Bovine Heart Protein Kinase Research Articles

Antiserum against the catalytic subunit of adenosine 3':5'-cyclic monophosphate-dependent protein kinase. Reactivity towards various protein kinases.

An antiserum against the catalytic subunit C of cyclic AMP-dependent protein kinase, isolated from bovine heart type II protein kinase, was produced in rabbits. Reaction of the catalytic subunit with antiserum and separation of the immunoglobulin G fraction by Protein A-Sepharose quantitatively removed the enzyme from solutions. Comparative immunotitration of protein kinases showed that the amount of antiserum required to eliminate 50% of the enzymic activity was identical for pure catalytic subunit, and for holoenzymes type I and type II. The reactivity of the holoenzymes with the antiserum was identical in the absence or the presence of dissociating concentrations of cyclic AMP. Most of the holoenzyme (type II) remains intact when bound to the antibodies as shown by quantification of the regulatory subunit in the supernatant of the immunoprecipitate. Titration with the antibodies also revealed the presence of a cyclic AMP-independent histone kinase in bovine heart protein kinase I preparations obtained by DEAE-cellulose chromatography. Cyclic AMP-dependent protein kinase purified from the particulate fraction of bovine heart reacted with the antiserum to the same degree as the soluble enzyme, whereas two cyclic AMP-independent kinases separated from the particle fraction neither reacted with the antiserum nor influenced the reaction of the antibodies with the cyclic AMP-dependent protein kinase. Immunotitration of the protein kinase catalytic subunit C from rat liver revealed that the antibodies had rather similar reactivities towards the rat liver and the bovine heart enzyme. This points to a relatively high degree of homology of the catalytic subunit in mammalian tissues and species. Broad applicability of the antiserum to problems related to cyclic AMP-dependent protein kinases is thus indicated.

Magnetic resonance studies of the effect of the regulatory subunit on metal and substrate binding to the catalytic subunit of bovine heart protein kinase.

Magnetic resonance studies of the effect of the regulatory subunit on metal and substrate binding to the catalytic subunit of bovine heart protein kinase.

Specificity of bovine heart protein kinase for the Δ-stereoisomer of the metal—ATP complex

Specificity of bovine heart protein kinase for the Δ-stereoisomer of the metal—ATP complex

Cyclic AMP-dependent ATPase activity of bovine heart protein kinase.

The adenosine 3",5"-monophosphate (cAMP)-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity of cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) from bovine heart is characterized. That the ATPase activity is intimately associated with the catalytic subunit of the enzyme is suggested by the following: (i) the similar dependences of ATPase and protein kinase activities on cAMP; (ii) the dissociation of ATPase activity from the holoenzyme on addition of cAMP and its co-elution with the catalytic subunit on gel filtration chromatography; (iii) the similarity of the relative effectiveness of divalent metal ions in ATPase and protein kinase catalysis; and (iv) the correspondence of kinetically determined Km(MgATP) and Ki(MgADP) values with thermodynamic dissociation constants determined by equilibrium dialysis. The hydrolysis of ATP is stimulated 10- to 20-fold by cAMP in the holoenzyme. The molar specific activity of the catalytic subunit ATPase is approximately 0.7 min-1 with Km(MgATP) = 5 muM. MgADP is a competitive inhibitor of the reaction with a Ki value of approximately muM. The order of the relative effectiveness of metal ions for both ATPase and peptide kinase activities is Mg2+ greater than Mn2+ greater than Ca2+. A possible interpretation of these observations is that the role that the metal ion plays is more directly manifested in bond-breaking than in bond-forming.

Effects of adenosine 3′ : 5′-monophosphate and guanosine 3′ : 5′-monophosphate on calcium uptake and phosphorylation in membrane fractions of vascular smooth muscle

