Ca2+ is known to undergo transient alterations of intracellular concentration in nerve and other cells in response to diverse stimuli and to exert many of its actions by binding to a protein termed calmodulin (CaM). Calmodulin alters the activity of many enzymes including several CaM-dependent protein kinases and a CaM-dependent protein phosphatase termed calcineurin (CN). Our laboratory has recently prepared in rabbits polyclonal antibodies to native bovine brain CN and its constituent subunits A and B. Antibodies to bovine brain CN B have been shown to cross-react with a 16,000 Mr protomer in extracts of PC-12 rat pheochromocytoma cells. Further, we have purified an apparently unique, narrow substrate specificity CaM-dependent myosin light chain kinase (MLCK) from bovine brain. We propose to characterize our antibody population in detail and to use these antibodies to effect a rapid purification of calcineurin by immune affinity chromatography and to compare the kinetic, physical and regulatory properties of this phosphatase with that generated by chromatography on immobilized CaM. The antibodies will be used to effect a selective removal of CN from brain and PC-12 cell extracts and to identify endogenous substrates by altered Ca2+-dependent dephosphorylation. Endogenous substrates of calcineurin will also be sought by identifying thiophosphorylated proteins from brain extract which bind to immobilized native calcineurin. The narrow substrate specificity CaM-dependent MLCK from bovine brain will be purified to homogeneity and characterized. The MLCK will be compared to that isolated from highly purified bovine cerebral cortex microvessels. The bovine brain MLCK will be tested for ability to phosphorylate the light chains of native bovine brain myosin and for the consequences of their phosphorylation with respect to actin-stimulated myosin ATPase activity. The calcineurin analog from PC-12 cells and a second CaM-dependent phosphatase form observed by our laboratory in this cell line will be characterized physically, chemically, and immunologically. The effect of Nerve Growth Factor on the immunocytochemical localization of PC-12 calcineurin will be determined. Finally, the effect of fusion of liposome-encapsulated inhibitory anti-CN antibodies with PC-12 cells on protein phosphorylation and expression of its differentiated functions will be sought.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS011252-14
Application #
3394437
Study Section
Neurology B Subcommittee 1 (NEUB)
Project Start
1977-03-01
Project End
1989-02-28
Budget Start
1987-03-01
Budget End
1988-02-29
Support Year
14
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Medicine & Dentistry of NJ
Department
Type
Schools of Medicine
DUNS #
622146454
City
Piscataway
State
NJ
Country
United States
Zip Code
08854
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Wolff, D J; Gribin, B J (1994) Interferon-gamma-inducible murine macrophage nitric oxide synthase: studies on the mechanism of inhibition by imidazole agents. Arch Biochem Biophys 311:293-9
Wolff, D J; Datto, G A; Samatovicz, R A (1993) The dual mode of inhibition of calmodulin-dependent nitric-oxide synthase by antifungal imidazole agents. J Biol Chem 268:9430-6
Wolff, D J; Datto, G A; Samatovicz, R A et al. (1993) Calmodulin-dependent nitric-oxide synthase. Mechanism of inhibition by imidazole and phenylimidazoles. J Biol Chem 268:9425-9
Wolff, D J; Datto, G A (1992) Identification and characterization of a calmodulin-dependent nitric oxide synthase from GH3 pituitary cells. Biochem J 285 ( Pt 1):201-6
Bartelt, D C; Fidel, S; Farber, L H et al. (1988) Calmodulin-dependent multifunctional protein kinase in Aspergillus nidulans. Proc Natl Acad Sci U S A 85:3279-83
Bartelt, D C; Moroney, S; Wolff, D J (1987) Purification, characterization and substrate specificity of calmodulin-dependent myosin light-chain kinase from bovine brain. Biochem J 247:747-56
Farber, L H; Wilson, F J; Wolff, D J (1987) Calmodulin-dependent phosphatases of PC12, GH3, and C6 cells: physical, kinetic, and immunochemical properties. J Neurochem 49:404-14
Bartelt, D C; Wolff, D J; Scheele, G A (1986) Calmodulin-binding proteins and calmodulin-regulated enzymes in dog pancreas. Biochem J 240:753-63
Wolff, D J; Sved, D W (1985) The divalent cation dependence of bovine brain calmodulin-dependent phosphatase. J Biol Chem 260:4195-202