Effect of adenosine 3',5'-cyclic monophosphate derivatives on alpha-amylase release, protein kinase and cyclic nucleotide phosphodiesterase activity from rat parotid tissue.

Several 8-substituted derivatives of cyclic AMP were tested for their effects on alpha-amylase release. None of the 8-substituted compounds were more active than N6,O2-dibutyryl- or N6-monobutyryl adenosine 3',5'-monophosphate in causing alpha-amylase release. The rat parotid was found to contain a high (105 muM) and a low (1.15 muM) Km cyclic AMP phosphodiesterase activity. All of the 8-substituted cyclic AMP compounds inhibited the hydrolysis of 1 muM cyclic AMP. However, there was only a partial correlation between the ability to cause alpha-amylase release and inhibit cyclic AMP hydrolysis. Extracts of parotid tissue contained a cyclic AMP-dependent protein kinase activity. None of the compounds were as effective as cyclic AMP in activating the protein kinase. As in the case of inhibition of cyclic AMP hydrolysis, the ability of the 8-substituted cyclic AMP compounds to increase protein kinase activity did not correlate with their effects on alpha-amylase release. It is concluded that factors in addition to the in vitro inhibition of cyclic AMP hydrolysis and activation of protein kinase are important in determining the net result of the 8-substituted cyclic AMP compounds on parotid gland function. These additional factors might include differences in the rate of uptake and differences in rats of conversion to compounds with modified activity.     

8-Substituted derivatives of adenosine 3',5'-cyclic phosphate require an unsubstituted 2'-hydroxyl group in the ribo configuration for biological activity.

Derivatives of adenosine 3',5'-cyclic phosphate (cAMP) with modifications in both the 2' and the 8 positions were synthesized and their enzymic activities as activators of cAMP-dependent protein kinase and as substrates for and inhibitors of cAMP phosphodiesterases were determined. Three types of derivatives were investigated: 8-substituted derivatives of O2'-Bt-cAMP, 8-substituted derivatives of 9-beta-D-arabinofuranosyladenine 3',5'-cyclic phosphate (ara-cAMP), and 8-substituted derivatives of 8,2'-anhydro-9-beta-D-arabinofuranosyladenine 3,'5'-cyclic phosphate (8,2'-anhydro-cAMP). The 8-substituted O2'-Bt-cAMP derivatives were synthesized by acylation of the preformed 8-substituted cAMP (8-HS-cAMP, 8-MeS-cAMP, and 8-PhCH2S-cAMP). 8-Br-O2'-tosyl-cAMP was sued as an intermediate for the preparation of 8,2'-anhydro-cAMP derivatives (8-HO-, 8-SH-, 8-H2N-, and 8-H3 CHN derivatives of 8,2'-anhydro-cAMP). 8-Substituted ara-cAMP derivatives were obtained by ring opening of 8-HO-8,2'-anhydro-cAMP with H+/H2O, NH3/MeOH, or MeONa/MeOH (to yield the 8-HO-, 8-H2N-, and 8-MeO-ara-cAMP derivatives). All of these doubly modified derivatives of cAMP are less than one-hundredth as active as cAMP at activating protein kinase and did not serve as substrates for the phosphodiesterase. These data show that the general inactivity of 2' derivatives of cAMP with kinase was not overcome by addition of an 8-substituent, even though many 8-substituted derivatives of cAMP activate the kinase more efficiently than does cAMP itself. In addition they show that while 2'-modification were tolerated by the phosphodiesterase, addition of an 8-substituent countermanded the allowable 2'-modification. The 8-substituted derivates of 02'-Bt-cAMP were found in general to be slightly better inhibitors of phosphodiesterase than the parent compounds containing no o2'-Bt substitution. As a group, the 8-substituted ara-cAMP derivatives were poorer inhibitors of phosphodiesterase than 8-substituted cAMP derivatives while the 8,2'-anhydro-cAMP derivatives were much poorer inhibitors than the 8-substituted ara-cAMP derivatives.     

