Purification and partial characterization of ATP pyrophosphohydrolase from fetal bovine epiphyseal cartilage.
ATP pyrophosphohydrolase was partially purified from fetal bovine epiphyseal cartilage. The purification was about 10- and 100-fold over the enzyme activities of matrix vesicle fraction and cell homogenate, respectively. The pyrophosphohydrolase and alkaline phosphatase were separated by a sequential application of Sepharose CL-6B and DEAE-cellulose column chromatographies. The purified enzyme migrated as a single band corresponding to the molecular weight of 230,000 in sodium dodecyl sulfate-polyacrylamide disc gel by electrophoresis. The enzyme absolutely required Zn2+ for its activity and appeared to bind Zn2+ strongly with an apparent affinity of p[Zn2+]0.5 = 13.4. The apparent Km for ATP was 0.18 mM. The enzyme was also reactive toward various nucleoside triphosphates including GTP, CTP, and UTP. In contrast, various phosphodiesters including RNA, UDP-glucose, NAD, and bis-p-nitrophenylphosphate were 5% or less as reactive as the nucleoside triphosphates. The pyrophosphohydrolase was inactive toward adenosine 3':5'-monophosphate or various phosphonates. UDP-glucose (1 mM), NAD (1 mM), or RNA (1 mg/ml) failed to inhibit the ATP pyrophosphohydrolase activity. These observations suggest that the ATP pyrophosphohydrolase of the cartilage is probably not a phosphodiesterase I. The matrix vesicle fraction, which probably also included some plasma membrane vesiculated during collagenase digestion, contained the highest specific activity of the enzyme as compared to other subcellular fractions of either epiphyseal or articular cartilage.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002356:Cartilage / Q000201:enzymology*; D002358:Cartilage, Articular / Q000201:enzymology; D002417:Cattle; D004838:Epiphyses / Q000201:enzymology*; D005333:Fetus; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D011755:Pyrophosphatases; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D000251:Adenosine Triphosphatases; D011755:Pyrophosphatases; C019757:ATP pyrophosphohydrolase