Properties of the regulatory subunit of cAMP‐dependent protein kinase type II from human brain

Abstract

The regulatory subunit type II (RII) of cAMP‐dependent protein kinase purified from human brain was represented by two proteins with apparent molecular masses of 51‐52 kD and 54 kD. Dephosphorylation of human RII containing 3 mol phosphate/mol protein did not change the electrophoretic pattern. One‐dimensional peptide mapping of 51‐52 kD and 54 kD proteins after digestion with St. aureus V8 protease evidenced to their being distinct proteins. The data obtained permit to assume that human RII of neural type is represented by two isoforms.

Incorporation of monoclonal antibodies in living rat pheochromocytoma PC12 cells

“…It is known that binding of cAMP of the R subunit of inactive cAMP‐dependent protein kinase leads to enzyme activation. MoAb used in the present study were previously shown to inhibit cAMP binding to the neural RII in vitro [1, 2]. So abolition of cAMP‐induced protein kinase activation in the presence of MoAb can indicate the formation of an immune complex.…”

“…This concentration is comparable with 0.5 μM RII endogenous concentration [15]taking into account the leakage of ∼1/3 of the enzyme (B). The intracellular concentrations of RII and MoAb were enough for their association ( K d for MoAb 6 and the homologous antigen is equal to 3.8×10 −9 M [1]). Formation of the immune complex manifested itself in inhibition of cAMP binding.…”

“…MoAb clone 6 raised against bovine brain RII [1]and mouse normal IgG were purified by chromatography on protein G‐Sepharose. Home‐made goat anti‐mouse IgG antibodies were purified in the same way and conjugated with horseradish peroxidase [12].…”

“…Cells were suspended in 50 μl of 5 mM Tris‐HCl, pH 7.4, containing 150 mM NaCl, 2 mM EDTA, 3 mM NaF, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, disrupted by sonication (3×10 s, 0°C) and centrifuged (10 000× g , 10 min, 4°C). The supernatant was assayed for protein kinase activity using histone H1 as substrate [1].…”

“…Recently we have developed a digitonin‐mediated permeabilization technique which allows insertion of proteins as large as immunoglobulins (160 kDa) into rat pheochromocytoma PC12 cells which retain their capacity to repair. Now we applied this method for incorporation of anti‐RII MoAb previously shown to inhibit completely the cAMP binding activity of neural RII in vitro [1, 2]. The present experiments were undertaken to ascertain that the incorporated MoAb form an immune complex with the target antigen inside the cells and to investigate the influence of the inserted MoAb on the activity of intracellular protein kinases.…”