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

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.