Liver Cyclic AMP-dependent Protein Kinase Research Articles

Selective inhibition of eukaryote protein kinases by anti-inflammatory triterpenoids.

The ursane triterpenoid alpha-amyrin and the lupane triterpenoid lupeol are potent inhibitors of the catalytic subunit (cAK) of rat liver cyclic AMP-dependent protein kinase (PKA) with IC50 values of 8 and 5 microM, respectively. The palmitate and linoleate esters of alpha-amyrin and lupeol are also potent inhibitors of cAK (IC50 values in the range of 4-9 microM). alpha-Amyrin, lupeol and lupeol linoleate are much less potent as inhibitors of rat brain Ca(2+)- and phospholipid-dependent protein kinase (PKC) (IC50 values 32, 82 and 35 microM; respectively) and alpha-amyrin linoleate and the palmitate esters of lupeol and alpha-amyrin are ineffective or very poor inhibitors of this protein kinase. These compounds are very poor or ineffective as inhibitors of chicken gizzard calmodulin-dependent myosin light chain kinase (MLCK). alpha-Amyrin inhibits plant Ca(2+)-dependent protein kinase (CDPK) (IC50 52 microM) but lupeol and the triterpenoid esters tested are ineffective. alpha-Amyrin and the linoleate and palmitate esters of alpha-amyrin and lupeol inhibit cAK in a fashion that is competitive with respect to both peptide substrate and ATP (Ki values in the range 2-6 microM). However, while lupeol is competitive with respect to ATP it is apparently non-competitive with respect to peptide substrate. alpha-Amyrin also inhibits CDPK competitively and alpha-amyrin, lupeol and lupeol linoleate are competitive inhibitors of PKC. alpha-Amyrin and the palmitate esters of lupeol and alpha-amyrin are competitive inhibitors of the potato high affinity cyclic AMP-binding phosphatase (Pase) but lupeol inhibits the Pase non-competitively. These hydrophobic triterpenoids are further examples of anti-inflammatory triterpenoids that are cAK inhibitors.

Inhibition of eukaryote protein kinases and of a cyclic nucleotide-binding phosphatase by prenylated xanthones

A series of prenylated xanthones are variously potent inhibitors of the catalytic subunit (cAK) of rat liver cyclic AMP-dependent protein kinase (PKA), rat brain Ca 2+and phospholipid-dependent protein kinase C (PKC), chicken gizzard myosin light chain kinase (MLCK), wheat embryo Ca 2+-dependent protein kinase (CDPK) and potato tuber cyclic nucleotide-binding phosphatase (Pase). The prenylated xanthones examined are mostly derivatives of α-mangostin in which the 3-hydroxyl and 6-hydroxyl are variously substituted with groups R or R′, respectively, or derivatives of 3-isomangostin (mangostanol) in which the 9-hydroxyl is substituted with groups R′ or the prenyl side chain is modified. The most potent inhibitors of cAK have non-protonatable and relatively small R′ and R groups. Conversely, the most potent inhibitors of PKC and MLCK have bulkier and basic R′ groups. Some prenylated xanthones are also potent inhibitors of CDPK. PKC and cAK are competitively inhibited by particular prenylated xanthones whereas the compounds that are the most potent inhibitors of MLCK and CDPK are non-competitive inhibitors. Prenylated xanthones having relatively small and non-protonatable R′ and R groups inhibit a high-affinity cyclic nucleotide binding Pase in a non-competitive fashion.

Inhibition of eukaryote serine/threonine-specific protein kinases by piceatannol.

The protein tyrosine kinase (PTK) inhibitor piceatannol is also an inhibitor of the rat liver cyclic AMP-dependent protein kinase (PKA) catalytic subunit (cAK), rat brain Ca(2+)- and phospholipid-dependent protein kinase C (PKC), avian gizzard Ca(2+)-calmodulin-dependent myosin light chain kinae (MLCK), and of wheat embryo Ca(2+)-dependent protein kinase (CDPK) (IC50 values 3, 8, 12, and 19 microM, respectively). However, a number of piceatannol-related compounds with fewer or no phenolic hydroxy substituents are inactive or very poor inhibitors of these serine/threonine protein kinases. Similarly, the PTK inhibitor ellagic acid is a potent inhibitor of cAK and of PKC (IC50 values 2 and 8 microM, respectively), whereas the non-phenolic perylene is ineffective as a protein kinase inhibitor. Ellagic acid is a competitive inhibitor of both cAK and of PKC but piceatannol inhibits these enzymes in a fashion that is competitive and non-competitive, respectively. Interaction with calmodulin may contribute to the inhibition of MLCK and CDPK by piceatannol.

