Phosphorylation of the alpha-subunit of (Na+ + K+)-ATPase by carbachol in tissue slices and the role of phosphoproteins in stimulus-secretion coupling.
This study examined the changes in protein phosphorylation in response to cholinergic (muscarinic) stimulation of salivary secretion in the rat submandibular gland. Carbachol stimulation was associated with phosphorylation in a number of protein bands as detected by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and autoradiography. The molecular masses (Mr) of two proteins, in which the amount of phosphorylation more than doubled in response to carbachol, were 22,000 and 96,000. The Mr 96,000 protein precipitated at 120,000 X g while most of the Mr 22,000 protein remained in the supernatant at this speed. The effect of carbachol on the phosphorylation of the Mr 22,000 and 96,000 proteins was blocked by atropine, indicating that the cholinergic receptor involved is muscarinic. The time course of phosphorylation of the Mr 22,000 protein consisted of a rapid increase in phosphorylation within the first min of carbachol stimulation. This increased phosphorylation persisted for less than 1 min. The increased phosphorylation of the Mr 96,000 protein also occurred within the first min but it persisted for at least 10 min. However, removal of the muscarinic agonist, carbachol, resulted in the rapid dephosphorylation of this protein. When the plasma membranes were purified, the Mr 96,000 protein was phosphorylated by ATP in the presence of Na+ and Mg2+. It was dephosphorylated by K+. This proves that the Mr 96,000 dalton protein is the alpha-subunit of the (Na+ + K+)-ATPase.
D000818:Animals; D002217:Carbachol / Q000494:pharmacology*; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008297:Male; D010042:Ouabain / Q000494:pharmacology; D010101:Oxygen Consumption / Q000187:drug effects; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D011919:Rats, Inbred Strains; D012471:Salivary Proteins and Peptides / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013363:Submandibular Gland / Q000187:drug effects / Q000378:metabolism*
D046911:Macromolecular Substances; D010750:Phosphoproteins; D012471:Salivary Proteins and Peptides; D010042:Ouabain; D002217:Carbachol; D000254:Sodium-Potassium-Exchanging ATPase