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)

A study of the effect of varying ionic strength on the glucose-induced quenching of tryptophan fluorescence of hexokinase isoenzymes A(P-I) and B(P-II) was carried out at pH 8.3 and pH 5.5. At p/ 8.3 both isoenzymes gave apparently linear Scatchard-type data plots even with protein concentrations and ionic strengths for which both dimeric and monomeric forms of hexokinase coexist in signiciant amounts. Taking inco account a 1% accuracy in the experimental measurements, we concluded that the intrinsic dissociation constants K(M) and K(D), for the binding of glucose to the monomeric and dimeric forms of HkB, are within a factor of two of each other, i.e. K(D)/K(M) less than or equal to 2. The values of K(M), estimated from the apparent K, were so greatly influenced by ionic strength that it is clear that it is meaningless to compare K(M) and K(D) values measured at different ionic strengths as has been done in the literature. Curvature in the pH 5.5. fluorescence-quenching plots for relatively low ionic strengths demonstrates cooperativity for glucose-binding to the dimer, positive for HkA but negative for HkB. In contrast, the binding is relatively non-cooperative at high ionic strength at this pH. These results were attributed to the well known effect of salt-neutralization of side chain electrical charges on the flexibility and compactness of proteins.
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PMID:Fluorescence-quenching study of glucose binding by yeast hexokinase isoenzymes. 2 68

A procedure is described to prepare uniformly labelled D-[14C]ribulose 1,5-bisphosphate enzymically from uniformly labelled D-[14C]glucose through the coupled reactions catalysed by hexokinase (EC 2.7.1.1), glucose 6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44) and 5-phosphoribulokinase (EC 2.7.1.19). All reagents utilized in the method are commercially available. The procedure is a reliable preparative-scale method for synthesizing the dibarium salt of D-[14C]ribulose 1,5-biphosphate with a specific radioactivity up to 7 mCi/mmol and a purity near 90%. The final product was free of other 14C-labelled sugars, sugar phosphate esters, Pi and nucleotides.
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PMID:Preparative-scale enzymic synthesis of D-[14C]ribulose 1,5-bisphosphate. 21 56

Several glycolytic enzymes were observed to have between 40-90% of their activities associated with the particulate fractions of lysed nerve endings. The enzymes showing high particulate activity in lysed nerve endings were hexokinase (EC 2.7.1.1), aldolase (EC 4.1.2.13), glucosephosphate isomerase (EC 5.3.1.9), phosphofructokinase (EC 2.7.1.11), glyceraldehyde-phosphate dehydrogenase (EC 1.2.1.12), pyruvate kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.27). With the exception of phosphofructokinase, 80% or more of the particle associated activity of each enzyme was solubilized by salt treatment indicating the association with particles was ionic. Sub-fractionation of lysed nerve endings showed hexokinase and fumarase (EC 4.2.1.2) had the highest specific activity in the same fractions which is consistent with observations indicating that hexokinase is associated with mitochondria. The other glycolytic zymes having high particulate activity, aldolase, glucosephosphate isomerase, phosphofructokinase, glyceraldehyde-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase, showed enrichment in fractions containing synaptosomal membranes, i.e. the fractions having highest specific activity of acetylcholinesterase (EC 3.1.1.7) and (Na+ + K+)-ATPase (EC 3.6.1.3).
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PMID:Association of glycolytic enzymes with particulate fractions from nerve endings. 62 35

Triethyloxonium tetrafluoroborate was used to O-alkylate nylon-tube thus producing the imidate salt of the nylon which was further made to react with 1,6-diaminohexane. 2. Hexokinase (EC 2.7.1.1) and glucose 6-phosphate dehydrogenase (EC 1.1.1.49) were immobilized on the amino-substituted nylon tube through glutaraldeyde and bisimidates. 3. The effect of varying the conditions of O-alkylation and the amount of enzyme immobilized on the activity of nylon tube-hexokinase derivatives was determined. 4. The effect of varying the amount of enzyme immobilized on the activity of nylon-tube-glucose 6-phosphate dehydrogenase derivatives was determined. 5. The thermal stability of nylon-tube-hexokinase and nylon-tube-glucose 6-phosphate dehydrogenase derivatives was studied. 6. Different ratios of hexokinase and glucose 6-phosphate dehydrogenase were co-immobilized on nylon tube, and the rate of conversion of glucose into 6-phosphogluconolactone was compared with the individual activities of the immobilized enzymes. 7. Hexokinase and glucose 6-phosphate dehydrogenase co-immobilized on nylon tube were used in the automated analysis of glucose.
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PMID:The preparation of nylon-tube-supported hexokinase and glucose 6-phosphate dehydrogenase and the use of the co-immobilized enzymes in the automated determination of glucose. 116 61

