Characterization of an 80-kDa phosphoprotein involved in parietal cell stimulation.
When isolated rabbit gastric glands were stimulated with histamine plus isobutylmethylxanthine, a redistribution of H+-K+-ATPase, from microsomes to a low-speed pellet, occurred in association with the phosphorylation of an 80-kDa protein (80K) in the apical membrane-rich fraction purified from the low-speed pellet. Histamine alone or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), but not carbachol, also stimulated both the redistribution of H+-K+-ATPase and phosphorylation of 80K. Under stimulated conditions, 80K copurified in the apical membrane fraction along with H+-K+-ATPase and actin; whereas purified microsomes from resting stomach were highly enriched in H+-K+-ATPase but contained neither 80K nor actin. Treatment of the apical membranes with detergents, salts, sonication, and so on, led us to conclude that 80K is a membrane protein, unlike actin; however, the mode of association of 80K with membrane differed from H+-K+-ATPase, an integral membrane protein. Isoelectric focusing and peptide mapping revealed that 80K consists of six isomers of slightly differing pI, with 32P occurring only in the three most acidic isomers and exclusively on serine residues. Moreover, stimulation elicited a shift in the amount of 80K isomers, from basic to acidic, as well as phosphorylation. We conclude that 80K is an apical membrane protein in the parietal cell and an important substrate for cAMP-dependent, but not calcium-dependent, pathway of acid secretion.
Urushidani
T
T
Department of Physiology-Anatomy, University of California, Berkeley 94720.
Hanzel
D K
DK
Forte
J G
JG
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D017506:H(+)-K(+)-Exchanging ATPase; D006632:Histamine / Q000494:pharmacology; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000302:isolation & purification / Q000502:physiology*; D008970:Molecular Weight; D010295:Parietal Cells, Gastric / Q000187:drug effects / Q000502:physiology*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000502:physiology*; D010766:Phosphorylation; D011817:Rabbits
D008565:Membrane Proteins; D010750:Phosphoproteins; D003994:Bucladesine; D006632:Histamine; D002217:Carbachol; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase; D015056:1-Methyl-3-isobutylxanthine