Properties of adenylate cyclase in mucosal cells of the rabbit ileum and the effect of cholera toxin

  • Sharp G
  • Hynie S
  • Ebel H
 et al. 
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Abstract

A study of the properties of adenylate cyclase in mucosal epithelial cells of rabbit ileum has been performed under control conditions and after stimulation by cholera toxin. A 3-5 fold stimulation of enzyme activity occurred after treatment with cholera toxin. Optimal conditions for activity were attained with Mg2+concentrations between 5 and 25 mM at a pH of 8.0-9.0. Linearity of reaction rate was achieved over 25 min at protein concentrations of the homogenate of up to 40 μg per 50 μl incubation mixture when an ATP regeneration system was employed. Mn2+at 3·10-4to 1·10-3M could replace Mg2+but was inhibitory at 5 mM. Ca2+was inhibitory at all concetrations above 10-5M. Michaelis-Menten plots were linear with regard to ATP concentrations under both sets of of conditions, V was 28 pmoles·mg-1protein·min-1for control and 90 pmoles·mg-1protein·min-1for toxin-treated enzyme. Kmfor both conditions were 4·10-4M. No evidence of cooperativity was detected. The mechanisms of activation by F-and cholera toxin were presumed different from an analysis of experiments involving combinations of F-and toxin and by a study of pyrophosphate inhibition. Isoproterenol at 10-5M was stimulatory to the control enzyme, while no effect of epinephrine or propranolol was detected. In contrast, isoproterenol, epinephrine and propranolol were inhibitory to the enzyme after stimulation by cholera toxin. © 1973.

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Authors

  • Geoffrey W G Sharp

  • Sixtus Hynie

  • Hans Ebel

  • David K. Parkinson

  • Patricia Witkum

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