Early on.. we reported the partial purification of prophenoloxidase-activating proteinase-1 (PAP-1) from the tobacco hornworm. Manduca sexta [Proc. Nail. 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 I to near homogeneity by hydroxylapatite. dextran sulfate. gel filtration, and lectin affinity chromatography. With 2.4 x 10(3)-fold purification and 20% yield. we obtained 63 mug PAP-1 from about 120 M. sexta prepupal cuticles (similar to400 g). The purified glycoprotein (M-r, = 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 0-10degreesC 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 30degreesC than 0-10degreesC because activated PO was unstable at a higher temperature. The K(M)5 is of PAP-1 toward IEARpNA and proPO were 201 +/- 18 muM and 16.6 +/- 3.0 mug/ml, respectively, and the absence of SPHs did not significantly affect K-M 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. (C) 2004 Elsevier Inc. All rights reserved.