Purification and characterization of the active form of tyrosine hydroxylase from mesangial cells in culture

Purification and characterization of the active form of tyrosine hydroxylase from mesangial cells in culture

Autor Arita, D. Y. Google Scholar
Di Marco, G. S. Google Scholar
Schor, N. Google Scholar
Casarini, D. E. Google Scholar
Instituição Universidade Federal de São Paulo (UNIFESP)
Resumo The capacity of mesangial cells (MC) to produce catecholamines (CAs) has been investigated in our laboratory. To study the CA cascade, it is necessary to examine some steps in their metabolic pathway. Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of these biogenic amines (dopamine (DA), norepinephrine (NE), and epinephrine (EPI)). Since the glomerular mesangium is their target in the regulation of renal sodium transport and renin secretion, the aim of the study was to determine the presence of TH in those cells in culture. the CA levels were detected in immortalized MC by high-performance liquid chromatography with electrochemical detection. the following concentrations were found in the intracellular region and in the medium, respectively: NE=284 +/- 31 and 134 +/- 22, EPI = 75 +/- 14 and 22 +/- 5, and DA=42 +/- 14, 40 +/- 20 pg/mg cell protein. the enzymatic activity of the cell lysate and medium was measured based on L-dopa formation. in the presence of o-phenanthroline, both samples presented 39% inhibition. the biopterin was detected in the intracellular and in the medium (64.87 and 631.99 pmol/mg protein, respectively) using high-performance liquid chromatography with ultraviolet detection. the cell lysate was submitted to a DEAE-Sephacel column, followed by gel filtration, and Heparin-Sepharose. TH was purified 613.16-fold with a specific activity of 466.0 pg/mg cell protein. Immunoblotting using monoclonal antibody revealed the presence of TH in the different purification steps. Purified TH was sequenced, presenting an alignment with amino-terminal sequence of mouse enzyme. Our results demonstrated the presence of active TH in MC, suggesting that these cells are able to produce CA in vivo, and establishing a convenient purification method for TH that can be applied to the study of the molecular properties of the enzyme modified in vivo by different physiological and pathophysiological stimuli.
Palavra-chave mesangial cells
catecholamines
tyrosine hydroxylase
purification
kidney
Idioma Inglês
Data de publicação 2002-01-01
Publicado em Journal of Cellular Biochemistry. New York: Wiley-liss, v. 87, n. 1, p. 58-64, 2002.
ISSN 0730-2312 (Sherpa/Romeo, fator de impacto)
Publicador Wiley-Blackwell
Extensão 58-64
Fonte http://dx.doi.org/10.1002/jcb.10277
Direito de acesso Acesso restrito
Tipo Artigo
Web of Science WOS:000177767500007
Endereço permanente http://repositorio.unifesp.br/handle/11600/26693

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