Abstract
Previously, we have derived murine hybridomas producing monoclonal antibodies against DNA methyltransferase from human placenta (Kaul, S., Pfeifer, G. P., and Drahovsky, D. (1984) Eur. J. Cell Biol. 34, 330-335). One of these monoclonal antibodies, M2B10, which undergoes immune complex formation also with DNA methyltransferase from P815 mouse mastocytoma cells, was used for the immunoaffinity purification of mouse and human DNA methyltransferases. In sodium dodecyl sulfate-polyacrylamide gels and in immunoblotting studies, the immunoaffinity-purified mouse DNA methyltransferase revealed 5-6 polypeptides of molecular masses 150-190 kDa. The immunoaffinity-purified human placental DNA methyltransferase was characterized by a polypeptide of 158 kDa, presumably representing the native enzyme molecule and by polypeptides of 105-108 kDa and 50-68 kDa, probably generated by a limited proteolysis of the native enzyme molecule. The immunoaffinity-purified DNA methyltransferases preferred hemimethylated DNA substrates over unmethylated ones, and among all unmethylated substrates tested, poly[(dG-dC).(dG-dC)] had the highest methyl-accepting activity. DNA polymers of at least 90 base pairs in length were required for the binding reaction of the immunoaffinity-purified human DNA methyltransferase, and this initial binding was apparently independent of the nucleotide composition of the DNA polymer and of the presence of S-adenosyl-L-methionine.
Highlights
Producing monoclonal antibodies against DNA meth- 2.1.1.37)
Cells-Recently, we have described the construction of murine hybridomas producing monoclonal antibodies directed against DNA methyltransferase from humanplacenta (21)
When the immunoglobulins were added directly to theDNA methyltransferase incubation mixtures withoutremoving the immune complexes from the solution no inhibitory effect was observed
Summary
Cells-Recently, we have described the construction of murine hybridomas producing monoclonal antibodies directed against DNA methyltransferase from humanplacenta (21). The monoclonal anti-human DNA methyltransferase antibodies, M2B10, M8F73A,and M11F1, werefound to cross-react with DNA methyltransferase from mouse P815 mastocytoma cells and were selected for the furtherstudies Both enzymes were bound into immune complexes which can be removed from the solution by the aid of goat anti-mouse immunoglobulin. About 30-50% of the applied DNA methyltransferase activity (depending on the enzyme preparation) was always recovered in theflowthrough volume (fraction VA, Table I), even if reapplied several times onto unused columns of the same immobilized antibody This portion of DNA methyltransferase failed to undergo immune complex formation with monoclonal antibodies M2B10, M8F73A,and MllFl in theliquid phase. The DNA methyltransferase activity in the minor 0.5 M NaCl heparin-agarose peak failed to complex with monoclonal antibodies M2B10, M8F73A, and MllFl and was not subjected to further purification. Further purification of fraction VB (Table I) on single-stranded DNA-agarose did not result in elimination of TABLEI
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