Phosphorylation of regulatory subunit of type I cyclic AMP-dependent protein kinase: biphasic effects of cyclic AMP in intact S49 mouse lymphoma cells

Intact S49 mouse lymphoma cells were used as a model system to study the effects of cyclic AMP (cAMP) and its analogs on the phosphorylation of regulatory (R) subunit of type I cAMP-dependent protein kinase. Phosphorylation of R subunit was negligible in mutants deficient in adenylate cyclase; low levels of cAMP analogs, however, stimulated R subunit phosphorylation in these cells to rates comparable to those in wild-type cells. In both wild-type and adenylate cyclase-deficient cells, R subunit phosphorylation was inhibited by a variety of N6-substituted derivatives of cAMP; C-8-substituted derivatives were generally poor inhibitors. Two derivatives that were inactive as kinase activators (N6-carbamoylmethyl-5'-AMP and 2'-deoxy-N6-monobutyryl-cAMP) were also ineffective as inhibitors of R subunit phosphorylation. Preferential inhibition by N6-modified cAMP analogs could not be ascribed simply to selectivity for the more aminoterminal (site I) of the two cAMP-binding sites in R subunit: Analog concentrations required for inhibition of R subunit phosphorylation were always higher than those required for activation of endogenous kinase; 8-piperidino-cAMP, a C-8-substituted derivative that is selective for cAMP-binding site I, was relatively ineffective as in inhibitor; and, although thresholds for activation of endogenous kinase by site I-selective analogs could be reduced markedly by coincubation with low levels of site II-selective analogs, no such synergism was observed for the inhibitory effect. The uncoupling of cyclic nucleotide effects on R subunit phosphorylation from activation of endogenous protein kinase suggests that, in intact cells, activation of cAMP-dependent protein kinase requires more than one and fewer than four molecules of cyclic nucleotide.     

Use of nucleotide photoaffinity probes to study hormone action

It has been clearly shown that the action of several hormones is differentially mediated intracellularly by nucleotides containing either adenosine or guanosine base units. To study the protein-nucleotide interactions involved in several complex biological systems our laboratory has synthesized several 8-azido-adenosine (8-N3 A) and 8-azidoguanosine (8-N3 G) derivatives of naturally occurring nucleotides. Modification of the nucleotides in the 8-position of the purine ring was done because: a) 8-substituted derivatives of cAMP and cGMP activated their respective protein kinases at physiological concentrations and were much less susceptible to hydrolysis by specific phosphodiesterases (PDE's) and b) substitution at the 8-position was much less likely to disturb the preferential and selective binding of adenosine versus guanosine nucleotides by enzymes that are specifically regulated by such interactions. This would allow studies of guanosine nucleotide specific binding in the presence of both adenosine nucleotides and adenosine nucleotide binding proteins, and vice-versa. In general, such has been the case and [32P] 8-N3 cAMP and [32P] 8-N3 cGMP have been used effectively to study their respectively activated protein kinases in several systems. Also, [32P] 8-N3 ATP has been used to study several ATPases and kinases while [gamma 32P] 8-N3 GTP has been shown effective for studies on tubulin and the G-regulatory protein (G/N) of adenylyl cyclase (A.C.). Several observations suggest that there must be important physical and energetic tie-ins between external hormone binding and the loading and unloading of specific internal nucleotide binding sites. These binding sites may be activator signals for protein kinases (e.g., cAMP protein kinase regulatory subunit), or cyclases (e.g., G/N proteins of A.C.) or catalytic sites involved in the production or hydrolysis of cyclic nucleotides. The thrust of this article is to detail the use of 8-azidopurine photoaffinity analogs of ATP, GTP, cAMP and cGMP as they may be used to study hormone-mediated events which may or may not involve cyclic nucleotides as a second messenger.     

Cyclic adenosine 3':5'-monophosphate in moss protonema: a comparison of its levels by protein kinase and gilman assays

