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Structural and Kinetic Alterations in Adenosine Deaminase Associated with the Differentiation of Rat Intestinal Cells¹

Laboratory of Cell Metabolism, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

Forms of the enzyme adenosine deaminase (ADase) in rapidly dividing versus differentiated epithelial cells of rat intestinal mucosa were compared. Two variants with isolectric points at pH ca. 4.85 (ADase I) and 4.80 (ADase II) were resolved by preparative and analytical isoelectric focusing. These forms, which could also be partially resolved by molecular exclusion chromatography, displayed apparent molecular weight values of 37,000 and 33,500, respectively. K_m values for adenosine for the two forms were significantly different (0.07 mM for ADase I and 0.38 mM for ADase II). Small differences were also observed in relative substrate specificity, while the pH-activity profiles for the two forms were essentially identical, with broad maxima between pH values of 6.5 and 9.0. The intensity of ADase I observed in analytical isoelectric focusing increased dramatically with respect to ADase II as the epithelial cells differentiated. The specific adenosine deaminase activity also increased with cellular differentiation by over 30-fold, expressed per mg DNA, or over 6-fold, expressed per mg protein. Cycloheximide, sharply reduced this increase in specific activity. Cycloheximide was also shown to prevent the increase in the activity of ADase I that is normally associated with differentiation in rat intestines. The data imply the formation of a particular enzyme variant characteristic of the differentiated cell by a mechanism related to active protein synthesis.

¹ This investigation was supported by USPHS Grant CA-14906 from the National Cancer Institute through the National Large Bowel Cancer Project, by Grant CA08748, and by Grant BC-202 from the American Cancer Society.

Received 1/17/77. Accepted 4/15/77.