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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR034602-19
Application #
3156878
Study Section
Physiology Study Section (PHY)
Project Start
1984-09-01
Project End
1994-08-31
Budget Start
1993-09-01
Budget End
1994-08-31
Support Year
19
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Illinois at Chicago
Department
Type
Schools of Medicine
DUNS #
121911077
City
Chicago
State
IL
Country
United States
Zip Code
60612
Barany, K; Barany, M; Giometti, C S (1995) Polyacrylamide gel electrophoretic methods in the separation of structural muscle proteins. J Chromatogr A 698:301-32
Barany, M; Barany, K (1993) Dissociation of relaxation and myosin light chain dephosphorylation in porcine uterine muscle. Arch Biochem Biophys 305:202-4
Barany, M; Barany, K (1993) Calponin phosphorylation does not accompany contraction of various smooth muscles. Biochim Biophys Acta 1179:229-33
Barany, M; Polyak, E; Barany, K (1992) Protein phosphorylation during the contraction-relaxation-contraction cycle of arterial smooth muscle. Arch Biochem Biophys 294:571-8
Barany, K; Polyak, E; Barany, M (1992) Protein phosphorylation in arterial muscle contracted by high concentration of phorbol dibutyrate in the presence and absence of Ca2+. Biochim Biophys Acta 1134:233-41
Barany, M; Rokolya, A; Barany, K (1991) Absence of calponin phosphorylation in contracting or resting arterial smooth muscle. FEBS Lett 279:65-8
Rokolya, A; Barany, M; Barany, K (1991) Modification of myosin light chain phosphorylation in sustained arterial muscle contraction by phorbol dibutyrate. Biochim Biophys Acta 1057:276-80
Barany, M; Rokolya, A; Barany, K (1991) Exchange of 20-kDa myosin light chain-bound phosphate during sustained contraction of arterial smooth muscle. Arch Biochem Biophys 287:199-203
Barany, K; Rokolya, A; Barany, M (1990) Stretch activates myosin light chain kinase in arterial smooth muscle. Biochem Biophys Res Commun 173:164-71
Barany, K; Rokolya, A; Barany, M (1990) Analysis of myosin light chain phosphopeptides in phorbol dibutyrate-contracted artery. Biochim Biophys Acta 1035:105-8

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