title: Macromolecular association of ADP-ribosyltransferase and its correlation with enzymic activity.
abstract: The macromolecular self-association of ADP-ribosyltransferase protein in solution was studied by several experimental techniques: quantitative gel filtration, electrophoretic analyses in non-denaturing gels, and cross-linking the enzyme protein with glutaraldehyde, dimethyl pimelimidate, dimethyl suberimidate, dimethyl 3,3'-dithiobisproprionimidate and tetranitromethane. The self-association of the polypeptide components obtained by plasmin digestion was also determined by using the above cross-linking agents. Monomers and cross-linked dimers of the enzyme protein, possessing enzymic activity, were separated in non-denaturing gels by electrophoresis. The basic polypeptide fragments, exhibiting molecular masses of 29 kDa and 36 kDa, self-associated, whereas the polypeptides with molecular masses of 56 kDa and 42 kDa associated only to a negligible extent, indicating that the peptide regions that also bind DNA and histones are probable sites of self-association in the intact enzyme molecule. Macromolecular association of the enzyme was indicated by a protein-concentration-dependent red-shift in protein fluorescence. The specific enzymic activity of the isolated ADP-ribosyltransferase depended on the concentration of the enzyme protein, and at 2.00 microM concentration the enzyme was self-inhibitory. Dilution of the enzyme protein to 30-40 nM resulted in a large increase in its specific activity. Further dilution to 1-3 nM coincided with a marked decrease of specific activity. Direct enzymic assays of electrophoretically separated monomers and cross-linked dimers demonstrated that the dimer appears to be the active molecular species that catalyses poly(ADP-ribose) synthesis. The NAD+ glycohydrolase activity of the enzyme was also dependent on protein concentration and was highest at 1-3 nM enzyme concentration, when polymerase activity was minimal, indicating that the monomeric enzyme behaved as a glycohydrolase, whereas poly(ADP-ribosyl)ation of enzyme molecules was maximal when the enzyme tends to be self-associated to the dimeric form.
authors: Department of Pharmacology, University of California, San Francisco 94143-0130.
authors: P I
authors: PI
authors: Bauer
authors: K G
authors: KG
authors: Buki
authors: A
authors: A
authors: Hakam
authors: E
authors: E
authors: Kun
mesh_terms: D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005341:Fibrinolysin / Q000494:pharmacology; D005976:Glutaral; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011489:Protein Denaturation; D013329:Structure-Activity Relationship
chemical_list: D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011065:Poly(ADP-ribose) Polymerases; D005341:Fibrinolysin; D005976:Glutaral