Serine proteinase inhibitors from eggs and larvae of tick Boophilus microplus: purification and biochemical characterization.
The present study describes the purification, characterization, and comparison of serine proteinase inhibitors during the development of egg and larva phases of the tick Boophilus microplus. Samples were collected of eggs between the first day of hatching and the beginning of eclosion (defined as El, E2, and E3) and of larvae between the first day of eclosion and the infectant phase (defined as L1, L2, and L3). Crude extracts of the samples (2.5% w/v in Tris-HCI buffer) were analyzed by SDS-PAGE, and showed three major protein bands of 42, 62, and 85 kDa, differing in intensity, from E1 to L3 samples. The total protein of the larva extracts was 34% less than that of the egg extracts, while no differences in active protein were detected. The apparent dissociation constant Ki determined for trypsin was 10-fold lower from E1 to L3 samples. Serine proteinase inhibitors from tick eggs and larvae (BmTls) were purified on trypsin-Sepharose column and analyzed by SDS-PAGE. The results showed a slight difference in protein pattern, with a protein band of 20 kDa in the E1 and E2 samples which did not appear in the other samples. The Ki for neutrophil elastase was 10-fold lower in L3 than E1. BmTI reverse-phase chromatography showed two and one major peaks in egg and larva samples, respectively. The N-terminal amino acid sequence of the L3 main peak from a C8 column showed a mix of BmTIs with the major sequence AVDFDKGCVPTADPGPCKG. Changes indicated by molecular weight and inhibition activity suggest different roles for BmTIs during the development process.
Andreotti
R
R
Embrapa Gado de Corte, C. Grande, MS, Brazil.
Malavazi-Piza
K C
KC
Sasaki
S D
SD
Torquato
R J
RJ
Gomes
A
A
Tanaka
A S
AS
D000595:Amino Acid Sequence; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D007814:Larva / Q000378:metabolism; D008969:Molecular Sequence Data; D010063:Ovum / Q000737:chemistry; D011506:Proteins / Q000032:analysis; D015842:Serine Proteinase Inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D013987:Ticks / Q000196:embryology / Q000378:metabolism*
D011506:Proteins; D015842:Serine Proteinase Inhibitors