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)

To establish optimum conditions for creatine kinase (EC 2.7.3.2) activity measurement with the creatine phosphate in equilibrium creatine reaction, we re-examined all kinetics factors relevant to an optimal and standardized enzyme assay at 30 and 25 degrees C. We determined the pH optimum in vaious buffers, considering the effect of the type and concentration of the buffer, as well as the influence of various buffer anions on the activity. The relation between activity and substrate concentration was shown and the apparent Michaelis constants of creatine kinase for creatine phosphate and ADP were evaluated. We tested the effect on creatine kinase measurement of the concentration of substrates (glucose and NADP+) in the auxillary and indicator reactions, especially the influence of the added auxiliary (hexokinase) and indicator (glucose-6-phosphate dehydrogenase) enzymes on the lag phase, at different temperatures. The NADP+ concentration proved to be the factor limiting the duration of constant reaction rate. We studied the inhibition of creatine kinase and adenylate kinase by AMP and established a convenient AMP concentration. For reactivation of creatine kinase, N-acetyl cysteine as sulfhydryl compound was introduced. Finally, we examined the relationship between activity and temperature.
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PMID:Creatine kinase in serum: 1. Determination of optimum reaction conditions. 0 40

I have re-examined optimum reaction conditions for measurement of creatine kinase (EC 2.7.3.2). The optimum pH is 6.45, and 2,2-bis(hydroxymethyl)-2,2',2''-nitrotriethanol acetate, 200 mmol/liter, is the buffer of choice. Thioglycerol, 20 mmol/liter, is superior for both in-assay reactivation and for storage stability of sera. Fluoride, 25 mmol/liter, a broad inactivator of adenylate kinase (EC 2.7.4.3), has little effect on creatine kinase and is superior to AMP for adenylate kinase inhibition in the assay of creatine kinase. Magnesium ion, ADP, and buffer concentrations are interdependent and their optima must be determined together. The hexokinase/glucose-6-phosphate dehydrogenase activity ratio should not exceed 1.6. The range of linearity is limited by the glucose-6-phosphate dehydrogenase and NAD+ concentrations. Glucose-6-phosphate dehydrogenase, ADP, and NAD+ are the constituents most likely to result in unacceptable blanks. Creatine kinase is inhibited noncompetitively by anions: acetate and fluoride inhibit slightly, but sulfates, nitrates, and excessive chlorides should be avoided.
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PMID:Creatine kinase: re-examination of optimum reaction conditions. 1 66

The activity of enzymes regulating the processes providing functional activity of leukocytes was studied in the exudate leukocytes of healthy rabbits and animals with alloxan diabetes. Rabbits with diabetes displayed a reduction of hexokinase, phosphoglucomutase, glucose-6-phosphate dehydrogenase and adenylate kinase activity. The activity of UDPH-pyrophosphorylase, UDPH-glycogentranspherase, 6-phosphogluconate dehydrogenase and glutathion reductase showed no significant changes in the exudate leukocytes in diabetes. A reduction of hexokinase and glucose-6-phosphate dehydrogenase limiting glycolysis and the pentose-phosphate cycle, respectively, providing energy for leukocytes and important in protein metabolism of these cells, is of great significance in the reduction of functional activity of leukocytes in the inflammatory focus in diabetes.
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PMID:[Enzymatic profile of the exudate leukocytes in diabetes mellitus]. 9 55

The problems encountered with a coupled enzyme assay for ATP using glucose, hexokinase and glucose-6-phosphate dehydrogenase are discussed and a modification where fructose and glucosephosphate isomerase were substituted for glucose is described. This modified assay was used successfully to measure the ATP synthesized by reversal of the sarcoplasmic reticulum ATPase. ATP synthesized by adenylate kinase contaminating the sarcoplasmic reticulum was easily corrected for by a subtraction procedure.
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PMID:A spectrophotometric assay of ATP synthesized by sarcoplasmic reticulum. 9 39

Extensor digitorum longus muscles of rats were removed and injected with a solution of Marcaine plus hyaluronidase. After incubation in Marcaine solution for 10 min, the muscles were grafted into their original beds. The grafts and the contralateral control muscles were removed from the rats at 0, 1-5, 7, 11, 36, and 69 days postoperatively. The muscles were then frozen in dry ice and isopentane and subsequently homogenized and centrifuged. The supernatant was analyzed for a number of enzymes, the regenerative patterns of which can be classified into 3 groups: (1) early increase in activity: hexokinase, glucose-6-phosphate dehydrogenase; (2) early decrease in activity with failure to recover to control levels: phosphorylase, phosphofructokinase, alpha-glycerophosphate dehydrogenase; and (3) early decrease followed by return to control levels: lactate dehydrogenase, pyruvate kinase, creatine phosphokinase, adenylate kinase. These patterns are not identical to those reported for embryogenesis of muscle. The data are discussed with regard to correlative histological studies of muscle regeneration.
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PMID:Developmental patterns of glycolytic enzymes in regenerating skeletal muscle after autogenous free grafting. 14 74

