THE SPORE WALL AND POLAR TUBE PROTEINS OF THE MICROSPORIDIAN NOSEMA GRYLLI:
THE MAJOR SPORE WALL PROTEIN IS RELEASED BEFORE SPORE EXTRUSION
V. V. Dolgikh, P. B. Semenov
All-Russian Research Institute for Plant Protection, St. Petersburg;
e-mail: dollslav@yahoo.com
Incubation of Nosema grylli spores in alkaline-saline solution (10 mM KOH, 170 mM KCl) leads to
solubilization of the major spore wall protein of 40 kDa (p40). Both the compounds of this solution are crucial for
p40 solubilization. After spore incubation in 170 mM KCl no proteins were released in the medium. In contrast, 10 mM
KOH causes a release of many spore proteins but only a small amount of p40. A long storage of spores (over a year)
in water or 0.02 % sodium azide results in a sharp decrease of p40 content. Specific polyclonal antibodies were
obtained by immunization of rabbits with isolated p40. The specificity of serum was confirmed by immunoblotting.
IFA showed reliable reaction on the envelopes of sporonts and sporoblasts, whereas only part of spores reacted with
antibodies. This distinction may be due to changing surface antigens during spore maturation. Solubilization of p40
under alkaline conditions could be associated with spore extrusion, since a subsequent transfer of spores to neutral
solution leads to their discharge. Subsequent wash of discharged spores with 1-3 % SDS, 9 M urea and treatment by
100 % 2-ME result in solubilization of protein of 56 kDa (p56). The maximum concentration of 2-ME is important for
isolation of pure p56. Evidence has been provided that p56 is a protein of N. grylli polar tubes. Treatment of
discharged spores by 2-ME in the presence of SDS results in solubilization of four additional proteins with molecular
weights about 46, 34, 21 and 15 kDa.
Key words: Microsporidia, polar tube, proteins, spore wall
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