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By John M. Lowenstein

ISBN-10: 0121818705

ISBN-13: 9780121818708

The severely acclaimed laboratory average, Methods in Enzymology, is without doubt one of the such a lot hugely revered guides within the box of biochemistry. on the grounds that 1955, every one quantity has been eagerly awaited, usually consulted, and praised by way of researchers and reviewers alike. The sequence comprises a lot fabric nonetheless correct this day - really a vital e-book for researchers in all fields of lifestyles sciences

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The addition of the ethanol takes 15-20 minutes. The mixture is centrifuged at a temperature of --12 ° at about 20,000 g for 15 minutes. The supernatant is discarded. The precipitate is washed out of the centrifuge bottles with small portions of buffer. The final volume of the resulting solution is 85 ml. The solution is dialyzed against 4 liters of buffer at 0 ° for 12 hours. The resulting solution contains 43 mg protein per milliliter. ,Step 3. Gel Filtration. A Sephadex G-100 column, 3 cm in diameter The meat may be stored at --15 ° at this stage.

Chem. 193, 265 (1951). P. D. J. Weitzman and P. Dunmore, unpublished. ~H. L. Kornberg, P. J. R. Phizackerley, and J. R. Sadler, Biochem. 77, 438 (1960). 24 aEACTIONS ON THE CYCLE [5] protein), and the mixture is stirred continuously. After 30 minutes the precipitate is removed by centrifugation for 20 minutes at 25,000 g. Step 3. Fractionation with Ammonium Sul]ate. 1 g of finely ground solid (NH4)2SO~ (55% saturation). After the preparation has stood for 30 minutes with continuous stirring, the precipitate is removed by centrifugation for 20 minutes at 25,000 g.

The enzyme from porcine heart is inhibited about 50% by 10 mM NaCN; at this concentration KCNS, NAN3, and Na2Mo04 are inactive. 11 p-Hydroxymercuribenzoate inhibits the enzyme from porcine heart. 7 pjl/, inhibits the enzyme from bovine heart completely when isocitrate is added to the reaction mixture last; a combination of isocitrate and Mn ++ affords partial protection against p-chloromercuribenzenesulfonate inhibition when added to the enzyme before the mercurial. The substrate, threo-D~-isocitrate, and not the threo-b8 isomer, is responsible for the protection against inhibition by p-chloromercuribenzenesulfonate.

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Citric Acid Cycle by John M. Lowenstein


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