Induction profiles and properties of a novel stress-induced peroxidase in Neurospora crassa

Abstract

Exposure of Neurospora crassa cells to heat shock and oxidative stress results in the synthesis of several stress-related proteins, including a peroxidase. Northern blot analysis of total RNA revealed a heat-inducible (HI)-peroxidase transcript of ∼10 kb, induced in response to heat shock and oxidative stress. The HI peroxidase was isolated from heat-shocked mycelium and purified to near homogeneity, and its properties were examined. Chromatography in size-exclusion matrices yielded an apparent molecular mass of ∼116 kDa for the native enzyme, whereas the estimate obtained by SDS-PAGE was 90–95 kDa. Studies of substrate saturation kinetics were conducted using the purified enzyme with ABTS [2,2′-azino-bis (3-ethylbenzthiazole-6-sulfonic acid)] and H₂O₂ as substrates. The experimentally estimated K _m, V _max, and K _cat values for ABTS were ∼36 μM, 5200 nmol mg⁻¹, and 8 s⁻¹, respectively, and those for H₂O₂ were 44 μM, 6640 nmol mg⁻¹, and 10 s⁻¹. O-dianisidine was a substrate for this enzyme, but guaiacol was not. HI peroxidase was found to be a glycoprotein, stable at temperatures up to 60°C.