Abstract

Background:In humans, pregnancy and lactation are associated with the production of catalytically active antibodies (abzymes) in serum and breast milk. However, the substrate specificities of the abzymes in these biological fluids, particularly breast milk, have not been studied extensively.Material/Methods: IgG fractions were isolated from human milk by subsequent steps of chromatographic purification on Protein-A Sepharose, DEAE-cellulose, and anti-IgG Sepharose. The nucleotide-hydrolyzing activity of electrophoretically homogeneous IgG antibodies was measured using 32P-labeled nucleotides and TLC.Results: We demonstrated by different methods that IgG antibodies from the serum and milk of clinically healthy human mothers are able to hydrolyze ribo- and deoxyribonucleoside-5’-mono, di- and triphosphates; this nucleotide-hydrolyzing activity was also present in Fab fragments of the IgG molecule. Affinity modification of the milk IgG oligomeric form by chemically reactive derivatives of ATP led to preferential modification of the L-chain. However, after separation of the subunits by SDS electrophoresis, an in-gel assay showed ATP-hydrolyzing activity in various oligomeric forms of IgG subunits (H2L2, H2L and HL), while the separated heavy (H) and light (L) chains were not catalytically active. The Km and Vmax values characterizing the interaction of IgG with nucleotides were estimated.Conclusions: Our findings speak in favor of the generation of a variety of polyclonal nucleotide-hydrolyzing antibodies by the immune system of clinically healthy mothers.

Keywords: Adenosine Monophosphate - analogs & derivatives, Adenosine Monophosphate - metabolism, Adenosine Triphosphatases - metabolism, Adenosine Triphosphate - analogs & derivatives, Adenosine Triphosphate - metabolism, Antibodies, Catalytic - blood, Antibodies, Catalytic - isolation & purification, Antibodies, Catalytic - metabolism, Hydrolysis, Immunoglobulin Fab Fragments - chemistry, Immunoglobulin Fab Fragments - isolation & purification, Immunoglobulin Fab Fragments - metabolism, Immunoglobulin G - isolation & purification, Immunoglobulin G - metabolism, Immunoglobulin Heavy Chains - metabolism, Immunoglobulin Light Chains - metabolism, Kinetics, Lactation, Milk, Human - chemistry, Milk, Human - immunology, Nucleotides - chemistry, Nucleotides - metabolism, Phosphorus Radioisotopes, Pregnancy, Serum - chemistry, Adenosine Monophosphate - metabolism, Adenosine Triphosphatases - metabolism, Adenosine Triphosphate - metabolism, Antibodies, Catalytic - metabolism, Hydrolysis, Immunoglobulin Fab Fragments - metabolism, Immunoglobulin G - metabolism, Immunoglobulin Heavy Chains - metabolism, Immunoglobulin Light Chains - metabolism, Kinetics, Lactation, Milk, Human - immunology, Nucleotides - metabolism, Phosphorus Radioisotopes, Pregnancy, Serum - chemistry