Shapiro J P, Silhacek D L, Niedz R P
U.S. Department of Agriculture, Agricultural Research Service, Orlando, FL 32803.
Arch Insect Biochem Physiol. 1992;20(4):315-31. doi: 10.1002/arch.940200407.
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 Ia and Ib) using native gradient pore-limiting electrophoresis. The high concentration of proteins Ia, 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 alpha subunit (M(r) 75,000), while the beta subunit (M(r) 71,000) was lacking from one of the three. Repeated passage through an anion exchange column yielded two of the three proteins (Ib and II) 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 alpha subunit was high in Met, His, and Thr, and the beta subunit was high in Lys. Both were high in Pro and had approximately 16% Phe+Tyr. Sequences of the 20 N-terminal amino acid residues of each subunit showed 45-60% homology between subunits. These coleopteran proteins also showed some sequential homology but no immunological cross-reactivity with storage proteins from the lepidopterans Galleria mellonella and Heliothis virescens.
先前的研究表明，在象鼻虫幼虫（Diaprepes abbreviatus）血淋巴中高浓度存在的蛋白质能够结合一种合成香豆素——7-氨基-3-苯基香豆素（香豆素-10）。之前鉴定出的两种天然蛋白质之一（蛋白质I），现在通过天然梯度孔径限制电泳显示可分离为两条不同的条带（蛋白质Ia和Ib）。幼虫血淋巴中蛋白质Ia、Ib和II的高浓度，它们在化蛹时从血淋巴中消失，以及化学交联显示出的明显六聚体结构，将它们鉴定为六聚体储存蛋白（hexamerins）。现在通过阴离子交换高效液相色谱法分离得到的至少一种Ib色谱形式显示能结合核黄素（Rb）。储存蛋白的部分分离混合物对Rb荧光的淬灭也证明了这种结合。脂转运蛋白不会淬灭Rb荧光。Rb通过热提取从全血淋巴中获得，并通过其荧光光谱以及带有荧光检测的反相高效液相色谱法进行鉴定。通过连续密度梯度超速离心、凝胶渗透高效液相色谱法和反相高效液相色谱法，分离得到了三种全蛋白共有的两个亚基。所有三种全蛋白都共享α亚基（相对分子质量75,000），而三种中的一种缺乏β亚基（相对分子质量71,000）。反复通过阴离子交换柱可得到三种蛋白质中的两种（Ib和II）的均一形式。用亚胺基二甲酯进行化学交联表明全蛋白具有六聚体结构。当在聚偏二氟乙烯膜上用生物素化的伴刀豆球蛋白A进行检测时，所有亚基和全蛋白都被染成高甘露糖糖蛋白。α亚基富含甲硫氨酸、组氨酸和苏氨酸，β亚基富含赖氨酸。两者都富含脯氨酸，且苯丙氨酸 + 酪氨酸含量约为16%。每个亚基的20个N端氨基酸残基序列显示亚基之间有45 - 60%的同源性。这些鞘翅目蛋白质与鳞翅目昆虫大蜡螟（Galleria mellonella）和棉铃虫（Heliothis virescens）的储存蛋白也显示出一些序列同源性，但没有免疫交叉反应性。
