Brownleader M D, Dey P M
Department of Biochemistry, University of London, Egham, Surrey, UK.
Planta. 1993;191(4):457-69. doi: 10.1007/BF00195747.
Extensin, a hydroxyproline-rich glycoprotein comprising substantial amounts of beta-L-arabinose-hydroxyproline glycosidic linkages is believed to be insolubilized in the cell wall during host-pathogen interaction by a peroxidase/hydroperoxide-mediated cross-linking process. Both extensin precursor and extensin peroxidase were ionically eluted from intact water-washed tomato (hybrid of Lycopersicon esculentum Mill. and L. peruvianum L. (Mill.) cells in suspension cultures and purified to homogeneity by a rapid and simple procedure under mild and non-destructive experimental conditions. The molecular weight of native extensin precursor was estimated to be greater than 240-300 kDa by Superose-12 gel-filtration chromatography. Extensin monomers have previously been designated a molecular weight of approximately 80 kDa. Our results indicate that salt-eluted extensin precursor is not monomeric. Agarose-gel electrophoresis, Superose-12-gel-filtration, extensin-peroxidase-catalysed cross-linking, Mono-S ion-exchange fast protein liquid chromatography (FPLC), and peptide-sequencing data confirmed the homogeneity of the extensin preparation. Evidence that the purified protein was extensin is attributed to the presence of the putative sequence motif--Ser (Hyp)4--within the N-terminal end of the protein. Treatment of extensin with trifluoroacetic acid demonstrated that arabinose was the principal carbohydrate. The amino-acid composition of the purified extensin was similar to those reported in the literature. The cross-linking of extensin in vitro upon incubation with extensin peroxidase and exogenous H2O2 was characteristic of other reported extensins. Furthermore, Mono-S ion-exchange FPLC of native extensin precursor resolved it into two isoforms, A (90%) and B (10%). The amino-acid compositions of extensin A and extensin B were found to be similar to each other and both extensins were cross-linked in vitro by extensin peroxidase.
伸展蛋白是一种富含羟脯氨酸的糖蛋白，含有大量的β-L-阿拉伯糖-羟脯氨酸糖苷键，据信在宿主-病原体相互作用过程中，通过过氧化物酶/氢过氧化物介导的交联过程在细胞壁中不溶解。伸展蛋白前体和伸展蛋白过氧化物酶都可以通过离子交换从完整的水洗番茄（番茄和秘鲁番茄的杂交种）悬浮培养细胞中洗脱出来，并在温和且无损的实验条件下通过快速简单的程序纯化至同质。通过Superose-12凝胶过滤色谱法估计天然伸展蛋白前体的分子量大于240-300 kDa。伸展蛋白单体先前被指定的分子量约为80 kDa。我们的结果表明，盐洗脱的伸展蛋白前体不是单体。琼脂糖凝胶电泳、Superose-12凝胶过滤、伸展蛋白过氧化物酶催化的交联、Mono-S离子交换快速蛋白质液相色谱（FPLC）和肽测序数据证实了伸展蛋白制剂的同质性。纯化的蛋白质是伸展蛋白的证据归因于蛋白质N末端存在假定的序列基序——Ser（Hyp）4。用三氟乙酸处理伸展蛋白表明阿拉伯糖是主要的碳水化合物。纯化的伸展蛋白的氨基酸组成与文献中报道的相似。伸展蛋白与伸展蛋白过氧化物酶和外源H2O2孵育后在体外的交联是其他报道的伸展蛋白的特征。此外，天然伸展蛋白前体的Mono-S离子交换FPLC将其解析为两种异构体，A（90%）和B（10%）。发现伸展蛋白A和伸展蛋白B的氨基酸组成彼此相似，并且两种伸展蛋白在体外都被伸展蛋白过氧化物酶交联。
