A comprehsensive study will be undertaken for studying myofilament protein phosphorylation in smooth muscle to understand its role in regulation of contractility. Specifically: 1) Determination of the amino acid sequence around the phosphorylation sites of the 20,000- dalton myosin light chain (LC) isoforms in smooth muscles which are contracting and relaxing under various conditions to correlate chemical anatomy with physiological role; 2) Quantitation of phosphorylation of LC isoforms in smooth muscles with different shortening velocity and tension to delineate the parameters that relate phosphorylation of KC isoforms to muscle contraction; 3) We will test the hypothesis that the initial phase of smooth muscle contraction is regulated by LC phosphorylation through protein kinase C. The alternative hypothesis will also be tested that the sustained phase of smooth muscle contraction is regulated by the thin filament proteins with special emphasis on caldesmon, the calmodulin binding protein; 4) Protein phosphorylation will be compared between arteries and veins under various conditions as well as between uteri from pregnant and nonpregnant rats; 5) the significance of protein phosphorylation will be further tested in muscles: a) with reduced ATP content, b) treated with phorbol esters, c) treated with okadaic acid; 6) The distinct phosphorylation sites of KC will be characterized with specific phosphoprotein phophatases. These experiments require 32p-labeling of the muscles so that changes in the [32p]phosphate content of a protein, as the result of a physiological stimulus, may be followed at the level of microgram protein quantities. The correlation of biochemical data with the physiological data is based on freezing the muscles at various stages of the contraction-relaxation cycle. The radioactive phosphorylated proteins are isolated by two-dimensional gel electrophoresis, dissected from the gels, and their [32p]phosphate content is determined by counting. The isolated proteins will also be subjected to phosphoamino acid analysis and phosphopeptide mapping. The knowledge gained from basic study on normal blood vessels could be applied to the characterization of diseased conditions of blood vessels. Studies on phosphorylation in normal uterus may form the basis for understanding the disorders of uterus.
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