused concentration-dependent Ca2+ releases from permeabilized cells and MC, but not from MT and SG. In an ATP-depleted condition, homogenates still took up a significant amount of Ca2+ but was not able to respond to IP3 and caffeine. These results suggest that the endoplasmic reticulum is a major Ca(2+)-storing organelle, which releases Ca2+ in response to IP3 and caffeine in bovine adrenal chromaffin cells.
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PMID:Inositol 1,4,5-trisphosphate- and caffeine-sensitive Ca(2+)-storing organelle in bovine adrenal chromaffin cells. 901 39

To elucidate the reasons for glycolytic deviation commonly found in brain tumors, hexokinase (HK) activity, mitochondria-HK binding, oxidative phosphorylation and mitochondrial ultrastructure were studied in 4 human xenografted gliomas. Lactate/pyruvate ratios were increased 3-4 fold and HK activity was of 2-4 fold lower than that of normal rat brain tissue, used as the control. The mitochondria-bound HK (mHK) fraction varied considerably and represented 9 to 69% of the total HK of that normal rat brain. The respiratory activity of glioma mitochondria, assessed by polarography and spectrophotometry, was within the normal range. However, the mitochondrial content of gliomas was lower than in the rat brain tissue, as revealed by the markedly decreased, activities of two unrelated mitochondrial enzymes, cytochrome c oxidase and citrate synthase in glioma homogenates. Electron microscopical studies confirmed the reduced number of mitochondria in 3 out of the 4 gliomas. Profound alterations of mitochondrial ultrastructure, namely of cristae and matrix densities, were observed in the 4 gliomas. The intercrista space was wider in all gliomas and the crista area was larger in 3 out of the 4 gliomas than in normal rat brain. Finally, the outer membrane of glioma mitochondria interacted intimately and extensively with the rough endoplasmic reticulum (RER) and/or nuclear membrane. These results suggest that, because of the very low content of normally functioning mitochondria, gliomas shift their energy metabolism towards a high-level glycolysis to generate their cellular ATP supply, probably through RER-mitochondria interactions and transformation-dependent redistribution of particulate HK from non-mitochondrial to mitochondrial receptors.
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PMID:Gliomas are driven by glycolysis: putative roles of hexokinase, oxidative phosphorylation and mitochondrial ultrastructure. 921 43

Although three germ cell-specific transcripts of type 1 hexokinase exist in murine male germ cells, only one form, HK1-sc, is found at the protein level. This single isoform localizes to three distinct structures in mouse spermatozoa: the membranes of the head, the mitochondria in the midpiece, and the fibrous sheath in the flagellum (Travis, A. J., Foster, J. A., Rosenbaum, N. A., Visconti, P. E., Gerton, G. L., Kopf, G. S., and Moss, S. B. (1998) Mol. Biol. Cell 9, 263-276). The mechanism by which one protein is targeted to multiple sites within this highly polarized cell poses important questions of protein targeting. Because the study of protein targeting in germ cells is hampered by the lack of established cell lines in culture, constructs containing different domains of the germ cell-specific hexokinase transcripts were linked to a green fluorescent protein and transfected into hexokinase-deficient M+R42 cells. Constructs containing a nonhydrophobic, germ cell-specific domain, present at the amino terminus of the HK1-SC protein, were targeted to the endoplasmic reticulum and the plasma membrane. Mutational analysis of this domain demonstrated that a complex motif, PKIRPPLTE (with essential residues italicized), represented a novel endoplasmic reticulum-targeting motif. Constructs based on another germ cell-specific hexokinase transcript, HK1-sa, demonstrated the specific proteolytic removal of an amino-terminal domain, resulting in a protein product identical to HK1-SC. Such processing might constitute a regulatory mechanism governing the spatial and/or temporal expression of the protein.
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PMID:A novel NH(2)-terminal, nonhydrophobic motif targets a male germ cell-specific hexokinase to the endoplasmic reticulum and plasma membrane. 1056 28

