Partial purification of phosphodiesterase I from microsomes of rat intestinal mucosa

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Abstract
Intestinal mucosa was homogenized in Krebs–Ringer phosphate buffer, pH 7.4, containing 6% Dextran. After centrifugation, the sediment was rehomogenized in 0.24 M sucrose and the homogenate centrifuged. The supernatant material from both steps was combined and centrifuged at high speed through a layer of 25% sucrose to yield a pellet of microsomes. This pellet was suspended in Tris–HCl buffer, pH 8.4, made isotonic in KCl, and the suspension was sonicated and centrifuged. A suspension of the sediment in Tris–HCl buffer was shaken with t-amyl alcohol to yield the soluble enzyme in the aqueous phase. The enzyme was purified further by chromatography on DEAE-cellulose using elution with a KCl gradient in Tris–HCl buffer, pH 8, made 6 M in urea. Phosphodiesterase I and alkaline phosphomonoesterase were eluted in peaks which overlapped only partially enabling the collection of phosphodiesterase I free of Phosphomonoesterase. The enzyme solution was concentrated and freed of urea by pressure dialysis.

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