Characterization of polynucleotide phosphorylase from Micrococcus luteus and isolation of the 13,000 base poly(A) product of the polymerization reaction.
A new purification procedure for polynucleotide phosphorylase from freeze-dried Micrococcus luteus cells gives approximately 20% yield of nearly homogeneous, primer-independent enzyme which is free of nucleic acid. The physicochemical properties of M. luteus polynucleotide phosphorylase are similar to those previously described for the enzyme from Escherichia coli in terms of Mr, subunit structure, and amino acid composition. The purified enzyme appears to be a trimer composed of three identical subunits (Mr 92,000), but it probably does not exist as such in the cell. Ferguson plot analyses of enzyme in cell extracts indicate that prior to purification the enzyme exists in oligomeric forms characterized by both higher charge and greater Mr. Changes in size and charge of oligomers which occur during purification are probably due to the dissociation of proteins and/or nucleic acids. Dissociation of the oligomers is achieved by dilution and electrophoresis, but reassociation does not occur after concentration. The poly(A) product of the initial polymerization stages migrates as a single band on both nondenaturing and urea-agarose gels. It is 13,000 +/- 2,000 nucleotides long, as measured by electron microscopy, and 8,000 nucleotides long by gel electrophoretic analysis. This poly(A) product remains bound to the enzyme after synthesis, yet can be easily obtained free of protein by proteinase K digestion.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D046911:Macromolecular Substances; D008837:Micrococcus / Q000201:enzymology*; D008854:Microscopy, Electron; D008970:Molecular Weight; D011061:Poly A / Q000302:isolation & purification*; D011117:Polyribonucleotide Nucleotidyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D046911:Macromolecular Substances; D011061:Poly A; D011117:Polyribonucleotide Nucleotidyltransferase