Localization of the beta-glucosidases in Neurospora crassa

The beta-glucosidases (EC 3.2.1.21) of Neurospora crassa were studied with respect to their location in conidia and young mycelia. Aryl-beta-glucosidase of conidia was nearly equally divided between extracellular and bound activity. Bound aryl-beta-glucosidase was almost all available to substrate. An induction procedure was used to maximize both beta-glucosidases in 4 to 6-hr cells. Aryl-beta-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-beta-glucosidase could be obtained by this treatment. Dilute HCl (0.1 n) destroyed the patent aryl-beta-glucosidase but not the cryptic aryl-beta-glucosidase or the cryptic cellobiase activity in intact cells. This suggested that most aryl-beta-glucosidase activity was exterior to the cell membrane but still within the mural space. The thermal stability of patent aryl-beta-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-beta-d-glucopyranoside by the enzymes in intact cells compared to enzymes in extracts.