Protein kinases of retinal rod outer segments: identification and partial characterization of cyclic nucleotide dependent protein kinase and rhodopsin kinase

Protein kinase activity of dark-adapted bovine rod outer segments is partitioned by centrifugation into soluble and membrane-bound fractions. The soluble kinases are separated by DEAE-cellulose chromatography into three peaks of activity, which can be classified by substrate specificity and cyclic nucleotide dependence into two categories. One peak of protein kinase activity has the characteristics reported for rhodopsin kinase (category one); it phosphorylates only bleached rhodopsin, and its activity is not affected by light, exogenous adenosine cyclic 3',5'--monophosphate (cAMP), guanosine cyclic 3',5'-monophosphate (cGMP), or a protein kinase inhibitor from skeletal muscle. Rhodopsin kinase has an apparent molecular weight of 68 000. The second category of kinase includes two peaks of activity which are stimulated severalfold by cAMP or cGMP but not by light. These protein kinases phosphorylate soluble proteins including histones and a protein kinase substrate prepared from rat intestine but not rhodopsin. The two peaks elute from DEAE-cellulose with 0.09 and 0.20 M KCl, suggesting that they are similar respectively to type I and type II cyclic nucleotide dependent protein kinases that have been characterized in other tissues. The activity of type I kinase is variable and much less than that of the type II enzyme; its molecular weight was not determined. The type II protein kinase has an apparent molecular weight of 165 000. This study confirms that different protein kinase enzymes catalyze selectively the phosphorylation of bleached rhodopsin and soluble proteins, and it repudiates the speculation in a previous publication [Farber, D. B., Brown, B. M., & Lolley, R. N. (1979) Biochemistry 18, 370-378] that a single protein kinase might catalyze both phosphorylation reactions.     

On a soluble system for studying light activation of rod outer segment cyclic GMP phosphodiesterase

Bovine rod outer segment (ROS) cyclic GMP phosphodiesterase (PDE) could be activated about 6-fold by light, an effect that could be simulated by isolated bleached rhodopsin. About 90% of PDE activity in ROS could be extracted with 10 mM Tris-HCl, pH 7.5, but light is ineffective in activating the soluble enzyme. However, bleached rhodopsin could activate it in the presence of a very low concentration of ATP, strongly suggesting the mediation of rhodopsin in the light activation of the enzyme in ROS. Direct evidence is presented to suggest that the phosphorylation of opsin (bleached rhodopsin) is unrelated to the activation of PDE by bleached rhodopsin and ATP. The reconstitution of the light activation of PDE in a soluble system presented here opens up a new direction to future investigations on the mechanism of light regulation of cyclic GMP levels in retina and its implication in the photoreceptor function.     

Induction of phosphodiesterase by cyclic adenosine 3':5'-monophosphate in differentiating Dictyostelium discoideum amoebae.

Cyclic adenosine 3':5'-monophosphate added to the starvation media of Dictyostelium discoideum amoebae induces both intracellular and extracellular phosphodiesterase activities of these cells. The induced enzyme activity appears several hours earlier than that in starved cells which have not been induced with cyclic nucleotide. In both cases, the appearance of enzyme is inhibited by cycloheximide, and actinomycin D, and daunomycin. The KmS for the extracellular enzyme(s) of nucleotide-induced and uninduced control cells are identical. The induction of enzyme activity seems specific for cyclic adenosine 3':5'-monophosphate since cyclic guanosine 3':5'-monophosphate, as well as other nucleotides, have no effect. No differences in the activity or excretion of either N-acetylglucosaminidase or the inhibitory of the extracellular phosphodiesterase are observed between cyclic adenosine 3':5'-monophosphate-induced and control cells. A direct activation of phosphodiesterase by cyclic adenosine 3':5'-monophosphate can be excluded, since the addition of this nucleotide to cell lysates has no effect on the enzyme activity.     

Cyclic AMP-dependent and -independent protein kinases of the water mold, Blastocladiella emersonii.

Protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) and cyclic adenosine 3',5'-monophosphate binding activities have been identified in zoospore extracts of the water mold Blastocladiella emersonii. More than 75% of these activities is found in the soluble fraction. Soluble protein kinase activity is resolved in three peaks(I, II and III) by DEAE-cellulose chromatography. Peak I is casein dependent and insensitive to cyclic AMP. Peak II is histone dependent and cyclic AMP independent; this enzyme is inhibited by the heat-stable inhibitor from bovine muscle. Peak III utilizes histone as substrate and is activated by cyclic AMP.     

