Purification and properties of an ommochrome-binding protein from the hemolymph of the tobacco hornworm, Manduca sexta.
A yellow-colored protein (YCP) was isolated from the hemolymph (i.e. blood) of fifth instar wandering stage larvae of Manduca sexta. The molecular mass of YCP was 31 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration chromatography suggested that native YCP was a monomer. The absorbance spectrum of YCP revealed maxima at 278 and 405 nm. Chromophore was released from YCP through denaturation of the protein with methanol and chloroform. In neutral solution and in acid, the released chromophore showed the absorbance characteristics of an ommochrome: ommatin D. In addition, the chromophore was sensitive to treatment with arylsulfatase as would be expected for ommatin D. The amino acid composition and the N-terminal sequence of YCP were determined. The YCP polypeptide chain was found to be glycosylated. Carbohydrate analysis suggested that Man and GlcNAc were present in a 3:1 ratio. Circular dichroism indicated that YCP consisted of 68% beta-pleated sheet with no alpha-helices being detected. An in vitro incubation of larval fat body in the presence of [35S]methionine indicated that this organ was the site of synthesis. Ommochromes arise in insects as end products of the metabolism of tryptophan. It is well-documented that 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 protein.
Department of Biochemistry, University of Arizona, Tucson 85721.
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Martel
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Law
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002352:Carrier Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D006458:Hemolymph / Q000502:physiology; D007301:Insect Hormones / Q000302:isolation & purification* / Q000378:metabolism; D019476:Insect Proteins; D007814:Larva; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths / Q000378:metabolism*; D010640:Phenothiazines / Q000302:isolation & purification; D010860:Pigments, Biological / Q000302:isolation & purification; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D000596:Amino Acids; D002241:Carbohydrates; D002352:Carrier Proteins; D007301:Insect Hormones; D019476:Insect Proteins; C071652:OBP protein, Manduca sexta; D010640:Phenothiazines; D010860:Pigments, Biological; C002772:ommochrome