By Willis A. Wood (Eds.)
The severely acclaimed laboratory typical, Methods in Enzymology, is without doubt one of the so much hugely revered courses within the box of biochemistry. considering the fact that 1955, every one quantity has been eagerly awaited, usually consulted, and praised through researchers and reviewers alike. The sequence comprises a lot fabric nonetheless appropriate at the present time - really an important booklet for researchers in all fields of lifestyles sciences
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Additional resources for Carbohydrate Metabolism - Part E
3. 2 m M ) is similar to that of hexokinase from various rat tissues. 7 The enz y m e is capable of phosphorylating fructose, but the apparent Km value for fructose is 80 m M . The inhibition constants of glucose 6-phosphate versus glucose and ATP 4 are at least one order of magnitude higher than those for hexokinase from m a m m a l i a n tissues. 7 The product inhibition pattern of the ascarid hexokinase suggests that the e n z y m e has a r a n d o m kinetic m e c h a n i s m 4 similar to that found in other h e x o k i n a s e s ?
J. Ballard, Bh~chem. J. 98, 347 (1966). -G. Blume and E. Beutler, J. Biol. Chem. 246, 6507 (1971). 6 M. A. Schell and D. B. Wilson, J. Biol. Chem. 252, 1162 (1977). 7 M. R. Henrich, J. Biol. Chem. 239, 50 (1964). METHODS IN ENZYMOLOGY, VOL. 90 Copyright © 1982by AcademicPress. Inc. All rights of reproduction in any form reserved. 1 mg of bovine serum albumin per milliliter Procedure. The assay solution consists of 10 tzl of [14C]galactose, 20 ~1 of solution 1, 50 tzl of solution 2, and 20 tzl of the sample to be assayed.
The mixture, suspended in 500 ml of buffer A, is centrifuged at 4500 g for 30 rain, and the precipitate is reextracted with 200 ml of buffer A followed by centrifugation. The volume of the combined extract is about 800 ml. Step 2. Fractionation with (NH4)2SO,. 0 M NaOH, is allowed to stand for 4-6 hr. The precipitate is removed by centrifugation at 13,000 g for 30 rain and discarded. 5 and kept for 4-6 hr before the precipitate containing phosphofructokinase activity is collected by centrifugation at 13,000 g for 30 min.
Carbohydrate Metabolism - Part E by Willis A. Wood (Eds.)