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)

Synaptosomes prepared and incubated in a variety of ways from rat cerebra exhibited intractable, unphysiologically low adenylate energy charge values (approximately 0.37-0.60), low total adenine nucleotide contents (approximately 8-10 nmol/mg protein), and much higher adenylate kinase apparent Keq values (approximately 3-8) as compared to intact brain tissue (values of approximately 0.90, 25 nmol/mg, and 0.74, respectively). Synaptosomes prepared from mouse, dog, and chicken cerebra had values essentially identical to those from rat. When incubated under oxygen in a physiological salt solution containing glucose, synaptosomes metabolized more glucose to lactic acid than to CO2, and the addition of 100 microM veratridine caused a two- to threefold stimulation of O2 uptake, lactate accumulation, and CO2 output. It is known that synaptosome fractions contain a substantial number (at least 30-45% by volume) of cytoplasm-containing particles devoid of mitochondria (henceforth termed "cytosolic particles"), and that approximately 80% of brain hexokinase is bound to the outer mitochondrial membrane. For the cytosolic particles, lacking oxidative phosphorylation, to maintain their "in vivo" ATP turnover would require about a 19-fold increase in the glycolytic rate, which is not possible due to limiting amounts of hexokinase, and thus these particles are postulated to be responsible for the high level of aerobic lactate accumulation and the intractable low energy charge values found in synaptosome fractions. The mitochondria-containing particles are postulated to have a normal energy charge, a submaximal glycolytic rate, and minimal lactate production, on the basis of the capacity of veratridine to stimulate synaptosomal O2 uptake and CO2 and lactate output. Calculations based on this "two populations of particles" hypothesis indicate that for synaptosome fractions in general, (1) the cytosolic particles contain approximately 35-64% of the total adenine nucleotides and maintain an energy charge of approximately 0.12; (2) the cytosolic particles and mitochondria-containing particles have adenylate kinase apparent Keq values of approximately 0.21-1.66 and 0.74, respectively, revealing that the higher apparent Keq values of the synaptosome fractions probably are not real departures from equilibrium: and (3) approximately 31-45% of synaptosome fraction protein is contained in debris, which, when taken into account, yields total adenine nucleotide contents in the cytosolic particles and mitochondria-containing particles of approximately 15-24 and approximately 11-19 nmol/mg of particle protein, respectively.
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PMID:Intractable unphysiologically low adenylate energy charge values in synaptosome fractions: an explanatory hypothesis based on the fraction's heterogeneity. 309 Feb 2

A considerable amount of experimental evidence exists suggesting that forebrain structures are involved in the pathogenesis of hypertension. In particular, the paraventricular nucleus of the hypothalamus (PVH) has been implicated in the development and maintenance of the elevated arterial pressure (AP) in several different experimental models of hypertension. The present study was done to determine whether the PVH contributed to the maintenance of the increased AP in deoxycorticosterone acetate-salt (DOCA) hypertension in the rat. In the first series of experiments, using the hexokinase histochemical method, increased metabolic activity was observed in the PVH of DOCA-salt hypertensive rats. In addition, the lateral septal nucleus, median preoptic nucleus, bed nucleus of the stria terminalis, subfornical organ, nucleus circularis, supraoptic nucleus and central nucleus of the amygdala were observed to have increased metabolic activity. In the second series of experiments, bilateral lesions of the PVH resulted in a transient reduction in the elevated AP of DOCA-salt hypertensive animals. However, within approximately a week, the level of AP was not significantly different from sham-PVH lesioned DOCA-salt hypertensive rats. These data suggest that the PVH may be one of several forebrain structures that contributes to the elevated sympathetic activity in DOCA-salt hypertension and when absent other pressor systems are recruited to maintain the elevated AP.
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PMID:Contribution of forebrain mechanisms in the maintenance of deoxycorticosterone acetate-salt hypertension. 324 87

The pressure-induced dissociation of the isozymes P1 and P2 of hexokinase was investigated by studies of the spectral shift of the intrinsic protein fluorescence and by the fluorescence polarization of dansyl conjugates. The free energy of association of the monomers at atmospheric pressure, Katm, was -14.2 kcal mol-1 at 20 degrees C and -11.4 kcal mol-1 at 0 degrees C. The positive enthalpy indicates that the association of the monomers is entropy-driven, overcoming the negative enthalpy of hydration of the subunit interfaces. At 0 degrees C and 1 bar, glucose stabilizes the association by -1.1 kcal mol-1 and the binding of both adenosine 5'-(beta, gamma-methylenetriphosphate) (AMPPCP) and glucose by an even larger amount, -1.34 kcal mol-1. Paradoxically, adenosine 5'-triphosphate (ATP), or AMPPCP, in the absence of glucose destabilizes the association by +0.34 kcal mol-1, while adenosine 5'-diphosphate (ADP) stabilizes it by -0.6 kcal mol-1. Comparison of dV0, the apparent standard volume of association, at different pHs and temperatures indicates that its value (115-160 mL mol-1) is strongly dependent upon the ionization of a group at the subunit interface with a pK near neutrality. Under dissociating pressures, trypsin action results in permanent dissociation of the dimer, confirming earlier observations of Colowick by less direct methods. The P1 and P2 enzymes differ in Katm and dV0 and markedly so in the effects of salt upon the stability of the dimer.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Dissociation of yeast hexokinase by hydrostatic pressure. 329 47

