Hegde R, Podder S K
Department of Biochemistry, Indian Institute of Science, Bangalore.
Eur J Biochem. 1992 Feb 15;204(1):155-64. doi: 10.1111/j.1432-1033.1992.tb16618.x.
This study elucidates some structural and biological features of galactose-binding variants of the cytotoxic proteins ricin and abrin. An isolation procedure is reported for ricin variants from Ricinus communis seeds by using lactamyl-Sepharose affinity matrix, similar to that reported previously for variants of abrin from Abrus precatorius seeds [Hegde, R., Maiti, T. K. & Podder, S. K. (1991) Anal. Biochem. 194, 101-109]. Ricin variants, subfractionated on carboxymethyl-Sepharose CL-6B ion-exchange chromatography, were characterized further by SDS/PAGE, IEF and a binding assay. Based on the immunological cross-reactivity of antibody raised against a single variant of each of ricin and abrin, it was established that all the variants of the corresponding type are immunologically indistinguishable. Analysis of protein titration curves on an immobilized pH gradient indicated that variants of abrin I differ from other abrin variants, mainly in their acidic groups and that variance in ricin is a cause of charge substitution. Detection of subunit variants of proteins by two-dimensional gel electrophoresis showed that there are twice as many subunit variants as there are variants of holoproteins, suggesting that each variant has a set of subunit variants, which, although homologous, are not identical to the subunits of any other variant with respect to pI. Seeds obtained from polymorphic species of R. communis showed no difference in the profile of toxin variants, as analyzed by isoelectric focussing. Toxin variants obtained from red and white varieties of A. precatorius, however, showed some difference in the number of variants as well as in their relative intensities. Furthermore, variants analyzed from several single seeds of A. precatorius red type revealed a controlled distribution of lectin variants in three specific groups, indicating an involvement of at least three genes in the production of Abrus lectins. The complete absence or presence of variants in each group suggested a post-translational differential proteolytic processing, a secondary event in the production of abrin variants.
本研究阐明了细胞毒性蛋白蓖麻毒素和相思子毒素的半乳糖结合变体的一些结构和生物学特征。报道了一种从蓖麻种子中分离蓖麻毒素变体的方法，该方法使用内酰胺基琼脂糖亲和基质，类似于先前报道的从相思豆种子中分离相思子毒素变体的方法[赫格德，R.，迈蒂，T.K.和波德，S.K.（1991年）《分析生物化学》194，101 - 109]。通过羧甲基琼脂糖CL - 6B离子交换色谱法对蓖麻毒素变体进行亚分级分离，然后通过SDS/PAGE、IEF和结合试验进一步表征。基于针对蓖麻毒素和相思子毒素各自单一变体产生的抗体的免疫交叉反应性，确定相应类型的所有变体在免疫上无法区分。在固定pH梯度上分析蛋白质滴定曲线表明，相思子毒素I的变体与其他相思子毒素变体不同，主要在于它们的酸性基团，并且蓖麻毒素的差异是电荷取代的结果。通过二维凝胶电泳检测蛋白质的亚基变体表明，亚基变体的数量是全蛋白变体数量的两倍，这表明每个变体都有一组亚基变体，这些亚基变体虽然同源，但在等电点方面与任何其他变体的亚基都不相同。从蓖麻多态物种获得的种子，通过等电聚焦分析，毒素变体谱没有差异。然而，从相思豆红色和白色品种获得的毒素变体在变体数量及其相对强度方面存在一些差异。此外，对相思豆红色类型的几个单粒种子分析的变体显示，凝集素变体在三个特定组中呈受控分布，这表明至少有三个基因参与了相思豆凝集素的产生。每组中变体的完全缺失或存在表明存在翻译后差异蛋白水解加工，这是相思子毒素变体产生过程中的一个次级事件。