From the protonema of the moss Funaria hygrometrica (L.) Sibth, a factor indistinguishable from cyclic adenosine 3′:5′-monophosphate (cAMP) has been isolated. The factor stimulated the activity of protein kinase from rabbit skeletal muscle and co-chromatographed with authentic cAMP in two solvent systems. Its ability to stimulate protein kinase activity was completely abolished by 3′:5′-cyclic nucleotide phosphodiesterase, the rate of inactivation being similar to that of authentic cAMP. Based on these properties, this factor is identified as 3′,5′-cAMP. Cyclic AMP could be readily removed from the cells and washing the cells with water reduced the endogenous level of cAMP by 2- to 3-fold. A comparison of cAMP levels by protein kinase and Gilman assays was made. The intracellular levels determined by protein kinase assay were about 7-fold lower than the values obtained by Gilman assay. This discrepancy was due to the presence of unidentified compounds which were completely degraded by 3′:5′-cyclic nucleotide phosphodiesterase. Although these displaced labeled cAMP in the Gilman assay, they did not stimulate the protein kinase activity. The protonema may contain cyclic nucleotides other than cAMP; these will not be detected in the protein kinase assay due to the specificity of this reaction. The crude extracts were found to be unsuitable for assaying cAMP by either method.     

2-substituted derivatives of adenosine and inosine cyclic 3',5'-phosphate. Synthesis, enzymic activity, and analysis of the structural requirements of the binding locale of the 2-substituent on bovine brain protein kinase.

A number of 2-substituted cyclic nucleotide derivatives were synthesized and investigated as activators of cAMP-dependent protein kinase and as substrates for and inhibitors of cAMP phosphodiesterase. Ring closure of 5-amino-1-beta-D-ribofuranosylimidazol-4-carboxamide cyclic 3',5'-phosphate (1) with various aldehydes according to a new procedure (Meyer, R. B., Jr., Shuman, D.A., and Robins, R. K. (1974), J. Am. Chem. Soc. 96, 4962) gave new derivatives of adenosine cyclic 3',5'-phosphate with the following 2-substituents: n-propyl, n-hexl, n-octyl, n-decyl, styryl, o-methoxyphenyl, and 2-thienyl. Alkylation of 2-mercaptoadenosine cyclic 3',5'-phosphate (20, Meyer et al., 1974) gave new cAMP derivatives with the following 2-substituent: ethylthio, n-propylthio, isopropylthio, allylthio, n-decylthio, and benzylthio. Deamination of 2-methyl-,2-n-butyl-, and 2-ethylthioadenosine cyclic 3',5'-phosphate. Using multiple regression analysis, a striking relationship was found between the relative potency of the compounds as activators of bovine brain cAMP-dependent protein kinase and parameters describing the hydrophobic, steric, and electronic character of the substituents on these compounds. All compounds were substrates for a cyclic nucleotide phosphodiesterase preparation from rabbit kidney. Additionally, the compounds were as a group, good inhibitors of the hydrolysis of cAMP by phosphodiesterase preparations from rabbit lung, beef heart, and dog heart.     

Cyclic nucleotide metabolism in the human erythrocyte

Erythrocytes, which show little or no guanylate or adenylate cyclase activity, take up cyclic nucleotides from blood. Studies were done by incubating human erythrocytes in isotonic medium with the dibutyryl derivatives of cAMP and cGMP and in a hypotonic medium in which the cells are partially hemolyzed and, therefore, freely permeable to cAMP and cGMP. At cAMP and cGMP concentrations of 50 microM and above, the amount of 14CO2 generated from 1-14C-glucose was decreased significantly. This effect was suppressed by 4.6 mM theophylline. Inosine and ribose, which are catabolites of cAMP and cGMP also decreased formation of 14CO2 from 1-14C-glucose. Accordingly, it is postulated that in the absence of theophylline, the activity of phosphodiesterase resulted in AMP and GMP formation. These mononucleotides enter into the purine salvage pathways to form ribose phosphate. Additionally, the production of lactate and 2,3-diphosphoglycerate (2,3-DPG) was measured in human erythrocytes after incubation with the dibutyryl derivatives of cAMP (bt2-cAMP) and cGMP (bt2-cGMP). At a concentration of 0.1 microM, bta2-cGMP inhibits lactate production at 60 min (p less than 0.01). Slight increases in 2,3-DPG were not statistically significant. Catabolism of cyclic nucleotides may prevent diffusion equilibria allowing for the erythrocyte's continuous uptake of cyclic nucleotides and providing a detoxification mechanism that could compensate for conditions in which elevations of these substances are observed.     