ATP was synthesized in presence of insulin or insulin and prostaglandin E2 by rat skeletal muscle particulate preparation enriched in plasmatic membranes. Isolation of the ATP was carried out using column chromatography on Dowex 1 x 8/cl-form, 100--200 mesh). ATP was formed within 1 min in a medium containing Tris-HCl buffer, pH 7.5, ADP, Mg2+, inorganic phosphate, NaF during NADH-related oxidation involving cytochrome c and O2 in amounts of 100--300 pmoles per mg of protein. Quantitative estimation of ATP in the lyophilized product was carried out by means of spectrophotometry at 340 nm of NADPH formed during a coupled enzymatic reactions involving hexokinase and glycose-6-phosphate dehydrogenase. This products identified by descending paper chromatography on Whatman NI in the system containing ethanol-ammonium citrate pH 4.4 and pH 7.5. Identification of ATP was also performed by thin-layer chromatography. The product was tested for content of ribose (orcinol method) and of inorganic phosphate after acid hydrolysis within 7.5 min at 100 degrees. In the product obtained adenine was identified by UV-spectrophotometry at 260 nm. A salt of ATP was synthesized from the product obtained.
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PMID:[Isolation, identification and quantitative determination of the ATP synthesized by a preparation of plasma membrane-enriched particles from rat skeletal muscles in the presence of insulin]. 703 65

Hoggett & Kellett [Eur. J. Biochem. 66, 65-77 (1976)] have reported that the binding of glucose to the monomer of hexokinase PII isoenzyme is independent of ionic strength, in contrast to the subsequent claim of Feldman & Kramp [Biochemistry 17, 1541-1547 (1978)] that the binding is strongly dependent on ionic strength. Since measurements with native hexokinase P forms are complicated by the fact that the enzyme exists in a monomer-dimer association-dissociation equilibrium, we have now studied the binding of glucose to the proteolytically-modified S forms which are monomeric. At pH 8.5, the affinity of glucose for both SI and SII monomers is independent of salt concentration over the range of KCl concentrations 0-1.0 mol . dm-3 and is in good agreement with that of the corresponding P forms in both low and high salt. These observations confirm that the binding of glucose to hexokinase P monomers is independent of ionic strength and that the affinity of glucose for the hexokinase PII monomer is about an order of magnitude greater than that for the dimer.
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PMID:The binding of glucose to yeast hexokinase monomers is independent of ionic strength. 705 60

Hydrophobic interaction chromatography (HIC) has been employed extensively in the separation of proteins by elution using a descending salt gradient, with and without the use of detergents or denaturing agents. In this study, a new hydrophobic interaction chromatographic support, Toyopearl Phenyl 650 S, was investigated in order to examine the distribution of multiple forms of rabbit reticulocyte hexokinase type I. These distinct forms of the enzyme, designated hexokinase Ia, Ia* and Ib, show similar kinetic and physical properties, similar molecular masses (ca. 100,000) and a different intracellular distribution. The results obtained using Toyopearl Phenyl 650 S of 20-50-microns particle diameter show that this HIC support allows very high resolution, comparable to that obtainable with HIC-HPLC columns but with the advantage of charging a higher amount of starting material even with a high protein concentration. These characteristics render Toyopearl Phenyl 650 S suitable for analytical and preparative purposes. Further, in the separation of multiple forms of rabbit reticulocyte hexokinase, the HIC method was shown to be superior to RP-HPLC, making possible the efficient separation of proteins with high molecular mass and their recovery in active forms. The Toyopearl Phenyl 650 S column was also shown to be more efficient than the ion-exchange chromatographic media previously used, allowing a quicker analysis of the multiple forms of rabbit reticulocyte hexokinase under different biological conditions.
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PMID:High resolution of multiple forms of rabbit reticulocyte hexokinase type I by hydrophobic interaction chromatography. 792 Nov 81

