Adenosine deaminase from calf spleen

Pfrogner, N.

doi:10.1016/0003-9861(67)90439-0

Cited by 33 publications

(2 citation statements)

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“…The placental enzyme differs from the enzymes from these tissues in that it is electrophoretically homogeneous. Pfrogner [5] reported that the purified calf spleen enzyme was electrophoretically homogeneous, but component isoenzymes may have been lost during purification as several peaks of activity were found in the ion exchange eluates, and the yield of purified enzyme was only 4O/,. The placental enzyme eluted from DEAE-Sephadex A-50 as a single sharp peak of activity indicating the absence of isoenzymes.…”

Section: Discussionmentioning

confidence: 99%

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Studies on Adenosine Deaminase

1971

European Journal of Biochemistry

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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...

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Section: Discussionmentioning

confidence: 99%

“…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]. …”

mentioning

confidence: 99%

Studies on Adenosine Deaminase

1971

European Journal of Biochemistry

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Characterization of Human Adenosine Deaminase

Kelley

Daddona

Weyden

1977

Novartis Foundation Symposia

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Purification and partial characterization of brain adenosine deaminase: Inhibition by purine compounds and by drugs

Centelles

Franco

Bozal

1988

J of Neuroscience Research

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Rat brain adenosine deaminase (E.C. 3.5.4.4.) was purified 667-fold from the supernatant fraction by the following techniques: heat treatment (60 degrees C), fractionation with ammonium sulfate, column chromatography on DEAE-Sepharose, and preparative gel electrophoresis. The purified enzyme was homogeneous by the criterion of polyacrylamide disc gel electrophoresis and isoelectric focusing. Amino acid composition is given. The isoelectric point of the enzyme (5.2) was determined by isoelectric focusing on agarose. The apparent molecular weight was estimated to be 39,000 (Stokes Radius [Rs] = 27.3 A) using a calibrated Sephacryl S-300 column. The study of the influence of the temperature on the initial reaction rates allowed calculation of Ea (8.9 Kcal/mole) and delta H (5.0 Kcal/mole) values. The variation of V and Km with pH suggests the existence of a sulfhydryl group and an imidazole group in the enzyme-substrate complex. The enzyme had a Km (adenosine) of 4.5 X 10(-5) M and was inhibited by inosine, guanosine, adenine, and hypoxanthine but not by other intermediates of purine metabolism. None of the inhibitors were active as substrates. The enzyme was also inhibited by dimethyl sulfoxide and ethanol. Inhibition by ethanol can account partially for the CNS depressant effects of levels 3 and 4 of alcohol intoxication. A number of drugs having therapeutic uses such as sedative, anxiolytic, analgesic, and relaxant are modulators of the enzyme. Among these, lidoflazine, phenylbutazone, and chlordiazepoxide are the most potent as inhibitors (Ki 30, 54, and 83 microM, respectively), whereas medazepam is the most potent as activator (Ka 0.32 mM). Thus, it is concluded that some drugs that inhibit adenosine uptake also modulate adenosine deaminase activity. Besides, since the enzyme is located extracellularly [Franco et al, 1986], these drugs can modulate the physiological effects exerted by extracellular adenosine.

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Adenosine deaminase from calf spleen

Cited by 33 publications

References 8 publications

Studies on Adenosine Deaminase

Studies on Adenosine Deaminase

Characterization of Human Adenosine Deaminase

Purification and partial characterization of brain adenosine deaminase: Inhibition by purine compounds and by drugs

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