Binding of adenosine 3',5'-monophosphate dependent protein kinase regulatory subunit to immobilized cyclic nucleotide derivatives.

Several cyclic nucleotide derivatives with aminoalkyl side chains attached to the purine ring were synthesized and their interactions with adenosine 3',5'-monophosphate (cAMP) dependent protein kinase were studied before and after immobilization to CNBr-activated Sepharose 4B. The soluble N6-substituted derivatives were as effective as cAMP itself in activating protein kinase and were more effective than 8-substituted cAMP derivatives, whereas the 2-substituted cAMP derivatives and the cGMP derivatives were the least effective. All of the synthetic derivatives tested were poor substrates for beef heart phosphodiesterase being hydrolyzed at rates less than 2% for that of cAMP itself. Utilizing methodology developed to evaluate the affinity of protein kinase for immogilized cyclic nucleotides it was found that all of the immobilized cyclic nucleotides interacted with protein kinase in a biospecific manner as judged by the following criteria: (1) the immobilized cyclic nucleotides competed with cAMP for the binding sites on protein kinase; (2) the analogous spacer-arm did not compete; and (3) the effects of enzyme concentration, MgATP, and cleavage of the cyclic phosphate ring on the interactions of protein kinase with the immobilized cyclic nucleotides were the same as previously shown for free cAMP. In addition, the immobilized ligands were bound with the same order of effectiveness as the analogous soluble ligand. The observed Ka for the activation of 0.005 muM protein kinase by N6-H2N(CH2)2-cAMP was increased from 0.23 to 3 muM by the process of immobilization. This increase was unaffected by the coupling density and spacer-arm length. The observed Kb for 0.10 muM protein kinase binding to immobilized N6-H2N(CH2)2-cAMP was increased as the molecular sieving exclusion limit of the matrix used was decreased indicating that at least part of this decrease in apparent affinity upon immobilization is due to exclusion of the enzyme from a portion of the matrix and therefore of the immobilized ligand molecules.     

Nuclear protein-kinase activity in perfused rat liver stimulated with dibutyryl-adenosine cyclic 3':5'-monophosphate.

Cytoplasmic and nuclear protein kinase activities from perfused rat liver have been studied in response to dibutyryl-adenosine cyclic 3':5'-monophosphate added at a concentration that stimulates hepatic gluconeogenesis (100 muM). Total nuclear protein kinase, as assayed using a mixed histone fraction as phosphate acceptor, is increased by 5-fold within 8 min of the addition of cyclic nucleotide to the perfusate. In contrast the total cytoplasmic protein kinase activity is decreased to 50% of the control value. The protein substrate specificity of the protein kinase that is present in the nucleus in response to dibutyryl-adenosine cyclic 3':5'-monophosphate stimulation is similar to that of cytoplasmic, adenosine cyclic 3':5'-monophosphate-dependent, protein kinase but is distinct from that of the enzyme(s) present in control nuclei. The predominant species to protein kinase from stimulated nuclei has a sedimentation constant of 3.9 S. This value is identical to that of the catalytic subunit of cytoplasmic adenosine 3':5'-monophosphate-dependent protein kinase. These data suggest that some of the effects of adenosine 3':5'-monophosphate on nuclear events may be mediated through its interaction with the inactive protein kinase holoenzyme in the cytoplasm and the subsequent redistribution of the active catalytic subunits generated by this interaction.     

Activation of bovine rod outer segment phospholipase C by arrestin.

Phospholipase C (PLC) enzyme activity in rod outer segment (ROS) membranes bleached in the presence of ATP and GTP was assayed using exogenously added [3H]phosphatidylinositol 4,5-bisphosphate vesicles as substrate. The addition of the soluble ROS protein arrestin (also known as S-antigen or 48K protein) to ROS membranes activated PLC 2-3.4-fold. This activation was dose-dependent, and maximal activation was observed at an arrestin concentration of congruent to 110-220 nM. PLC activation by arrestin was dependent on ROS protein concentration and free Ca2+. Soluble PLC (s-PLC) enzyme activity present in hypotonic extracts of bleached ROS was also activated 2-4-fold by arrestin. Maximum activation of s-PLC by arrestin was observed at free Ca2+ of 80 nM. Arrestin activation of s-PLC was not affected by urea-treated and extensively washed ROS membranes, suggesting that rhodopsin was not required for the observed effect of arrestin on s-PLC. The results are indicative of a direct interaction of arrestin with s-PLC, resulting in the activation of the latter. Based on these results and the documented binding of arrestin to bleached and phosphorylated rhodopsin, a model for the light activation of PLC in ROS is proposed.     

