Macromolecular association of ADP-ribosyltransferase and its correlation with enzymic activity.

摘要: The macromolecular self-association of ADP-ribosyltransferase protein in solution was studied by several experimental techniques: quantitative gel filtration, electrophoretic analyses non-denaturing gels, and cross-linking the enzyme with glutaraldehyde, dimethyl pimelimidate, suberimidate, 3,3′-dithiobisproprionimidate tetranitromethane. polypeptide components obtained plasmin digestion also determined using above agents. Monomers cross-linked dimers protein, possessing enzymic activity, were separated gels electrophoresis. basic fragments, exhibiting molecular masses 29 kDa 36 kDa, self-associated, whereas polypeptides 56 42 associated only to a negligible extent, indicating that peptide regions bind DNA histones are probable sites intact molecule. Macromolecular association indicated protein-concentration-dependent red-shift fluorescence. specific activity isolated depended on concentration at 2.00 microM self-inhibitory. Dilution 30-40 nM resulted large increase its activity. Further dilution 1-3 coincided marked decrease Direct assays electrophoretically monomers demonstrated dimer appears be active species catalyses poly(ADP-ribose) synthesis. NAD+ glycohydrolase dependent highest concentration, when polymerase minimal, monomeric behaved as glycohydrolase, poly(ADP-ribosyl)ation molecules maximal tends self-associated dimeric form.