Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.8 (polynucleotide phosphorylase)
 723 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mutations which affect the activity of polynucleotide phosphorylase (PNPase) map near 69 min on the bacterial chromosome. This region of the chromosome has been cloned by inserting the kanamycin-resistant transposon Tn5 near the argG and mtr loci at 68.5 min. Large SalI fragments of chromosomal DNA containing the Tn5 element were inserted into pBR322, and selection was made for kanamycin-resistant recombinant plasmids. Two of these plasmids were found to produce high levels of PNPase activity in both wild-type and host strains lacking PNPase activity. The pnp gene was further localized and subcloned on a 4.8 kilobase HindIII-EcoRI fragment. This fragment was shown to encode an 84,000-molecular weight protein which comigrated with purified PNPase during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The orientation of the pnp gene was determined by insertion of Tn5 into the 4.8 kilobase fragment cloned in pBR322. Some of the insertions had lost the ability to elevate the level of PNPase activity in the host bacterium. Restriction mapping of the positions of the Tn5 insertions and analysis of plasmid-encoded polypeptides in UV-irradiated maxi-cells indicated that the pnp gene is oriented in the counterclockwise direction on the bacterial chromosome.
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PMID:Cloning and orientation of the gene encoding polynucleotide phosphorylase in Escherichia coli. 630 41

The polynucleotide phosphorylase of Thermus aquaticus was purified using ammonium sulfate fractionation and column chromatography on DEAE-cellulose, heparin-Sepharose 4B and DEAE-Sephadex A25. The enzyme was purified 1500-fold and was 90-95% homogeneous as checked by polyacrylamide gel electrophoresis. It has a molecular weight of 275 000 and consists of four identical subunits. The Km values for the enzyme as determined in polymerization (ADP, GDP, UDP) and phosphorolytic reactions (poly A, poly U) are in the same concentration range as in the case of the enzyme deriving from mesophilic microorganisms. Furthermore, the enzyme is primer dependent and its activity is lost gradually at temperatures higher than 65 degrees C. In the base ratio of the copolymers followed the input base ratio polymerization reactions with polyUA, while with polyAG and polyUG a marked difference between the initial base ratio and the base composition of copolymers was observed.
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PMID:The purification of polynucleotide phosphorylase from Thermus aquaticus by the use of heparin-sepharose 4B affinity chromatography. 734 86