4-Aminodiphenylamine (N-phenyl-1,4-phenylenediamine, CAS 101-54-2) and its water-soluble HCl salt (CAS 2198-59-6) were demonstrated to be efficient mediators for glucose oxidase, lactate oxidase, xanthine oxidase, and lysine oxidase. Using cyclic voltammetry, single oxidative peak potentials were observed for scans ranging from 0 to 0.5 V vs Ag/AgCl. The half-wave potential for both preparations was 0.11 V vs Ag/AgCl at pH 7 and decreased 59 mV per unit pH increase. Peak current data were analyzed to estimate diffusivities of 0.8 x 10(-5) cm2/s for soluble 4-ADPA HCl, and 2.36 x 10(-5) cm2/s for 4-ADPA solubilized in 2.5 mM 2-hydroxypropyl-beta-cyclodextrin. The overall second-order kinetic constants (k) for the reaction of reduced glucose oxidase with oxidized 4-ADPA HCl and 4-ADPA in cyclodextrin were estimated to be 1.8 x 10(5) and 1.7 x 10(-5) M-1 s-1, respectively, using cyclic voltammetry measurements at varied scan rates and enzyme concentrations. Both preparations proved to be suitable electron acceptors for horseradish peroxidase, as indicated by changes in absorbance spectra upon oxidation or reduction. The electrochemical and spectral behavior of the preparations were applied in conjunction with glucose oxidase to devise mediated amperometric and hydrogen peroxide-coupled spectrophotometric assays for glucose. The results of both assays compared favorably with the hexokinase reference method.
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PMID:Dual functionalities of 4-aminodiphenylamine in enzymatic assay and mediated biosensor construction. 859 91

A unique cDNA for hexokinase (HK) was identified from poly(A)+ RNA of human reticulocytes by anchored polymerase chain reaction. This appeared to represent the cDNA for the red blood cell (RBC)-specific HK isozyme (HKR) described in our previous study (Murakami et al: Blood 75:770, 1990). Its nucleotide sequence was identical to HKI cDNA except for the 5' extreme end. It lacked the first 62 nucleotides of the HKI coding region: instead, it contained a unique sequence of 60 nucleotides at the beginning of the coding sequence as well as another unique sequence upstream of the putative translation initiation site. It lacked the porin-binding domain which facilitates binding to the mitochondria, thus explaining the exclusive cytoplasmic localization of HKR. It was the major cDNA derived from reticulocytes, consistent with the observation that HKR activity is predominant in reticulocytes. Northern blot analysis showed that it was expressed in the reticulocytes and in the K562 erythroleukemic cell line, but not in a lymphocytic cell line. In the extract of K562 cells, HKR activity co-eluted with the HKR of human RBCs on a MonoQ column (Pharmacia, Piscataway, NJ) chromatography, using a salt gradient elution. The separate genetic control of the RBC-specific HK isozyme explains the clinical reports of two types of HK deficiency, one in which the HK activity was reduced exclusively in the RBC (HKR defect) and another with general decrease of HK activity in several tissues (HKI defect).
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PMID:Identification of the cDNA for human red blood cell-specific hexokinase isozyme. 941 10

Evidence is presented that intermediates of the oxidative pentose phosphate pathway (OPPP) are channeled from one pathway enzyme to the next. CO2 produced from [1-14C]glucose in the presence of unlabelled pathway intermediates contained much more radioactivity than predicted by a model in which pathway-produced intermediates are in equilibrium with identical molecules in the bulk phase. This was the case whether glucose 6-phosphate (Glc6P), 6-phosphogluconolactone, or 6-phosphogluconate was added. Assumptions involved in calculating the amount of 14CO2 predicted for free mixing of 14C-labelled and unlabelled intermediates are discussed, together with the following results. (a) 14CO2 production by pea nodules in the presence of 3 mM 6-phosphogluconate was higher than in its absence. (b) Apparent channeling of intermediates was much higher for purified yeast enzymes than for yeast extract. (c) 6-Phosphogluconate and 6-phosphogluconolactone were channeled between yeast Glc6P dehydrogenase and 6-phosphogluconate dehydrogenase despite the absence of 6-phosphogluconolactonase in the purified yeast enzyme mixture. (d) When purified yeast hexokinase was physically separated from Glc6P dehydrogenase and 6-phosphogluconate dehydrogenase by a dialysis membrane, there was no apparent channeling. (e) Poly(ethylene glycol), high salt and detergents had little effect on apparent channeling of OPPP intermediates, which is consistent with a stable complex of enzymes. On the other hand, density gradient centrifugation experiments suggested a more transient interaction between the enzymes. Taken together, the results support channeling of OPPP pathway intermediates.
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PMID:Evidence for channeling of intermediates in the oxidative pentose phosphate pathway by soybean and pea nodule extracts, yeast extracts, and purified yeast enzymes. 920 16

Rats were fasted for 48 h, but infused with either NaCl or the sodium salt of monoethyl succinic acid (EMS), both delivered at a rate of 80 mumol/g body weight per day. The infusion of EMS, as compared to NaCl, failed to affect paraovarian adipose tissue or liver weight, liver or muscle glycogen, and insulinemia. It accentuated the starvation-induced fall in body weight, and decreased both liver and muscle protein content. Nevertheless, the succinate ester increased plasma D-glucose concentration, delayed the rise in ketonemia, maintained a higher glucokinase/hexokinase activity ratio in liver and pancreatic islets, and allowed for a more efficient stimulation of insulin release by D-glucose or 2-ketoisocaproate in isolated pancreatic islets. These findings indicate that monoethyl succinate displays a significant nutritional value when infused in starved rats.
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PMID:Nutritional value of succinic acid monoethyl ester in starvation. 926 86


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