Transitional endoplasmic reticulum (tER) consists of confluent rough and smooth endoplasmic reticulum (ER) domains. In a cell-free incubation system, low-density microsomes (1.17 g cc(-1)) isolated from rat liver homogenates reconstitute tER by Mg(2+)GTP- and Mg(2+)ATP-hydrolysis-dependent membrane fusion. The ATPases associated with different cellular activities protein p97 has been identified as the relevant ATPase. The ATP depletion by hexokinase or treatment with either N-ethylmaleimide or anti-p97 prevented assembly of the smooth ER domain of tER. High-salt washing of low-density microsomes inhibited assembly of the smooth ER domain of tER, whereas the readdition of purified p97 with associated p47 promoted reconstitution. The t-SNARE syntaxin 5 was observed within the smooth ER domain of tER, and antisyntaxin 5 abrogated formation of this same membrane compartment. Thus, p97 and syntaxin 5 regulate assembly of the smooth ER domain of tER and hence one of the earliest membrane differentiated components of the secretory pathway.
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PMID:Role of p97 and syntaxin 5 in the assembly of transitional endoplasmic reticulum. 1093 Apr 51

Activities of hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PDH), fructose-6-phosphate kinase (F6PK), glutamate dehydrogenase (GlutDH), aspartate aminotransferase (AAT), malate dehydrogenase (MDH) and glycerol-3-phosphate dehydrogenase (GPDH) were determined in tissue extracts of testes and ovaries of adult Dipetalogaster maximus (Uhler) and Triatoma infestans (Klug) (Hemiptera: Reduviidae), insect vectors of Chagas disease. The fine structure organization of the same organs were studied by electron microscopy. Results allow the following inferences: in testes from both species, most of the glucose would be utilized through the glycolytic pathway. Amino acid catabolism for energy purposes appears to be unimportant. The number of mitochondria and the development of the rough endoplasmic reticulum in cells of the spermatogenic line indicate the occurrence of active oxidative metabolism and protein synthesis; in ovaries, levels of G6PDH indicate the existence of an active pentose pathway which would supply the NADPH required for fat and ecdysteroid synthesis. Amino acid catabolism appears to be relatively more important in ovary than in testis. Fat and glycogen are stored in follicular cells of D. maximus; oocytes of both species contain numerous fat droplets. Abundant mitocondria are present in follicular cells and oocytes. A well developed rough endoplasmic reticulum and free ribosomes are also conspicuous in these cells. The malate/aspartate H-transfer system seemed to be relatively more important than the glycerophosphate shuttle in ovaries as well in testes.
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PMID:Comparative study of enzymes in testes and ovaries from adult Dipetalogaster maximus (Uhler) and triatoma infestans (Klug) (Hemiptera: Reduviidae). correlation with fine structural organization. 1175 15

The cAMP-dependent protein kinase anchoring protein, d-AKAP1, has two N-terminal splice variants. The shorter forms (N0, d-AKAP1a, and -1c) target to mitochondria, and the longer forms (N1, d-AKAP1b, and -1d) with 33 additional residues N-terminal to N0 target to the endoplasmic reticulum (ER) (Huang, L. J., Wang, L., Ma, Y., Durick, K., Perkins, G., Deerinck, T. J., Ellisman, M. H., and Taylor, S. S. (1999) J. Cell Biol. 145, 951-959). In d-AKAP1a, translation may initiate from both Met-34 or Met-49 producing two molecules both targeted to mitochondria. The shorter molecule contains the 15-residue targeting motif, homologous to the N-terminal mitochondrial targeting motif of hexokinase I. Extensive mutagenesis showed that one hydrophobic surface of the 15-residue hexokinase-homologous segment contained the key elements for mitochondrial targeting. The same 15 residues are also part of the ER-targeting signal, but for ER targeting multiple hydrophobic residues are required that encompass both surfaces of the helix. The different involvement of the same helical motif for targeting to the two organelles appears to reflect different modes of interaction with the two organelles. This is the first example of a bifunctional helical element that is required for both ER and mitochondrion targeting.
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PMID:A 15-residue bifunctional element in D-AKAP1 is required for both endoplasmic reticulum and mitochondrial targeting. 1199 83


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