A highly effective method consisting of two affinity chromatography steps and ion-exchange and gelfiltration chromatography steps was developed for purification of autoantibodies from human sera with DNA-hydrolyzing activity. Antibody Fab fragment, which had been purified 130-fold, was shown to catalyze plasmid DNA cleavage. The flow linear dichroism technique was used for quantitative and qualitative studying of supercoiled plasmid DNA cleavage by these autoantibodies in comparison with DNase I and EcoRI restriction endonuclease. The DNA autoantibody Fab fragment was shown to hydrolyze plasmid DNA by Mg2+-dependent single-strand multiple nicking of the sub-strate. Kinetic properties of the DNA autoantibody Fab fragment were evaluated from the flow linear dichroism and agarose gel electrophoresis data and revealed a high affinity (KmS = 43 nM) and considerable catalytic efficiency (k8caPp/KbS = 0.32 min-l'nM'1) of the reaction. Antibodies able to catalyze a variety of chemical transformations were developed in the last decade, using the strategy of raising antibodies to haptens that resemble the transition state of reactions, as was suggested by Jencks (1) (for review see refs. 2 and 3). An increasing number of autoantibodies with catalytic activity toward natural substrates (4-6), as well as antiidiotypic antibodies exhibiting a catalytic function (7), have also been described recently. To date, however, the antibodymediated catalysis is usually characterized by relatively low rate enhancements, indicating the existence of at least two very important problems. The first one, especially important in the case of naturally occurring antibodies, is the purity of the antibody preparation used for the assay, since even a trace enzyme contaminant may mimic a high antibody turnover number (8). The second problem is the development of sensitive, continuous, and accurate methods for detection and monitoring of such low activity (9, 10). Here we describe a reproducible method of purification of DNA-hydrolyzing autoantibodies from human sera. We also demonstrate the advantages of the flow linear dichroism (FLD) technique for quantitative and qualitative characterization of the interaction of these catalytic antibodies with supercoiled (sc) plasmid DNA. EXPERIMENTAL PROCEDURESChemicals. All chemicals were from Sigma and Merck. Enzymes were obtained from Boehringer Mannheim.Plasmid DNA. Plasmid pUC19 was isolated as described elsewhere (11). More than 95% of the isolated plasmid DNA was in the sc form, judging by 1% agarose gel electrophoresis (AGE). Antibody Isolation. Antibodies in 5 ml of serum were precipitated twice with 50% saturated ammonium sulfate; this was followed by chromatography on a Pharmacia HR 5/5 staphylococcal protein A fast-performance liquid chromatography (FPLC) column, as described in ref. 13 (p. 310).Antibody Purification. Samples isolated with protein A were dialyzed twice for 4 hr against 500 vol of buffer A (20 mM Tris HCl, pH 9.0) at 4°C and applied to a Pharmacia HR 5/5 Mono Q FPLC column...

The catalytic (C) subunit of protein kinase A (PKA) is regarded as a framework for the protein kinase family. Its sequence is composed of a conserved core (residues 40 300) between two segments at the amino and carboxyl termini of the protein. Since the various protein kinases differ in their specificity, it seems reasonable to assume that these nonhomologous segments may be involved in endowing each kinase with its individual specificity. Here we present data to show that the cluster of acidic amino acids (328DDYEEEE334) at the carboxyl-terminal "tail" of the C subunit, specifically Tyr330, contributes to its substrate recognition. This is based on three complementary lines of evidence: (i) on a conformation-sensitive cleavage of the C subunit by a kinase-splitting membranal proteinase that specifically recognizes this cluster, to demonstrate the occurrence in solution of "open" (cleavable) and "closed" (noncleavable) conformations of the C subunit; (ii) on analysis of the three-dimensional structures of the open and closed conformations of the C subunit, showing an approximately 7-A movement of the phenolic hydroxyl of Tyr330 to reach (in the closed conformation) an approximately 3-A distance from the nitrogen atoms of the Arg residue at position p-3 of the PKA consensus sequence; and (iii) on single-site mutations of the C subunit (e.g. Y330A) that show a significant contribution of Tyr330 to the Km of PKA for its substrates/inhibitors and to its catalytic efficacy (Vmax/Km).

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Monoclonal anti-DNA autoantibody BV 04-01 catalyzed hydrolysis of DNA in the presence of Mg2+. Catalysis was associated with BV 04-01 IgG, Fab, and single-chain-antibody (SCA) proteins. Cleavage of both ss and dsDNA was observed with efficient hydrolysis of the C-rich region of A7C7ATATAGCGCGT2, as well as a preference for cleaving within CG-rich regions of dsDNA. Data on specificity of ssDNA hydrolysis and kinetic data obtained from wild-type SCA, and two SCA mutants were used to model the catalytically active antibody site using the previously resolved X-ray structure of BV 04-01. The resulting model suggested that the target phosphodiester bond is activated by induction of conformational strain. In addition, the antibody-DNA complex contained a Mg2+ coordination site composed of the L32Tyr and L27dHis side chains and a DNA 3'-phosphodiester group. Induction of strain along with the metal coordination could be part of the mechanism by which this antibody catalyzes DNA hydrolysis. Sequence data for BV 04-01 V(H) and V(L) genes suggested that the proposed catalytic-antibody active site was germline-encoded. This observation suggests that catalytic activity might represent an important-rarely examined-function for some antibody molecules.

Catalysis by antibodies could be a frequent phenomenon if the immune system generates a sufficiently diverse number of antibody-active sites, some of which may possess catalytic activity. A catalytic antibody can be expected to do more damage than one that simply binds antigen. The best biochemical marker of systemic lupus erythematosus (SLE) is presence of autoantibodies to DNA. In the present article, we describe the DNA-hydrolyzing activity of DNA-binding autoantibodies purified from SLE patients. The substrates employed were supercoiled plasmid, radiolabeled plasmid fragments, and oligonucleotides. Hydrolysis of DNA by the antibodies was indicated by the appearance of fragments visualized by ethidium bromide staining of agarose gels or autoradiography of polyacrylamide gels. Changes in linear dichroism values were also indicative of DNA hydrolysis. The antibody activity was purified by protein A-sepharose chromatography, high-performance liquid chromatography gel filtration, and DNA-affinity chromatography. Scrupulous control studies were done to demonstrate that DNA-hydrolyzing activity really belongs to the antibodies. Purified Fab fragments showed hydrolyzing activity, whereas the Fc fragment was inactive. The specificity of DNA cleavage was investigated, and the rate parameters of hydrolysis by antibodies and conventional nucleases were compared.