Localization of α-L-Arabinofuranosidase and Acid Phosphatase in Mycelium of Sclerotinia fructigena

Abstract

α-L-Arabinofuranosidase (AF) and acid p-nitrophenyl phosphatase (AP) were secreted by Sclerotinia fructigena grown in a liquid pectin/ammonium tartrate medium. ‘Gentle’ mechanical manipulation of mycelium solubilized most of the AF and much of the AP, while brief acid treatment considerably inactivated both enzymes. Both enzymes were present predominantly in a soluble form in homo-genates prepared for subcellular fractionation, but some particulate activity of both was recovered from a sucrose density gradient in a fraction which also contained mitochondria. Azo-dye techniques with appropriate 1-naphthyl derivatives as substrates and p-(acetoxymercuric) aniline diazotate as capturing agent produced similar staining patterns for both enzymes in the light and electron microscopes, but the distribution of β-glycerophosphatase activity as visualized by the Gomori technique was more variable. A proportion of the activity of the enzymes remaining after fixation was located between the plasmalemma and the hyphal wall, in vacuoles in the cytoplasm, and in spherosome-like bodies. Some evidence was obtained for structure-linked latency of both enzymes and for their secretion by a process of reverse pinocytosis.