Department of Botany, University of Iowa, Iowa City, Iowa 52242.
Plant Physiol. 1991 Aug;96(4):1329-37. doi: 10.1104/pp.96.4.1329.
Mandelonitrile lyase (MDL, EC 4.1.2.10), which catalyzes the reversible dissociation of (R)-(+)-mandelonitrile to benzaldehyde and hydrogen cyanide, was purified to apparent homogeneity from mature black cherry (Prunus serotina Ehrh.) seeds by conventional protein purification techniques. This flavoprotein is monomeric with a subunit molecular mass of 57 kilodaltons. Glycoprotein character was shown by its binding to the affinity matrix concanavalin A-Sepharose 4B with subsequent elution by alpha-methyl-d-glucoside. Upon chemical deglycosylation by trifluoromethanesulfonic acid, the molecular mass was reduced to 50.9 kilodaltons. Two-dimensional gel analysis of deglycosylated MDL revealed the presence of several subunit isoforms of similar molecular mass but differing slightly in isoelectric point. Polyclonal antibodies were raised in New Zealand white rabbits against deglycosylated and untreated MDL. Antibody titers were determined by enzyme linked immunosorbent and dot immunobinding assays, while their specificities were assessed by Western immunoblot analysis. Antibodies raised against untreated lyase recognized several proteins in addition to MDL. In contrast, antisera raised against deglycosylated MDL were monospecific and were utilized for developmental and immunocytochemical localization studies. SDS-PAGE and immunoblotting analysis of seed proteins during fruit maturation showed that MDL first appeared in seeds shortly after cotyledons began development. In cotyledon cells of mature seeds, MDL was localized primarily in the cell wall with lesser amounts in the protein bodies, whereas in endosperm cells, this labeling pattern was reversed. N-terminal sequence data was gathered for future molecular approaches to the question of MDL microheterogeneity.
杏仁腈裂解酶（MDL，EC4.1.2.10），能够催化（R）-（+）-苦杏仁腈可逆地分解为苯甲醛和氰化氢，从成熟的黑樱桃（Prunus serotina Ehrh.）种子中通过常规蛋白质纯化技术被纯化到明显的均一性。这种黄素蛋白是单体，亚基分子量为 57 千道尔顿。通过其与伴刀豆球蛋白 A-Sepharose 4B 的亲和基质结合，并随后用α-甲基-D-吡喃葡萄糖苷洗脱，显示出糖蛋白的特性。经三氟甲磺酸化学去糖基化后，分子量降低至 50.9 千道尔顿。去糖基化 MDL 的二维凝胶分析显示，存在几种亚基同工型，分子量相似，但等电点略有不同。针对去糖基化和未经处理的 MDL，在新西兰白兔中产生了多克隆抗体。通过酶联免疫吸附和点免疫结合测定法测定抗体滴度，而通过 Western 免疫印迹分析评估其特异性。针对未经处理的裂解酶产生的抗体除了 MDL 外还识别几种蛋白质。相比之下，针对去糖基化 MDL 产生的抗血清是单特异性的，用于发育和免疫细胞化学定位研究。种子蛋白在果实成熟过程中的 SDS-PAGE 和免疫印迹分析表明，MDL 首先在子叶开始发育后不久出现在种子中。在成熟种子的子叶细胞中，MDL 主要定位于细胞壁中，而在蛋白质体中则较少，而在内胚乳细胞中，这种标记模式则相反。为了进一步研究 MDL 微异质性的问题，收集了 N 端序列数据。
