Antibodies against the fungal enzyme mannitol dehydrogenase

Abstract

Affinity-purified, electrophoretically homogeneous NADP +-mannitol dehydrogenase (MtDH; EC 1.1.1.138), isolated from fruit bodies of Agaricus bisporus (Lange) Sing., was used to produce polyclonal antibodies. Antiserum against MtDH and the affinity-purified antibody inhibited enzyme activity in a dose-dependent manner, with about 15 mol of purified antibody required for 50% inhibition of 1 mol MtDH. Immunological specificity of the antisera was demonstrated by double immunodiffusion, the dotimmunobinding assay, and immunoblotting. Controls (preimmune sera and preadsorbed antisera) were negative. An enzyme-linked immunosorbent assay (ELISA) was established to quantitate immunobinding in various cell fractions of the fungus. The bound protein was found predominantly in the soluble fraction (150,000 g), where it contributed 5% to the total protein of the fruit body and 1% to that of the mycelium. Though only to a minor extent, the anti-MtDH antiserum also bound to the supernatants obtained following treatment of a “mixed membrane fraction” and the cell wall fraction (54,000 g and 4,000 g sediments, respectively; both rigorously washed with buffer) with digitonin or sodium dodecyl sulfate/heat. In sporocarp extracts, the antibody bound specifically to a protein of M r 30K (corresponding to the subunit molecular weight of MtDH) as shown by immunoblotting, whereas mycelial extracts contained three (30K, 26K, 24K) protein bands, which cross-reacted with the anti-MtDH antiserum.