Inhibition of the catalytic properties of Staphylococcus aureus nuclease by monoclonal antibodies.
Monoclonal antibodies (Mab) specific for Staphylococcus aureus nuclease (nuclease) were examined for their capacity to inhibit the enzyme-mediated cleavage of DNA. Within a panel of 22 anti-nuclease Mab produced by hybridoma cell lines derived from SJL/J, A/J or BALB/c mice, only five were capable of modifying nuclease activity. Of the five, only one protected DNA from enzymatic degradation whereas the others reduced the rate of the enzymatic reaction. When mixed together, partially inactivating Mabs were frequently more efficient inhibitors than when used individually. It was shown by competitive binding assay that nuclease could be bound simultaneously to more than one Mab. Mixtures of five inactivating Mabs were able to completely block the nuclease activity. Although the actual mechanism for Mab nuclease inactivation is not known, the present data are consistent with simple steric hindrance for the formation of the DNA-nuclease complex by bulky Mab molecules bound to epitopes close to, but distinct from, nuclease catalytic sites. A mathematical model for Mab binding and inactivation of nuclease, taking into account multiple binding events for one or two Mabs interacting with nuclease, was used to derive affinities and maximum reductions of the enzymatic rate (details on the derivation of the equations and on the hypotheses of the model are given in an appendix). This analysis showed that the observed cooperative effects were dependent on the formation of multi-molecular complexes in which nuclease is bound simultaneously to two (or more) different Mabs. It also shows that the formation of cyclic complexes, if allowed, might result in very high apparent affinities. Since in screening of hybridoma fusions, the probability of finding such pairs of monoclonal antibodies would be low, this phenomenon may explain the fact that no Mab, or mixture of Mabs, matched the polyclonal antisera in capacity to block nuclease enzymatic activity.
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism; D000939:Epitopes; D008836:Micrococcal Nuclease / Q000009:adverse effects / Q000276:immunology* / Q000378:metabolism; D013211:Staphylococcus aureus / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D004247:DNA; D008836:Micrococcal Nuclease