Oligomeric protein structure of beef spleen exonuclease.
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.
Mitkova
A V
AV
Institute of Molecular Biology, Bulgarian Academy of Sciences, 113 Sofia, Bulgaria.
Bakalova
A T
AT
Stoynov
S S
SS
Dolapchiev
L B
LB
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D002417:Cattle; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification; D004220:Disulfides / Q000737:chemistry; D004795:Enzyme Stability / Q000502:physiology; D005092:Exonucleases / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification; D006031:Glycosylation; D008970:Molecular Weight; D011487:Protein Conformation
D000906:Antibodies; D004268:DNA-Binding Proteins; D004220:Disulfides; D006023:Glycoproteins; D005092:Exonucleases; C022650:spleen exonuclease