Artículo
Kinetic characterization of human thyroperoxidase. Normal and pathological enzyme expression in Baculovirus System: A molecular model of functional expression
Belforte, Fiorella Sabrina
; Targovnik, Alexandra Marisa
; Gonzalez-Lebrero, Rodolfo Martin
; Osorio Larroche, Carolina
; Citterio, Cintia Eliana
; González Sarmiento, Rogelio; Miranda, Maria Victoria
; Targovnik, Hector Manuel
; Rivolta, Carina Marcela
Fecha de publicación:
03/2015
Editorial:
Elsevier Ireland
Revista:
Molecular and Cellular Endocrinology
ISSN:
0303-7207
Idioma:
Inglés
Tipo de recurso:
Artículo publicado
Clasificación temática:
Resumen
BACKGROUND: Human thyroperoxidase (hTPO) is a membrane-bound glycoprotein located at the apical membrane of the thyroid follicular cells which catalyzes iodide oxidation and organification in the thyroglobulin (TG) tyrosine residues, leading to the thyroid hormone synthesis by coupling of iodotyrosine residues. Mutations in hTPO gene are the main cause of iodine organification defects (IOD) in infants. METHODS: We investigated the functional impact of hTPO gene missense mutations previously identified in our laboratory (p.C808R, p.G387R and p.P499L). In order to obtain the whole wild-type (WT) coding sequence of hTPO, sequential cloning strategy in pGEMT vector was carried out. Then, site-directed mutagenesis was performed. WT and mutant hTPOs were cloned into the pAcGP67B transfer vector and the recombinant proteins were expressed in Baculovirus System, purified and characterized by SDS-PAGE and Western blot. Moreover, we report for the first time the kinetic constants of hTPO, of both WT and mutant enzymes. RESULTS: The functional evaluation of the recombinant hTPOs showed decreased activity in the three mutants with respect to WT. Regarding to the affinity for the substrate, the mutants showed higher Km values with respect to the WT. Additionally, the three mutants showed lower reaction efficiencies (Vmax/Km) with respect to WT hTPO. CONCLUSIONS: We optimize the expression and purification of recombinant hTPOs using the Baculovirus System and we report for the first time the kinetic characterization of hTPOs.
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Articulos(INIGEM)
Articulos de INSTITUTO DE INMUNOLOGIA, GENETICA Y METABOLISMO
Articulos de INSTITUTO DE INMUNOLOGIA, GENETICA Y METABOLISMO
Articulos(IQUIFIB)
Articulos de INST.DE QUIMICA Y FISICO-QUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Articulos de INST.DE QUIMICA Y FISICO-QUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Articulos(NANOBIOTEC)
Articulos de INSTITUTO DE NANOBIOTECNOLOGIA
Articulos de INSTITUTO DE NANOBIOTECNOLOGIA
Citación
Belforte, Fiorella Sabrina; Targovnik, Alexandra Marisa; Gonzalez-Lebrero, Rodolfo Martin; Osorio Larroche, Carolina; Citterio, Cintia Eliana; et al.; Kinetic characterization of human thyroperoxidase. Normal and pathological enzyme expression in Baculovirus System: A molecular model of functional expression; Elsevier Ireland; Molecular and Cellular Endocrinology; 404; 3-2015; 9-15
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