Inhibition of eukaryote protein kinases by isoquinoline and oxazine alkaloids.

The aporphine isoquinoline alkaloid apomorphine is a potent inhibitor of the catalytic subunit (cAK) of rat liver cyclic AMP-dependent protein kinase (PKA), myosin light chain kinase (MLCK), and Ca(2+)- and phospholipid-dependent protein kinase C (PKC) (IC50 values 1, 11, and 8 microM, respectively). However, a number of O-methylated analogues of apomorphine are inactive or poor inhibitors of cAK. The benzophenanthridine isoquinoline alkaloid sanguinarine is a potent inhibitor of cAK but is a relatively poor inhibitor of PKC (IC50 values 6 and 217 microM respectively). However a number of methylated analogues of sanguinarine are inactive as cAK inhibitors. The aporphine isoquinoline alkaloids (+)-boldine and bulbocapnine are non-competitive inhibitors of MLCK with respect to both peptide substrate and ATP. The inhibition of cAK, MLCK, and PKC by apomorphine and sanguinarine is competitive with respect to ATP as substrate. The oxazine alkaloids darrow red, nile blue A, and oxazine 170 are variously effective as inhibitors of cAK, MLCK, PKC, and CDPK (IC50 values 4-65 microM). Ca2+ binds to apomorphine and (+)-boldine which, together with nile blue A and oxazine 170, are potent inhibitors of calmodulin (CaM)-dependent MLCK (IC50 values 11, 12, 4, and 7 microM, respectively), and interact with dansyl-CaM.

Purification and characterization of basic proteins with in vitro antifungal activity from seeds of cotton, Gossypium hirsutum

Basic proteins from seeds of cotton ( Gossypium hirsutum) were purified by a procedure involving cation exchange chromatography on carboxymethyl cellulose (CM52) and reversed phase high performance liquid chromatography (HPLC). The basic protein fraction from these seeds contains a multiplicity of vicilin-derived 9–11 kDa proteins, a 16.3 kDa protein that has an N-terminal sequence nearly identical to that of a cotton cysteine-rich small vicilin protein and a 46.3 kDa protein having an N-terminal sequence with 61% identity and 75% similarity to the cDNA-based sequence of a γ-conglutin from Lupinus angustifolius. Edman N-terminal sequencing, electrospray ionization mass spectrometry (ESMS) and literature cDNA-based sequencing data were used to define the sequences of the vicilin-derived proteins. The inferred N- and C-terminal sequences of the 9–11 kDa proteins correspond to N-terminally truncated forms of cysteine-rich small vicilin proteins (such as the 16.3 kDa protein) suggesting that both could be processed from the vicilin ( α-globulin) preproprotein. The various cotton basic proteins were found to have selective growth inhibitory activity in vitro against the filamentous fungi Botrytis cinerea, Alternaria brassicicola, Chalara elegans and Fusarium oxysporum. These proteins differ, however, from numerous other seed antifungal proteins in being neither substrates nor inhibitors of signal transduction elements such as wheat germ Ca 2+-dependent protein kinase (CDPK), rat liver cyclic AMP-dependent protein kinase (PKA) catalytic subunit (cAK), rat brain Ca 2+- and phospholipid-dependent protein kinase (PKC) and chicken gizzard calmodulin-dependent myosin light chain kinase (MLCK).

