Soluble tyrosinases from pharate pupal integument of the tobacco hornworm,Manduca sexta (L.)

Two soluble phenoloxidases were partially purified from pharate pupal integument of Manduca sexta by gel filtration or sucrose density gradient centrifugation. Thiourea was used to retard the formation of higher molecular weight aggregates. Subsequent analysis by gel filtration HPLC showed that the apparent molecular weights were about 300 kD and >700 kD. The smaller phenoloxidase was further purified by anion exchange HPLC. In comparative studies with mushroom tyrosinase and a fungal laccase, the Manduca phenoloxidases were identified as tyrosinases, since they exhibited monophenol mono-oxygenase activity (EC 1.14.18.1) and catechol oxidase activity (EC 1.10.3.1). Both enzyme preparations catalyzed ortho-hydroxylation of tyrosine at a relatively show rate, oxidized o-diphenols at a much faster rate than p-diphenols or monophenols, had broad pH optima from about pH 5.5–7.5, and were completely inhibited by 1 μM phenylthiourea, N-β-alanyldopamine (NBAD) and N-β-alanylnorepinephrine (NBANE), which are both abundant in pupal cuticle, were oxidized to o-quinones by the tyrosinases, with the former catecholamine oxidizing at a 10-fold higher rate than the latter. NBANE was synthesized from NBAD, apparently by spontaneous tautomerization of the o-quinone to a p-quinone methide, which then reacted with water to form the β-hydroxylated product. The possible role of tyrosinase in insect integument is discussed.