Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.1.1 (hexokinase)
 5,274 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

HEPES-based biological buffer is subject to photooxidation upon exposure to fluorescent illumination. Thereby hydrogen peroxide is generated, which interferes with amperometric oxidoreductase-based biosensors for glucose or adenosine triphosphate (ATP). These biosensors operate at an oxidation potential above 500 mV vs. the standard calomel electrode (SCE) and involve hydrogen peroxide as the electroactive molecule detected at the electrode surface. False-positive detection of ATP was observed in HEPES buffer utilizing an amperometric microbiosensor based on the co-immobilization of glucose oxidase and hexokinase for detection of ATP in biological specimens. Electrochemical, mass spectrometric, (31)P NMR, and (1)H NMR studies indicate that complexation of ATP and HEPES induced by the presence of Ca(2+) in HEPES buffer decreases the photooxidation of HEPES. Consequently, the hydrogen peroxide background concentration is reduced, thereby leading to erroneous ATP detection at the dual-enzyme microbiosensor, which determines an increase in ATP via a reduced hydrogen peroxide signal.
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PMID:The interference of HEPES buffer during amperometric detection of ATP in clinical applications. 1836 90

To determine the effects of ammonia load on glucose metabolism in ruminant small intestinal tissues, duodenal mucosal cells (DMC) were isolated from growing female sheep (n = 10; 46. 0 +/- 0. 8 kg of BW) fed diets differing in CP content: high (19. 4%) vs. low (13. 1%). Ammonia concentration in the duodenal digesta fluid was greater for sheep fed a high CP diet compared with those fed a low CP diet (16. 4 +/- 1. 0 vs. 9. 1 +/- 1. 8 mM). The isolated primary mucosal cells were incubated for 90 min with [2-(13)C] glucose (3 mM) and ammonium chloride (0, 0. 1, 1, 5, 10, 20, or 50 mM) in Krebs-Ringer HEPES buffer. It was hypothesized that DMC would increase glucose carbon utilization for the synthesis of nonessential AA when the ammonia concentration in the incubation media increased. However, utilization of glucose carbon for alanine synthesis decreased linearly (P = 0. 03) as the ammonia concentration in the incubation media increased. Furthermore, glucose disappearance and utilization of glucose carbon for aspartate synthesis were not affected (P > 0. 47) by the ammonia concentration. Contrarily, in vitro glucose disappearance was greater (P = 0. 03) for DMC isolated from sheep fed a low CP diet vs. a high CP diet [14. 6 +/- 1. 6 vs. 8. 6 +/- 1. 3 nmol.(10(6) cells)(-1).(90 min) (-1)], and hexokinase activity was greater (P = 0. 01) in the mucosa of sheep fed a low CP diet compared with a high CP diet (1. 22 +/- 0. 05 vs. 1. 04 +/- 0. 02 mUnit/mg of protein). These observations indicate that ammonia load does not affect the extent of glucose utilization by DMC, and that glucose carbon may not play a significant role for the synthesis of alanine, aspartate, or glutamate when DMC are exposed to increased concentrations of ammonia.
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PMID:Effects of ammonia load on glucose metabolism by isolated ovine duodenal mucosa. 1846 43

A CE-based method for hexokinase inhibitor screening was developed in the present paper. In this method, hexokinase activity was assayed via electrophoretically mediated microanalysis (EMMA), which combines on-column hexokinase-mediated reaction and measurement of produced adenosine 5'-diphosphate (ADP) via electrophoretical separation and UV detection. Enzyme inhibition can be read out directly from the reduced peak area of ADP in comparison with a reference electropherogram obtained in the absence of any inhibitor. Conditions for on-column enzyme reaction and separation of adenosine 5'-triphosphate (ATP) and ADP were optimized. The optimal buffer composition for enzymatic reaction was 25 mM HEPES buffer (pH 7.5) containing 5 mM MgCl(2), whereas the optimal buffer composition for separation was 100 mM Tris-phosphate buffer (pH 5.5) containing 0.02% (m/v) hexadimethrine bromide (HDB). Fortunately, discontinuous buffer system can be adapted easily in the EMMA method. The time for separation was reduced dramatically to less than 3 min by reversing the direction of EOF via dynamically coating the capillary wall with the cationic polyelectrolyte HDB. Moreover, the peak tailing of ATP was also reduced by HDB coating. The Z' factor as high as 0.98 was obtained, indicating a high quality of the screening data. The present method is simple, robust and cost-effective.
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PMID:Hexokinase inhibitor screening based on adenosine 5'-diphosphate determination by electrophoretically mediated microanalysis. 1930 67