Please use this identifier to cite or link to this item: http://repositorio.unicamp.br/jspui/handle/REPOSIP/107258
Type: Artigo de periódico
Title: Cytotoxicity Of Okadaic Acid And Kinetic Characterization Of Protein Tyrosine Phosphatase Activity In V79 Fibroblasts
Author: Aoyama H.
Melo P.S.
Granjeiro P.A.
Haun M.
Ferreira C.V.
Abstract: Protein phosphorylation and dephosphorylation mediate signal transduction events that control several important cellular processes. The aim of this work was to determine some kinetic properties of a protein phosphatase obtained from non-metabolizing V79 Chinese hamster cells. The effect of okadaic acid, a tumour promoter, on these cell cultures was also determined. Phosphatase activity was assayed in V79 cells which were lysed with 0.1 M imidazole buffer (pH 7.4). Enzyme activity was determined using p-nitrophenylphosphate and tyrosine phosphate (TyrP) as substrates. Maximum phosphatase activity was obtained at pH7·4. The apparent K(m) (Michael's constant) and specificity constant for TyrP were 0·06 nm and 57, respectively. Phosphatase activity was inhibited by 100μM pCMB (p-chloro-mercuribenzoate; 70%), 10 mM fluoride (30%), 10 mM phosphate (40%), 100 μM m-vanadate (80%) and 100 μM o-vanadate (90%). Tartrate (5 mM) had no effect. The low K(m) value for TyrP and the high level of inhibition by vanadate suggest that the V79 phosphatase is a protein tyrosine phosphatase. The cytotoxic effect of okadaic acid was evaluated and the IC50 was 15, 20, 35 and 45 nM for nucleic acid content, neutral red uptake, MTT and protein phosphatase assays, respectively. These results agree with the kinetic data, indicating that the V79 phosphatase is a protein tyrosine phosphatase, as enzyme activity was stimulated when the cells were treated with okadaic acid. Serine/threonine protein phosphatases are completely inhibited by okadaic acid. The phosphatase enzyme system may be useful for studying cellular adaptation to apoptosis, oxidative stress and other conditions.
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Rights: fechado
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Address: http://www.scopus.com/inward/record.url?eid=2-s2.0-0033851565&partnerID=40&md5=2ddef6b04fd758e58f63349005a0c722
Date Issue: 2000
Appears in Collections:Unicamp - Artigos e Outros Documentos

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