Purification and characterization of Manduca sexta prophenoloxidase-activating proteinase-1, an enzyme involved in insect immune responses

Early on, we reported the partial purification of prophenoloxidase-activating proteinase-1 (PAP-1) from the tobacco hornworm, Manduca sexta [Proc. Natl. Acad. Sci. USA 95 (1998) 12220]. PAP-1 requires an auxiliary factor for generating active phenoloxidase (PO) [Insect Biochem. Mol. Biol. 33 (2003) 197; Insect Biochem. Mol. Biol. 34 (2004) 731]. To further characterize their roles in the proteolytic activation of prophenoloxidase (proPO), we purified PAP-1 to near homogeneity by hydroxylapatite, dextran sulfate, gel filtration, and lectin affinity chromatography. With 2.4 × 103-fold purification and 20% yield, we obtained 63 μg PAP-1 from about 120 M. sexta prepupal cuticles (∼400 g). The purified glycoprotein (Mr = 39,810 ± 20; pI = 5.6) had the highest amidase activity at pH 8.0 and a low salt concentration. The optimal conditions for proPO activation by PAP-1 and SPHs were: pH 8.0-8.4, PAP:SPH = 1.5:1, and 0-10 °C for 40-50 min. While PAP-1 and SPHs are reasonably heat stable, PO activity generated after 1 h incubation was lower at 20 or 30 °C than 0-10 °C because activated PO was unstable at a higher temperature. The KMs of PAP-1 toward IEARpNA and proPO were 201 ± 18 μM and 16.6 ± 3.0 μg/ml, respectively, and the absence of SPHs did not significantly affect KM for the synthetic substrate. PO activity and proPO cleavage were reduced in reaction mixtures containing the same amounts of proPO, PAP-1, and SPHs but increasing concentrations of NaCl. Ionic strength of the reaction buffer may reduce proPO-PAP-SPH interactions, proPO processing, and PO assembly.