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By Willis A. Wood

The severely acclaimed laboratory usual, Methods in Enzymology, is among the so much hugely revered courses within the box of biochemistry. seeing that 1955, each one quantity has been eagerly awaited, often consulted, and praised via researchers and reviewers alike. The sequence comprises a lot fabric nonetheless appropriate this day - actually a vital ebook for researchers in all fields of lifestyles sciences.

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The frozen (NH4)2SO~ precipitate could be stored up to a week with no loss of activity. Step 3. Dialysis. 1 M Tris, 5 mM EDTA, 5 mM MgS04, 10 mM fl-EtOH, and 50 mM glucose (buffer B). This volume was generally about 250 ml. 1 M KC1 buffer with two changes of buffer. No loss of activity was noted on dialysis. 1~R. C. Hard, Fed. Proc. Fed. Amer. Soc. Exp. Biol. 30, 292 (1971). 34 KINASES [51 Step 4. Batchwise DEAE-Sephadex Treatment. The dialyzate was further diluted to 1 liter with buffer B. 5 g of protein, of a slurry (supernatant decanted) of DEAE-Sephadex equilibrated with buffer B.

Biophys. 154, 332 (1973). [4] Glucokinase from Yeast By P. K. MAITRA D-Glucose + A T P - * D-glucose 6-phosphate + A D P Assay Method Principle. 4 Glucose-6-phosphate dehydrogenase, 140 units per milliliter of saturated (NH~)~S0~ solution Procedure. To 1 ml of triethanolamine buffer-MgC12 mixture in a cuvette the following additions are made: 10 ~l of glucose, 5 t,1 of ATP, 2 ~l of NADP, and 1 t~l of the glucose-6-phosphate dehydrogenase suspension. It is convenient to prepare daily a mixture of all the above ingre:iients except the dehydrogenase.

Fractions with a minimum specific activity of 60 units of glucose-ATP phosphotransferase activity per E~8o were pooled, and the enzyme was precipitated with solid (NH~)~SO4 to 65% saturation. The fluffy white precipitate of the enzyme was taken up in a minimum volume of the Tris dialysis buffer, transferred to a thin cellophane bag and dialyzed against this buffer for an hour, whereupon it dissolved. Step 6. Sephadex G-IO0 Chromatography. The protein solution from this step was layered carefully on a column (175 ml; 55 cm length) of Sephadex G-100 resin equilibrated with the Tris dialysis buffer and eluted with the same buffer.

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