Ca2+-calmodulin-, cyclic AMP- and cyclic GMP-induced phosphorylation of proteins in purified microcillus membranes of rabbit ileum

Mark Donowitz, M. E. Cohen, R. Gudewich

Research output: Contribution to journalArticle

Abstract

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+.

Original languageEnglish (US)
Pages (from-to)573-581
Number of pages9
JournalBiochemical Journal
Volume219
Issue number2
StatePublished - 1984
Externally publishedYes

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Phosphorylation
Cyclic GMP
Calmodulin
Ileum
Cyclic AMP
Rabbits
Membranes
Peptides
Cyclic Nucleotides
Proteins
Microvilli
Cyclic GMP-Dependent Protein Kinases
Ion Transport
Cytosol
Mucous Membrane
Ions

ASJC Scopus subject areas

  • Biochemistry

Cite this

Ca2+-calmodulin-, cyclic AMP- and cyclic GMP-induced phosphorylation of proteins in purified microcillus membranes of rabbit ileum. / Donowitz, Mark; Cohen, M. E.; Gudewich, R.

In: Biochemical Journal, Vol. 219, No. 2, 1984, p. 573-581.

Research output: Contribution to journalArticle

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abstract = "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+.",
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