Specific inhibition of cyclic amp-dependent protein kinase by warangalone and robustic acid

The prenylated isoflavone warangalone from the insecticidal plant Derris scandens is a selective and potent inhibitor of rat liver cyclic AMP-dependent protein kinase catalytic subunit (cAK) (IC 50 3.5 μM). The inhibition of rat liver cAK by warangalone is non-competitive with respect to both ATP and the synthetic peptide substrate (LRRASLG) employed in this study. Warangalone is a poor inhibitor of avian calmodulin-dependent myosin light chain kinase (MLCK), rat brain Ca 2+- and phospholipid-dependent protein kinase C (PKC) and wheat embryo Ca 2+-dependent protein kinase (CDPK). The related plant derived prenylisoflavones are also potent cAK inhibitors. Thus, 8-γ-γ-dimethylallylwighteone, 3′-γ-γ-dimethlallylwighteone and nallanin are inhibitors of cAK with IC 50 values in the range 20–33 μM. The prenyl-substituted isoflavones tested in this study are ineffective or poor as inhibitors of PKC. Thus nallanin is a poor PKC inhibitor (IC 50 value of 120 μM). The related isoflavones biochanin A and genistein are poor inhibitors of cAK (IC 50 values 100 μM and 126 μM, respectively). Genistein inhibits MLCK (IC 50 value 14 μM) but biochanin A is a poor MLCK inhibitor (IC 50 value 300 μM). The D. scandens prenyl-isoflavones and related isoflavones are ineffective inhibitors of wheat embryo Ca 2+-dependent protein kinase (CDPK). The 4-methoxy-3-phenyl-coumarin robustic acid is a potent inhibitor of rat liver cAK (IC 50 value 10 μM) but is a poor inhibitor of rat brain PKC, avian MLCK and wheat embryo CDPK. The coumarins 5-methoxypsoralen and 4,4′-di- O-methyl scandenin are poor cAK inhibitors (IC 50 values 240 and 248 μM, respectively). All of the non-prenylated coumarins examined are ineffective as inhibitors of the eukaryote signal-regulated protein kinases cAK, MLCK, PKC and CDPK. The selective, high affinity interaction of warangalone and robustic acid with cAK may contribute to their biological effects in vivo and to the insecticidal activity of the plant D. scandens.

Differential inhibition of eukaryote protein kinases by condensed tannins

Condensed tannins, isolated from a variety of plant sources, were characterized according to the constituent flavans, being based on procyanidin and/or prodelphinidin and having a cis or trans stereochemistry at positions 2 and 3. All the tannin preparations are potent inhibitors of rat liver cyclic AMP-dependent protein kinase catalytic subunit (cAK) with IC 50 values (concentrations for 50% inhibition) ranging from 0.009 to 0.2 μM. The tannin preparations are very good inhibitors of rat brain Ca 2+- and phospholipid-dependent protein kinase C (PKC) (IC 50 values in the range 0.3–7 μM), wheat embryo Ca 2+-dependent protein kinase (CDPK) (IC 50 values in the range 0.8–7 μM) and of calmodulin (CaM)-dependent myosin light chain kinase (MLCK) (IC 50 values in the range 7–24 μM). One of the most effective preparations, that from the leaves of Ribes nigrum, has IC 50 values with respect to cAK, PKC, CDPK and MLCK of 0.009, 0.6, 2.0 and 16 μM, respectively. In general, the order with respect to sensitivity to inhibition by these condensed tannins is cAK > PKC > CDPK > MLCK. The Ribes nigrum preparation is a competitive inhibitor of cAK with respect to both ATP and synthetic peptide substrate. These condensed tannin preparations are the most potent plant-derived inhibitors of cAK yet found.

Selective inhibition of cyclic AMP-dependent protein kinase by amphiphilic triterpenoids and related compounds

