Localization of the β-Glucosidases in Neurospora crassa

Abstract

The β-glucosidases (EC 3.2.1.21) of Neurospora crassa were studied with respect to their location in conidia and young mycelia. Aryl-β-glucosidase of conidia was nearly equally divided between extracellular and bound activity. Bound aryl-β-glucosidase was almost all available to substrate. An induction procedure was used to maximize both β-glucosidases in 4 to 6-hr cells. Aryl-β-glucosidase was entirely bound but still mostly (90%) detectable, whereas cellobiase was mostly internal and cryptic. A freeze-thaw cycle or treatment with phenethyl alcohol or deoxycholic acid made the cellobiase detectable without releasing it from the cell. A 10 to 20% increase in cell-bound aryl-β-glucosidase could be obtained by this treatment. Dilute HCl (0.1 n ) destroyed the patent aryl-β-glucosidase but not the cryptic aryl-β-glucosidase or the cryptic cellobiase activity in intact cells. This suggested that most aryl-β-glucosidase activity was exterior to the cell membrane but still within the mural space. The thermal stability of patent aryl-β-glucosidase and released cellobiase was found to be higher than in corresponding cell-free extracts. Measurements of K m suggested a slightly lower affinity for substrate p -nitrophenyl-β- d -glucopyranoside by the enzymes in intact cells compared to enzymes in extracts.