Evidence for catecholamine-stimulated adenylate cyclase activity in frog and rabbit corneal epithelium and cyclic AMP-dependent protein kinase and its protein substrates in frog corneal epithelium |
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Authors: | P S Reinach M A Kirchberger |
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Affiliation: | 2. Department of Ophthalmology, Mount Sinai School of Medicine of the City University of New York, New York, NY 10029, U.S.A.;3. Department of Physiology and Biophysics, Mount Sinai School of Medicine of the City University of New York, New York, NY 10029, U.S.A. |
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Abstract: | Evidence was obtained for catecholamine-stimulated adenylate cyclase activity in particulate fractions of frog and rabbit corneal epithelium. Epinephrine (10(-5)M) stimulated adenylate cyclase by 22 and 53% in the frog and rabbit, respectively. The corresponding changes were statistically significant (P less than 0.01) when the data was analyzed using paired variates. Preincubation with 10(-4)M propranolol eliminated any stimulatory effect by 10(-5)M isoproterenol. Adenylate cyclase activity derived from either source was activated several fold by either 10 mM NaF or 10(-5)MGpp (NH)p. Soluble fractions of homogenized frog corneal epithelium contained cyclic AMP-dependent protein kinase activity which was half-maximally stimulated by about 6 nM cyclic AMP. Evidence was also obtained for the presence of protein substrates of cyclic AMP dependent protein kinase in frog corneal epithelium. With exogenous cyclic AMP and protein kinase, a rapid 32P labelling of proteins having approximate molecular weights of 56, 46, 23 and 21 K was obtained with sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. A less marked and slower increase in phosphoprotein formation was observed when corneal membranes were incubated with cyclic AMP in the absence of added protein kinase. |
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Keywords: | frog cornea cyclic AMP protein phosphorylation adenylate cyclase protein kinase active Cl transport epinephrine β-adrenergic system epithelium |
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