A set of plant- and animal-derived amphiphilic triterpenoids have been shown to be potent and selective inhibitors of the catalytic subunit of rat liver cyclic AMP-dependent protein kinase (cAK). Thus plant-derived 18α- and 18β-glycyrrhetinic acid, ursolic acid, oleanolic acid and betulin and animal-derived lithocholic acid, 5-cholenic acid and lithocholic acid methyl ester are inhibitors of cAK with IC 50 values (concentrations for 50% inhibition) in the range 4–20 μM. These compounds are ineffective or relatively ineffective as inhibitors of various other eukaryote signal-regulated protein kinases namely wheat embryo Ca 2+-dependent protein kinase (CDPK), avian calmodulin-dependent myosin light chain kinase (MLCK) and rat brain Ca 2+- and phospholipid-dependent protein kinase C(PKC). These naturally occurring triterpenoids have a common structural motif involving polar residues located at opposite ends of an otherwise non-polar triterpenoid nucleus. A variety of triterpenoids not possessing this structural motif are relatively inactive as inhibitors of cAK and of CDPK, PKC and MLCK. The terpenoid amphiphilic compound crocetin is also a potent and relatively selective inhibitor of cAK (IC 50 value for cAK 3.0 μM). 12-Hydroxystearic acid and 10-hydroxydecanoic acid do not inhibit CDPK, PKC or MLCK but are selective inhibitors of cAK (IC 50 values 127 and 138 μM, respectively), consistent with a simple model for amphiphile inhibition of cAK involving two polar groups separated by a non-polar region. However, laurylgallate and 15-pentadecanolide are also potent and selective inhibitors of cAK (IC 50 values 1.5 and 20 μM, respectively) although the structures of both of these compounds involve a large non-polar portion associated with only one polar region. Crocetin and the plant-derived amphiphilic triterpenoids described here are the most potent non-aromatic plant-derived inhibitors of cAK yet found.

Inhibition of signal-regulated protein kinases by plant-derived hydrolysable tannins

A variety of hydrolysable tannins purified from Phyllanthus amarus are potent inhibitors of rat liver cyclic AMP-dependent protein kinase catalytic subunit (cAK) with IC 50 values (concentrations for 50% inhibition) in the range 0.2–1.7 μM. The three most effective compounds of this series of hydrolysable tannins have five phenolic substituents. These three compounds are also the most effective inhibitors of wheat embryo Ca 2+-dependent protein kinase (CDPK), rat brain Ca 2+- and phospholipid-dependent protein kinase C (PKC) and Ca 2+-calmodulin-dependent myosin light chain kinase (MLCK). The order of sensitivity for protein kinase inhibition by the hydrolysable tannins studied is cAK>CDPK>PKC>MLCK. Thus the IC 50 values for protein kinase inhibition by the most potent compound are 0.2 μM (for cAK), 1.8 μM (for CDPK), 26 μM (for PKC) and 56 μM (for MLCK) when protein kinase affinity is measured using synthetic peptide substrates. These hydrolysable tannin inhibitors found are the most specific and potent plant-derived inhibitors of cAK yet found.

CAMP-dependent protein kinase and anoxia survival in turtles: purification and properties of liver PKA.

The catalytic subunit of turtle (Trachemys scripta elegans) liver cyclic AMP-dependent protein kinase (PKAc) was purified to homogeneity with a final specific activity of 65,783 pmol phosphate transferred.min-1.mg protein-1. Subunit molecular weight was 42-43 kDa as determined by SDS-PAGE and Sephacryl S-300 chromatography. The isolectric point was pH 6.41 +/- 0.02. Turtle liver PKAc showed highest activity with kemptide as its substrate; activity with other artificial substrates, histone IIA and protamine, was only 21 and 11%, respectively, of the activity with kemptide. Km values were 83 +/- 6.5 microM for Mg.ATP and 11.7 +/- 0.5 microM for kemptide and enzyme activity was strongly reduced by inhibitors of mammalian PKA (H-89, PKA-1) but not by inhibitors of other protein kinases. The enzyme was also inhibited by salts, especially fluoride salts (I50 about 30 mM), and showed a sharp break in the Arrhenius plot (0-45 degrees C) with activation energy increasing by 4-fold from 27.9 +/- 1.85 to 115 +/- 2.5 kJ/mol for temperatures above versus below 15 degrees C. Temperature effects may be important in suppressing PKA function, and therefore PKA-mediated responses, in vivo to enhance anoxic survival time during winter hibernation under water. Analysis of the effects of in vivo anoxia exposure at 7 degrees C on PKA in turtle organs showed a rapid 2.3-fold increase in the amount of active enzyme in liver within 1 h of anoxic submergence accompanied by a 60% increase cAMP levels; with longer anoxia (5 or 20 h) the percentage of active PKA was suppressed to 2.1-3.7% of the total.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific inhibition of cyclic AMP-dependent protein kinase by the antimalarial halofantrine and by related phenanthrenes.

