Neuronal N-acetyl-β-D-glucosaminidase. Evidence for its biosynthesis in vitro

Abstract

Neuronal cell bodies, isolated in bulk from 8-day-old rat cerebral cortices, were incubated in the presence of a 3H-labelled amino acid mixture, and subcellular fractions isolated by differential centrifugation. The particulate fractions were frozen/thawed in 0.20 M-sucrose/0.1 M-KCl [Selling et al. (1973) Biochim. Biophys. Acta 315, 128-146] and the profiles of acid-insoluble radioactivity and N-acetyl-beta-D-glucosaminidase (glucosaminidase) activity compared in the resulting non-sedimentable fractions by DEAE-cellulose chromatography and cellulose acetate electrophoresis. Radioactivity and glucosaminidase activity co-migrated to a significant extent. Electrophoresis revealed that after 1 min of incubation 42% of the radioactivity of the non-sedimentable microsomal fraction after freezing and thawing co-migrated with an intensely fluorescent band of glucosaminidase activity. Since the pellet fraction obtained on freezing/thawing the microsomal fraction contained up to 75% of the RNA, 95% of the radioactivity and 45% of the glucosaminidase, a detailed study of the association between its radioactivity and nascent glucosaminidase activity was undertaken. After 1 and 2 min of incubation, followed by centrifugation of the microsomal pellet on 35-60% (w/v) sucrose density gradients, radioactivity and glucosaminidase activity exhibited parallel profiles in the region of heavy polyribosomes and at the top of the gradient which contains spontaneously released nascent polypeptide chains. DEAE-cellulose chromatography of these chains revealed glucosaminidase A to be the principal nascent glucosaminidase component, with glucosaminidases B and C as minor peaks. After 2 min of incubation, all of the glucosaminidase components appeared labelled, and glucosaminidase A exhibited two distinct sub-components. The pattern of glucosaminidase labelling in the soluble and microsomal fractions suggested that newly formed glucosaminidase molecules traverse both the cellular sap and the lumen of the endoplasmic reticulum. Only glucosaminidase A reacted specifically with concanavalin A and radioactive glucosaminidase A could be successfully regenerated by treatment with alpha-methyl-D-glucoside. Glucosaminidase A and a substantial portion of the radioactivity associating with it could be readily converted into glucosaminidase B by re-chromatography on DEAE-cellulose and by reaction of the concanavalin A-glucosaminidase A complex with methyl glucosides.