Structural and functional analysis of poly(ADP ribose) polymerase: an immunological study.
Poly(ADP ribose) polymerase (EC 2.4.2.30) was studied using monoclonal antibodies for three different epitopes on the enzyme. The epitopes were mapped in relation to the functional domains of the protein and the inhibitory properties of the antibodies. The intranuclear and interspecies immunoreactivity of the enzyme was also investigated. The epitope of antibody 2 was mapped to the 17 kDa fragment generated by chymotryptic digestion of the C-terminal 54 kDa NAD-binding domain. Antibody 9 binds to the N-terminal 29 kDa fragment of the DNA binding domain and inhibits the enzyme activity by 80%. This antibody was used to purify poly(ADP ribose) polymerase by immunoaffinity chromatography. The third antibody binds to a central 36 kDa fragment that possesses part of the DNA-binding domain and the automodification domain. This antibody increases the enzymatic activity by 30%. An analysis of the species cross-reactivity of the antibodies was carried out by immunoblot analysis of nuclear proteins. Antibody 10 binding was detected in rat FR3T3 cells, Chinese hamster ovary cells (CHO) and epidermoid carcinoma lung human cells (CALU-1). The other two antibodies are specific for the human and bovine enzymes. Western blot analysis showed the association of poly(ADP ribose) polymerase with residual nuclear material obtained after nuclease treatment and high-salt extraction. Immunofluorescence studies with the three different monoclonals demonstrated that accessibility of the epitopes varies in the nucleus.
Lamarre
D
D
Centre de recherche en cancérologie de l'Université Laval à l'Hôtel-Dieu de Québec, Canada.
Talbot
B
B
de Murcia
G
G
Laplante
C
C
Leduc
Y
Y
Mazen
A
A
Poirier
G G
GG
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D002417:Cattle; D002467:Cell Nucleus / Q000201:enzymology; D003429:Cross Reactions; D000939:Epitopes; D005455:Fluorescent Antibody Technique; D007163:Immunosorbent Techniques; D010446:Peptide Fragments / Q000276:immunology; D011065:Poly(ADP-ribose) Polymerases / Q000276:immunology*; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D011065:Poly(ADP-ribose) Polymerases