Purification and properties of carboxypeptidase G2 from Pseudomonas sp. strain RS-16. Use of a novel triazine dye affinity method.
A folate-degrading enzyme, carboxypeptidase G2, has been purified on a large scale from Pseudomonas sp. strain RS-16. Homogeneous enzyme was obtained by a three-step procedure involving ion-exchange chromatography and a novel triazine dye (affinity) chromatography step which utilizes Zn2+ to promote adsorption of the enzyme. Enzyme was selectively eluted by the use of a chelating agent (EDTA) and a step change in pH. The enzyme is a dimeric protein (Mr 83000) with two identical subunits of 41800 and contains four atoms of zinc per enzyme molecule, which are required for full activity. The enzyme follows Michaelis-Menten kinetics with Km values of 4.0 microM for folate, 8.0 microM for methotrexate and 34.0 microM for 5-methyltetrahydrofolate, the predominant form of reduced folate found in plasma.
D001665:Binding Sites; D002268:Carboxypeptidases / Q000302:isolation & purification*; D055598:Chemical Phenomena; D002621:Chemistry; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D004396:Coloring Agents; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008968:Molecular Conformation; D011549:Pseudomonas / Q000201:enzymology* / Q000254:growth & development; D014227:Triazines; D015032:Zinc / Q000494:pharmacology; D011623:gamma-Glutamyl Hydrolase / Q000302:isolation & purification*
D004396:Coloring Agents; D008670:Metals; D014227:Triazines; D002268:Carboxypeptidases; D011623:gamma-Glutamyl Hydrolase; D015032:Zinc