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.
Stimulation of secretion in exocrine glands by agonists involving cAMP as second messenger leads to the phosphorylation of the ribosomal protein S6 (protein I) and two other particulate proteins with apparent molecular masses of 24 kDa (protein II) and 22 kDa (protein III) [Jahn, R., Unger, C. & Söling, H. D. (1980) Eur. J. Biochem. 112, 345-352]. This report describes the purification and characterization of protein III. Solubilization studies indicate that protein III is an intrinsic membrane protein. 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 (greater than 8.7). For determination of the amino acid composition of protein III 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 III 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-Ala-Asp-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 III were sequenced. So far no sequence homology with other known peptides or proteins could be found.
Thiel
G
G
Abteilung Klinische Biochemie, Zentrum Innere Medizin, Universität Göttingen, Federal Republic of Germany.
Schmidt
W E
WE
Meyer
H E
HE
Söling
H D
HD
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000242:Cyclic AMP / Q000502:physiology*; D007545:Isoproterenol / Q000494:pharmacology; D046911:Macromolecular Substances; D008297:Male; D008861:Microsomes / Q000378:metabolism*; D008970:Molecular Weight; D010306:Parotid Gland / Q000187:drug effects / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D051381:Rats; D011919:Rats, Inbred Strains; D012471:Salivary Proteins and Peptides / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; C511598:Bpifa2 protein, rat; D046911:Macromolecular Substances; D012471:Salivary Proteins and Peptides; D000242:Cyclic AMP; D007545:Isoproterenol