Adenosine deaminase was purified 525-fold from the maternal component of early gestation bovine placenta by acetone fractionation, salt fractionation, ion exchange chromatography and gel filtration. The final enzyme preparation was homogeneous when subjected to sedimentation analysis in the ultracentrifuge and had an ~2 0 ,~ value of 4.3 S. I mg of enzyme protein deaminated 252 pmoles of adenosine per min a t 30 "C, and there was no loss in activity on storage in the presence of 1 mM 2-mercaptoethanol a t 2-4 "C for 12 months. The molecular weight by gel filtration was 35480 and the energy of activation was 6.66 kcal/mole. The molecular properties of the placental enzyme are generally similar to those reported for adenosine deaminase purified from calf intestinal mucosa and calf serum, but differ in that only one protein band is observed on cellulose acetate electrophoresis a t pH 5.9 and 8.6.Comparative studies of adenosine deaminase activity in different tissues of a number of mammalian species have shown that the activity of the enzyme is always high in the placenta [i], and that there is a characteristic variation in activity during gestation in the cow, cat and guinea pig [2]. I n these species placental adenosine deaminase activity increased sharply early in gestation and then gradually decreased until term [2]. The rise in adenosine deaminase activity occurred in the maternal component (juxtaplacental endometrium) of the placentas of these species and there was little variation in adenosine deaminase activity of the fetal components during gestation.Adenosine deaminase has been purified from the maternal component of the bovine placenta obtained early in gestation when the activity of the enzyme is a t its peak. Some molecular properties of the enzyme have been examined and compared with the properties of the enzyme isolated from other tissues of the samef species, e.g. intestinal mucosa [3,4], spleen [5,6] and serum [7].
EXPERIMENTAL PROCEDURES
MaterialsScphadcx G-200, G-100 and DEAE-Sephadex A-50 were obtained from Pharmacia. CM-cellulose CMll was a Whatman product. The ion exchangers were cycled before use according to the manufacturer's instructions. All reagents and buffers were analytical grade. Cytochrome c, ribonuclease, chymo-
MethodsThe protein concentration of the crude aqueous extract was measured by the method of Lowry et al.[8] using bovine serum albumin as standard. Column eluates were monitored by measuring ultraviolet absorption a t 280 nm. The protein content of combined eluates and other enzyme fractions was determined from the absorbancies a t 280 and 260 nm [9].Adenosine deaminase activity throughout purification was measured spectrophotometrically as previously described [lo] by following the decrease in absorbance a t 265 nm resulting from the conversion of adenosine to inosine. The enzyme (5 to 20 pl) was added to 3 ml of solution of 0.1 mM adenosine in 50 mM 3,3-dimethylglutarate buffer pH 7 in a cuvette of 1 cm pathlength. Absorbance changes were followed with a Gilford Absorba...
