Purification and characteristics of a specific alkaline phosphatase from the integument of the mediterranean fruit fly, Ceratitis capitata

✍ Scribed by Christina Psachoulia; Kostas Bourtzis; Dr. Vassilis J. Marmaras

• Book ID: 102745815
• Publisher: John Wiley and Sons
• Year: 1989
• Tongue: English
• Weight: 743 KB
• Volume: 11
• Category: Article
• ISSN: 0739-4462
• DOI: 10.1002/arch.940110403

✦ Synopsis

A specific alkaline phosphatase (ALPase) from t h e integument of white pupae has been purified 500-fold. The purification procedure included solubilization with Triton X-100, butanol extraction, fractionation with ammonium sulfate, and chromatography o n concanavalin A-Sepharose, Sephadex G-200, and Sepharose 6B. Two peaks with enzyme activity were observed. The major peak had a molecular weight of approximately 180,000, while t h e minor peak, which had identical kinetic parameters and substrate specificity as those of t h e major o n e , was eluted in a high molecular weight form (about 900,000), probably cross-linked with chitin, since t h e enzyme was separated from t h e chitin only by lysozyme treatment. The enzyme hydrolyzes only tyrosine phosphate and P-glycerophosphate, with apparent K, s of 0.35 mM and 0.22 mM, respectively, but not serine phosphate, threonine phosphate, ATP, and AMP. The optimum pH was in t h e alkaline range, with a peak at pH 9.4. The divalent cations Mn2+, Mg2+, and Ba2+ had stimulatory actions, while Cu2+ exerted a very strong inhibitory action o n the enzyme activity. The ALPase was inhibited by L-tyrosine in a dose-dependent fashion. At a concentration of 2 mM, L-tyrosine totally inhibited t h e enzyme activity, while L-phenylalanine inactivated t h e enzyme about 25%. The accumulated evidence that ALPase is involved in t h e sclerotization process of insect integument is discussed.