A Boophilus microplus vitellin-degrading cysteine endopeptidase.
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.
Seixas
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A
Centro de Biotecnologia, Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, P.O. Box 15005, 91501-970, Porto Alegre, RS, Brasil.
Dos Santos
P C
PC
Velloso
F F
FF
Da Silva Vaz
I
I
Masuda
A
A
Horn
F
F
Termignoni
C
C
D000818:Animals; D003546:Cysteine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D004527:Egg Proteins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000145:classification / Q000378:metabolism; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D026863:Ixodidae / Q000166:cytology / Q000201:enzymology* / Q000254:growth & development*; D007814:Larva / Q000201:enzymology; D010053:Ovary / Q000201:enzymology; D010063:Ovum / Q000201:enzymology; D013379:Substrate Specificity; D013696:Temperature
D004527:Egg Proteins; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D003546:Cysteine Endopeptidases