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 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.
D000818:Animals; D001120:Arginine / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002384:Catalysis; D002466:Cell Nucleolus / Q000201:enzymology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000502:physiology; D003512:Cyclohexanones / Q000494:pharmacology; D003593:Cytoplasm / Q000201:enzymology; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006651:Histocytochemistry / Q000379:methods*; D047908:Intracellular Signaling Peptides and Proteins; D008114:Liver Neoplasms, Experimental / Q000201:enzymology; D010455:Peptides / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D013861:Thiocyanates
D002352:Carrier Proteins; D003512:Cyclohexanones; D005452:Fluoresceins; D006146:Guanidines; D047908:Intracellular Signaling Peptides and Proteins; D010455:Peptides; D013861:Thiocyanates; C015482:protein kinase modulator; C015462:polyarginine; D001120:Arginine; D000242:Cyclic AMP; D011494:Protein Kinases; D016650:Fluorescein-5-isothiocyanate; D019791:Guanidine