Partial purification and properties of guanosine 3':5'-monophosphate-dependent protein kinase from pig lung.

Guanosine 3':5'-monophosphate(cyclic GMP)-dependent protein kinase which catalyzes the phosphorylation of histone was purified about 200-fold from the soluble fraction of pig lung by pH 5.5 precipitation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration. The apparent Ka values for guanosine 3':5'-monophosphate and adenosine 3':5'-monophosphate were determined to be about 17 and 360 nM, respectively. Mg2+ was essential for the activity exhibiting biphasic stimulation behavior and neither Mn2+ nor Ca2+ could substitute for Mg2+. However, these divalent ions markedly inhibited the protein kinase activity stimulated by cyclic GMP in the presence of Mg2+.     

Comparison of mode of activation of guanosine 3':5'-monophosphate-dependent and adenosine 3':5'-monophosphate-dependent protein kinases from silkworm.

Guanosine 3':5'-monophosphate (cyclic GMP)-dependent protein kinase (protein kinase G) partially purified from silkworm pupae was selectively activated by cyclic GMP at lower concentrations. Nevertheless, the enzyme seemed to differ from adenosine 3':5'-monophosphate-dependent protein kinase (protein kinase A) with respect to the mode of response to cyclic nucleotides. The catalytic activity and cyclic GMP-binding activity were not dissociated by cyclic GMP in a manner similar to that described for protein kinase A. The enzyme was not inhibited by regulatory subunit of protein kinase A nor by protein inhibitor. A sulfhydryl compound such as 2-mercaptoethanol or glutathione was essential for the activation by cyclic GMP, and an extraordinary high concentration of either Mg2+ (100 mM) or Mn2+ (25 mM) was needed for maximal stimulation by cyclic GMP. A polyamine such as spermine, spermidine, or putrescine could substitute partly for the cation. Kinetic analysis indicated that Km for ATP was decreased whereas Ka for cyclic GMP was increased significantly at high concentrations of the cation. The effect of the cation to decrease Km for ATP was not evident in the absence of a sulfhydryl compound. These characteristics of protein kinase G described above were not observed for protein kinase A which was obtained from the same organism.     

Changes in cyclic AMP-dependent protein dinase activity in Tetrahymena pyriformis during the growth cycle.

An adenosine 3':5'-monophosphate-dependent protein kinase II (ATP:protein phosphotransferase, EC 2.7.1.37) was partially purified from the cytosol fraction of an exponentially growing culture of Tetrahymena pyriformis. Protein kinase II represented approximately 90% of the cytosolic protein kinase activity. The enzyme had a high degree of substrate specificity for calf thymus and Tetrahymena histones as compared to casein, protamine and phosvitin. The enzyme incorporated the terminal phosphate of ATP into serine and threonine residues of all the histone fractions. The apparent Km of the enzyme for adenosine 3':5'-monophosphate (cyclic AMP) was 1-10-minus 8 M. Protein kinase II was also activated by other cyclic nucleotides with apparent Km values in the range 2.k-10-minus 6 M. Ther specific activity of the cyclic AMP-dependent protein kinase of Tetrahymena decreases markedly from initial high values during the transition from the lag to early log phase of growth. This is followed by a shrp increase in the activity of the enzyme as the log phase of growth progresses. The specific activity of the enzyme increases rapidly during the heat-induced synchronization of Tetrahymena cells. The capacity for rapid phosphorylation of multiple classed of organelle-specific phosphoproteins and the level of cyclic AMP were maximal in Tetrahymena during the earliest phase of growth. These results demonstrate that the cell cycle of Tetrahymena may be coordinated by marked variations in the level of cyclic AMP which in turn regulate the cyclic AMP-dependent protein kinase.     

Comparison of cyclic nucleotide specificity of guanosine 3',5'-monophosphate-dependent protein kinase and adenosine 3',5'-monophosphate-dependent protein kinase.