A new type of aqueous two-phase system composed of an ethylene oxide and propylene oxide random co-polymer, UCON 50-HB-5100, as the upper phase polymer and either dextran or hydroxypropyl starch as the lower phase polymer has been characterized and used to purify 3-phosphoglycerate kinase (EC 2.7.2.3) and hexokinase (EC 2.7.1.1) from bakers' yeast. The UCON 50-HB-5100 polymer has a cloud point of 55 degrees C at which temperature it phase separates from water. This cloud point can be lowered to 40 degrees C by the addition of 0.2 M sodium sulfate salt. The low cloud point of this UCON polymer makes it possible to obtain the target enzymes in a water and buffer solution, and to recover and recycle the UCON 50-HB-5100 polymer. The phase diagrams for the systems UCON 50-HB-5100/Dextran T500 and UCON 50-HB-5100/hydroxypropyl starch have been determined. Yeast homogenate was first partitioned in a system composed of a top phase containing UCON 50-HB-5100 and a bottom phase containing either dextran or hydroxypropyl starch. The top phase containing the enzyme free of cell debris was removed and the temperature increased above the cloud point of the UCON until a new two phase system composed of water as the top phase and a concentrated liquid UCON 50-HB-5100 bottom phase was formed. The water phase containing the enzyme was removed and the bottom phase containing the UCON 50-HB-5100 could be recycled to perform a second extraction.
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PMID:Enzyme purification using temperature-induced phase formation. 136 89

We recently proposed that extracellular ATP (ATPo) may be involved in CTL-mediated cytotoxicity by acting in concert with yet unidentified cellular components (ATPo receptors/ATPo-binding proteins, ectoprotein kinases). The TCR-triggered ATPo accumulation by CTL has been demonstrated, whereas the resistance of CTL to ATPo was explained by the action of highly active ecto-ATPases or by the absence of relevant ATP-binding proteins. However, no data were available to discriminate between the possibilities of: i) ATPo acting alone as a "hit" molecule because of the cell-permeabilizing properties of ATP4- or ii) ATPo acting as a "messenger" (as MgATP2-) in concert with other molecules. Comparing ATPo-induced and CTL-mediated cell lysis, we found that ATPo-induced lysis of some target cells is greatly decreased at neutral and acidic pH, whereas Ca(2+)-dependent CTL-mediated lysis of the same cells is barely affected. In agreement with the observed pH dependency, at low Mg2+ concentrations, which favor ATP4- over MgATP2-, maximal ATPo-induced lysis was observed. However, CTL-mediated cytotoxicity in both Ag-specific and retargeting assays was markedly reduced at low Mg2+ concentrations. These results suggest that ATPo acting alone as a "hit" molecule cannot fully account for the extracellular Ca(2+)-dependent lethal hit delivery by CTL or that ATP4- is active at very low concentrations. This conclusion was further supported by studying the lytic effect of ATPo and CTL on the anti-TCR mAb-coupled SRBC. CTL were efficient in the SRBC lysis, whereas no lysis of SRBC by ATPo was detected. The resistance of SRBC to ATPo is not caused by a high ATPo degradation, because the ecto-ATPase activity of SRBC was much lower than in ATPo-resistant CTL OE4 cells and comparable with EL4 tumor cells, which were easily lysed by ATPo. These data suggested the need for careful consideration of the pH and cation composition of the media used for studying ATPo effects. The caveats in the use of ATP-degrading enzymes to implicate the role of extracellular ATPo in the CTL-mediated cytotoxicity are described here. A clarification of the previously described cytotoxicity inhibition by hexokinase, which is caused by an inhibitory salt effect, is presented. It is suggested that if Ca(2+)-dependent lysis of SRBC and of other target cells by CTL does involve extracellular ATP, it may function as a "messenger" in concert with other extracellular molecules.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Comparative studies of the cytotoxic T lymphocyte-mediated cytotoxicity and of extracellular ATP-induced cell lysis. Different requirements in extracellular Mg2+ and pH. 194 Mar 62

The hexokinase (HK) of the human red blood cell (RBC) was separated into two distinct major isozymes by fast protein liquid chromatography using a linear salt gradient on a MonoQ column. The first isozyme (HKI) eluted as a sharp peak at the same position as HKI of human liver. The second isozyme eluted between HKI and HKII of human white blood cells, and it appeared to be unique to the RBC (it was designated HKR). From a gel filtration column, HKR eluted before HKI, suggesting that it was larger than HKI by several kilodaltons. In a mitochondria-enriched fraction from human reticulocytes, no HKR was found; thus, HKR was not a mitochondrial enzyme. Despite these differences in chromatographic behavior, size, and mitochondrial binding, both forms behaved kinetically as HKI. RBC from normal blood contained HKI and HKR at an equal activity, but in reticulocyte-rich RBC, HKR dominated. When RBC of increasing age was separated by buoyant density ultracentrifugation, the total HK activity decayed in a biphasic manner, with half-lives respectively of approximately 15 and approximately 51 days. When isolated by MonoQ column from each age-separated fraction, HKR was the major form in the youngest RBC, and decreased rapidly with cell age, with a t 1/2 of approximately 10 days, representing a negligible activity in the oldest RBC. Instead, HKI was relatively stable through the entire life span of the RBC, with a t 1/2 of approximately 66 days. Thus, HKR appears to be an RBC-specific isozyme that is predominant in the reticulocyte and is then rapidly degraded. During maturation of the RBC, the fast decay of HKR contributes to the early sharp decline of HK activity and the slow decay of HKI to the later gradual decline.
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PMID:An isozyme of hexokinase specific for the human red blood cell (HKR) 238 63

