Abstract
Myosin purified from rabbit alveolar macrophages has been shown previously to be phosphorylated on the rod portion of the heavy chain and on the 20-kDa light chains (Trotter, J.A. (1982) Biochem Biophys. Res. Commun. 106, 1071-1077). Phosphorylation of the 20-kDa light chains by endogenous kinase activity is associated with a significant enhancement of the actin-activated MgATPase activity (Trotter, J.A., and Adelstein, R.S. (1979) J. Biol. Chem. 254, 8781-8785), whereas the function of heavy-chain phosphorylation is unknown. The isolated heavy chains of myosin purified from freshly harvested cells contain between 0.4 and 1.5 mol of PO4/mol of heavy chain, all esterified to serine residues. Using myosin phosphorylated by incubating living unstimulated macrophages in the presence of 32Pi, two-dimensional thin-layer mapping of tryptic peptides derived from heavy chains yields four phosphopeptides, which are phosphorylated to different extents. Limited trypsin digestion of similar radioactive myosin removes all radioactivity from the heavy chain while reducing its apparent molecular mass by less than 10 kDa. It is concluded that the heavy chain of macrophage myosin is phosphorylated on as many as four serines within 10 kDa of the tip of the tail.
Highlights
In order to probe the possibility that macrophage myosin has been shown previoustloybe phosphorylated on theis regulated by reversible phosphorylation of the heavy chain, rod portion of the heavy chain and o2n0-tkhDe a light in addition to its regulation by light chain phosphorylation, chains
Macrophage myosin is phosphorylated on the heavy chain, both agents used in polyacrylamide gel electrophoresis were from Bio-Rad
Quantitation of Heavy-chain Phosphate-As previously noted, myosin purified from resting rabbit alveolar macrophages has phosphate covalently bound both to the heavy chain and to the 20-kDa light chains [7]
Summary
In order to probe the possibility that macrophage myosin has been shown previoustloybe phosphorylated on theis regulated by reversible phosphorylation of the heavy chain, rod portion of the heavy chain and o2n0-tkhDe a light in addition to its regulation by light chain phosphorylation, chains Thereis evidence purified from rabbit alveolar macrophages by a modification of a previously described method [4].The extraction solution contained benzoyl-L-arginine methyl ester (0.1 mg/ml), soybean trypsin inhibitor (0.1 mg/ml), leupeptin (2 pg/ml), phenylmethylsulfonyl fluoride (0.18 mg/ml), pepstatin (10pg/ml), and TPCK (0.1 mg/ml) The latter threeprotease inhibitors were dissolvedin avolume of dimethyl that myosin from myeloid leukemic cells may be regulated by sulfoxide equal to 0.5% of the final solution volume and were added independent phosphorylation of both heavy chains and light to the rapidly stirring extraction solution at room temperature. In preparation for polyacrylamide gel electrophoresis, the digestion was stopped by transferring an aliquot of the incubation mixture to a tube containing ice-cold 10% trichloroacetic acid, whichwas subsequently processed as described below. The F-actinconcentration was determined spectrophotometrically, using 1.15 cm" as the extinction coefficient for a 1 mg/ml solution a t 280nm [29] and correcting for light scattering a t 340 nm
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
