Storage proteins of the larval root weevil Diaprepes abbreviatus (Coleoptera: Curculionidae): Riboflavin binding and subunit isolation
✍ Scribed by Jeffrey P. Shapiro; Randall P. Niedz; Donald L. Silhacek
- Publisher
- John Wiley and Sons
- Year
- 1992
- Tongue
- English
- Weight
- 949 KB
- Volume
- 20
- Category
- Article
- ISSN
- 0739-4462
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✦ Synopsis
Proteins present at high concentrations in hemolymph of the larval weevil Diaprepes abbreviatus were previously shown to bind a synthetic coumarin, 7-amino-3-phenyl coumarin (coumarin-10). One of the two native proteins previously identified (protein I) is now shown to separate into two distinct bands (proteins la and Ib) using native gradient pore-limiting electrophoresis. The high concentration of proteins la, Ib, and II in larval hemolymph, their disappearance from hemolymph upon pupation, and an apparent hexameric structure shown by chemical crosslinking identify them as hexameric storage proteins (hexamerins). At least one chromatographic form of Ib isolated by anion exchange HPLC is now shown to bind riboflavin (Rb). Binding was also demonstrated by quenching of Rb fluorescence by a partially isolated mixture of the storage proteins. Lipophorin did not quench Rb fluorescence. Rb was heat-extracted from whole hemolymph and identified by its fluorescence spectra and by reverse phase HPLC with fluorescence detection.
The two subunits shared by the three holoproteins have been isolated by sequential density gradient ultracentrifugation, gel permeation HPLC, and reverse phase HPLC. All three holoproteins shared the a subunit (M, 75,000), while the p subunit (M, 71,000) was lacking from one of the three, Repeated passage through an anion exchange column yielded two of the three proteins (Ib and Il} in homogeneous form. Chemical crosslinking with dimethylsuberimidate indicated a hexameric structure for the holoproteins. All subunits and holoproteins stained as high mannose glycoproteins when probed with biotinylated concanavalin A on PVDF membranes. The a subunit was high in Met, His, and Thr, and the p subunit was high in Lys. Both were high in Pro and had approximately 16% Phe + Tyr. Sequences of the 20 N-terminal amino