Electrophoretic separation and identification of phenoloxidases in hemolymph and midgut of adult Anopheles stephensi mosquitoes.
Anopheles mosquitoes frequently respond to invading malaria parasites with a rejection mechanism consisting of phenoloxidase-mediated melanization of ookinetes in the mosquito midgut epithelium. The relative roles of hemolymph vs. midgut phenoloxidase in this rejection mechanism is unclear. We have separated and identified phenoloxidase isozymes from midgut and hemolymph of Anopheles stephensi by native gel electrophoresis followed by zymography. The isozymes from the 2 sites had distinctively different electrophoretic characteristics. Hemolymph possessed 2 phenoloxidase-positive bands, both of which were bifunctional molecules that oxidized monophenol as well as o-diphenol substrates. Midgut extract possessed 3 bands that migrated more rapidly than those of the hemolymph. None of these midgut bands had detectable monophenoloxidase activity; they possessed, however, a broad spectrum of diphenoloxidase activity in their ability to oxidize both o- and p-diphenol substrates, as well as the laccase substrate syringaldazine. The 2 most rapidly migrating midgut PO bands could be distinguished from the more slowly migrating band through their insensitivity to inhibition by the chelating agent tropolone. A question to be resolved in the future relates to the relative roles of hemolymph vs. midgut phenoloxidase in mosquito defense against invasive parasites.
Sidjanski
S
S
Department of Medical and Molecular Parasitology, New York University School of Medicine, New York 10016, USA.
Mathews
G V
GV
Vanderberg
J P
JP
D000818:Animals; D000852:Anopheles / Q000201:enzymology*; D002498:Centrifugation; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006458:Hemolymph / Q000201:enzymology*; D007303:Insect Vectors / Q000201:enzymology*; D007527:Isoenzymes / Q000032:analysis* / Q000097:blood / Q000302:isolation & purification; D008970:Molecular Weight; D014442:Monophenol Monooxygenase / Q000032:analysis* / Q000097:blood / Q000302:isolation & purification
D007527:Isoenzymes; D014442:Monophenol Monooxygenase