The effects of adenosine 3′ : 5′-monophosphate (cyclic AMP), guanosine 3′ : 5′-monophosphate (cyclic GMP) and exogenous protein kinase on Ca uptake and membrane phosphorylation were studied in subcellular fractions of vascular smooth muscle from rabbit aorta. Two functionally distinct fractions were separated on a continuous sucrose gradient: a light fraction enriched in endoplasmic reticulum (fraction E) and a heavier fraction containing mainly plasma membranes (fraction P). While cyclic AMP and cyclic GMP had no effect on Ca uptake in the absence of oxalate, both cyclic nucleotides inhibited the rate of oxalate-activated Ca uptake when used at concentrations higher than 10 −5 M. The addition of bovine heart protein kinase to either fraction produced an increase in the rate of oxalate-activated Ca uptake which was further augmented by cyclic AMP. Cyclic GMP caused smaller stimulations of protein kinase-catalyzed Ca uptake than cyclic AMP. Mg-dependent phosphorylation, attributable to endogenous protein kinase(s), was inhibited in fraction E by low concentrations (10 −8 M) of both cyclic AMP and cyclic GMP. In fraction P, an inhibition by cyclic AMP occurred also at a concentration of 10 −8 M, while with cyclic AMP a concentration of 10 −5 M was required for a similar inhibition. Bovine heart protein kinase stimulated the phosphorylation of the membrane fractions much more than Ca uptake. In fraction E, in the presence of bovine protein kinase, both cyclic AMP and cyclic GMP stimulated phosphorylation up to 200%. Under these conditions, no stimulation was observed in fraction P. These results are compatible with the hypothesis that in vascular smooth muscle soluble rather than particulate protein kinases are involved in the regulation of intracellular Ca concentration.

Characterization of the protein kinases in a transplantable islet cell tumor of the syrian hamster

The protein kinase activities of a transplantable, insulin-producing hamster islet cell tumor were characterized using gel filtration, sucrose density gradient centrifugation and acrylamide gel electrophoresis. The post-microsomal supernatant fluid contains 70–80% of the protein kinase activity present in crude homogenates. A cAMP-dependent protein kinase, PK I ( M r 170,000), represents 25% of the soluble protein kinase activity assayed with protamine as substrate. It dissociates in the presence of cAMP into a cAMP-binding protein, R 2 ( M r 90,000) and a catalytic subunit C ( M r 33,000). The dissociation induced by cAMP seems to be facilitated by the addition of Mg 2+ and ATP. The regulatory subunit, R 2, changes its gel filtration pattern in the presence of 0.5 M NaCl suggesting dissociation into a smaller subunit, R 1 ( M r 44,000). By analogy with purified beef heart protein kinase (Erlichman et al., 1973) and skeletal muscle protein kinase (Beavo et al., 1974), an R 2C 2 structure is proposed for the cAMP-dependent insulinoma protein kinase, PK I. The presence in crude homogenates of a free cAMP-binding protein indistinguishable from the R 2 derived by dissociation of PK I, suggests that PK I is partially dissociated in vivo. A cAMP-independent (casein) kinase ( M r 210,000) elutes with PK I on columns of Sepharose 6B. Another cAMP-independent protein kinase, PK II ( M r 88,000), is the predominant form of soluble protein kinase accounting for approximately 75% of the soluble protein kinase activity detected using protamine as substrate. This cAMP-independent protein kinase changes its gel filtration pattern in the presence of 0.5 M NaCl giving rise to a form which appears to have the same M r (33,000) as the catalytic subunit of PK I. Studies comparing the catalytic subunit C of PK I with PK II and its salt-induced smaller molecular form demonstrate facile association of C with the cAMP-binding protein of purified bovine heart protein kinase to yield a hybrid holoenzyme, whereas PK II and its smaller form fail to recombine in this fashion. The 33,000 dalton forms derived from PK I (by cAMP) and PK II (by salt) also show different substrate specificities. It would appear, therefore, that pK II is a cAMP-independent protein kinase unrelated to PK I.

Effect of calcium on cyclic AMP-dependent and cyclic GMP-dependent endogenous protein phosphorylation in mouse brain cytosol

In the presence of Ca ++ (>1mM), cyclic AMP (10μM) inhibited, and cyclic GMP (10μM) stimulated, the phosphorylation of a protein in the brain cytosol fraction of A/Jax mice. Lower concentrations of cyclic AMP stimulated phosphorylation of this protein, while lower concentrations of cyclic GMP had no effect. This protein (called protein 49) co-migrated on SDS - polyacrylamide gels with the endogenously phosphorylated band of partially purified bovine heart protein kinase, and was estimated to have an apparent molecular weight of 49,000. The significance of this Ca ++-dependent effect of the two cyclic nucleotides is discussed.

A protein kinase inhibitor in brown adipose tissue of developing rats.

A heat-stable protein was extracted from brown adipose tissue of infant rats that inhibited both purified bovine heart protein kinase and a crude preparation of protein kinase from brown fat. It did not act as an adenosine triphosphatase nor did it exert its effect by proteolytic action. Inhibition of protein kinase was affected in both the presence and the absence of cyclic AMP. Most of the inhibitory activity was present in the 100000g supernatant fraction of tissue homogenates. Inhibitory activity was highest perinatally and it declined 10 days after birth. It is suggested that the protein kinase inhibitor may play a role in regulating cyclic AMP actions.