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 nmolmg^-1, and 8 s^-1, respectively, and those for H₂O₂ were 44 μM, 6640 nmolmg^-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.