ABSTRACT
The 5'-->3' exonuclease from beef spleen is a 160-kDa tetramer consisting of four subunits of two types. Partial reduction of the tetramer led to one stable intermediate state of the enzyme with Mr = 80 kDa. The aim of this paper was to attribute the exonucleolytic activity to one of the two monomers, to the dimer or to the tetramer. The different forms of the exonuclease were separated by SDS-polyacrylamide gel electrophoresis, transferred on an Immobilon-P membrane and subsequently renaturated. Antibodies monospecific against each of the two monomers as well as against the dimer were isolated and their inhibitory effect on the holoenzyme determined. It was found that after renaturation the two monomers did not possess any exonuclease activity while the 80-kDa dimer showed a lower recovery of the specific activity of the enzyme (20.8+/-0.23 nkat/nmol, (n = 5)) in comparison with the 160-kDa tetramer (64.8+/-0.75 nkat/nmol (n = 5)). It was demonstrated that the antibodies monospecific against the dimer caused 53% maximum inhibition of the 160-kDa exonuclease. The antibodies monospecific against 25- and 55-kDa monomers did not inhibit the activity of the holoenzyme. No single-strand endonuclease activity of the spleen exonuclease was observed when using supercoiled Bluescript KS+ plasmid DNA as a substrate. This data suggest the emergence of an 80 kDa active form of beef spleen exonuclease upon association of two monomers of the tetrameric enzyme.
Subject(s)
Exodeoxyribonucleases/chemistry , Exodeoxyribonucleases/metabolism , Spleen/enzymology , Animals , Binding Sites , Cattle , Chromatography, Affinity , Exodeoxyribonuclease V , Exodeoxyribonucleases/isolation & purification , Kinetics , Macromolecular Substances , Molecular Weight , Protein Structure, QuaternaryABSTRACT
A one-step purification of beef spleen exonuclease in the form of a DNA-enzyme complex is described. The purity of the exonuclease was verified by two-dimensional gel electrophoresis. It possesses molecular mass 160 kDa and pI 6.92. The one-dimensional sodium dodecyl sulfate gel after reduction with beta-mercaptoethanol suggests that the 160-kDa exonuclease consists of four polypeptide chains of two different types with molecular masses 55 and 25 kDa. The tetrameric structure of the exonuclease is supported by intermolecular disulfide bonds, and their partial reduction leads to the formation of one stable intermediate with molecular mass 80 kDa formed by binding one 55-kDa with one 25-kDa subunit into a dimer. During two-dimensional gel electrophoresis, the dimer showed pI 6.92 while monomers showed pI 6.78 for the 55-kDa and pI 6.29 for the 25-kDa. Two other intermediate states of two big and one small (135 kDa) and two small and one big subunit (105 kDa) were also visualized. They are unstable and easily dissociated into one 80-kDa dimer and either one 55-kDa or one 25-kDa monomer. The immunoblotting analysis with specific polyclonal antibodies against 160-kDa protein confirmed the subunit structure of the exonuclease. It was found that both monomers are glycosylated.
Subject(s)
Exonucleases/chemistry , Protein Conformation , Animals , Antibodies/immunology , Antibodies/pharmacology , Cattle , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/isolation & purification , Disulfides/chemistry , Enzyme Stability/physiology , Glycoproteins/chemistry , Glycoproteins/isolation & purification , Glycosylation , Molecular WeightABSTRACT
Polyclonal antibodies against the exonuclease from Crotalus adamanteus venom (the 140-kDa protein) inhibit both the exonucleolytic and the single-strand-specific endonucleolytic activities, present in the exonuclease preparation. The antibodies also diminish the ability of the enzyme to split the negatively supercoiled Bluescript KS+ in the AT-rich fragment near-by the transcription termination site of the Ampicillin gene. Therefore the single-strand-specific endonucleolytic activity was attributed to the protein molecule of the exonuclease. The processivity of the exonucleolytic action was found to be less than 3 monomers as indicated by the heparin trapping method.