Effects of Cyclic AMP on the Activity of Soluble Protein Kinases in Lemna paucicostata

Three protein kinases which phosphorylate histone were isolatedfrom cellular extract of Lemna plants. They were separated byelution from DEAE-Sephacel column and referred to as PI, PITand PHI. The PI protein kinase activity was partially inhibitedby 10µM cyclic AMP, cyclic GMP or cyclic IMP, while thePII enzyme was activated in the presence of these cyclic nucleotides.The PIII enzyme was cAMPindependent, but slightly inhibitedby cyclic CMP and cyclic UMP. The molecular weights of thesethree protein kinases were 165,000, 85,000 and 145,000, respectively,as estimated from Sephacryl S-300 gel filtration. A single cyclicAMP-binding protein was detected in the PII enzyme fractionby using the photoaffinity cAMP-analogue, 8-N₃-cAMP. The proteinwhich specifically bound [³H]-8-N₃-cAMP had an apparent molecularweight of 48,000 as determined by SDS-polyacrylamide gel electrophoresis.The phosphorylation of cellular proteins in Lemna was examinedby SDS-polyacrylamide gel electrophoresis. Four phosphorylatedpolypeptides were detected, the phosphorylations of which werestimulated by cAMP. The molecular weights of these four polypeptideswere 59,000, 19,000, 16,000 and 14,000, respectively. (Received January 26, 1983; Accepted April 13, 1983)     

Synthesis and enzymic activity of 8-acyl and 8-alkyl derivatives of guanosine 3, 5-cyclic phosphate.

Several new 8-alkyl and 8-acyl derivatives of quanosie 3',5'-cyclic phosphate (cGMP) and inosine 3',5'-cyclic phosphate (cGMP) were prepared by direct alkylation or acylation of the parent cyclic nucleotide via free radicals generated in situ. These compounds have been examined for their ability to stimulate a cGMP-dependent protein kinase, and several of the cGMP derivatives were as active in this regard as cGMP. These compounds proved to be quite ineffective when tested for their ability to activate an adenosine 3',5'-cyclic phosphate (cAMP) dependent protein kinase. In addition, these 8-substituted cGMP derivatives are not substrates for a phosphodiesterase preparation from rabbit kidney, but do show inhibition of the hydrolysis of cAMP by crude phosphodiesterase preparations from rabbit lung and beef heart.     

The effect of cyclic AMP on the growth and morphology of a normal human fibroblast parent strain and its transformed progeny line.

A normal human diploid fibroblast cell strain, Lederle 130 (Led 130), and its virus-transformed progeny line, transformed Led 130, were subjected to 0.75 and 1.5 mM concentrations of adenosine-5'-monophosphate (AMP), cyclic AMP (cAMP) and dibutyryl cyclic AMP (Bt2cAMP). While cAMP was markedly inhibitory to neoplastic cells at 1.5 mM, Bt2-cAMP was even more effective at this concentration, producing 85% inhibition by 4 days and 91% inhibition by 6 days. Bt2-cAMP was the only nucleotide to reverse morphological transformation effects in the neoplastic fibroblasts. Normal fibroblasts were inhibited in growth rate to a comparable extent by all nucleotides, and were not altered morphologically.     

Adenosine cyclic 3',5'-monophosphate uptake and regulation of membrane protein kinase in intact human erythrocytes

The uptake of adenosine cyclic 3',5'-monophosphate (cAMP) and stimulation of membrane-associated protein kinase in mature human erythrocytes were investigated. cAMP transport across the membrane was temperature dependent, and cAMP binding to the isolated membrane had less temperature dependence. More than 99% of the [3H]-cAMP taken up by erythrocytes was nonmembrane bound. Maximal stimulation of membrane protein kinase and maximal occupancy of membrane cAMP binding sites by extracellular cAMP cccurred at 30 degrees C within 30 min after initiation of the incubation of erythrocytes with cAMP. The concentration of extracellular cAMP that gave half-maximal stimulation of membrane protein kinase was 5.4 X 10-4 M, a value consistent with the concentrations of cAMP (5.2 X 10-4 M) found to occupy half-maximally the membrane cAMP binding sites in erythrocytes. Extracellular cAMP and to a lesser extent guanosine cyclic 3',5'-monophosphate and inosine cyclic 3',5'-monophosphate stimulated membrane protein kinase in erythrocytes. The cAMP uptake by human erythrocytes as well as cAMP binding to membranes in the erythrocyte was blocked by an inhibitor [4,4'-bis(isothiocyano)stilbene-2,2-disulfonate] of the anion channel. These studies indicate that cAMP can be transported across membranes into human erythrocytes and can bind to membranes to activate membrane protein kinase. It appears that there is a shared transport channel for cAMP and anion transport.     

