Five major basic polypeptides with characteristics typical of proline-rich proteins, accumulated in parotid glands after long term isoprenaline treatment of Balb C mice. They were studied by two-dimensional gel electrophoresis and designated Bl", B2'", B2", B3" and B4" on the basis of PI-dependent mobility. They were not observed in the glands of normal mice and were precipitated when glands were homogenized in 10% trichloroacetic acid unlike the three isoprenaline-induced proline-rich proteins of murine parotid glands reported previously.Isoprenaline induced six proline-rich in vitro translation products which were absent normally. Four of these species had PI-dependent mobilities almost identical to BI", B2", B3" and B4", indicating not only precursor/ product relationships, but also that isoprenaline induced the accumulation of the proteins by regulating the mRNA.Identical salivary counterparts of the basic glandular proline-rich proteins were not detected whereas a series of smaller and more basic isoprenaline-induced polypeptides were observed in saliva (major speices BIS -B4'). The glandular proline-rich proteins were secreted from parotid tissue in vitro and the data indicate that prolinerich proteins are synthesised as precursors and processed into salivary form in the parotid glands after secretion. The relationships between the B-type in vitro translation products, parotid gland precursors and salivary proteins were also confirmed immunologically.Rodent parotid glands are a useful system for studying how P-adrenergic stimulation can effect a complex series of cellular changes requiring new gene expression. Repeated daily injections of rodents with the B-adrenergic agonist isoprenaline increase the size of the parotid glands [I, 21. This is due to enlargement of the secretory acinar cells but although DNA synthesis is also initiated [3, 41 the result is an increase in ploidy rather than new cell divisions [5, 61. Accompanying the morphological changes is an induction of members of the proline-rich protein family [7 -121 which are useful markers for 8-adrenergic action upon specific genes. Proline-rich proteins are classed into acidic and basic subgroups, are secreted into saliva [13] and in humans constitute 70% of total salivary protein [14].Proline-rich protein expression is different in rodents from that in humans. The proteins are present only in small amounts [9, 121 in normal rat parotid glands but are induced by isoprenaline to constitute 50% of total glandular protein [9, 101. In rats, one glycosylated acidic proline-rich protein [8] and six basic proline-rich proteins [9] have been identified and Correspondence to C. J. McDonald, Department of Biochemistry, The University of Sheffield, Western Bank, Sheffield S10 2TN, EnglandAbbreviations. NEPHGE, non-equilibrium pH gradient electrophoresis; pX", parotid gland polypeptide of molecular mass X kDa; pX, translation product of X kDa; pXs salivary polypeptide of X kDa; pX"*, initial in vitro secretion product of X kDa; PA' ", final in vitro secret...

622nd MEETING, LEICESTER 95 I As isoprenaline is known to modulate the expression of mRNAs for members of the 'proline-rich' protein (PRP) family in rats (Meunzer et ul.. 1979) and induce at least three PRPs in mice (Mehansho et al., 1985), a rabbit anti-PRP IgG preparation was used to identify PRP in vitro products. All six 'early' in vitro products were immunoprecipitated while the 'late' species was not. Fig. 1 illustrates the translational activity of the isoprenaline regulated mRNA for the PRP of M , 41 000 during the course of isoprenaline treatment.To analyse the effect of isoprenaline upon parotid gland gene expression further, a cDNA library in pBR322 was made from isoprenaline 'stimulated' parotid poly(A+ )RNA. Sixty cDNA clones derived from abundant isoprenaline regulated mRNAs were identified by differential colony hybridization using [32P]cDNA probes made from 'stimulated' and 'non-stimulated' parotid poly(A+ )RNA. The cDNAs were then analysed with restriction endonucleases and grouped into families of homologous sequences by colony cross-hybridization using selected cloned cDNAs as probes after labelling with [32P]dCTP by nick translation.The cDNAs for these isoprenaline regulated sequences represent useful tools for studying P-adrenergic regulation of mRNA abundance.