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Direct cytochemical localization of catalytic subunits dissociated from cAMP-dependent protein kinase in Reuber H-35 hepatoma cells. I. Development and validation of fluorescinated inhibitor
A specific and sensitive procedure has been developed that reliably localizes intracellular sites of free catalytic unit (C) dissociated from cAMP-dependent protein kinase. The method is based on a FITC conjugate (F:PKI) of affinity column-purified heat-stable protein inhibitor (PKI) of free C. The...
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Lenguaje: | English |
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The Rockefeller University Press
1982
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2112138/ https://www.ncbi.nlm.nih.gov/pubmed/6288732 |
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collection | PubMed |
description | A specific and sensitive procedure has been developed that reliably localizes intracellular sites of free catalytic unit (C) dissociated from cAMP-dependent protein kinase. The method is based on a FITC conjugate (F:PKI) of affinity column-purified heat-stable protein inhibitor (PKI) of free C. The fidelity of this cytochemical probe was determined using cultures of Reuber H-35 hepatoma cells that had been stimulated for 2 h with 0.1 mM DBcAMP, or with diluent, then fixed with anhydrous acetone at -30 degrees C. In these preparations the F:PKI probe complexed with free C in cytoplasm, nucleolus, and, to a minor extent, in nucleoplasm. Binding of the F:PKI molecule to free C was competitively diminished by arginine analogues, guanidinium HCI and polyarginine, each used over a 2-log dose range. When the inhibitor's arginine residues were blocked by reaction with cyclohexanedione it no longer inhibited phosphotransferase activity of free C, and when fluorescinated it failed to localize C in stimulated cells. Similarly, when F:PKI was preabsorbed with excess pure C it no longer functioned as a cytochemical stain. Affinity column-purified antibody to free C also reduced significantly the ability of F:PKI to complex with C in cell cultures stimulated with 0.1 mM DBcAMP. 1 microgram of antibody reduced by approximately 10% the binding of F:PKI to all cell compartments while 5 microgram of antibody diminished binding by greater than 50%. Together, these results indicate that the F:PKI binds specifically, perhaps exclusively, to the catalytic units of cAMP- dependent protein kinase. The cytochemical procedure, unlike its biochemical counterparts, is able to locate the dissociation of cAMP- dependent protein kinase in individual cells of functionally or histologically complex cultures. Also, it reveals variations in the time- and dose-dependent activation of the kinase amongst clonal cells stimulated with cyclic nucleotide analogues or hormones. |
format | Text |
id | pubmed-2112138 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 1982 |
publisher | The Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-21121382008-05-01 Direct cytochemical localization of catalytic subunits dissociated from cAMP-dependent protein kinase in Reuber H-35 hepatoma cells. I. Development and validation of fluorescinated inhibitor J Cell Biol Articles A specific and sensitive procedure has been developed that reliably localizes intracellular sites of free catalytic unit (C) dissociated from cAMP-dependent protein kinase. The method is based on a FITC conjugate (F:PKI) of affinity column-purified heat-stable protein inhibitor (PKI) of free C. The fidelity of this cytochemical probe was determined using cultures of Reuber H-35 hepatoma cells that had been stimulated for 2 h with 0.1 mM DBcAMP, or with diluent, then fixed with anhydrous acetone at -30 degrees C. In these preparations the F:PKI probe complexed with free C in cytoplasm, nucleolus, and, to a minor extent, in nucleoplasm. Binding of the F:PKI molecule to free C was competitively diminished by arginine analogues, guanidinium HCI and polyarginine, each used over a 2-log dose range. When the inhibitor's arginine residues were blocked by reaction with cyclohexanedione it no longer inhibited phosphotransferase activity of free C, and when fluorescinated it failed to localize C in stimulated cells. Similarly, when F:PKI was preabsorbed with excess pure C it no longer functioned as a cytochemical stain. Affinity column-purified antibody to free C also reduced significantly the ability of F:PKI to complex with C in cell cultures stimulated with 0.1 mM DBcAMP. 1 microgram of antibody reduced by approximately 10% the binding of F:PKI to all cell compartments while 5 microgram of antibody diminished binding by greater than 50%. Together, these results indicate that the F:PKI binds specifically, perhaps exclusively, to the catalytic units of cAMP- dependent protein kinase. The cytochemical procedure, unlike its biochemical counterparts, is able to locate the dissociation of cAMP- dependent protein kinase in individual cells of functionally or histologically complex cultures. Also, it reveals variations in the time- and dose-dependent activation of the kinase amongst clonal cells stimulated with cyclic nucleotide analogues or hormones. The Rockefeller University Press 1982-06-01 /pmc/articles/PMC2112138/ /pubmed/6288732 Text en This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/). |
spellingShingle | Articles Direct cytochemical localization of catalytic subunits dissociated from cAMP-dependent protein kinase in Reuber H-35 hepatoma cells. I. Development and validation of fluorescinated inhibitor |
title | Direct cytochemical localization of catalytic subunits dissociated from cAMP-dependent protein kinase in Reuber H-35 hepatoma cells. I. Development and validation of fluorescinated inhibitor |
title_full | Direct cytochemical localization of catalytic subunits dissociated from cAMP-dependent protein kinase in Reuber H-35 hepatoma cells. I. Development and validation of fluorescinated inhibitor |
title_fullStr | Direct cytochemical localization of catalytic subunits dissociated from cAMP-dependent protein kinase in Reuber H-35 hepatoma cells. I. Development and validation of fluorescinated inhibitor |
title_full_unstemmed | Direct cytochemical localization of catalytic subunits dissociated from cAMP-dependent protein kinase in Reuber H-35 hepatoma cells. I. Development and validation of fluorescinated inhibitor |
title_short | Direct cytochemical localization of catalytic subunits dissociated from cAMP-dependent protein kinase in Reuber H-35 hepatoma cells. I. Development and validation of fluorescinated inhibitor |
title_sort | direct cytochemical localization of catalytic subunits dissociated from camp-dependent protein kinase in reuber h-35 hepatoma cells. i. development and validation of fluorescinated inhibitor |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2112138/ https://www.ncbi.nlm.nih.gov/pubmed/6288732 |