Investigations into the molecular size and shape of tomato extensin

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Abstract
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×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 µM and sedimentation velocity data generated a Kd between 1 and 10 µM. 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.