Biotinyl-l-3-(2-naphthyl)-alanine hydrazide derivatives of N-glycans: versatile solid-phase probes for carbohydrate-recognition studies

Biotinyl-oligosaccharides are a relatively new generation of saccharide probes that enable immobilization of desired oligosaccharides on streptavidin matrices for studies of carbohydrate-protein interactions. Here we describe the facile preparation of biotinyl-l-3-(2-naphthyl)- alanine hydrazide (BNAH) derivatives of oligosaccharides, containing a strong UV absorbing and fluorescent group, in which the ring of the reducing-end monosaccharide is nonreduced. We evaluate reactivities of immobilized BNAH- N -glycans with plant lectins that recognize aspects of the oligosaccharide core or outer-arms. We make some comparisons with 2-amino-6-amidobiotinyl-pyridine (BAP) derivatives obtained by reductive amination, and 6-(biotinyl)-aminocaproyl-hydrazide (BACH) derivatives which have a longer spacer-arm. N -Glycan-BNAH and-BAP derivatives have, overall, comparable reactivities with lectins which recognize N -glycan outer-arms or the trimannosyl core, but only BNAH and BACH derivatives are bound by lectins which recognize the non- reduced core. Moreover, with Pisum sativum agglutinin (PSA) which additionally requires the fucosyl- N- glycan-asparaginyl core for high affinity binding, the immobilized BNAH derivative (which is an alanine hydrazide beta-glycoside) can substitute for the natural beta- glycosylasparaginyl core, whereas the BACH derivative (aminocaproyl- hydrazide-beta-glycoside) is less effective. BNAH is a derivatization reagent of choice, therefore, for solid phase carbohydrate-binding experiments with immobilized N -glycans.      

Mechanism of activation of protein kinase I from rabbit skeletal muscle. Mapping of the cAMP site by spin-labeled cyclic nucleotides.

Binding of adenosine 3':5'-monophosphate (cAMP) to protein kinase (type I) from rabbit skeletal muscle has been investigated using spin-labeled cAMP derivatives. Different compounds were synthesized with the spin label attached by spacer chains of different length at different positions on the adenine base. Immobilization of the spin label, determined by comparing the electron-spin resonance spectra recorded in the presence of the kinase with those of the free ligand in solutions of different viscosities, gave information about the geometry of the cAMP site. Strong immobilization of the N-6 substituents up to a spacer length of seven atoms indicates a rather deep cleft of the cAMP site. The depth of this cleft differs, however, when the spin label is attached to the different positions at the adenine (N-6, C-2 and C-8). Whereas the N-6 derivatives indicate a rather deep site, the C-2 derivatives reveal a significantly smaller depth and C-8 substituents (syn conformation) obviously occupy a very shallow surface with almost no immobilation. In addition the binding affinities of the spin-labeled cAMP derivatives have been determined, together with those of a series of (diamagnetic) C-2 derivatives bearing hydrophobic alkyl chains of different length. The latter results helped to clarify the differences between the regions near to C-2 and N-6, respectively, of the cAMP site. N-6 spin-labeled derivatives have also been investigated in the presence of ATP and protein kinase. These results are interpreted as indicative of a conformational change at the cAMP site upon formation of the holoenzyme, due to binding of ATP, leaving cAMP less strongly immobilized.     

Endogenous phosphorylation of sarcoplasmic reticulum fragments of rabbit fast skeletal muscles