The phenanthrenemethanol antimalarial halofantrine is a potent inhibitor of bovine heart and rat liver cyclic AMP-dependent protein kinase catalytic subunit (cAK) (IC50 values 2.1 microM and 0.6 microM, respectively). The inhibition of rat liver cAK by halofantrine is non-competitive with respect to both ATP and to the synthetic peptide substrate employed (LRRASLG). Halofantrine is a poor inhibitor of calmodulin-dependent myosin light chain kinase (MLCK) and wheat embryo Ca(2+)-dependent protein kinase (CDPK) and does not inhibit rat brain Ca(2+)- and phospholipid-dependent protein kinase C (PKC). In contrast, the acridine-based antimalarial quinacrine and a variety of quinoline-based antimalarials are very poor inhibitors of cAK, the best inhibitor being chloroquine (IC50 for bovine heart cAK, 80 microM). Quinacrine and the quinoline-based antimalarials variously inhibit CDPK, PKC and MLCK albeit at relatively high concentrations (about 1 to 4 x 10(-4) M), the best inhibitors found being primaquine, pentaquine and mefloquine (IC50 values for MLCK 49, 103 and 33 microM, respectively). A number of phenanthrene derivatives having a 9-hydroxy or 9-keto substituent, namely phenanthrenequinone, 6(5H)-phenanthridinone and 9-phenanthrol are potent inhibitors of bovine heart cAK (IC50 values 8, 10 and 10 microM, respectively) and of MLCK (IC50 values 6, 53 and 10 microM, respectively). The selective, high affinity interaction of halofantrine with cAK may contribute to biological effects in vivo of this clinically-employed antimalarial compound.

Inhibition of rat liver cyclic AMP-dependent protein kinase by flavonoids.

Rat liver cyclic AMP-dependent protein kinase catalytic subunit (cAK), assayed using the synthetic peptide substrate, LRRASLG, is inhibited by a range of plant-derived flavonoids. In general, maximal inhibitory effectiveness (IC50 values 1 to 2 microM) requires 2,3-unsaturation and polyhydroxylation involving at least two of the three flavonoid rings. 3-Hydroxyflavone (IC50 value 4 microM), 3,5,7,2',4'-pentahydroxyflavone (IC50 = 10 microM) and 5,7,4'-trihydroxyflavone (IC50 = 7 microM) represent somewhat less active variations from this pattern. Flavonoid O-methylation or O-glycosylation greatly decreases inhibitory effectiveness, as does 2,3-saturation. Various flavonoid-related compounds, notably gossypol (IC50 = 10 microM), also inhibit cAK. Flavonoids and related compounds are in general much better inhibitors of cAK than of avian Ca(2+)-calmodulin-dependent myosin light chain kinase or of plant Ca(2+)-dependent protein kinase. Tricetin (IC50 = 1 microM) inhibits cAK in a fashion that is non-competitive with respect to both peptide substrate and ATP (Ki value 0.7 microM). When histone III-S is used as a substrate, inhibition of cAK requires much higher flavonoid concentrations.

Inhibition of wheat embryo calcium-dependent protein kinase and other kinases by mangostin and γ-mangostin

The hull of the fruit of the mangosteen tree ( Garcinia mangostana) contains four inhibitors of plant Ca 2+-dependent protein kinase. Two of these inhibitors have been purified and identified as the xanthones 1,3,6-trihydroxy-7-methoxy-2,8-bis(3-methyl-2-butenyl)-9 H-xanthen-9-one (mangostin) and 1,3,6,7-tetrahydroxy-2,8-bis(3-methyl-2-butenyl)-9 H-xanthen-9-one (γ-mangostin). Both xanthones also inhibit avian myosin light chain kinase and rat liver cyclic AMP-dependent protein kinase. This is the first report of inhibition of plant and animal second messenger-regulated protein kinases by plant-derived xanthones.

