The long-term objective of this research is to understand the role of smooth muscle tropomyosin in the regulation of contraction. Although this role is presently unclear, it is thought that tropomyosin's end-to-end interaction plays a key part in the cooperative nature of regulation. In order to better understand the molecular basis of this end-to-end interaction, a specific goal of this proposal is to investigate the source of the large heterogeneity in gizzard tropomyosin's end-to-end interaction observed by hydroxylapatite chromatography. This question will be addressed by using several techniques including viscosity, chromatography, electrophoresis, amino acid analysis, and sedimentation. A second specific aim is to test the hypothesis that tropomyosin's end-to-end interaction is responsible for its cooperative behavior. This will be accomplished by determining the effect of gizzard tropomyosin with different end-to-end interactions on its cooperative binding to actin and its cooperative regulation of actomyosin ATPase activity. Caldesmon, a new smooth muscle protein, imparts a thin filament Ca2+- sensitivity to regulation which requires tropomyosin. In order to understand the mechanism thereby these two proteins work in concert, another goal of this project is to determine the effect of tropomyosin, with different end-to-end interactions, on the binding of caldesmon to the thin filament and correlate this with caldesmon's regulation of actomyosin ATPase activity. Finally, since the direct interaction between tropomyosin and caldesmon may play a key role in regulation, this grant proposes to investigate the direct interaction between tropomyosin, with different end-to- end interactions, and caldesmon with viscosity, spin labels, fluorescence labels, sedimentation, and chemical cross-linking.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
2R01AR030917-04A3
Application #
3155922
Study Section
Biophysics and Biophysical Chemistry B Study Section (BBCB)
Project Start
1982-04-01
Project End
1992-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
4
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Boston Biomedical Research Institute
Department
Type
DUNS #
058893371
City
Watertown
State
MA
Country
United States
Zip Code
02472
Graceffa, Philip; Lee, Eunhee; Stafford, Walter F (2013) Disulfide cross-linked antiparallel actin dimer. Biochemistry 52:1082-8
Mabuchi, K; Li, Y; Carlos, A et al. (2001) Caldesmon exhibits a clustered distribution along individual chicken gizzard native thin filaments. J Muscle Res Cell Motil 22:77-90
Graceffa, P (2000) Phosphorylation of smooth muscle myosin heads regulates the head-induced movement of tropomyosin. J Biol Chem 275:17143-8
Graceffa, P (1999) Movement of smooth muscle tropomyosin by myosin heads. Biochemistry 38:11984-92
D'Angelo, G; Graceffa, P; Wang, C A et al. (1999) Mammal-specific, ERK-dependent, caldesmon phosphorylation in smooth muscle. Quantitation using novel anti-phosphopeptide antibodies. J Biol Chem 274:30115-21
Graceffa, P (1997) Arrangement of the COOH-terminal and NH2-terminal domains of caldesmon bound to actin. Biochemistry 36:3792-801
Graceffa, P; Adam, L P; Morgan, K G (1996) Strong interaction between caldesmon and calponin. J Biol Chem 271:30336-9
Graceffa, P (1995) Cross-linking and fluorescence study of the COOH- and NH2-terminal domains of intact caldesmon bound to actin. J Biol Chem 270:30187-93
Szczesna, D; Graceffa, P; Wang, C L et al. (1994) Myosin S1 changes the orientation of caldesmon on actin. Biochemistry 33:6716-20
Stafford, W F; Chalovich, J M; Graceffa, P (1994) Turkey gizzard caldesmon molecular weight and shape. Arch Biochem Biophys 313:47-9

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