Guanosine 3',5'-monophosphate-dependent protein kinase (cyclic GMP-dependent protein kinase) and adenosine 3',5'-monophosphate-dependent protein kinase (cyclic AMP-dependent protein kinase) exhibited a high degree of cyclic nucleotide specificity when hormone-sensitive triacylglycerol lipase, phosphorylase kinase, and cardiac troponin were used as substrates. The concentration of cyclic GMP required to activate half-maximally cyclic dependent protein kinase was 1000- to 100-fold less than that of cyclic AMP with these substrates. The opposite was true with cyclic AMP-dependent protein kinase where 1000- to 100-fold less cyclic AMP than cyclic GMP was required for half-maximal enzyme activation. This contrasts with the lower degree of cyclic nucleotide specificity of cyclic GMP-dependent protein kinase of 25-fold when histone H2b was used as a substrate for phosphorylation. Cyclic IMP resembled cyclic AMP in effectiveness in stimulating cyclic GMP-dependent protein kinase but was intermediate between cyclic AMP and cyclic GMP in stimulating cyclic AMP-dependent protein kinase. The effect of cyclic IMP on cyclic GMP-dependent protein kinase was confirmed in studies of autophosphorylation of cyclic GMP-dependent protein kinase where both cyclic AMP and cyclic IMP enhanced autophosphorylation. The high degree of cyclic nucleotide specificity observed suggests that cyclic AMP and cyclic GMP activate only their specific kinase and that crossover to the opposite kinase is unlikely to occur at reported cellular concentrations of cyclic nucleotides.     

A link between rhodopsin and disc membrane cyclic nucleotide phosphodiesterase. Action spectrum and sensitivity to illumination.

Frog (Rana pipiens) rod outer segment disc membranes contain guanosine 3',5'-cyclic monophosphate phosphodiesterase (EC 3.1.4.1.c) which, in the presence of ATP, is stimulated 5- to 20-fold by illumination. The effectiveness of monochromatic light of different wavelengths in activating phosphodiesterase was examined. The action spectrum has a maximum of 500 nm, and the entire spectrum from 350 to 800 nm closely matches the absorption spectrum of rhodopsin, which is apparently the pigment which mediates the effects of light on phosphodiesterase activity. trans-Retinal alone does not mimic light. Half-maximal activation of the phosphodiesterase occurs with a light exposure which bleaches 1/2000 of the rhodopsins. Half-maximal activation can also be achieved by mixing 1 part of illuminated disc membranes in which the rhodopsin is bleached with 99 parts of unilluminated membranes. Regeneration of bleached rhodopsin by addition of 11-cis-retinal is illuminated disc membranes reverses the ability of these membranes to activate phosphodiesterase in unilluminated membranes. If the rhodopsin regenerated by 11-cis-retinal is illuminated again, it regains the ability to activate phosphodiesterase. These studies show that the levels of cyclic nucleotides in vetebrate rod outer segments are regulated by minute amounts of light and clearly indicate that rhodopsin is the photopigment whose state of illumination is closely linked to the enzymatic activity of disc membrane phosphodiesterase.     

Regulation by light of cyclic nucleotide-dependent protein kinases and their substrates in frog rod outer segments

Cyclic nucleotides (both cAMP and cGMP) stimulate the phosphorylation of several proteins of 65-70, 50-52, 21, 13, and 12 kD in rod outer segments (ROS) of the frog retina. Subcellular fractionation showed that phosphopeptides of 67, 21, 13, and 12 kD were soluble and phosphopeptides of 69, 67, 50-52, and 12 kD were membrane associated at physiological ionic strength. Components I and II, 13 and 12 kD, respectively, are the major cyclic nucleotide-dependent phosphoproteins of ROS and have been reported to be phosphorylated in the dark and dephosphorylated in the light. Under unstimulated conditions, phosphorylated Components I and II were found in the soluble fraction. Cyclic nucleotide stimulation of phosphorylation resulted in increased phospho-Components I and II in the soluble fraction, and phospho-Component II on the membrane. Light had no effect on the phosphorylation level of soluble Components I and II, but it caused a depletion within 1 s of the membrane-bound phospho-Component II. A half-maximal decrease in membrane-bound Component II was seen at 5 x 10(5) rhodopsins bleached per outer segment. The cyclic nucleotide-dependent protein kinase(s) were found primarily in the peripheral membrane fraction of ROS proteins. 8-bromo cyclic AMP was two orders of magnitude more effective than 8-bromo cyclic GMP at stimulating Component I and II phosphorylation. An active peptide of the Walsh inhibitor of cAMP-dependent protein kinase [PKI(5-22)amide] blocked the phosphorylation with an IC50 of 10 nM. Photoaffinity labeling studies with 8-N3-cAMP and 8-N3-cGMP revealed the presence of a 52-kD band specifically labeled with 8-N3-cAMP, but no specific 8-N3-cGMP labeling. These data suggest that cyclic nucleotide-dependent protein phosphorylation in ROS occurs via the activation of a cAMP-dependent protein kinase.     