The dynamic properties of a series of in vitro reaction systems with increasing complexity and containing phosphofructokinase as central enzyme have been investigated. An experimental strategy and a principal mathematical treatment was elaborated to search for the minimum requirements with respect to the enzyme composition of a reaction system for generating limit cycle behaviour. As a criterion, such models have been developed which permit experimental realization by application of a specially designed flow-through equipment. In addition to phosphofructokinase, the following enzymes have been stepwise included into the reaction systems composing the Models 1 through 6: pyruvate kinase, adenylate kinase, hexokinase, and glucose 6-phosphate isomerase. It turned out that only a minimum dynamic system containing phosphofructokinase and pyruvate kinase as well as excesses of adenylate kinase and glucose 6-phosphate isomerase for maintaining equilibrium conditions between the respective reacting species, acquires the property of limit cycle behaviour and, hence, to generate sustained self-oscillations. The approach permits to compute the region of the experimentally variable parameters (influx rates of fructose 6-phosphate and ATP, maximum rate of pyruvate kianse) for which self-oscillatory behaviour can be predicted.
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PMID:Dynamic properties of in vitro enzyme systems containing phosphofructokinase. 15 82

From a 2.7-A resolution electron density map we have built a model of the polypeptide backbone of a monomer of yeast hexokinase B (EC 2.7.1.1). This map was obtained from a third crystal form of hexokinase, called BIII, which exhibits space group P212121 and which contains only one monomer per asymmetric unit. The 51,000 molecular weight monomer has an elongated shape (80 A by 55 A by 50 A) and is divided into two lobes by a deep central cleft. The polypeptide chain is folded into three structural domains, one of which is predominantly alpha-helical and two of which each contain a beta-pleated sheet flanked by alpha-helices. Both glucose and AMP bind to these crystals and produce significant alterations in the protein structure. Glucose binds in the deep cleft, as was observed previously in the BII crystal of the dimeric enzyme. AMP, however, binds to a site that is different from the major intersubunit ATP binding site observed in the crystalline dimer. The AMP is found near one of the beta-pleated sheets. From our current interpretation of this electron density map we conclude that neither of the two nucleotide binding regions has the same structure as has been observed for the nucleotide binding regions of the dehydrogenases, adenylate kinase, and phosphoglycerate kinase, although some similarities exist.
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PMID:The structure of a yeast hexokinase monomer and its complexes with substrates at 2.7-A resolution. 16 23

5-Acetyl-4-methyl-1-(beta-D-ribofuranosyl)-imidazole-5'-phosphate reacts with diphenylphospho chloridate forming the asymmetrical pyrophosphate ester. This in turn reacts with tri-n-butyl-ammonium phosphate yielding 5-acetyl-4-methyl-imidazole-riboside-5'-diphosphate and with tri-n-butylammonium pyrophosphate to give the nucleotide triphosphate. 5-Acetyl-4-methyl-imidazole-riboside-5'-pyrophosphate shows in the test with pyruvate kinase a reaction rate three times slower than that of ADP; but the same Km as that of ADP. The ATP analogue is only about 10% as effective as ATP itself in the test with hexokinase, 3-phosphoglycerate kinase and gloconate kinase. Adenylate kinase and NAD" kinase show no activity when ATP is replaced by the nucleotide-triphosphate-analogue. In presence of ATP the analogue strongly inhibits the reaction of adenylate kinase.
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PMID:[Synthesis and properties of 5-acetyl-4-methyl-1-(beta-d-ribofuranosyl)-imidazole-5' di-and-triphosphate]. 16 88

1. The concentration of adenylate kinase in carp muscle is about 0.3 mg/g. An improved isolation procedure makes use of a dilute solution of the substrates, ATP and AMP, to elute the enzyme from a phosphocellulose column in overall yields of 60% before crystallization. By the hexokinase--pH-stat assay the specific activity is 3550 units/mg. The preparation has been found to be essentially homogeneous by dodecylsulfate gel electrophoresis, isoelectrofocusing and gel filtration. 2. The molecular weight has been determined to be 22000 by several methods. The absorbance of a 1% solution at 280 nm is 6.9 and the isoelectric point by electrofocusing is pH 5.9. 3. The crystals of carp adenylate kinase have the space group P4-1-22 or P4-3-22. 4. The amino acid composition has been determined. There is no tryptophan, no cystine. There is one amino acid residue each of cysteine and histidine which are at or close to the catalytic center. 5. Several peptides derived by tryptic hydrolysis have been isolated and identified with corresponding peptides of porcine adenylate kinase. Consideration is given to histidine and cysteine being a part of the active site.
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PMID:Crystalline adenylate kinase from carp muscle. 16 48

The adenylate kinase system offers a mechanism for the rapid provision of energy by catalysing the production of ATP from ADP. Fluormetric micromethods were developed for determination of the activity of this enzyme using either formation of ADP or ATP, in each case measured by coupling to suitable dehydrogenase reactions. Both procedures yielded results in good agreement, but when ADP formation was measured an interfering phosphatase splitting of ATP had to be corrected for. Therefore, ADP was preferred as the substrate and its conversion to ATP was determined in a coupled hexokinase-glucose-6-phosphate dehydrogenase reaction yielding stoichiometric amounts of NADPH which were measured by the native fluorescence of this form of the nucleotide. The sensitivity and reproducibility of our micro-method permitted assay of small samples (50-500 ng) such as a layer of cerebellar cortical nerve cells and of insulin producing cells from the islets of Langerhans. Although not reaching the high values in muscle, these cells showed significantly higher activities than parenchymatous cells from the liver and the exocrine pancreas. The sensitivity attained is more than required for assay of clinical fine needle biopsies and is quite satisfactory for detection and estimation of adenylate kinase contaminants in enzyme preparations.
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PMID:Fluorometric microassays of adenylate kinase, an enzyme important in energy metabolism. 20 11


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