Analysis and modification of thiols in the hemolymph juvenile hormone binding protein of Manduca sexta.
Hemolymph-induced in vitro modifications of the hemolymph juvenile hormone binding protein from the tobacco hornworm, Manduca sexta, were analyzed by polyacrylamide gel electrophoresis, Western blots, and equilibrium dialysis. Upon hemolymph melanization, a complex reaction involving polymerization of phenolic compounds, multiple forms of the protein were detected. Under melanizing conditions, one slower- and several faster-migrating proteins were observed. When the hemolymph was treated with 100 mM catechol, a melanin precursor, two well-defined forms of the binding protein appeared. When the protein was incubated with excess catechol (> 100 mM) a single faster-migrating form appeared. Both fast and slow forms bound juvenile hormone I with similar efficiency. These results were duplicated using the thiol modifying reagents p-chloromercurobenzoate and N-ethylmaleimide, suggesting modification of cysteine residues. Using differential alkylation, it was determined that hemolymph juvenile hormone binding protein contained two cystine and two cysteine residues. One of the cysteines is exposed and readily accessible for modification. Since modification of the exposed free thiol did not alter binding, it presumably resides outside the hormone-binding domain. The analyses support the observation that there is a single form of the hemolymph juvenile hormone-binding protein in our strain of M. sexta.
Park
Y C
YC
Department of Entomology, University of Wisconsin-Madison 53706.
Goodman
W G
WG
D000478:Alkylation; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000097:blood*; D002396:Catechols / Q000494:pharmacology; D002729:Chloromercuribenzoates / Q000494:pharmacology; D003956:Dialysis; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D006458:Hemolymph / Q000378:metabolism*; D019476:Insect Proteins; D007460:Iodoacetamide; D007461:Iodoacetates; D019807:Iodoacetic Acid; D008543:Melanins / Q000378:metabolism; D009036:Moths; D013438:Sulfhydryl Compounds / Q000097:blood*; D020245:p-Chloromercuribenzoic Acid
D002352:Carrier Proteins; D002396:Catechols; D002729:Chloromercuribenzoates; D019476:Insect Proteins; D007461:Iodoacetates; D008543:Melanins; D013438:Sulfhydryl Compounds; C035606:juvenile hormone-binding protein, insect; D020245:p-Chloromercuribenzoic Acid; C034221:catechol; D005033:Ethylmaleimide; D019807:Iodoacetic Acid; D007460:Iodoacetamide