Diadenosine tetraphosphate binding protein from human HeLa cells: purification and characterization.
The ubiquitous dinucleotide P1,P4-di(adenosine-5') tetraphosphate (Ap4A) has been proposed to be involved in DNA replication and cell proliferation, DNA repair, platelet aggregation, and vascular tonus. A protein binding specifically to Ap4A is associated with a multiprotein form of DNA polymerase alpha (pol alpha 2) in HeLa cells. The Ap4A binding protein from HeLa cells has been purified to homogeneity starting from pol alpha 2 complex. The Ap4A binding protein is hydrophobic and is resolved from the pol alpha 2 complex by hydrophobic interaction chromatography on butyl-Sepharose and subsequently purified to homogeneity by chromatography on Mono-Q and Superose-12 FPLC columns. The Ap4A binding activity elutes as a single symmetrical peak upon gel filtration with a molecular mass of 200 kDa. Upon polyacrylamide gel electrophoresis under nondenaturing conditions, the purified protein migrates as a single protein of 200 kDa. Upon electrophoresis under denaturing conditions, the binding activity is resolved into two polypeptides of 45 and 22 kDa, designated as A1 and A2, respectively. A1 and A2 can be cross-linked using the homobifunctional cross-linking agent disuccinimidyl suberate. The cross-linked protein migrates as a single protein of 210 kDa on polyacrylamide gels under denaturing conditions, suggesting that these two polypeptides are subunits of a single protein. The purified protein binds Ap4A efficiently, and by Scatchard analysis, we have determined a dissociation constant of 0.25 microM, indicating high affinity of Ap4A binding protein to its ligand. ATP is not required for the binding activity. The nonionic detergent Triton X-100 is necessary for stabilizing the purified protein. Amino acid composition analysis indicates that A1 and A2 are distinct.
Vishwanatha
J K
JK
Department of Biochemistry, University of Nebraska Medical Center, Omaha 68198-4525.
Wei
Z
Z
D000596:Amino Acids / Q000032:analysis; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D015226:Dinucleoside Phosphates / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells / Q000737:chemistry; D006801:Humans; D046911:Macromolecular Substances; D008970:Molecular Weight; D046912:Multiprotein Complexes
C032578:AP4A binding protein, human; D000596:Amino Acids; D002352:Carrier Proteins; D015226:Dinucleoside Phosphates; D046911:Macromolecular Substances; D046912:Multiprotein Complexes; C020733:diadenosine tetraphosphate