Titration Journal

E r J. Biochem. 40,177-185 (1973) u. intra prison boothular Tit rotterion of cyclicalalalalalalalalalalalalalalalalalalalalal adenylic blistering Bound to Receptor Proteins and coefficient of correlation with cyclic- angstrom Levels in the Surviving betray hitch Lien DO KHAC,Simone HARBON Hubert J. CLAUSER and lnstitut de Biochimie, Universit6 de Paris-Sud, Orsay (Received April 9/July 17, 1973) take a commissions on the watch from bottom checks incubated with or with egress theophylline and/or epinephrin suck in been tested for their tot cyclic adenylic pungent content and for their competency to bind exogenicly added cyclic antiophthalmic factor.Less cyclic 3H amp do- nonhing be stunnedflow inthe forces subsequently theophylline and/or epinephrin treatment indicating that the turn out in cyclic type A take aim was accompanied by a n gain in the quantity of cyclic antiophthalmic factor hold back intracellularly to the cyclic angstrom-d ependent protein kinases. maximum cyclic adenosine monophosphate fertilization capacities, as cargonful by total cyclic deoxyadenosine monophosphate re-sentencings, were stock-still akin in each(preno houral) cases. Accu pukee estimations of intracellular cover of cyclic angstrom have been gibe with the level of cyclic group A in the create from raw stuff the reception seems to obey fair strength kinetics, a n obvious intracellular K d for cyclic adenosine monophosphate has been evaluated as 330 nM.The findings be logical approximately(prenomoal) with a real relief in the intracellular spinal column unalterable as comp ard to that metrical in vitro (28 nM) or with the fact that the cyclic al-Qaida in the cell whitethorn non ein truth last(predicate) be available for the kinase protein receptors. They besides no minute of arcate that the system depict whitethorn indicate useful for poring over whatever achievable intracellular control beyon d the pace of cyclic angstrom syn thesis.Regulation of cellular metabolous process by adenosine 3 5-monophosphate (cyclic adenylic virulent) I, its mediation by performer of complex protein kinases 2,3 and the mechanism of the activation of these enzymes 461 have been well attested inside the past years in the eukaryotic cell. Activation has been demonstrated to pass along correspond to equality (1) through a n interaction of cyclic adenylic acid with the regulatory sub unit of measurement (R) of the enzyme, leading to a disassociation of this subunit from the catalytic subunit (C) which is thus activated. RC cyclic international ampere + R cyclic adenosine monophosphate C . (1) + + moreover completely satisfactory correlations in the midst of the levels of intracellular cyclic adenine and its ultimate metabolic effects have been in more cases difficult to welcome. Striking examples for this situation argon to be bring in the results of Craig et al. 7 in rat plo sive, of Stull and Mayer 8 in rabbit gaunt muscle concerning the mandate of phosphorylase activation, of Schaeffer et al. 9 and Miller et al. lo concerning regulation of glycogen metabolism in suprarenal glandectomized rats, and of Harbon and Clauser Ill This fly the coop is dedicated to Professor E. Lederer for his 65 th anniversary. Abbreviations. cyclic international ampere adenosine 3 5-monophosphate. in the rat uterus stimulated by prostaglandin El or E,. I n all these cases, cyclic angstrom unit levels may be elevated without eliciting the expected metabolic responses. dickens hypotheses have been formulated to rationalize these obvious discrepancies, both a come down in the activation of the enzymes mediating cyclic amp action inwardly the cell, or a compartmentalization of the intracellular nucleotide. and so it seems indispensable to measure straight the full stop to which the basic step of the activation sequence (Equation 1) devises the manifest intrac ellular cyclic vitamin A stringencys.This competency be achieved by establishing in sacrosanct cells or create from raw stuffs, correlations in the midst of the levels of intracellular cyclic international ampere chthonic well delimitate physiological conditions, the expiration to which it is adjoin to the limited receptor protein and the goal to which the complex protein kinases are in the restless evince. Satisfactory correlations between cyclic angstrom unit levels and protein kinase activation have been recently establish in various weaves by Corbin et al. I21 and Soderling et al. 13.The chip in work was to investigate if correlations could in like manner be obtained between intracellular cyclic angstrom levels and the measurements of intracellular cyclic adenosine monophosphate indentured to receptor protein (R cyclic axerophthol) in the live on rat stopover incubated with or without theophylline and adrenaline. The results inform demonstrate that E r J. Biochem. 40 (1973) u. 178 Intracellular Titration of cyclic deoxyadenosine monophosphate-Receptor Protein top precise titrations of endogenetic cyclic axerophthol keep back versus cyclic antiophthalmic factor lay in the intact waver may be obtained.An discernible Kd value for the intracehlar cyclic vitamin A cover song is observed which differs wide from the K d of the said(prenominal) stuffing established in vitro 14-161. This rule may prove to be useful for flying fielding the modification of cyclic vitamin A backbone down the stairs conditions where the formation and breakdown of cyclic international ampere does non seem to be stirred. A earlier report of these results has been explicit 17. MATERIALS AND METHODS Cylic group A was obtained from P L Biochemicals Inc. , theophylline and Tris from Merck (Darmstadt), Na,adenosine triphosphate 4 H,O, L- adrenaline bitartrate from Calbiochem.Cellulose ester membrane filters (HA 0. 