Purification of terminal riboadenylate transferase from calf thymus gland.
A poly(A) polymerase has been purified from the soluble protein fraction of calf thymus gland. The activity is cytoplasmic and nonparticulate. Mn-2+ATP is the preferred substrate. On the basis of disc gel electrophoresis in sodium dodecyl sulfate-acrylamide gels, gel filtration, and sedimentation velocity in sucrose gradients, the enzyme has a molecular weight of 62,000 and appears to consist of one polypeptide chain. The enzyme preparation is shown to be nearly homogeneous by disc gel electrophoresis and isoelectric-focusing. The activity has a pI of about 7.4. The specific activity of the enzyme is about 1700 mumol per hour per mg of protein, giving a turnover number of about 1800 mol of substrate per mol of enzyme min- minus 1. The activity is highly specific for ATP and is inhibited by other ribonucleoside triphosphates. It is sensitive to high levels of RNA-polymerase inhibitors. Km for oligoadenylate is 50 muM in the presence of Mn-2+ and 200 muM in Mg-2+ and equivalent Vmax is achieved with either metal ion. The initiator function may be filled by a variety of oligoribonucleotides having a free 3'-OH.
D000227:Adenine Nucleotides; D000818:Animals; D002417:Cattle; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification* / Q000378:metabolism; D004355:Drug Stability; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D006882:Hydroxyapatites; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D009928:Organ Specificity; D051381:Rats; D013154:Spleen / Q000201:enzymology; D013950:Thymus Gland / Q000201:enzymology*
D000227:Adenine Nucleotides; D006882:Hydroxyapatites; D008345:Manganese; D012321:DNA-Directed RNA Polymerases; D008274:Magnesium