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Purification and characteristics of a specific alkaline phosphatase from the integument of the mediterranean fruit fly, Ceratitis capitata
Author(s) -
Psachoulia Christina,
Bourtzis Kostas,
Marmaras Vassilis J.
Publication year - 1989
Publication title -
archives of insect biochemistry and physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.576
H-Index - 66
eISSN - 1520-6327
pISSN - 0739-4462
DOI - 10.1002/arch.940110403
Subject(s) - ceratitis capitata , sephadex , chromatography , biochemistry , enzyme , enzyme assay , phosphate , biology , alkaline phosphatase , chitin , integument , divalent , chemistry , botany , organic chemistry , chitosan , pest analysis , tephritidae
Abstract A specific alkaline phosphatase (ALPase) from the 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 on 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 the minor peak, which had identical kinetic parameters and substrate specificity as those of the major one, was eluted in a high molecular weight form (about 900,000), probably cross‐linked with chitin, since the enzyme was separated from the chitin only by lysozyme treatment. The enzyme hydrolyzes only tyrosine phosphate and β‐glycerophosphate, with apparent K m s of 0.35 mM and 0.22 mM, respectively, but not serine phosphate, threonine phosphate, ATP, and AMP. The optimum pH was in the alkaline range, with a peak at pH 9.4. The divalent cations Mn 2+ , Mg 2+ , and Ba 2+ had stimulatory actions, while Cu 2+ exerted a very strong inhibitory action on 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 the enzyme activity, while L‐phenylalanine inactivated the enzyme about 25%. The accumulated evidence that ALPase is involved in the sclerotization process of insect integument is discussed.