Brownleader M D, Byron O, Rowe A, Trevan M, Welham K, Dey P M
School of Applied Science, South Bank University, London, U.K.
Biochem J. 1996 Dec 1;320 ( Pt 2)(Pt 2):577-83. doi: 10.1042/bj3200577.
The molecular characteristics of soluble extensin from tomato have been investigated. An apparent molecular mass greater than 240 kDa has been previously observed with the shape-dependent method of gel-filtration chromatography [Brownleader and Dey (1993) Planta (Berlin) 191, 457-469]. Tomato extensin is a heavily glycosylated protein that does not migrate into SDS/polyacrylamide gels. This shape-dependent behaviour raises doubts about agreement between the observed apparent mass and the absolute value. The molecular mass measured with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) was 72.3 kDa, with no evidence of any other species except a doubly charged ion. The sample was therefore considered to be monodisperse under the conditions used. Electron microscopy of soluble extensin showed the presence of particles 40-50 nm in length and 2.0-2.5 nm in width. A minority of these particles showed a central 'kink'. A number of smaller and generally wider particles (20 nm x 2-4 nm) were considered to be folded monomers and larger particles were thought to be dimers. Sedimentation analysis showed that extensin exists in a rapid monomer-dimer equilibrium in the concentration range and buffer used. Sedimentation equilibrium data gave a Kd of 8.5 microM and sedimentation velocity data generated a Kd between 1 and 10 microM. The concentration dependence of the measured sedimentation coefficient was used, together with hydrodynamic bead modelling, to define plausible shapes for monomer and dimer. This suggests that monomeric extensin is an elongated rod of length 40 nm and width 2 nm, which forms staggered dimers of average length 50 nm and width 3 nm. Extensin is an integral component of the primary cell wall. The physical characteristics (size, shape and form) of the rod-like extensin have been evaluated in this paper so that the role that extensin plays in primary cell wall architecture and during plant disease resistance can be more fully understood.
对番茄可溶性伸展蛋白的分子特性进行了研究。先前使用凝胶过滤色谱的形状依赖法观察到其表观分子量大于240 kDa[Brownleader和Dey（1993年），《植物（柏林）》191卷，457 - 469页]。番茄伸展蛋白是一种高度糖基化的蛋白质，不会迁移到SDS/聚丙烯酰胺凝胶中。这种形状依赖行为让人对观察到的表观质量与绝对值之间的一致性产生怀疑。用基质辅助激光解吸/电离飞行时间质谱（MALDI - TOF MS）测得的分子量为72.3 kDa，除了双电荷离子外没有任何其他物种的证据。因此，在所使用的条件下，该样品被认为是单分散的。可溶性伸展蛋白的电子显微镜观察显示存在长度为40 - 50 nm、宽度为2.0 - 2.5 nm的颗粒。这些颗粒中的少数显示出中央“扭结”。一些较小且通常更宽的颗粒（20 nm×2 - 4 nm）被认为是折叠单体，而较大的颗粒被认为是二聚体。沉降分析表明，在所用的浓度范围和缓冲液中，伸展蛋白以快速的单体 - 二聚体平衡存在。沉降平衡数据给出的解离常数（Kd）为8.5 μM，沉降速度数据得出的Kd在1至10 μM之间。将测得的沉降系数的浓度依赖性与流体动力学珠模型一起用于确定单体和二聚体的合理形状。这表明单体伸展蛋白是长度为40 nm、宽度为2 nm的细长杆状，形成平均长度为50 nm、宽度为3 nm的交错二聚体。伸展蛋白是初生细胞壁的一个组成部分。本文评估了杆状伸展蛋白的物理特性（大小、形状和形态），以便更全面地了解伸展蛋白在初生细胞壁结构和植物抗病过程中所起的作用。