The amounts of rat liver cyclic AMP-dependent protein kinase I and II are differentially regulated by diet

1. The fluctuations in rat hepatocyte volume and protein content in response to dietary perturbations (starvation, protein restriction, refeeding) were accompanied by corresponding fluctuations in the amount of the regulatory (R) and catalytic (C) subunits of cyclic AMP-dependent protein kinase. Thus the intracellular concentration of this key enzyme was adjusted to be near constant. 2. The adjustment of cellular R was accomplished almost exclusively by regulating cytosolic RI (R subunit of type I kinase). The preferential down-regulation of cytosolic RI in response to starvation/protein restriction indicates that particulate RI and cytosolic as well as particulate RII are more resistant to breakdown during general catabolism in the hepatocyte. 3. The diet-induced fluctuations of kinase subunits were uniformly distributed in all populations of parenchymatous hepatocytes, regardless of their size and density. It is thus possible to isolate hepatocytes with uniformly altered RI/RII ratio from livers of rats with different feeding regimens. 4. The binding of endogenous cyclic AMP to RI and RII was similar in livers with high RI/RII ratio (fed rats) and low RI/RII ratio (fasted rats) as well as in hepatocytes isolated from fasted rats. Under the conditions of the experiment (short-term stimulation by glucagon), therefore, neither the dietary state nor the RI/RII ratio seemed to affect the apparent affinity of the isoreceptors for cyclic AMP. However, RI appeared to show a slightly higher co-operativity of intracellular cyclic AMP binding than did RII in all states.

Synthetic fragments of protamines as model substrates for rat liver cyclic AMP-dependent protein kinase

Nine synthetic peptides reproducing either exactly or with suitable substitutions three phosphorylatable sites of the protamines thynnine Z1 and galline have been prepared and tested as phosphate acceptors for the rat liver cyclic AMP-dependent protein kinase (type I). The most significant results obtained can be summarized as follows: 1. The hexapeptide: Arg-Arg-Ser-Thr-Val-Ala gives two phpsphorylated products, containing only Ser-P and both Ser-P and Thr-P, respectively. The relative amount of the di-phosphorylated derivative is not dependent on the incubation time but rather on the concentration of the substrates. 2. Both the replacements of ornithine for the two N-terminal arginines of glutamic acid for Val 5 in the above peptide completely prevent phosphorylation, without conferring any inhibitory activity to the modified peptides, thus supporting the view that the N-terminal guanido groups and the C-terminal hydrophobic residue(s) are both required for the binding at the catalytic site rather than for the subsequent transphosphorylation reaction. 3. The replacement of alanine for Ser 3 gives rise to a peptide whose Thr 4 residue is still phosphorylated with an efficiency comparable to that of the unmodified peptide. The di-substituted peptide: Arg-Arg-Ala-Ser-Val-Ala however exhibits a dramatically lower K m value indicating that serine is a much better target than threonine whenever it is not adjacent to the N-terminal arginine couple. 4. The importance of the distance between the target residue and the N-terminal basic determinants is also evidenced by the phosphorylation of the dodecapeptide: Pro-(Arg) 5-Ser-Ser-Arg-Pro-Val-Arg exhibiting a K m value about 20-times higher than that of salmine A1, whose phosphorylation site is comprised within an identical amino acid sequence, including however three rather than two adjacent serine residues. 5. The tetradecapeptide: (Arg) 4-Tyr-Gly-Ser-(Arg) 6-Tyr is completely unaffected by the kinase though a very similar site is found phosphorylated in native iridines, probably through a cyclic AMP-independent mechanism.

