Isolation and characterization of a yellow-colored protein from the hemolymph of the tobacco hornworm, Manduca sexta.

Persistent Link:
http://hdl.handle.net/10150/185522
Title:
Isolation and characterization of a yellow-colored protein from the hemolymph of the tobacco hornworm, Manduca sexta.
Author:
Martel, Ralph Roland.
Issue Date:
1991
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
A yellow-colored protein (YCP) has been isolated from the hemolymph of fifth instar, wandering stage larvae of Manduca sexta. The molecular mass of reduced and denatured YCP was 31 kDa. Gel filtration chromatography suggested that native YCP was a monomer. The absorbance spectrum of YCP contained maxima at 278 nm and 405 nm. The amino acid composition and the N-terminal sequence of YCP were determined. Circular dichroism indicated that YCP consisted of 68% $\beta$-pleated sheet and 32% random coil. The YCP polypeptide chain was found to be glycosylated. Carbohydrate analysis suggested that mannose and N-acetylglucosamine were present in a 3:1 ratio. Chromophore was released from YCP through treatment with methanol and chloroform. In neutral solution and in acid, the released chromophore showed the absorbance characteristics of the ommochrome, onmmatin D. In addition, the chromophore was sensitive to treatment with arylsulfatase as would be expected for ommatin D. The polypeptide chain of YCP was synthesized by the larval fat body and was detectable in hemolymph throughout the life cycle. However, only during the fifth instar did YCP polypeptide levels in the hemolymph increase significantly. The highest hemolymph concentration was observed on the first day of pupation, whereafter it gradually decreased. The association of chromophore with the YCP polypeptide was transient. In fifth instar wandering stage larvae and in female moths, YCP polypeptide and chromophore were detectable in the hemolymph. During the wandering stage, increasing amounts of chromophore became associated with the YCP polypeptide. However, in feeding fifth instar larvae and in male moths, the YCP polypeptide but not the chromophore was detectable. No representatives of seven other insect orders contained hemolymph proteins that cross-reacted with anti-YCP antiserum. However, each of four other lepidopteran examined had on immunologically-related hemolymph protein of approximately 31 kDa. Ommochromes arise in insects as end products of the metabolism of tryptophan. As such, ommochromes occur in both the tissues and the excreta of insects. We propose that in M. sexta, one such tryptophan metabolite is found in the hemolymph associated with a specific 31 kDa protein.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Dissertations, Academic; Biochemistry; Entomology.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Biochemistry; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Law, John H.

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleIsolation and characterization of a yellow-colored protein from the hemolymph of the tobacco hornworm, Manduca sexta.en_US
dc.creatorMartel, Ralph Roland.en_US
dc.contributor.authorMartel, Ralph Roland.en_US
dc.date.issued1991en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractA yellow-colored protein (YCP) has been isolated from the hemolymph of fifth instar, wandering stage larvae of Manduca sexta. The molecular mass of reduced and denatured YCP was 31 kDa. Gel filtration chromatography suggested that native YCP was a monomer. The absorbance spectrum of YCP contained maxima at 278 nm and 405 nm. The amino acid composition and the N-terminal sequence of YCP were determined. Circular dichroism indicated that YCP consisted of 68% $\beta$-pleated sheet and 32% random coil. The YCP polypeptide chain was found to be glycosylated. Carbohydrate analysis suggested that mannose and N-acetylglucosamine were present in a 3:1 ratio. Chromophore was released from YCP through treatment with methanol and chloroform. In neutral solution and in acid, the released chromophore showed the absorbance characteristics of the ommochrome, onmmatin D. In addition, the chromophore was sensitive to treatment with arylsulfatase as would be expected for ommatin D. The polypeptide chain of YCP was synthesized by the larval fat body and was detectable in hemolymph throughout the life cycle. However, only during the fifth instar did YCP polypeptide levels in the hemolymph increase significantly. The highest hemolymph concentration was observed on the first day of pupation, whereafter it gradually decreased. The association of chromophore with the YCP polypeptide was transient. In fifth instar wandering stage larvae and in female moths, YCP polypeptide and chromophore were detectable in the hemolymph. During the wandering stage, increasing amounts of chromophore became associated with the YCP polypeptide. However, in feeding fifth instar larvae and in male moths, the YCP polypeptide but not the chromophore was detectable. No representatives of seven other insect orders contained hemolymph proteins that cross-reacted with anti-YCP antiserum. However, each of four other lepidopteran examined had on immunologically-related hemolymph protein of approximately 31 kDa. Ommochromes arise in insects as end products of the metabolism of tryptophan. As such, ommochromes occur in both the tissues and the excreta of insects. We propose that in M. sexta, one such tryptophan metabolite is found in the hemolymph associated with a specific 31 kDa protein.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectDissertations, Academicen_US
dc.subjectBiochemistryen_US
dc.subjectEntomology.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineBiochemistryen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorLaw, John H.en_US
dc.contributor.committeememberWells, Michael A.en_US
dc.contributor.committeememberTischler, Marc E.en_US
dc.contributor.committeememberGrimes, William J.en_US
dc.contributor.committeememberMacKenzie, Neil E.en_US
dc.identifier.proquest9136853en_US
dc.identifier.oclc711689887en_US
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