Immunochemical studies on human, bovine and pig brain regulatory subunits of cAMP‐dependent protein kinase type II

Abstract

Regulatory subunits type II (RII) purified from human, pig and bovine brains were compared using 11 monoclonal antibodies (MoAb) against bovine brain RII. Bovine RII has at least 5 antigenic sites located in the N‐terminal 1‐110 residues. Immunochemical difference detected between human and animal RII was more pronounced than between pig and bovine RII. Certain MoAb influenced R‐cAMP binding and holoenzyme formation. RII of the three species responded to MoAb attachment in a similar fashion. The results suggest that anchoring of neural protein kinase via the N‐terminal part of RII may influence the enzyme activity.

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.…”

“…Formation of the immune complex manifested itself in inhibition of cAMP binding. So, the inserted MoAb 6 cross‐reacted with RII of PC12 cells and produced remarkably the same effect in vivo as in vitro on isolated bovine, pig and human brain RII [2].…”

“…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.…”

Protein Kinase A: Regulation and Receptor‐Mediated Delivery of Antisense Oligonucleotides and Cytotoxic Drugs

“…Also, we have developed a procedure for more detailed elucidation of the role of cAMP‐dependent protein kinases (of intracellular cAMP‐dependent protein kinase, in particular) in the regulation of cell metabolism—a procedure that makes it possible to modulate these enzyme activities in live cells. To this end, we obtained monoclonal antibodies to the regulatory subunit RIIβ isolated from bovine brain 31,32. Depending on their effects on RIIβ, these antibodies can be classified into three groups: inhibiting, activating, and so‐called neutral antibodies—those that do not interfere in the reaction of RIIβ with cAMP or the catalytic subunit of protein kinase (Table 5).…”

“…To this end, we obtained monoclonal antibodies to the regulatory subunit RIIβ isolated from bovine brain. 31,32 Depending on their effects on RIIβ, these antibodies can be classified into three groups: inhibiting, activating, and so-called neutral antibodies-those that do not interfere in the reaction of RIIβ with cAMP or the catalytic subunit of protein kinase (TABLE 5). Attachement of activating antibodies to RIIβ enhanced the ability of the latter to bind cAMP.…”