NUCLEOTIDES REVEAL POLYNUCLEOTIDE PHOSPHORYLASE-ACTIVITY FROM CONVENTIONALLY PURIFIED GROEL

GroEL, as conventionally purified, can be incubated with nucleotides t o produce high molecular weight material with an absorption maximum at 260 nm. This material is most clearly demonstrated when samples are s ubjected to gel filtration under conditions where GroEL is monomeric. There is a time-dependent increase in the high molecular weight materi al that occurs on incubation with ADP or, more slowly, with ATP, This material is generated during incubation, and none is present in the in itial samples. Experiments with nucleases, proteases, radiolabeled nuc leotides, and chemical cleavage reagents demonstrate that the high mol ecular weight material is polyadenylic acid whose formation is inhibit ed by phosphate. These results are consistent with the GroEL samples c ontaining polynucleotide phosphorylase activity. Nondenaturing gels st ained with acridine orange, after incubation in ADP, reveal that the a ctivity producing the poly(A) coelectrophoreses with authentic polynuc leotide phosphorylase. Conditions that remove the tryptophan-like fluo rescence from preparations of GroEL also remove the PNPase activity, T hus, this activity is not associated with GroEL itself, The results ar e consistent with reports that GroEL can associate with RNase E and wi th other studies showing that RNase E and PNPase can form complexes, T hus, the present experiments support suggestions that GroEL can partic ipate in multiprotein complexes that are involved in mRNA processing a nd degradation.