Epinephrine-stimulated phosphorylation of pyruvate kinase in hepatocytes

Incubation of rat liver parenchymal cells with 10 −5 m epinephrine or norepinephrine resulted in a rapid incorporation of 32P into pyruvate kinase. Inclusion of α-adrenergic blocking agents (phenoxybenzamine or phentolamine) in the hepatocyte incubation medium prior to addition of epinephrine suppressed the subsequent phosphorylation of pyruvate kinase. On the other hand, inclusion of the β-adrenergic antagonist, propranolol, in the hepatocyte incubation medium prior to addition of epinephrine did not suppress the epinephrine-elicited phosphorylation of pyruvate kinase. Exogenous addition of either cyclic AMP or cyclic GMP to the hepatocyte incubation medium also resulted in increased phosphorylation of pyruvate kinase. To investigate whether the same amino acid residue(s) of liver pyruvate kinase was being phosphorylated in each instance, 32P-labeled pyruvate kinase was isolated from hepatocytes after incubation in the presence or absence of either glucagon or epinephrine. In addition, purified liver pyruvate kinase was phosphorylated in vitro with a rat liver cyclic AMP-dependent protein kinase. Each 32P-labeled pyruvate kinase was then subjected to tryptic digestion, two-dimensional thin-layer peptide mapping, and autoradiography. Each 32P-labeled pyruvate kinase sample yielded 44 to 48 tryptic peptides upon staining with ninhydrin and 4 peptides that contain 32P as detected by autoradiography. Furthermore, the same 4 peptides of pyruvate kinase were radiolabeled in each instance. Thus phosphorylation of pyruvate kinase in vitro with [γ- 32P]ATP or upon addition of either glucagon or epinephrine to hepatocytes incubated with 32 P i resulted in phosphorylation of the same amino acid residues.

Purification and characterization of the catalytic subunit of adenosine 3':5'-cyclic monophosphate-dependent protein kinase from bovine liver

1. The catalytic subunit of bovine liver cyclic AMP-dependent protein kinase (EC2.7.1.37) was purified essentially by the method of Reimann & Corbin [(1976) Fed. Proc. Fed. Am. Soc. Exp. Biol. 35, 1384]. 2. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, sedimentation-velocity centrifugation and sedimentation-equilibrium centrifugation showed that the catalytic subunit was monodisperse. Polyacrylamide-gel isoelectric-focusing electrophoresis revealed the presence of at least three isoenzyme forms of catalytic subunit activity with slightly different pI values (6.72, 7.04 and 7.35). 3. Physical properties of the catalytic subunit were determined by several different methods. It had mol.wt. 39000-42000, Stokes radium 2.73-3.08 nm, so20.w 3.14S, f/fo 1.19-1.23 and, assuming a prolate ellipsoid, axial ration 4-5. 4. Amino acid analysis was performed on the catalytic subunit. It had one cysteine residue/molecule which was essential for activity. Inhibition by thiol-specific reagents was partially prevented by the presence of ATP-Mg2+. 5. The circular-dichroic spectrum showed the catalytic subunit contained 29% alpha-helical form, 18% beta-form and 53% aperiodic form. Near-u.v. circular dichroism showed the presence of aromatic residues whose equivalent molar ellipticity was greatly altered by the addition of ATP-Mg2+. 6. Kinetic experiments showed that the catalytic subunit had an apparent Km for ATP of 7 muM. 5'-Adenylyl imidodiphosphate inhibitied competitively with ATP with a Ki of 60 muM. The kinetic plot for histone (Sigma, type II-A) was biphasic showing 'high'-and 'low'-Km segments. Under assay conditions the specific activity of the catalytic subunit was 3 X 10(6) units/mg of protein. Of various metal ions tested, the catalytic subunit was most active with Mg2+.7. When assayed with histone (Sigma, type II-A) as substrate, the activity of the catalytic subunit was increased by non-ionic detergents or urea. No such activation was observed with casein as substrate.

Effect of polyanions and polycations on adenosine 3′,5′- monophosphate-binding and protein kinase activity

Histone, protamine, poly-L-arginine, and poly-L-lysine enhance the binding of adenosine 3′,5′-monophosphate (cyclic AMP) to rat liver cyclic AMP-dependent protein kinase as determined by Millipore filtration assay. Poly-L-glutamic acid and poly-L-aspartic acid suppress cyclic AMP-binding stimulated by histone. Poly-L-glutamic acid and poly-L-aspartic acid are effective against protein kinase and result in decrease in initial reaction velocity when histone is used as a protein substrate. Incubation of cyclic AMP-dependent protein kinase with 6 μg poly-L-glutamic acid produces half-maximal inhibition of cyclic AMP-dependent protein kinase when 30 μg histone is used as substrate.