RESULTS

Preparation of Pyruvate Carboxylase-The enzyme has been concentrated approximately go-fold from chicken liver by procedures summarized in Table I. The extract of washed, lyophilized mitochondria was used as the starting material. Fresh livers were cut into small pieces and homogenized in 4 volumes (milliliters per g) of 0.25 M sucrose in a mraring Blendor at reduced speeds for 45 seconds. The homogenate was centrifuged at 700 xg for 15 minutes and the supernatant layer carefully removed and discarded. The heavy mitochondrial layer can then be decanted as a poorly packed layer, leaving behind the more firmly sedimented unbroken cells and other debris. The mitochondrial layer was diluted to the original volume of the homogenate with water and centrifuged for 20 tiutes at 12,000 X g. The resulting precipitate was suspended evenly in 0.25 the original volume of water and lyophilized. All of the above procedures were carried out at O-3”. This treatment not only removes much of the water-soluble protein from the mitochondria but also renders pyruvate carboxylase readily extractable by mild procedures. The enzyme appears to be released only slowly from mitochondria kept in sucrose when subjected to sonic disintegration., 4s shown in Table I, little activity is lost in the preparation of the washed mitochondrial fraction. The lyophilized material is stable for several weeks when stored in a vacuum at-20”. Two grams of the lyophilized material were extracted twice at 2” with 10 volumes of 0.04 M Tris-HCl buffer, pH 7.8, and the clear supernatant layers obtained by centrifuging at 20,000 x g for 10 minutes were combined. The protein fraction precipitating between 25 and 33% saturation with ammonium sulfate at 2” was collected by centrifugation and dissolved in 10 ml of 0.04 M Tris-HCl, pH 7.8. The precipitate contains essentially all of the active material from the original extract but is quite unstable at this stage, particularly in solution. Some protective