Purification and characterisation of bovine spleen ADPase.
ADPase has been purified from bovine spleen. Electrophoresis revealed a minor contaminent in the preparation that may represent ADPase that has been decreased in size by limited proteolysis during extraction and purification. The native enzyme behaves on SDS/PAGE as a 100-kDa protein but ADPase is a glycoprotein and so its electrophoretic behaviour may be anomalous. The apparent molecular mass is decreased to 70 kDa after removal of carbohydrate by treatment with a glycosidase. The use of a cross-linking reagent followed by electrophoresis suggests that the enzyme is composed of a single subunit. The specific activity of the purified material was 115 U/mg protein. The enzyme catalyses the hydrolysis of nucleoside di- and tri-phosphates but nucleoside monophosphates are not acted upon. The Km for ADP (approx. 10-15 microM) is unaffected by the state of purification of the enzyme, but catalytic activity of the purified material is stimulated by inclusion of detergent in the assay system and by calcium ions. Maximum activity is seen at pH 8.0-8.5 with ADP as substrate but the optimum shifts to 7.5-8.0 for ATP hydrolysis.
Moodie
F D
FD
Department of Biochemistry, King's College, London, England.
Baum
H
H
Butterworth
P J
PJ
Peters
T J
TJ
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D001011:Aorta / Q000201:enzymology; D001081:Apyrase / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange; D003902:Detergents / Q000494:pharmacology; D007700:Kinetics; D008168:Lung / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology*; D009131:Muscle, Smooth, Vascular / Q000201:enzymology; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D013154:Spleen / Q000201:enzymology*
D003902:Detergents; D011092:Polyethylene Glycols; D017830:Octoxynol; D000251:Adenosine Triphosphatases; D001081:Apyrase