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.

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Abstract
A specific and sensitive procedure was developed that reliably localizes intracellular sites of free catalytic unit (C) dissociated from cAMP-dependent protein kinase. The method is based on a FITC (fluorescein isothiocyanate) 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 [rat] Reuber H-35 hepatoma cells that had been stimulated for 2 h with 0.1 mM dibutyryl cAMP, or with diluent, then fixed with anhydrous acetone at -30.degree. 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 analogs, guanidinium HCl 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 preabdsorbed 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 dibutyryl cAMP. One .mu.g of antibody reduced by .apprx. 10% the binding of F:PKI to all cell compartments while 5 .mu.g of antibody diminished binding by > 50%. The F:PKI probably 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 among clonal cells stimulated with cyclic nucleotide analogs or hormones.

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