Purification and characterization of a 22‐kDa microsomal protein from rat parotid gland which is phosphorylated following stimulation by agonists involving cAMP as second messenger

Abstract

Stimulation of secretion in exocrine glands by agonists involving cAMP as second messenger leads to the phosphorylation of the ribosomal protein S6 ( It could be extracted from the endoplasmic reticulum membrane only with Triton X-100, SDS or concentrated formic or acetic acid. The purification of this protein involved extraction of the microsomes with Triton X-100, removal of the detergent by acetone precipitation, extraction of water-soluble proteins, lipids and lipoproteins, and preparative SDS polyacrylamide gel electrophoresis. The protein has a basic PI (> 8.7). For determination of the amino acid composition of protein 111 and for sequencing of its amino-terminal portion, the protein was electroeluted out off the gel, the detergent removed and the protein finally purified by reversed-phase HPLC. Protein 111 could be phosphorylated in vitro by the catalytic subunit of the CAMP-dependent protein kinase to a degree of approximately 0.14 mol phosphate/mol protein. The only phosphopeptide obtained after in vitro phosphorylation and subsequent tryptic or chymotryptic digestion was identical with the phosphopeptide obtained after stimulation of intact rat parotid gland lobules with isoproterenol. The sequence of this peptide was Lys-Leu-Ser(P)-Glu-AlaAsp-Asn-Arg. It was confirmed by an analysis of the synthetic peptide following in vitro phosphorylation with CAMP-dependent protein kinase. The first 41 N-terminal residues of protein I11 were sequenced. So far no sequence homology with other known peptides or proteins could be found.