Ca²⁺-calmodulin-, cyclic AMP- and cyclic GMP-induced phosphorylation of proteins in purified microcillus 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 M(r) values of 137000, 77000, 58000, 53000 and 50000. The increases in phosphorylation caused by Ca²⁺-calmodulin were: M(r)-137000 peptide, 111 ± 26%; M(r)-77000 peptide, 71 ± 17%; M(r)-58000 peptide, 51 ± 8%; M(r)-53000 peptide, 113 ± 20%. These increases were maximal at 1 μM-calmodulin and 0.3-0.9 μM free Ca²⁺; concentrations of Ca²⁺ causing half-maximal effects on phosphorylation for the different peptides were 0.06-0.12 μM. Cyclic AMP and cyclic GMP increased phosphorylation of two peptides, of M(r) 137000 and 85000. The concentrations of cyclic nucleotides giving half-maximal phosphorylation of the M(r)-137000 peptide were 0.3 μM-cyclic AMP and 4.6 μM-cyclic GMP, and for the M(r)-85000 peptide, 3.9 μM-cyclic AMP and 0.05 μM-cyclic GMP. The maximal increase in phosphorylation of the M(r)-137000 peptide was 200% for cyclic AMP and 95% for cyclic GMP and that of the M(r)-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⁺.