Stimulation of rhodopsin phosphorylation by guanine nucleotides in rod outer segments

Porcine rod outer segment (ROS) proteins were phosphorylated in the presence of [gamma-32P]ATP and Mg2+, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and detected by autoradiography. The phosphorylation of rhodopsin, the major protein-staining band (Mr approximately 34 000-38 000), was markedly and specifically increased by exposure of rod outer segments to light; various guanine nucleotides (10 microM) including GMP, GDP, and GTP also specifically increased rhodopsin phosphorylation (up to 5-fold). Adenine nucleotides (cyclic AMP, AMP, and ADP at 10 microM) and 8-bromo-GMP (10 microM) or cyclic 8-bromo-GMP (10 microM) had no detectable stimulatory effect on rhodopsin phosphorylation. GTP increased the phosphorylation of rhodopsin at concentrations as low as 100 nM, and guanosine 5'-(beta, gamma-imidotriphosphate), a relatively stable analogue of GTP, was nearly as effective as GTP. Maximal stimulation of rhodopsin phosphorylation by GTP was observed at 2 microM. GMP and GDP were less potent than GTP. Both cyclic GMP and GMP were converted to GTP during the time period of the protein phosphorylation reaction, suggestive of a GTP-specific effect. Transphosphorylation of guanine nucleotides by [32P]ATP and subsequent utilization of [32P]GTP as a more effective substrate were ruled out as an explanation for the guanine nucleotide stimulation. With increasing concentrations of ROS proteins, the phosphorylation of rhodopsin was nonlinear, whereas in the presence of GTP (2 microM) linear increases in rhodopsin phosphorylation as a function of added ROS protein were observed. These results suggest that GTP stimulates the phosphorylation of rhodopsin by ATP and that a GTP-sensitive inhibitor (or regulator) of rhodopsin phosphorylation may be present in ROS.     

Activity of protein kinase dependent on adenosine 3':5'-monophosphate and of its thermostable protein inhibitor in rat hepatoma (HTC) cells. Unlikely role in the permissive action of glucocorticoids.

Normal expression of a variety of hormonal effects which depend on cyclic AMP (adenosine 3':5'-monophosphate) requires the presence of glucocorticoids. Our hypothesis was that glucocorticoids control directly or indirectly the activity of cyclic-AMP-dependent protein kinase. This has been investigated in cultured hepatoma (HTC) cells in which N6,O2'-dibutyryladenosine 3':5'-monophosphate increases the activity of tyrosine transaminase only after glucocorticoid treatment. In these cells, we have determined the concentration and half-life of protein kinase, the sensitivity of this enzyme in vitro to cyclic AMP and to its thermostable protein inhibitor, the state of dissociation of protein kinase holoenzyme in vivo and its sensitivity, in the intact cell, to dibutyryladenosine 3':5'-monophosphate and to the inhibitor diamide, and we have also determined the concentration of endogenous thermostable protein inhibitor of protein kinase. None of these parameters were influenced by glucocorticoids under conditions where these hormones stimulate the activity of tyrosine transaminase and restore sensitivity to dibutyryladenosine 3':5'-monophosphate. It is concluded that the permissive action of glucocorticoids probably results from a control of cyclic-AMP-dependent processes exerted at a level beyond the protein kinase system.     

