Subcellular location of stimulus-affected endogenous phosphoproteins in the rat parotid gland.
Rat parotid minces were labeled with [32P]Pi, stimulated with isoproterenol, homogenized in sucrose, and fractionated on continuous sucrose density gradients. We analyzed the resulting fractions by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiograms were made from the gels. Comparison of fractions from control and isoproterenol-stimulated minces revealed seven phosphoproteins that were affected by isoproterenol. The subcellular location of these proteins was determined by comparing their distribution in the sucrose gradients with that of a number of enzymes that are characteristic of specific organelles. Isoproterenol decreased the phosphorylation of two cytoplasmic proteins (Mr 16,000 and 18,000) and increased the phosphorylation of a third (Mr 14,000). The phosphorylation of two endoplasmic reticulum proteins was increased by isoproterenol (Mr 20,500 and 22,500), as was an Mr 31,000 protein which was probably the S6 ribosomal protein. The phosphorylation of a secretory granule protein (Mr 24,000) was decreased by isoproterenol. We then developed a purification scheme for parotid secretory granules. By using this method, we demonstrated that the phosphorylation of the Mr 24,000 was also decreased by carbamylcholine. Granules purified by this method also contained a small number of other phosphoproteins whose phosphorylation was increased only by isoproterenol. Secretory granule-associated stimulus-affected phosphoproteins were found in the particulate fraction when the granules were hypotonically lysed, and were not extracted from the particulate fraction by washing with 0.6 M KCl.
D015720:5'-Nucleotidase; D000818:Animals; D002499:Centrifugation, Density Gradient; D003594:Cytoplasmic Granules / Q000648:ultrastructure*; D005952:Glucose-6-Phosphatase / Q000032:analysis; D008854:Microscopy, Electron; D008970:Molecular Weight; D008995:Monoamine Oxidase / Q000032:analysis; D009245:NADH Dehydrogenase / Q000032:analysis; D009708:Nucleotidases / Q000032:analysis; D010306:Parotid Gland / Q000201:enzymology / Q000648:ultrastructure*; D010750:Phosphoproteins / Q000032:analysis* / Q000096:biosynthesis; D010761:Phosphorus Radioisotopes; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis; D013385:Succinate Dehydrogenase / Q000032:analysis; D013834:Thiamine Pyrophosphatase / Q000032:analysis
D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D013385:Succinate Dehydrogenase; D008995:Monoamine Oxidase; D009245:NADH Dehydrogenase; D009708:Nucleotidases; D015720:5'-Nucleotidase; D005952:Glucose-6-Phosphatase; D013834:Thiamine Pyrophosphatase; D000254:Sodium-Potassium-Exchanging ATPase