A new affinity adsorbent for guanyloribonuclease. Guanylyl-(2'-5')-guanosine coupled to aminohexyl-Sepharose.
Guanylyl-(2'-5')-guanosine binds to RNase T1 in 1:1 stoichiometry with a dissociation constant of 0.22 mM at pH 5.0 and 25 degrees C. This nucleotide, coupled to aminohexyl-Sepharose 4B, is able to serve as an affinity adsorbent for guanyloribonuclease [EC 3.1.4.8]. The strength of interaction between the adsorbent and various guanyloribonucleases at pH 5.0 was found to decrease in the following order: RNase N1 greater than RNase F1 greater than RNase T1 greater than RNase St. The bound enzymes can be released from the adsorbent either by increase of ionic strength or by increasing the pH from 5.0 to 7.5. The interaction between RNase T1 and the adsorbent is weakened by the presence of a low concentration of 2', 3'-, or 5'-GMP, which are competitive inhibitors of the enzyme. RNase F1 was purified to homogeneity by use of this affinity adsorbent.
D002846:Chromatography, Affinity / Q000379:methods; D005670:Fusarium / Q000201:enzymology; D006150:Guanine Nucleotides; D006151:Guanosine / Q000031:analogs & derivatives; D006157:Guanosine Monophosphate / Q000031:analogs & derivatives; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D006163:Ribonuclease T1 / Q000302:isolation & purification*; D012260:Ribonucleases / Q000302:isolation & purification*; D012685:Sepharose
D006150:Guanine Nucleotides; D006151:Guanosine; D006157:Guanosine Monophosphate; D012685:Sepharose; D012260:Ribonucleases; D006163:Ribonuclease T1