一氧化氮信号通路与血管功能

一氧化氮-环磷鸟苷-依赖蛋白激酶信号通路是血管功能调制的关键机制之一,本文对这一机制近年来来研究进展,特别是相关信号分子二聚体化的氧化还原调节及生理意义,及可溶性鸟苷酸环化酶催化产生的环磷肌苷作为一个新的血管收缩信使分子作简要介绍。     

Guanosine 3':5'-monophosphate-dependent protein kinase from bovine cerebellum. Purification and characterization.

Guanosine 3':5'-monophosphate (cyclic GMP)-dependent protein kinase was assayed with calf thymus histone as substrate and partially purified from the soluble fraction of bovine cerebellum. The enzyme was selectively activated by cyclic GMP at lower concentrations; the Ka value for cyclic GMP was 1.7 times 10- minus 8 M whereas that for adenosine 3':5'-monophosphate (cyclic AMP) was 1.0 times 10- minus 6 M. The Km value for ATP was 1.0 times 10- minus 5 M. A high concentration of Mg-2+ (100 mM) was needed for maximum stimulation by cyclic GMP and maximum reaction rate. The pH optimum was 7.5 to 8.0. The isoelectric point was pH 5.7. The molecular weight was about 140,000 as estimated by gel filtration. The enzyme was unable to activate muscle glycogen phosphorylase kinase, and was clearly distinguishable from cyclic AMP-dependent protein kinase in kinetic and catalytic properties. Comparative data on cyclic GMP-dependent and cyclic AMP-dependent protein kinases in this tissue are presented.     

Photoaffinity labelling of central-nervous-system myelin. Evidence for an endogenous type I cyclic AMP-dependent kinase phosphorylating the larger subunit of 2'',3''-cyclic nucleotide 3''-phosphodiesterase.

Endogenous cyclic AMP-stimulated phosphorylation of a 49700-Mr Wolfgram protein component in rabbit central nervous system was investigated by using photoaffinity labelling and 2',3'-cyclic nucleotide 3'-phosphodiesterase activity staining after electroblotting on to nitrocellulose paper. Photoaffinity labelling with 8'-azidoadenosine 3',5'-cyclic monophosphate showed a cyclic AMP-binding protein that appeared to be intrinsic to the myelin membrane and appeared to represent the R-subunit of a type I cyclic AMP-dependent protein kinase. This photoaffinity-labelled protein was of larger apparent Mr than the protein showing cyclic AMP-stimulated phosphorylation. Blotting of one-dimensional sodium dodecyl sulphate/polyacrylamide-gel electrophoretograms followed by staining for 2',3'-cyclic nucleotide 3'-phosphodiesterase activity showed two activity bands corresponding to the two components of the Wolfgram protein doublet. Cyclic AMP-stimulated protein phosphorylation corresponded to the upper component of this doublet. Electroblotting of two-dimensional non-equilibrium pH-gradient electrophoretograms also showed co-migration of cyclic AMP-stimulated protein phosphorylation with enzyme activity. It is proposed that central-nervous-system myelin contains an endogenous type I cyclic-AMP dependent protein kinase that phosphorylates the larger subunit of 2',3'-cyclic nucleotide 3'-phosphodiesterase.     

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.     

Interaction of 8-substituted derivatives and adenosine-3',5'-cyclophosphate esters with protein kinase from pig brain]

A synthesis of previously unknown 8-substituted derivatives and alkyl esters of cyclic adenosine-3',5'-monophosphate, containing reactive groups, was carried out. The interaction of the compounds obtained with a homogeneous preparation of protein kinase from pig brain was studied. It was found that all compounds, with the exception of neutral esters of 3',5'-AMP, activate the enzyme and competitively inhibit 3H-labelled 3',5'-cAMP binding by the regulatory subunit of protein kinase. The activating effect and affinity of 8-(beta-aminoethylamino)-3',5'-cAMP for protein kinase was 10 times lower than that for 3',5'-cAMP and other 8-substituted derivatives of the cyclic nucleotide. It was found that 8-(N-chloroacetylaminoethylamino)-3',5'-cAMP interaction with the enzyme is of irreversible type, which suggest covalent blocking of the nucleophilic group of the 3',5'-cAMP binding site of protein kinase. The data obtained indicate that the 3',5'-cAMP molecule is bound to the regulatory site of protein kinase in the syn-conformation. The previously made assumption on the crucial importance of the negative charge in the 3',5'-cyclophosphate system for the interaction of cyclic AMP with the regulatory subunit of protein kinase has been thus confirmed.