Mammalian poly(A)-binding protein II. Physical properties and binding to polynucleotides.

Abstract

The 49-kDa poly(A)-binding protein II (PAB II) was purified to homogeneity from calf thymus. The 70-kDa poly(A)-binding protein I (PAB I) was obtained in different fractions of the same preparation. Whereas PAB II stimulated poly(A) polymerase, PAB I was an inhibitor. In analytical ultracentrifugation, the predominant form of PAB II was a monomer of 50.3 kDa. A sedimentation constant of only 2.2 S indicated a distinctly non-spherical shape. Binding was specific for single-stranded purine polyribonucleotides. The dependence of the dissociation constant on the length of oligoriboadenylate indicated a binding site size of 12 nucleotides. A single site was bound with a KD of 2 x 10(-9) M, as determined by nitrocellulose filter binding assays. From fluorescence quenching and gel retardation experiments, the packing ratio on poly(A) was estimated as 23 nucleotides/protein monomer.