Isolation and purification of three antifungal proteins from sorghum endosperm.

Three proteins potently inhibitory to the growth of Fusarium moniliforme,
the grain mould pathogen have been identified from sorghum endosperm,
using aqueous extraction, gel filtration and ion-exchange chromatography
coupled with bioassay. The aqueous extract (Tris buffer, pH 6.8) of sorghum
endosperm flour was separated into two fractions by passing through Sephadex
G-50. The void volume fraction, showing antifungal activity was further fractionated
by ion-exchange chromatography on SP Sephadex C-50. The most potent
fraction was that which bound to the SP Sephadex column at pH 6.0. A less
potent fraction with an 18 kDa protein as major component was released on
shifting the pH to 8.5. The bound fraction at pH 8.5 was a much more potent
inhibitor of fungal growth than the unbound fraction; and it was composed of
two major polypeptides with molecular weights of 26 and 30 kDa. All three polypeptides
were separated on SDS-PAGE gels, transferred onto Immobilon and
tested for antifungal activity. The test showed that the 26 and 30 kDa proteins
retained the antifungal activity even after electrotransfer. Antibodies raised
against the electroeluted proteins were shown to abolish the antifungal activity of
the endosperm extracts. These proteins appeared only in the endosperm of
sorghum grains as revealed by dot immunobinding assay. The 26 and 30 kDa
antifungal proteins were also present in pearl millet and maize, while the 18 kDa
protein was found only in sorghum. Cross-reactivity of antibodies with the
aqueous extracts of wheat, rice and ragi was not seen.