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Localization and characterization of phosphodiesterase II in intestinal mucosa Flanagan, Peter Rutledge


PDase II activity was determined using a synthetic substrate, the 2,4-dinitrophenyl ester of thymidine 3'-phosphate. The enzyme activity was estimated in fractions obtained by differential centrifugation of homogenates of epithelial cells fromt.the small intestinal mucosa of guinea pigs and rats. In guinea pig preparations PDase II occurred with highest specific activity in those fractions rich in succinate dehydrogenase and acid phosphatase. A lysosomal location for the guinea pig enzyme was indicated by its structure-linked latency and by its association with particles which underwent a characteristic decrease in equilibrium density when Triton WR-1339 was injected into the animals. With rat preparations a much greater proportion of the PDase II activity was found in the soluble fraction after uult-ra;c;entrifugation. The rat enzyme exhibited a lower degree of latency and administration of Triton WR-1339 had no effect. The rat enzyme activity in these crude preparations further differed from that of the guinea pig in other respects; it was more labile at 60°C, exhibited a slightly lower pH optimum, had a higher molecular weight as determined by gel filtration chromatography and displayed a much smaller tendency to aggregate under Llow salt conditions. Both enzymes were purified by chromatography on DEAE-cellulose, CM-cellulose and agarose, the extensive purification (550 fold) of the rat enzyme being largely due to its behaviour oh the latter material where it was found to bind tenaciously in low ionic strength solutions. On the other hand, only a fifteen-fold purification of the guinea pig enzyme was obtained because of its tendency tofform insoluble aggregatesdduring the chromatographic steps. In the main, the properties of the partially purified enzymes were quite similar. Both displayed pH optima between pH 6 and 7, were inhibited in solutions of high ionic strength, were unaffected' by divalent cations or EDTA, were similarly inactivated by heating at a temperature of 60°G displayed discontinuous Arrhenius plots _5 and exhibited Km values of the order 2-5x10 M for dTpDNP. In most casestfche differences between the enzymes were just differences of degree and could probably be accounted for byethe different extents to which the enzymes were purified. A more extensive characterization of the highly purified rat PDase was carried out. The fall-off in PDase II reaction rate observed at high enzyme levels with dTpDNP as substrate was found to be due to competitive inhibition of the enzyme by dTp, a reaction product which showed a of 2x10 M. The isoelectric point of PDase II was estimated by electrofocusing but since multiple peaks of activity were found at pH 3.4, 4.2-4.5, and pH 7.2 a conclusive result was not obtained. Polyacrylamide gel electrophoresis of purified rat PDase II indicated that the pattern obtained was, in part, dependent on whether the preparation was fresh or not; freshly purified PDase II contained up to 10 bands in gels stained for protein whereas only 1-2 bands were obtained when the preparations were "aged". A molecular weight of 150000-170000 for the enzyme was estimated in experiments performed by gel-filtration chromatography on dextran and agarose gels. Investigation of the interaction with, and hydrolysis by, rat PDase II of a number of possible phosphodiester substrates indicated that'-, the enzyme required a nucleoside 3'-phosphoryl residue for the initiation of hydrolysis which then proceeded in a 5'+3' direction. Finally, the effect of some enzyme inhibitors was investigated. PDase II activity was inhibited in the presence; of NEM, PCMB, PCMPS and iodoacetic acid. It was further found that the inactivation by iodoacetic acid could be prevented by the presence of a PDase substrate or, better still, by dTp. This is good evidence that iodoacetate alkylates an essential residue at the active center of PDase II and is the first time that such an effect has been shown for a PDase.

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