Ca2+-calmodulin-, cyclic AMP- and cyclic GMP-induced phosphorylation of proteins in purified microvillus membranes of rabbit ileum.
Evidence is available to suggest that Ca2+-calmodulin and cyclic nucleotides are involved in the regulation of ion transport in rabbit ileum. Since both Ca2+-calmodulin and cyclic nucleotides exert many of their effects by phosphorylation, the effects of Ca2+-calmodulin and cyclic nucleotides on phosphorylation of purified microvillus membrane from rabbit ileal mucosa were evaluated. Ca2+-calmodulin increased phosphorylation of five microvillus-membrane peptides, with Mr values of 137000, 77000, 58000, 53000 and 50000. The increases in phosphorylation caused by Ca2+-calmodulin were: Mr-137000 peptide, 111 +/- 26%; Mr-77000 peptide, 71 +/- 17%; Mr-58000 peptide, 51 +/- 8%; Mr-53000 peptide, 113 +/- 20%. These increases were maximal at 1 microM-calmodulin and 0.3-0.9 microM free Ca2+; concentrations of Ca2+ causing half-maximal effects on phosphorylation for the different peptides were 0.06-0.12 microM. Cyclic AMP and cyclic GMP increased phosphorylation of two peptides, of Mr 137000 and 85000. The concentrations of cyclic nucleotides giving half-maximal phosphorylation of the Mr-137000 peptide were 0.3 microM-cyclic AMP and 4.6 microM-cyclic GMP, and for the Mr-85000 peptide, 3.9 microM-cyclic AMP and 0.05 microM-cyclic GMP. The maximal increase in phosphorylation of the Mr-137000 peptide was 200% for cyclic AMP and 95% for cyclic GMP, and that of the Mr-85000 peptide was 220% for cyclic AMP and 120% for cyclic GMP. These studies demonstrate the existence of Ca2+-calmodulin-, cyclic AMP- and cyclic GMP-dependent protein kinases and substrate proteins in purified rabbit ileal microvillus membranes and that Ca2+ can regulate phosphorylation of these proteins over the presumed physiological concentration range of cytosol free Ca2+.
D000818:Animals; D002147:Calmodulin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000494:pharmacology*; D006152:Cyclic GMP / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D007082:Ileum / Q000187:drug effects / Q000378:metabolism*; D066298:In Vitro Techniques; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D008871:Microvilli / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation; D011817:Rabbits; D014268:Trifluoperazine / Q000494:pharmacology
D002147:Calmodulin; D008565:Membrane Proteins; D014268:Trifluoperazine; D000242:Cyclic AMP; D006152:Cyclic GMP