Purification and identification of a 28-kDa calcium-regulated heat-stable protein. A novel secretagogue-regulated phosphoprotein in exocrine pancreas.
This study reports the purification and identification of a novel 28 kDa phosphoprotein from rat pancreatic acini, previously described as being highly regulated by calcium mobilizing secretagogues, which we have designated calcium-regulated heat-stable protein 28 (CRHSP-28). Internal amino acid sequences of purified CRHSP-28 were obtained following trypsin digestion and found to match with >95% identity the predicted amino acid sequence of a novel cDNA recently identified as being highly expressed in human breast carcinomas. Verification that this cDNA codes for human CRHSP-28 was demonstrated by the ability of antiserum raised against purified rat CRHSP-28 to recognize the recombinant human protein when expressed in bacteria. Furthermore, this antibody was found to specifically react with CRHSP-28 in rat acini following one- and two-dimensional electrophoresis and underwent a marked acidic shift in mobility after cholecystokinin stimulation, a phenomenon indicative of an increase in its phosphorylation. CRHSP-28 is predicted to be extremely hydrophilic, is phosphorylated entirely on serine residues, and bears little homology to any known proteins. Finally, the distribution of the CRHSP-28 protein in various rat tissues revealed that although it was present at low levels in almost all tissues, it was most highly expressed in pancreas, followed by the gastric, intestinal, and colonic mucosa. In view of its relative abundance throughout the digestive system and its apparent regulation by calcium-mobilizing agents, this protein may provide valuable insight into the mechanism(s) of calcium signaling in these tissues.
Groblewski
G E
GE
Department of Physiology, University of Michigan, School of Medicine, Ann Arbor, Michigan 48109, USA.
Wishart
M J
MJ
Yoshida
M
M
Williams
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010179:Pancreas / Q000737:chemistry*; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification*; D010766:Phosphorylation; D051381:Rats; D014018:Tissue Distribution
D010750:Phosphoproteins; C102584:calcium-regulated heat-stable protein 28; D002118:Calcium