HistochemicaI Journal 24, 737-746 (1992) Electron microscopic immunocytochemical localizakion of proline-rich proteins in normal parotid salivary glands mouse H. MANSOURI, ~ G. H. COPE, TM N. DIVECHA z and C. J. MCDONALD 2 *Department of Biomedical Science, and 2The Krebs Institute for Biomolecular Research, Department of Molecular Biology & Biotechnology, University of Sheffield, Sheffield SIO 2TN, UK Received 13 September 1991 and in revised form 16 February 1992 Summary Rabbit polyclonal antibodies against isoproterenol-induced mouse proline-rich proteins (PRPs) were used to localize PRPs in the parotid salivary glands of normal adult BALB/c mice. The antibodies recognized both acidic-type and basic-type PRPs. Immunoblotting experiments revealed that the glands contained an acidic-type and a basic-type PRP. Parotid gland tissue was fixed with Karnosky's fixative and embedded in Lowicryl resin at low temperature. PRPs were localized at the electron microscope level using an indirect post-embedding staining technique with protein A-gold. The secretion granules of the acinar cells were strongly labelled. Pre-absorption of the antibody with purified acidic-type and basic-type PRPs indicated that the basic-type PRP is mainly located at the periphery of the granules but that the acidic-type PRP is more evenly distributed within the granules. Pre-absorption of the antibody with a-amylase did not affect the staining pattern, suggesting minimal cross-reactivity. PRPs were also detected within the rough endoplasmic reticulum and the Golgi apparatus of acinar cells, within the granules of the proacinar cells and in the lumena of the ducts, but not within the intercalated or striated duct cell granules. Introduction Proline-rich proteins (PRPs) are an unusual family of proteins in which proline, glycine and glutamine make up 70-80% of the amino acid residues (Bennick, 1982, 1987). A number of different acidic, basic and glycosylated forms have been isolated and characterized (Bennick & Connell, 1971; Levine & Keller, 1977; Kauffman & Keller, 1983; Kauffman et aI., 1986). These are synthesized principally by the salivary glands and are major constituents of the saliva in some species. The parotid gland is a particularly rich source, and in man about 70% of the proteins secreted by this gland are PRPs (Bennick, 1982, 1987). The salivary glands of rodents normally contain only small amounts of PRP. Originally these were believed to be granule membrane proteins (Amsterdam et al., 1971) but later they were shown to be secretory proteins only loosely bound to the granule envelope (Wallach et al., 1975a, b; Robinovitch et al., 1980). The amount of PRP produced by the parotid gland can be increased dramatically by the administration of isoproterenol, a *To whom correspondence should be addressed. 0018-2214/92 1992 Chapman & Hall sympatheticomimer which also causes hypertrophy and hyperplasia (Seyle et al., 1961; Schneyer, 1962). Injections of isoproterenol increase the expression of PRP genes (Roberts et aI., 1991a, b, c) and after only 3-4 days newly synthesized PRPs make up more than 50% of the secretory products of the glands of mice (Robinovitch et al., 1977). One acidic and six basic PRPs have been identified in the hypertrophied glands of rats (Muenzer et al., 1979a, b) and three different glycosylated forms (Mehansho et al., 1985) and five non-glycosylated forms (Bannister et al., 1989) have been isolated from the parotid glands of mice. The secretion granules of rodents often appear inhomogeneous in electron micrographs, and cyto- chemical studies suggest that the electron-translucent cortex of these granules is rich in carbohydrate and lipoidal moieties (Simson et al., 1974; Simson, 1976). After isoproterenol treatment the granules usually become enlarged and appear uniformly electron-translu- cent (Simson e~ a]., 1974). This change in appearance is probably due to increased amounts of PRP and reduced amounts of other secretory proteins within the granules, but the distribution of secretory proteins in normal and