Cyclic AMP-stimulated phosphorylation of bovine tracheal smooth muscle contractile and non-contractile proteins

Abstract

1. 1.|Various proteins isolated from bovine tracheal smooth muscle were examined as phosphate acceptor substrates for a cyclic AMP-dependent protein kinase isolated from the same tissue. A fraction prepared in a manner similar to that of skeletal muscle troponin was the best substrate of the presumptive contractile proteins isolate. Actomyosin and tropomyosin were relatively poor substrates. 2. 2.|An assay was developed for the rapid detection in a large number of samples of the muscle specific substrate for the protein kinase on which we reported previously. 3. 3.|Using this assay, the muscle specific substrate found in bovine tracheal smooth muscle was partially purified resulting in a preparation which when resolved by polyacrylamide gel electrophoresis showed a single peak of 32P incorporated, and which could be further characterized. 4. 4.|Our findings suggest that the substrate contains a protein subunit of molecular weight 19 000, which can be phosphorylated at serine and threonine residues, in the presence of cyclic AMP and protein kinase. The phosphate is in a covalent ester linkage with these residues. 5. 5.|A phosphoprotein phosphatase was isolated from the bovine tracheal smooth muscle. 6. 6.|Bovine tracheal smooth muscle contains cyclic AMP dependent protein kinase and phosphoprotein phospahatase activity as well as the muscle specific substrate, suggesting that these elements may be part of a mechanism which regulates smooth muscle tone.