Poly(A) binding proteins located at the inner surface of resealed nuclear envelopes.
We have used a photoreactive cross-linking reagent, poly(A/8-N3-A) (a poly(A) of average molecular mass of 100 kDa in which 5-10% of the A residues are replaced by 8-N3-A), to label poly(A) binding proteins of rat liver nuclear envelopes. This reagent was prepared by polymerizing a mixture of ADP and 8-N3-ADP with polynucleotide phosphorylase. The purified poly(A) was labeled in the 5'-position with a 32P group. In nuclear envelopes prepared by a low salt DNase I procedure, the poly(A/8-N3-A) labeled a protein-nucleic acid complex of approximately 270 kDa, which on degradation with RNase U2 or NaOH at pH 10 yielded two polypeptides of approximately 50 and 30 kDa. These photoreaction products were markedly decreased when resealed nuclear envelopes or non-nuclear envelope proteins were irradiated in the presence of poly(A/8-N3-A). The affinity labeling was intensified when resealed vesicles were made leaky by freezing or ultrasonication, suggesting that the poly(A) binding proteins are accessible from the nucleoplasmic but not the cytoplasmic face of the envelope. Moreover binding was specific for poly(A). Alternative reagents, random poly(A/8-N3-A,C,G,U) of about 100 kDa and poly(dA) (molecular mass between 350 and 515 kDa), showed a very low affinity for poly(A) recognition proteins in the low salt DNase I-treated nuclear envelopes; the 270-kDa band was labeled only weakly. The binding site was not protected by poly(A,C,G,U), weakly by poly(dA), and distinctly by poly(A).
Institut für Biochemie, Johann Wolfgang Goethe Universität, Frankfurt am Main, West Germany.
D
D
Prochnow
N
N
Riedel
P S
PS
Agutter
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H
Fasold
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002458:Cell Fractionation; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000378:metabolism; D008297:Male; D008970:Molecular Weight; D009685:Nuclear Envelope / Q000378:metabolism* / Q000648:ultrastructure; D011061:Poly A / Q000378:metabolism*; D039102:Poly(A)-Binding Proteins; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D002352:Carrier Proteins; D039102:Poly(A)-Binding Proteins; D011061:Poly A; D012313:RNA