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)

A continuous, coupled, spectrophotometric assay is described in which the enzyme ATP sulfurylase is employed to measure the concentration of inorganic pyrophosphate (PPi) at equilibrium with known concentrations of inorganic orthophosphate (Pi) in the presence of excess inorganic pyrophosphatase (PPitase). In agreement with previous reports, the apparent equilibrium constant (Keq,app) of the PPi hydrolysis reaction was shown to decrease as the concentration of Mg2+ is increased. At pH 7.3, 30 degrees C, in the presence of 150 mM NaCl and 1 mM free Mg2+, Keq,app (calculated as [Pi]t2/[PPi]t) was 1950. Measurements of Keq,app at different total concentrations of Mg2+ and Pi permitted the determination of K0, the dissociation constant of the Mg-Pi complex. In 0.05 M Tris-Cl, pH 8.0, at 30 degrees C, K0 was 3.6 mM. In the presence of excess ATP sulfurylase, yeast PPitase catalyzed PPi formation from Pi with a specific activity (Vmax) of 9 units X mg protein-1 at pH 8.0, 30 degrees C, and 1 mM free Mg2+. Half-maximum reverse reaction velocity was observed at a total Pi concentration of 18 mM. (Under the same conditions, Vmax of the PPi hydrolysis reaction was 530 units X mg protein-1.) A radiochemical end point ("reaction-to-completion") assay for measuring unknown concentrations of PPi was devised. In the presence of excess 35S-adenosine-5'-phosphosulfate ([35S]APS) as the cosubstrate, 35SO2-4 formation was stoichiometric with added PPi. (The 35SO2-4 and [35S]APS are separated by adsorption of the latter onto charcoal.) The sensitivity of the assay can be adjusted by varying the specific radioactivity of the [35S]APS. In the absence of interfering substances, as little as 2 pmol of PPi per 1.0 ml assay volume can be measured. The sensitivity of the assay is reduced in the presence of ATP plus perchlorate (which synergistically inhibit the enzyme). However, if the bulk of the ATP is removed from perchloric acid extracts of tissues with glucose and hexokinase, initial intracellular levels as low as 1 microM can be measured. The possibility that most of the cellular PPi extracted with perchloric acid was originally enzyme bound is discussed.
 ...
PMID:ATP sulfurylase-dependent assays for inorganic pyrophosphate: applications to determining the equilibrium constant and reverse direction kinetics of the pyrophosphatase reaction, magnesium binding to orthophosphate, and unknown concentrations of pyrophosphate. 302 16

The aim of the study was to set up a novel fully enzymatic method for screening glucose and 1,5-anhydro-D-glucitol (1,5-AG) in one cuvette. We have determined glucose and 1,5-AG, based on glucokinase (GK) converting glucose to G6P, a compound that can be catalyzed ultimately into 6-PGA by G-6PD and its coenzyme NADP(+), and then calculated glucose concentration according to absorbance variety. Furthermore, pyranose oxidase was used to oxidize 1,5-AG with the formation of 1, 5-anhydro-fructose and H(2)O(2). Measurement was done according to Trinder's reaction principle. The mean within-run and day-to-day precision (CV) of this method for glucose was 0.88% and 1.4%, and also that for 1,5-AG was 1.05% and 1.94%, respectively. The mean recovery rate of two targets was 100.2% and 101.6%, respectively. The correlation (R(2)) between the results of 1,5-AG obtained with our proposed method (y) and those obtained with LanaAG method (x) was 0.999 (y=1.002x-0.675 micromol/l; n=86), and the correlation (R(2)) of glucose between the results obtained with our GK method (y) and those obtained with recommendatory hexokinase method (x) was 0.9999 (y=1.0043x+0.1229 mmol/l; n=86). The reference range (95%) of serological glucose and 1,5-AG was 3.7 to 5.7 mmol/l (4.70+/-0.51 mmol/l) and 83.1 to 240.7 micromol/l (161.9+/-40.2 micromol/l), respectively; and there was no difference with age and sex (P>0.05). This newly developed method was dependable and steady-going, with analysis automatization, and allows quicker and easier measurement of serum glucose and 1,5-AG in one identical reaction cuvette in-phase than previously described methods.
 ...
PMID:A novel fully enzymatic method for determining glucose and 1,5-anhydro-D-glucitol in serum of one cuvette. 1833 75