Purification of phosphodiesterase II from rat and guinea-pig intestinal mucosa.
Phosphodiesterase II from extracts of intestinal mucosa of rat and guinea pig was purified by chromatography on DEAE-cellulose, CM-cellulose and agarose. The rat enzyme was purified 350-550-fold, with recoveries ranging up to 46%. The best purification of the guinea-pig enzyme was 15-fold, and the recovery was only 2.6%, the large loss occurring during chromatography on DEAE-cellulose and agarose. The poor results with the guinea-pig enzyme reflect the difficulty in obtaining a truly soluble material. Repeated sonication of the crude guinea-pig preparations yielded material that was initially soluble but tended to re-aggregate quickly. Purification of the rat phosphodiesterase II increased its thermostability, the temperature of half-inactivation being increased from 54degrees to 60degreesC. Both enzymes had a Km value of 4 X 10(-5) M with thymidine 3'-(2,4-dinitrophenyl) phosphate as substrate and showed similar pH optima for activity. Both enzymes were inhibited slightly in 0.1 M-MgC12 or 2M-urea and much more strongly in 2M-(NH4)2SO4 or 6M-NaC1. The guinea-pig enzyme was usually inhibited more than the rat enzyme. The Arrhenius plots of the two enzymes differed slightly in slope, but both were biphasic, showing breaks between 30degrees and 40degreesC. It was concluded that the two enzymes were markedly similar in behaviour and that the differences found were related to the different degrees of purification attained by the procedures described.
D000818:Animals; D002847:Chromatography, Agarose; D002848:Chromatography, DEAE-Cellulose; D004492:Edetic Acid / Q000494:pharmacology; D006168:Guinea Pigs; D006358:Hot Temperature; D006868:Hydrolysis; D007413:Intestinal Mucosa / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification*; D051381:Rats
D010726:Phosphodiesterase Inhibitors; D004492:Edetic Acid; D010727:Phosphoric Diester Hydrolases; D008274:Magnesium