Abstract
Hemolymph of fifth instar Manduca sexta larvae collected under non-sterile conditions exhibited the presence of a novel high molecular weight protein complex, which was absent from the hemolymph collected aseptically. The high molecular weight complex consisted of, at least prophenol oxidase, phenol oxidase, and an interleukin 1-like molecule, thereby demonstrating the generation of this complex as a consequence of a host defense response. While the native phenol oxidase and the interleukin 1-like molecule possessed molecular weights of about 80,000 and 17,000, respectively, the complex had a molecular weight of about 400,000. Apart from prophenol oxidase, phenol oxidase, and interleukin 1, dopachrome isomerase and other, as of yet unidentified, proteins may be part of the complex as judged by the presence of additional bands observed during SDS-polyacrylamide gel electrophoresis. The significance of the assembly of this defense complex for insect host defense strategies is discussed.
Highlights
Invertebrate organisms have developed a plethora of defense reactions to fight invading parasites and pathogens
We have been examining the control mechanisms associated with the prophenol oxidase cascade and discovered: (a) a protease inhibitor controlling the prophenol oxidase cascade [9], (b) quinone isomerase that inactivates deleterious 4-alkylquinones that are formed during phenol oxidase action [10], and (c) phospholipid-mediated activation of prophenol oxidase [11]
Some authors have suggested that it is part of a recognition system [6]. This hypothesis needs to be proven there is no doubt that the generation of phenol oxidase is a consequence of final reactions triggered by the host defense system
Summary
Animals and Collection of Hemolymph—Larva of M. sexta were raised on a synthetic diet [19]. To collect hemolymph under aseptic conditions, the larvae were surface sterilized with 70% alcohol, and one of the posterior legs was cut with sterile scissors. Assay of Phenol Oxidase and Prophenol Oxidase Activity—Phenol oxidase activity was assayed as described previously [21] using a reaction mixture (1 ml) containing enzyme protein, 2 mM dopamine, and 50 mM sodium phosphate buffer, pH 6.0. The blots were blocked in Tris-buffered saline (TBS; 20 mM Tris-HCl, 150 mM NaCl (pH 7.5)) containing 0.1% Tween 20 (TBST) and 5% (w/v) nonfat dry milk. They were washed in TBST, incubated for 2 h in primary antibody After washing in TBS, the blots were reacted with substrate (p-nitroblue tetrazolium chloride and 5-bromo-4-chloro3-indoyl phosphate p-toluidine salt)
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