The purified membrane fragments of sarcoplasmic reticulum (SR) of rabbit fast skeletal muscles were found to incorporate 32P from[gamma-32P]ATP in endogenous membrane substrates and in histone H1. The existence of membrane-bound protein kinase of SR was demonstrated by steady state binding of [3H]-cAMP to the SR membranes. The constant of [3H]cAMP binding to the membranes is 2.5 +/- 0.003 x 10(6) M-1, the number of binding sites is 6.1 +/- 0.8 pmol per 1 mg of protein. The endogenous phosphorylation of SR components was inhibited by cAMP and cGMP at concentrations of 10(-7)-10(-6) and depended on Mg2+ and Ca2+. The thermostable protein inhibitor of cAMP-dependent protein kinase inhibited the endogenous phosphorylation of SR membranes by 30-40%. The protein phosphoproduct of SR membranes revealed the properties of a phosphoester. The membrane-bound protein kinase was active towards the exogenous substrate--histone H1. Phosphorylation in the presence of histones was independent of cyclic nucleotides, Mg2+ and Ca2+. Fractionation of 32P-labelled solubilized membranes in polyacrylamide gel in the presence of Na-SDS showed that the radioactivity is bound to protein zones with molecular weights of 95 000 and 6000.     

Fate of Cyclic Nucleotides in PC12 Cell Cultures: Uptake, Metabolism, and Effects of Metabolites on Nerve Growth Factor-Induced Neurite Outgrowth

The fate of cyclic AMP (cAMP), dibutyryl-cAMP (Bt2-cAMP), and the (Sp)-isomer of adenosine 3',5'-monophosphorothioate [(Sp)-cAMPS] was studied in the PC12 culture medium by means of HPLC. In the absence of PC12 cells, cAMP and Bt2-cAMP were rapidly degraded by nonspecific esterases and cyclic nucleotide phosphodiesterase both originating from the serum commonly used as a culture medium ingredient, whereas (Sp)-cAMPS was completely stable. Since 5'-AMP, adenosine, inosine, and hypoxanthine appeared in the culture medium after incubation with cAMP or Bt2-cAMP, we have determined their effect on nerve growth factor (NGF)-induced neurite outgrowth. 5'-AMP, adenosine, and inosine were indeed potent agents in producing a potentiating effect on NGF-induced early neurite outgrowth at a concentration of 1 mM. Thus, cAMP metabolites had the capacity to induce an effect that has been described as cAMP-specific. In serum-free culture medium and in the presence of cells, all cyclic nucleotides were taken up by PC12 cells. Uptake was highly correlated with the hydrophobic nature of the compounds, and was accompanied by a simultaneous excretion of metabolites. On incubation with cAMP, NGF had a pronounced effect on the metabolic pattern found in the culture medium. In particular, dephosphorylation of 5'-AMP was specifically enhanced. This effect of NGF on the degradation of cAMP was also apparent when cAMP metabolites were incubated with PC12 cells. Whereas 5'-AMP degradation was greatly increased, NGF had no effect on the metabolism of the other purine compounds.     

Cyclic nucleotide-dependent protein kinases in airway smooth muscle

Because of the potential importance of cyclic nucleotide-dependent protein kinases in the regulation of airway smooth muscle tone, we have examined some of the characteristics of these enzymes in the soluble fraction of canine trachealis homogenates. In the absence of added cAMP, the heat-stable cAMP-dependent protein kinase inhibitor (PKI) abolished only a half of the 32P incorporation into mixed histones. The remaining activity appeared to be contributed by a cyclic nucleotide-independent enzyme. Phosphotransferase activity was enhanced 5-fold by 5 microM cAMP but only 70% of the cAMP-stimulated activity could be inhibited by PKI. The sensitivity of the cyclic nucleotide-dependent, PKI-resistant enzyme to cAMP, cGMP, and Mg2+ indicated that it was cGMP-dependent protein kinase. Because of the large amount of cyclic nucleotide-independent activity, and the ability of cAMP to activate cGMP-dependent protein kinase, the traditional "-cAMP/+cAMP" ratio did not provide an accurate assessment of the in vivo activation state of cAMP-dependent protein kinase. However, a modified assay was developed which allowed the precise measurement of cAMP-dependent, cGMP-dependent, and cyclic nucleotide-independent protein kinase activities. Using this new method, the cAMP-dependent protein kinase activity ratio of 0.239 in untreated trachealis strips was increased to 0.355 and 0.386 by prior exposure of the intact tissue to the smooth muscle relaxants isoproterenol and prostaglandin E2, respectively. The results of this study are consistent with the proposed role of cAMP-dependent protein kinase in the regulation of smooth muscle contractile function.     

A comparison of the uptake, metabolism, and action of cyclic adenine nucleotides in cultured hepatoma cells.

