Enzymatic characterization of a cubilin-related serine proteinase from the hard tick Haemaphysalis longicornis.
In the present study, we performed enzymatic characterization of Haemaphysalis longicornis serine proteinase (HlSP) with a view to shed light on the mechanisms of blood digestion in the hard ticks. Escherichia coli-expressed recombinant HlSP (rHlSP) was shown to potently hydrolyze the synthetic substrates Bz-(DL)-Arg-pNA, Z-Ala-Ala-Leu-pNA and Suc-Ala-Ala-Ala-pNA and yielded an activity of 31.5, 88.2 and 18.3 mumol/min/mg protein, respectively at an optimum temperature of 25 degrees C. However, the enzyme showed little activity to hydrolyze the substrates Suc-Arg-Pro-Phe-His-Leu-Leu-Val-Tyr-MCA and Pyr-Phe-Leu-pNA. The optimum pH for the enzyme was shown to be 4.0 to 5.0. Several inhibitors such as antipain, leupeptin and phenylmethylsulfonyl fluoride (PMSF), specific for serine proteinase were shown to inhibit enzyme activity by 20-82%, while E-64 (specific for cysteine proteinases) and pepstatinA (specific for aspartic proteinases) had shown only little inhibitory effects on it. This is the first report on enzymatic characterization of a functional serine proteinase from the hard ticks.
Miyoshi
Takeharu
T
National Institute of Animal Health, National Agricultural Research Organization, Ibaraki, Japan.
Tsuji
Naotoshi
N
Islam
Khyrul M
KM
Kamio
Tsugihiko
T
Fujisaki
Kozo
K
D000818:Animals; D000976:Antipain / Q000494:pharmacology; D002863:Chromogenic Compounds; D004926:Escherichia coli; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D026863:Ixodidae / Q000201:enzymology*; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D010436:Pepstatins / Q000494:pharmacology; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D013379:Substrate Specificity; D013696:Temperature
D002863:Chromogenic Compounds; D005456:Fluorescent Dyes; D007976:Leupeptins; D010436:Pepstatins; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D000976:Antipain; D010664:Phenylmethylsulfonyl Fluoride; D012697:Serine Endopeptidases; D007930:Leucine; C032854:leupeptin; C024974:E 64; C031375:pepstatin