Abstract
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.
Highlights
Of Mr, subunit structure, andamin oacid composition
Dissociation of the oligomers is enzyme is large (- 13,000 nucleotides by electron microscopy) achieved bdyilution and electrophoresis, but reassocia-compared to thepreviously reported values of 200 to “greater tion does not occur after concentration
Polynucleotide phosphorylase from Micrococcus luteus has only a 1.1-to 2-fold stimulation when assayed for polymeribeen isolated in two forms: aprimer-independent form in zation in the presence of (AP)~AA. nalysis of the protein by which polymerization activity is stimulated only 1.1-to 2-fold SDS gel electrophoresis had shown [11] that 90% of the by small oligonucleotides (Form I), and a primer-dependent protein co-migrated with the u subunit of RNA polymerase form generated by partial proteolysis in which polymerization (M,= 85,000 to 92,000 [40]).The remaining 10%has a slightly activity is stimulated up to20-fold by oligonucleotide primer lower molecular weight, M, = 85,000, which has been attrib(Form T) [1,2,3,4,5]
Summary
Properties of Purified Polynucleotide Phosphorylase synthesis, yet can be obtained free of protein by proteinase K digestion. Samples of 20 pg were applied to 3.5% polyacrylamide gels and subjected to electrophoresis in SDScontaining buffer as described by Davies and Stark [25].Gels 2 and 2, primer-independent polynucleotide phosphorylase (I);gels 3 and 4, trypsin-treated enzyme (2’) after incubationfor 10min in the presence of 0.14M P-mercaptoethanol[55];gels and 6, trypsin-treated enzyme after incubationfor 10min in the presence of 2 mM N-ethylmaleimide [55].Molecular weight standards are shown on the left. End Group Analysis--In contrast to the E. coli enzyme, neither the primer-independent form of M. hteus polynucleotide phosphorylase nor the primer-dependent form of the enzyme obtained by limited proteolysis with trypsin contain a detectable free amino end group (Table 11).This suggests that proteolysis removes a peptide at thecarboxyl end of the molecule It is another indication of the homogeneity of the product and low level of proteolysis that characterizes this purification procedure.
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