Intracellular radioactivity following incubation of HTC or RLC cells in [³H]cAMP exceeds that following incubation in either [³H]mono- or dibutyryl cAMP by 30-fold, yet little [³H]cAMP is found within the cells. Even at early times (30 min) the label derived from [³H]cAMP is predominantly found in ADP or ATP, suggesting it mostly enters the cell as the nucleoside. Significant intracellular concentrations of monobutyryl cAMP (2–10 μm) result from incubation of both cell lines in either N⁶ mono- or dibutyryl cAMP. A very small percentage of this label is in cAMP, and within 2 h of incubation > 65% of the label is again found in ADP or ATP.Liver cytosol contains three major cAMP-dependent protein kinases, designated A, B, and C, as resolved by DEAE-Sephadex chromatography. cAMP is the most effective in vitro activator (10- to 16-fold stimulation) of kinases A and B, the preponderant forms, in the order cAMP > N⁶ monobutyryl cAMP ? dibutyryl cAMP. Kinase C, a minor fraction, was stimulated two to threefold with the order cAMP ≥ N⁶ monobutyryl cAMP > dibutyryl cAMP. HTC and RLC cell cytosol protein kinase has Chromatographic and cyclic nucleotide activation properties similar to those of liver fraction C.The activation state of the protein kinases of HTC and RLC cells incubated in the various cyclic nucleotides was also studied. The ability of such nucleotides to occupy regulatory protein binding sites in intact cells (as determined by the inhibition of subsequent in vitro binding of [³H]cAMP) was of the order N⁶ monobutyryl cAMP > dibutyryl cAMP > cAMP > untreated cells. Correspondingly, the ratio of basal protein kinase activity in cyclic nucleotide treated:control cells was higher in cells incubated in monobutyryl cAMP > dibutyryl cAMP > cAMP. This in vivo activation suggests that little additional stimulation would be obtained by adding cAMP to extracts prepared from such cells. This activation can be expressed as the ratio ? cAMP: + cAMP (a ratio of 1 being maximal activation). The highest such ratio was seen in cells which had been incubated in monobutyryl cAMP > dibutyryl cAMP > cAMP > untreated cells. The studies indicate that all three cyclic nucleotides are capable of activating protein kinase in intact RLC and HTC cells; however the monobutyryl derivative is the most effective, and the degree of stimulation is greater in RLC than in HTC cells.RLC cell tyrosine aminotransferase activity is increased two to threefold by butyrylated cAMP derivatives (but not by cAMP) whereas the HTC cell enzyme is not induced. The rate of replication of both lines is unaltered by the butyrylated compounds.Since HTC and RLC cells accumulate and metabolize cAMP and its derivatives equally, and since they both contain a protein kinase with similar in vivo and in vitro activation properties, it is suggested that the effects of butyrylated cAMP derivatives on cell replication and tyrosine aminotransferase induction are mediated separately, either by distinct protein kinases, or at a point distal to protein kinase, or by a mechanism independent of protein kinase.     

Effects of cAMP-binding site mutations on intradomain cross-communication in the regulatory subunit of cAMP-dependent protein kinase I

Each protomer of the regulatory subunit dimer of cAMP-dependent protein kinase contains two tandem and homologous cAMP-binding domains, A and B, and cooperative cAMP binding to these two sites promotes holoenzyme dissociation. Several amino acid residues in the type I regulatory subunit, predicted to lie in close proximity to each bound cyclic nucleotide based on affinity labeling and model building, were replaced using recombinant techniques. The mutations included replacement of 1) Glu-200, predicted to hydrogen bond to the 2'-OH of cAMP bound to site A, with Asp, 2) Tyr-371, the site of affinity labeling with 8-N3-cAMP in site B, with Trp, and 3) Phe-247, the position in site A that is homologous to Tyr-371 in site B, with Tyr. Each mutation caused an approximate 2-fold increase in both the Ka(cAMP) and Kd(cAMP); however, the off-rates for cAMP and the characteristic pattern of affinity labeling with 8-N3-cAMP differed markedly for each mutant protein. Furthermore, these mutations affect the cAMP binding properties not only of the site containing the mutation, but of the adjacent nonmutated site as well, thus confirming that extensive cross-communication occurs between the two cAMP-binding domains. Photoaffinity labeling of the native R-subunit results in the covalent modification of two residues, Trp-260 and Tyr-371, by 8-N3-cAMP bound to sites A and B, respectively, with a stoichiometry of 1 mol of 8-N3-cAMP incorporated per mol of R-monomer (Bubis, J., and Taylor, S. S. (1987) Biochemistry 26, 3478-3486). A stoichiometry of 1 mol of 8-N3-cAMP incorporated per R-monomer was observed for each mutant regulatory subunit as well, even when 2 mol of 8-N3-cAMP were bound per R-monomer; however, the major sites of covalent modification were altered as follows: R(Y371/W), Trp-371; R(E200/D), Tyr-371, and R(F247/Y), Tyr-371.     

