Enzymatic characterization of a cubilin-related serine proteinase from the hard tick Haemaphysalis longicornis
2004
Miyoshi, T. (National Inst. of Animal Health, Tsukuba, Ibaraki (Japan)) | Tsuji, N. | Islam, M.K. | Kamio, T. | Fujisaki, K.
In the present study, we performed enzymatic characterization of Haemaphysalis longicornis serine proteinase (HISP) with a view to shed light on the mechanisms of blood digestion in the hard ticks. Escherichia coil-expressed recombinant H1SP (rH1SP) 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 micro mol/min/mg protein, respectively at an optimum temperature of 25 deg C. However, the enzyme showed little activity to hydrolyze the substratese 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.
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