Proenzyme of Manduca sexta phenol oxidase: purification, activation, substrate specificity of the active enzyme, and molecular cloning.

Abstract

Phenol oxidase (PO) was isolated as a proenzyme (pro-phenol oxidase, pro-PO) from the hemolymph of Manduca sexta larvae and purified to homogeneity. Pro-PO exhibits a Mr of 130,000 on gel filtration and two bands with an apparent Mr of -100,000 on SDS/PAGE, as well as sizeexclusion HPLC. Activation of pro-PO was achieved either by specific proteolysis by a cuticular protease or by the detergent cetylpyridinium chloride at a concentration below the critical micellar concentration. A cDNA clone for M. sexta pro-PO was obtained from a larval hemocyte cDNA library. The clone encodes a polypeptide of -80,000 Da that contains two copper-binding sites and shows high sequence similarity to POs, hemocyanins, and storage proteins of arthropods. The M. sexta pro-PO, together with other arthropod pro-POs, contains a short stretch of amino acids with sequence similarity to the thiol ester region of a-macroglobulins and complement proteins C3 and C4.Melanin biosynthesis occurs widely, not only in animals but also in plants and fungi (1). Phenol oxidase (PO), which possesses both tyrosinase activity (monophenol monooxygenase; monophenol, L-dopa:oxygen oxidoreductase; EC 1.14.18.1), as well as o-diphenol oxidase (1,2-benzenediol:oxygen oxidoreductase; EC 1.10.3.1), is responsible for initiating the biosynthesis of melanin (2, 3). In arthropods and especially in insects, PO is uniquely involved in another important biochemical process-cuticular sclerotization (hardening). Sclerotization is vital for the survival of all insects, as it affords protection to the soft invertebrate body (3-8). During sclerotization, PO-generated quinones participate in the quinone tanning process or serve as substrates for quinone isomerases that convert quinones to quinone methides for quinone methide sclerotization (9-13). Certain quinone methides are converted by another cuticular enzyme, quinone methide isomerase, to 1,2-dehydro-N-acyldopamine derivatives (14). These compounds are further oxidized by PO to reactive quinone methide imine amides (15, 16), which serve as the reactive intermediates of a,f3-sclerotization. Quinones, quinone methides, and quinone methide imine amides react with cuticular proteins and chitin, forming eventually cross-linked cuticle.Apart from participating in sclerotization and melanization of cuticle, POs are also known to be involved in two other physiologically important processes-defense reactions (arthropod immunity) and wound healing. During invasion by a foreign organism, pro-phenol oxidase (pro-PO) present in the hemocytes is released and activated to produce melanin pigments for deposition on the intruder (17)(18)(19). The damage that can be caused by the foreign organisms is thus limited by encapsulation and melanization. Similarly during wounding of insect cuticle, PO causes massive deposition of melanin pigment at the wound site to prevent hemolymph loss and to block the entry of opportunistically invading microorganisms (20).Given such important functions, it could be expected that PO would be one o...

Transepithelially Transported Pro-phenoloxidase in the Cuticle of the Silkworm, Bombyx mori

“…Since the first cDNA cloning of insect proPOs in 1995 (11,45,46), the number of reports on cloned proPO cDNAs in literature has been increasing. A phylogenetic tree of proPOs of insects was constructed from the reported sequences (Fig.…”

Effects of Pink Bollworm Resistance to <I>Bacillus thuringiensis</I> on Phenoloxidase Activity and Susceptibility to Entomopathogenic Nematodes

“…When wandering larvae emerged, all larvae present in the diet cup were counted, and six wandering larvae were selected at random to assay for phenoloxidase (PO) activity. Basal PO activity (without the addition of an elicitor) was assayed by using a method adapted from Hall et al (1995) and Fabrick et al (2004). Brießy, each larva was chilled on ice and a proleg was removed with a micro-scissors.…”

Hemolymph Melanization in the Silkmoth Bombyx mori Involves Formation of a High Molecular Mass Complex That Metabolizes Tyrosine

“…It is generally assumed that processing of PPO to PO confers the enzymatic activity needed to cause hemolymph to melanize (1,(12)(13)(14)(15). Two lines of investigation, however, raise questions as to whether this is fully correct.…”