A blotting method is described to detect enzymes that do not normally yield a colored product. The method can be used for dot blotting as well as blotting after gel electrophoresis of many enzymes if the reactions they catalyze can be coupled to an oxidase or a dehydrogenase. The latter, designated "auxiliary enzymes," are preimmobilized on membranes of nitrocellulose or positively charged nylon and the reaction they catalyze is coupled with reduction of tetrazolium salt to yield colored formazan on areas of the transfer membrane occupied by the blotted enzymes. In the examples reported here, preimmobilized glucose oxidase, L-amino acid oxidase, xanthine oxidase, malate dehydrogenase, and a mixture of hexokinase and glucose-6-phosphate dehydrogenase were used as auxiliary enzymes to detect blotted invertase, leucine aminopeptidase, purine nucleoside phosphorylase, fumarase, and adenylate kinase, respectively. Detection limits varied, but never exceeded 100 ng for these enzymes. After blotting from polyacrylamide gels, the fumarase assay was the most sensitive of those investigated, detecting 10 ng of enzyme used for electrophoresis. Invertase, a glycoprotein, was detected with higher sensitivity on nitrocellulose membranes when concanavalin A was present on the membrane in addition to the auxiliary enzyme, glucose oxidase. On blots from isoelectric focusing gels, the assay detected two isozymes of purine nucleoside phosphorylase in a sample from calf spleen and at least five isozymes of this enzyme in lysates from human red cells.
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PMID:Activity staining of blotted enzymes by reaction coupling with transfer membrane-immobilized auxiliary enzymes. 245 38

Histochemical localization and photodensitometric quantification of the metabolic enzyme, hexokinase (HK), were used to study changes in brain metabolic activity that occur during the development of (5 days) and recovery from (7 days) dehydration. In water-deprived (WD) rats, HK activity increased after 2 days in the subfornical organ (SFO, 22%), nucleus circularis (NC, 36%), parvo- and magnocellular divisions of the paraventricular nucleus (pPVH, 17%; mPVH, 46%) and supraoptic nucleus (SON, 46%). Activity in SFO declined to control levels at 3 days but increased again thereafter. In pPVH, mPVH, and SON, activity was elevated until the end of the experiment. In NC, activity returned to control levels within 2 days of drinking by the rats. In salt-loaded (2% NaCl in water) rats, changes were similar to those of WD rats up to 2 days of dehydration (SFO, 25%; NC, 20%; pPVH, 16%; mPVH, 38%; SON, 50%). Activity in SFO and pPVH returned to control levels after 3 days and remained unchanged. In mPVH, SON, and NC, activity remained elevated and declined to control levels when salt-free water was provided. Results confirm that water deprivation is a stronger dehydrating stimulus than salt loading. In addition, metabolic activity, as measured by HK activity, varies daily during periods of dehydration and rehydration. These changes cannot always be predicted from results obtained only at the end of a period of dehydration. It is concluded that it is necessary to study dehydration-induced changes in brain metabolism on a daily basis to more fully understand the roles of discrete brain regions in the regulation of body fluids.
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PMID:Regional alterations in hexokinase activity within rat brain during dehydration and rehydration. 271 47

These studies addressed the question of the in vivo distribution of rat brain hexokinase (HK), and whether physiologically relevant changes in the glycolytic rate are accompanied by changes in the distribution of HK. Homogenates of fresh tissue showed only 11-15% of the overt (assayable without added detergent) HK to be soluble (found in high-speed centrifugation supernatant fractions) when homogenization was begun within 15-20 s of sacrifice. Freeze-blown rat brain tissue also was used, coupled with a new technique wherein it was homogenized as it thawed in a buffered sucrose solution containing 1 mM EDTA. In tissue sampled 15 min (anesthetized) or 60 min (waking) after ip Nembutal injection (40 mg/kg), 23% of the overt HK and 79% of the total lactate dehydrogenase were soluble. The average phosphocreatine content of these and similar homogenates had decreased only 23% from in vivo levels, while ATP had decreased by 65%, due to the combined effects of a high level of endogenous ATPase, chelation of Mg2+ by EDTA, and the greater stability of Mg-ATP2- relative to Mg-ADP1-. These data indicated that the tissue experienced, at most, the equivalent of 6 s of complete ischemia prior to the completion of homogenization. Synaptosomes derived from rat and chicken cerebra were incubated at 37 degrees C in a physiological salt solution containing 10 mM glucose. Addition of veratridine has been shown to stimulate glycolysis and oxidative phosphorylation two- to threefold (H. T. Kyriazi and R. E. Basford (1986) J. Neurochem., in press), but did not alter the HK distribution, as 21% was found in the supernatant fractions of both control and veratridine-stimulated synaptosomes treated with digitonin. These results indicate that in brain tissue, large net movements of HK on and off the outer mitochondrial membrane do not occur, and thus play no role in the regulation of glycolysis.
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PMID:An examination of the in vivo distribution of brain hexokinase between the cytosol and the outer mitochondrial membrane. 294 9


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