Characterization of apyrase activity from the salivary glands of the cat flea ctenocephalides felis

• Published in: Insect biochemistry and molecular biology 1998-12, Vol.28 (12), p.1025-1030
• Main Author: Cheeseman, M.T.
• Format: Article
• Language: English
• Subjects: Animals; apyrase; Apyrase - metabolism; blood-feeding; cat flea; Cats; Ctenocephalides felis; Pulicidae; salivary gland; Salivary Glands - enzymology; Siphonaptera - enzymology
• ISSN: 0965-1748; 1879-0240
• DOI: 10.1016/S0965-1748(98)00093-9

Apyrase activity (ATP diphosphohydrolase, EC 3.6.1.5) was detected in salivary glands of the cat flea Ctenocephalides felis. Whole extracts of salivary glands contain approximately 21 ng of protein, 145 U/mg ADP′ase and 158 U/mg ATP′ase activity; AMP is not hydrolysed by salivary gland extracts. DEAE-Sepharose CL-6B anion exchange chromatography, and Cibacron Blue affinity chromatography each give a single coincident peak of ADP/ATP′ase activity. Biogel P-100 gel filtration of salivary gland homogenates made in buffer containing Triton and protease inhibitors, separated enzymatic activity into 57 kD and 44 kD peaks of ADP/ATP′ase activity. Partially purified ADP/ATP′ases are dependent on divalent cations and activation increases between 0.125 mM and 5.0 mM calcium. At 5 mM, magnesium is almost equally effective as calcium in activating ADP/ATP′ase but manganese and zinc are less so, and EDTA abolishes activity. ADP/ATP′ases have a pH optima of 7–9. The K m for ADP hydrolysis by whole extracts and partially purified enzyme is approximately 66 μM ADP. The co-purification of ADP′ase and ATP′ase activity by three physiochemical techniques and parallelism between ADP and ATP hydrolysis under varying conditions of pH and activating cation indicates enzymatic activity is attributable to true apyrase(s).