Diastereomers of adenosine 3',5'-monothionophosphate (cAMP[S]) antagonize the activation of cGMP-dependent protein kinase

cGMP-dependent protein kinase contains four cGMP-binding sites which are homologous to the four cAMP-binding sites of cAMP-dependent protein kinase. The interaction of the diastereomers of adenosine 3',5'-thionophosphate, (PS)-cAMP[S] and (PR)-cAMP[S], with cGMP-dependent protein kinase has been studied. Autophosphorylation of cGMP-dependent protein kinase is stimulated by cAMP and (PS)-cAMP[S] with apparent KA values of 7 microM and 94 microM, respectively. cAMP-stimulated autophosphorylation is inhibited competitively by (PR)-cAMP[S] with a Ki value of 15 microM. The phosphorylation of the peptide substrate (Leu-Arg-Arg-Ala-Ser-Leu-Gly) is stimulated by cGMP (approx. KA 1 microM) and cAMP (approx. KA 98 microM) but neither by the (PR) nor (PS) stereoisomer of cAMP[S]. (PR)-cAMP[S] and (PS)-cAMP[S] inhibit competitively cAMP-or cGMP-stimulated phosphorylation of the peptide substrate with Ki values of 52 microM and 73 microM, respectively. (PS)-cAMP[S] stimulates the phosphorylation of the peptide substrate by an autophosphorylated enzyme. Binding of [3H]cGMP to cGMP-dependent protein kinase is inhibited by (PS)-cAMP[S] and (PR)-cAMP[S] with IC50 values of 200 microM and 15 microM, respectively. These results show that both diastereomers of cAMP[S] bind to cGMP-dependent protein kinase. (PR)-cAMP[S] has properties of a pure antagonist whereas (PS)-cAMP[S] has properties of a partial agonist. The results provide further evidence that autophosphorylation of the enzyme affects the interaction between the cGMP-binding sites and the catalytic center of the enzyme by facilitating the activation of the phosphotransferase reaction.     

Partial purification and characterization of adenosine- and guanosine-3',5'-monophosphate phosphodiesterases from human lung tissue.

Crude extracts of human lung tissue were examined for cyclic adenosine- and guanosine-3',5'-monophosphate (cAMP and cGMP) phosphodiesterase activities. Nonlinear reciprocal plots were observed for each substrate. DEAE-Sephadex chromatography of the extracts revealed four main fractions of activity, which were further purified by Sephadex gel filtration. The phosphodiesterase activity of the resulting individual fractions was partially characterized with respect to substrate specificity, kinetic parameters, apparent molecular weight (gel filtration), thermal stability at 30 and 37 degrees C, effect of the cyclic nucleotide not utilized as substrate, and the possible influence of Ca2+-dependent protein activator. The results indicate that the tissue contains phosphodiesterases with strict specificity and a high apparent affinity for each of the two cyclic nucleotides (the Km values determined were approximately 0.3-0.4 muM). The high affinity cAMP phosphodiesterase activity was enriched in two of the purified fractions; both activities probably represent fragments of the native high affinity cAMP specific enzyme. A third purified phosphodiesterase showed mixed substrate specificity. The Km value recorded for hydrolysis of either substrate with this enzyme was approximately 25 muM. A fourth, irregularly occurring, phosphodiesterase activity also showed mixed substrate specificity. The Km value registered for hydrolysis of either substrate with this fraction was approximately 0.4 muM. There was no evidence for a Ca2+-dependent specific activation by a boiled lung tissue supernatant of any of the purified enzymes.