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Purification of phosphodiesterase II from rat and guinea-pig intestinal mucosa.

ABSTRACT: 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.

SUBMITTER: Flanagan PR

PROVIDER: S-EPMC1172883 | biostudies-other | 1976 Jun

REPOSITORIES: biostudies-other

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Project description:Background and purposeMelatonin is involved in the regulation of colonic motility, and sensation, but little is known about the influence of melatonin on 5-hydroxytryptamine (5-HT) release from colonic mucosa. A tachykinin NK₂ receptor-selective agonist, [β-Ala⁸]-neurokinin A(4-10) [βAla-NKA-(4-10)] can induce 5-HT release from guinea pig colonic mucosa via NK₂ receptors on the mucosal layer. The present study was designed to determine the influence of melatonin on 5-HT release from guinea pig colonic mucosa, evoked by the NK₂ receptor agonist, βAla-NKA-(4-10).Experimental approachThe effect of melatonin was investigated on the outflow of 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) from muscle layer-free mucosal preparations of guinea pig colon, using high-performance liquid chromatography with electrochemical detection.Key resultsMelatonin caused a sustained decline in the βAla-NKA-(4-10)-evoked 5-HT outflow from the muscle layer-free mucosal preparations, but failed to affect its metabolite 5-HIAA outflow. The specific MT₃ receptor agonist, 5-methoxycarbonylamino-N-acetyltryptamine mimicked the inhibitory effect of melatonin on βAla-NKA-(4-10)-evoked 5-HT outflow. A MT₃ receptor antagonist prazosin shifted the concentration-response curve of melatonin to the right in a concentration-dependent manner and depressed the maximum effect, but neither a combined MT₁/MT₂ receptor antagonist luzindole, nor a MT₂ receptor antagonist N-pentanoyl-2-benzyltryptamine modified the concentration-response curve to melatonin.Conclusions and implicationsMelatonin inhibits NK₂ receptor-triggered 5-HT release from guinea pig colonic mucosa by acting at a MT₃ melatonin receptor located directly on the mucosal layer, without affecting 5-HT degradation processes. Possible contributions of MT₁/MT₂ melatonin receptors to the inhibitory effect of melatonin appear to be negligible. Melatonin may act as a modulator of excess 5-HT release from colonic mucosa.

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Purification of phosphodiesterase II from rat and guinea-pig intestinal mucosa.

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