Stereochemical and kinetic studies on the action of the catalytic subunit of bovine cardiac muscle adenosine 3',5'-monophosphate dependent protein kinase using metal ion complexes of ATP.beta.S

Biochemistry ◽  
1980 ◽  
Vol 19 (6) ◽  
pp. 1176-1182 ◽  
Author(s):  
D. W. Bolen ◽  
Juerg Stingelin ◽  
H. Neal Bramson ◽  
E. T. Kaiser
Keyword(s):  
Protein Kinase ◽  
Cardiac Muscle ◽  
Metal Ion ◽  
Kinetic Studies ◽  
Catalytic Subunit ◽  
Dependent Protein Kinase ◽  
Metal Ion Complexes ◽  
Dependent Protein

scholarly journals Chemical cross-linking of cyclic AMP-dependent protein kinase and its dissimilar subunits

1983 ◽  
Vol 209 (3) ◽  
pp. 581-586 ◽  
Author(s):  
J P Charlton ◽  
C H Huang ◽  
L C Huang
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Ternary Complex ◽  
Kinetic Studies ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Cross Linking ◽  
Dependent Protein Kinase ◽  
Native Form ◽  
Dependent Protein

Previous kinetic studies have demonstrated that the activation of cyclic AMP-dependent protein kinase by cyclic AMP involves the formation of a ternary complex of cyclic AMP, the regulatory subunit (R) and the catalytic subunit (C). It is suggested that only this ternary complex breaks down to liberate the enzymically active catalytic subunit. We have performed cross-linking experiments with the holoenzyme and its dissimilar subunits in the presence of MgATP and various concentrations of cyclic AMP. Results from these cross-linking studies indicate that regulatory subunits exist as dimers in the native form. Moreover, dissociation of the holoenzyme or the reconstituted enzyme is promoted by cyclic AMP, and the effect of MgATP is to stabilize the enzyme in the tetrameric form. The success in cross-linking the regulatory and the catalytic subunits of protein kinase with the lysine-specific bifunctional cross-linking reagent dimethyl suberimidate may be attributed to the presence of a large number of lysine residues in the enzyme.

Phosphorylation of skeletal and cardiac muscle C-proteins by the catalytic subunit of cAMP-dependent protein kinase

1986 ◽  
Vol 64 (7) ◽  
pp. 622-630 ◽  
Author(s):  
Megan S. Lim ◽  
Michael P. Walsh
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinase ◽  
Atpase Activity ◽  
Cardiac Muscle ◽  
Catalytic Subunit ◽  
Dependent Protein Kinase ◽  
C Protein ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein ◽  
C Proteins

Catecholamines are known to influence the contractility of cardiac and skeletal muscles, presumably via cAMP-dependent phosphorylation of specific proteins. We have investigated the in vitro phosphorylation of myofibrillar proteins by the catalytic subunit of cAMP-dependent protein kinase of fast- and slow-twitch skeletal muscles and cardiac muscle with a view to gaining a better understanding of the biochemical basis of catecholamine effects on striated muscles. Incubation of canine red skeletal myofibrils with the isolated catalytic subunit of cAMP-dependent protein kinase and Mg-[γ-32P]ATP led to the rapid incorporation of [32P]phosphate into five major protein substrates of subunit molecular weights (MWs) 143 000, 60 000, 42 000, 33 000, and 11 000. The 143 000 MW substrate was identified as C-protein; the 42 000 MW substrate is probably actin; the 33 000 MW substrate was shown not to be a subunit of tropomyosin and, like the 60 000 and 11 000 MW substrates, is an unidentified myofibrillar protein. Isolated canine red skeletal muscle C-protein was phosphorylated to the extent of ~0.5 mol Pi/mol C-protein. Rabbit white skeletal muscle and bovine cardiac muscle C-proteins were also phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, both in myofibrils and in the isolated state. Cardiac C-protein was phosphorylated to the extent of 5–6 mol Pi/mol C-protein, whereas rabbit white skeletal muscle C-protein was phosphorylated at the level of ~0.5 mol Pi/mol C-protein. As demonstrated earlier by others, C-protein of skeletal and cardiac muscles inhibited the actin-activated myosin Mg2+-ATPase activity at low ionic strength in a system reconstituted from the purified skeletal muscle contractile proteins (actin and myosin). Phosphorylation of skeletal or cardiac C-proteins had no effect on their inhibition of this actomyosin Mg2+-ATPase activity. Furthermore, cardiac C-protein inhibited the Mg2+-ATPase activity of desensitized cardiac actomyosin; in this case, phosphorylation of cardiac C-protein enhanced its inhibitory effect on the actomyosin Mg2+-ATPase. These observations suggest that C-proteins of fast- and slow-twitch skeletal muscle fibers and cardiac muscle fibers are phosphorylated in response to catecholamines and other agents which induce cAMP formation and that, at least in the heart, this phosphorylation may affect actin–myosin interaction and the contractile state of the muscle.

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