Studies on Adenosine Deaminase

Abstract

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...

Identification of a membrane adenosine deaminase binding protein from human placenta

“…However, although most human tissues exhibit varying levels of type A ADase, relatively few have an excess of soluble binding protein. In distinction to these data the soluble ADase isolated in homogeneous form from the maternal component of bovine placenta of early gestation has been demonstrated to be exclusively type C enzyme (Sim & Maguire, 1971). This result, which implies the complete absence of ADase binding protein, may reflect the difference in species or gestational age.…”

Presence of Adenosine in the Human Term Placenta: DETERMINATION OF ADENOSINE CONTENT AND PATHWAYS OF ADENOSINE METABOLISM

“…Adenosine deaminase was purified from bovine placenta, as described previously (20), to a specific activity of 252 at 30°C and pH 7. Adenylate deaminase was purified from rat skeletal muscle by the method of Smiley et al (21) and had a specific activity of 62 when assayed at 30°C in 0 .…”