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A Boophilus microplus vitellin-degrading cysteine endopeptidase

Published online by Cambridge University Press:  24 April 2003

A. SEIXAS,
P. C. DOS SANTOS,
F. F. VELLOSO,
I. DA SILVA VAZ,
A. MASUDA,
F. HORN and
C. TERMIGNONI
A. SEIXAS
Affiliation:
Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, P.O. Box 15005, 91501-970, Porto Alegre, RS, BrasilDepartamento de Bioquímica, Universidade Federal do Rio Grande do Sul, P.O. Box 15005, 91501-970, Porto Alegre, RS, Brasil
P. C. DOS SANTOS
Affiliation:
Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, P.O. Box 15005, 91501-970, Porto Alegre, RS, BrasilDepartamento de Bioquímica, Universidade Federal do Rio Grande do Sul, P.O. Box 15005, 91501-970, Porto Alegre, RS, BrasilPresent address: Department of Biochemistry, Virginia Tech, Blacksburg, VA, USA 24061.
F. F. VELLOSO
Affiliation:
Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, P.O. Box 15005, 91501-970, Porto Alegre, RS, Brasil
I. DA SILVA VAZ
Affiliation:
Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, P.O. Box 15005, 91501-970, Porto Alegre, RS, BrasilDepartamento de Patologia Clínica Veterinária, Universidade Federal do Rio Grande do Sul, P.O. Box 15005, 91501-970, Porto Alegre, RS, Brasil
A. MASUDA
Affiliation:
Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, P.O. Box 15005, 91501-970, Porto Alegre, RS, BrasilDepartamento de Biologia Molecular e Biotecnologia, Universidade Federal do Rio Grande do Sul, P.O. Box 15005, 91501-970, Porto Alegre, RS, Brasil
F. HORN
Affiliation:
Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, P.O. Box 15005, 91501-970, Porto Alegre, RS, BrasilDepartamento de Biofísica, Universidade Federal do Rio Grande do Sul, P.O. Box 15005, 91501-970, Porto Alegre, RS, Brasil
C. TERMIGNONI
Affiliation:
Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, P.O. Box 15005, 91501-970, Porto Alegre, RS, BrasilDepartamento de Bioquímica, Universidade Federal do Rio Grande do Sul, P.O. Box 15005, 91501-970, Porto Alegre, RS, Brasil
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Abstract

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Here we describe the purification and characterization of a vitellin (VT) degrading cysteine endopeptidase (VTDCE) from eggs of the hard tick Boophilus microplus. A homogeneous enzyme preparation was obtained by chromatographic fractionation on ion-exchange and gel filtration columns and an autolysis step. This step consisted of incubation of a semipurified enzyme (after the first ion-exchange chromatography) at pH 4·0 that dissociated the enzyme from VT, to which VTDCE is naturally tightly associated. The enzyme purity was confirmed by capillary and native gel electrophoresis, and SDS–PAGE suggested the enzyme is a dimer of 17 and 22 kDa. VTDCE was active upon several synthetic substrates, with a preference for a hydrophobic or a basic residue in P1, and a hydrophobic residue in P2. VTDCE also hydrolysed haemoglobin, albumin, gelatin and vitellin. VTDCE is inactive in the absence of DTT and was totally inhibited by E-64, indicating it is a cysteine endopeptidase. Our results suggest that VTDCE is a major enzyme involved in yolk processing during B. microplus embryogenesis.

Keywords

tickBoophilus micropluscysteine endopeptidasevitellinembryogenesis

Information

Type
Research Article
Information
Parasitology , Volume 126 , Issue 2 , February 2003, pp. 155 - 163
Copyright
2003 Cambridge University Press