Glucose 6-phosphate Volume

A seed culture of S. cerevisiae ATCC 24860 (American Type Culture Collection, Manassas, VA, USA) was grown in a media of 5g glucose, and 0.5 g yeast extract, respectively, 1.5 g KH2P04 and 2.25 g Na2P04 phosphate buffer up to a total volume of distilled water, 500 ml. The media was sterilised at 121 °C for 15 min. The stock culture of the microorganisms was transferred to the broth media for preparation of seed culture. 

After removal of the toluene, the mixture was pasteurized at 80° for 5 minutes, cooled, adjusted to pH 7.8 and 2.5 volumes of 95% alcohol added. The mixture was allowed to remain at 4° for 3 hours and the precipitate, containing most of the inorganic and esterified phosphate, was removed by filtration and the alcohol was removed by distillation in vacuo at about 30°. The solution was then made up to 600 ml. and passed through columns of Amberlite IR-100 and Amberlite IR-4. This treatment removed all the electrolytes, including the remaining traces of n-glucose-l-phosphate. After washing the columns with water, the volume had increased to about three liters. The solution was concen-... 

Monooxygenase Assays. Incubation media contained the following (final concentrations) 0.05M phosphate buffer, pH 7.A, glucose-6-phosphate (G-6-P, 2.3 mM), G-6-P dehydrogenase (3 units), NADP (0.23 mM), and KC1 (2.8 mM), and various tissue preparations. Substrates were added in small volumes (25 yl or less) of MeOH. Samples (1.1 ml) were shaken in a thermostated (usually at 22°C) water bath and reactions terminated by enzyme denaturation. Specific analytical procedures for aldrin epoxi-dation (13), 1 CH30-p-nitroanisole 0-demethylation (1A), and 3H-benzo(a)pyrene oxidation (15) have been described. 

Disodium hydrogen phosphate dihydrate (25.5 g), potassium dihydrogen phosphate (9.0 g), ammonium chloride (1.0 g) and sodium chloride (0.5 g) were dissolved in water and the volume adjusted to 900 mL. The solution was sterilized by autoclaving (121 °C, 20 min) and allowed to cool to room temperature. Magnesium sulfate (240.5 mg), kanamycin (50 mg), thiamine (10 mg) and glucose (5 g) were dissolved in water and adjusted to 100 mL volume. This mixture was sterilized by filtration through a 0.2 pm filter and added to the salt solution. 

Add to both sample and blank 50 pi glucose-6-phosphate (pH 6.5) - giving a total aliquot volume of 105 pi. 

Gluco5e-6-phosphate [sodium salt 54010-71-8] M 260.1. Can be freed from metal impurities as described for glucose-l-phosphate. Its barium salt can be purified by solution in dilute HCl and pptn by neutralising the soln. The ppte is washed with small volumes of cold water and dried in air. 

The rest of the Calvin cycle is involved in interconversion of carbohydrates to make glucose (or starch) and the regeneration of the ribulose-bisphosphate acceptor. The reactions are also found in the pathways for gluconeogenesis and the pentose phosphate shunt (see Volume 1, Chapters 10 and 12). The first step is the phosphorylation of 3-phosphoglycerate by the same reactions involved in gluconeogenesis. 

Liquid Culture Medium Dissolve 15 g of peptonized milk, 5 g of water-soluble yeast extract, 10 g of anhydrous glucose, and 2 g of anhydrous potassium dihydrogen phosphate in about 600 mL of water. Add 100 mL of filtered tomato juice (filtered through Whatman No. 1 filter paper, or equivalent), and adjust to pH 6.5 by the dropwise addition of 1.0 N sodium hydroxide. Add, with mixing, 10 mL of the Polysorbate 80 Solution. Dilute with water to a final volume of 1000 mL. Add 10-mL portions of this Liquid Culture Medium to test tubes, cover to prevent contamination, and sterilize by heating in an autoclave at 121° for 15 min. Cool the tubes rapidly to keep color formation to a minimum, and store at 10° in the dark. 

Glucose Standard Solution Dissolve 36.0 mg of anhydrous dextrose in Phosphate Buffer in a 1000-mL volumetric flask, dilute to volume with water, and mix. 

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