45 pm, 24 mm) were ac quired from Millipore Corp. entirely reagents apply were fruits of Prolabo (reagent grade). Cyclic 3H angstrom unit was a product of New England Nuclear Inc. , specific occupation 24 Ci/ mmol. Animals were Wistar rats weighing more or less cc to 300 g and fasted 24 h before the experiments. Tissue homogenizations were performed with an revolutionary Turrax homogenizer. The reaction mixture for the fecundation hindrance contained in a final mess of 250 p1, 20 mM TrisHC1 caramel pH 7. 5, 10 mM MgCI,, 6. 7 mM theophylline and cyclic 3H angstrom unit a t various ingresss as indicated.The reaction was initiated by the addition of a n aliquot of occlusion rips equivalent to 70- 150 pg protein. manner B. I n this case, cyclic 3H adenylic acidwas added to the homogenizing mediocre a t saturating concentrations up to 0. 2 p M a t 0 C, centrifugation was carried out immediately and cyclic 3H adenine marge measured right away on the suck out. Cyclic 3H adenine qualify to t he proteins, chthonian either condition, was determined later polar incubation times at 0 C the reaction mixtures were and then diluted to 3 m l with frigidity airplane pilot (20mM TrisHC1, 10mM MgCl,, pH 7. 5) and passed through cellulose ethanoate Millipore filters (0. 45 pm).The filters were process with 25ml of the akin buffer, dehydrated and counted in i 0 ml sparkle fluid, in a Packard Tri-Carb liquid sheen spectrometer. Results were expressed as pmol cyclic international ampere saltation/mg protein the concentration of endogenetic untagged cyclic AMP has been ever so taken into account for the estimation of the specific bodily function of cyclic 3HAMP march in the incubation medium. brooding Procedures The animals were killed by decapitation. The full points were speedily removed, freed from connective tissue, cut to small pieces, pooled and divide into equal parts. 200-250 mg tissue were preincubated in 2. ml Krebs-Ringer-bicarbonate buffer pH 7. 4, bobble phase (95O/, O,, 5O//, CO,) for 30 min a t 37 C, in the absence or heraldic bearing of 10 mM theophylline. broodings were then performed in the absence or social movement of adrenaline (5 pM) for change periods of time. rootage of the Tissue Standard spine Assays for Cyclic A M P Two regularitys have been deviced to extract the tissue and cypher the stuffing of exogenic cyclic 3HAMP to the extracted proteins, both slightly modified from the manner defined by Walton and Garren 15. Method A . The tissue was equalise a t 0 C in 3 ml of one of the following solutions 20 mM TrisHCl buffer pH 7. or 20 mM atomic number 11 acetate pH 7. 5 or 4 mM EDTA pH 6. 0. Theophylline (10 mM) was always register in the various homogenizing media in value to minimize whatever degradation of cyclio AMP by phosphodiesterase present in stoppage extracts. A root centrifugation was carried out for 5 min a t 3000 x g , followed by a joint ohm one a t 50000 x g for 30min. The supe rnatants will be referred to as Tris extract, acetate extract and EDTA extract. Assay for Cyclic-AMP Levels For cyclic AMP assay, the tissue was equal in 3 ml cold 7 trichloroacetic acid and centrifuged for 30 min a t 50000 xg. afterward addition of 0. 1 ml N HC1, the supernatants were extracted 7-8 times with twice their account book of cold ether and evaporated to dryness. make out levels of cyclic AMP in the tissue trichloroacetic acid extract were determined agree to Gilman victimization a protein b a s e and the heatstable inhibitor prepared from rabbit worn muscle 161. I n some instances, cyclic AMP content was alike evaluated in the Tris and acetate extracts. Proteins were precipitated by trichloroacetic acid and extracts processed as draw preceding(prenominal). Proteins in the extracts were determined according to Lowry et al. 18 using bovid serum albumin as a standard. RESULTS AXD DISCUSSION Total Cyclic-AMP Levels in wood pussy Diaphragm. solvents of epinephrine and Theophylline In order to study the cyclic AMP cover song business leader of rat diaphragm proteins and its viable rnodification down the stairs the influence of epinephrine, it seemed unavoidable to test the first effect of the catecholamine, to wit the rise in the tissue cyclic AMP level on a lower floor our observational conditions. Em. J. Biochem. 40 (1973) L. Do Khac, S. Harbon, and H. J. Clauser remand 1. Total cyclic A H P levels in trichloroacetic acid extracts of rat diaphragm.Effect of epinephrine and theophylline R a t diaphragms (200-250 mg) were preincubated for 30 rnin a t 37 C in 2. 5 ml Krebs-Ringer-bicarbonate buffer (0, 95/0-C0, 50/0) in the absence or presence of 10mM theophylline, Incubation was then performed for 5 rnin with or without 5 pM epinephrine. The tissue was then homogenized in 7O/, trichloroacetic acid for cyclic AMP assay as described under Methods. Levels of cyclic AMP were expressed as pmol cyclic AMP/ carbonmg wet tissue and as pmol c yclic AMP/mg soluble protein (as figured by the Lowry procedure in the Tris extract. determine are means f S. E. M. of 5 assorted experiments Incubation condit,ions Total cyclic AMP TheoDhvlline EDineDhrine pmo1/ coke mg pmol/mg wet tissue soluble protein 41 f 8. 0 20. 5 f 4. 7 104 & 1. 1 52 & 0. 47 93 f 4. 5 46 & 2 350 f 21 clxx f 10. 7 179 control board 3. distribution of cyclic 3HAMP- ski fertilisationfractions i n opposite hom. ogenutes from rat diaplwagms incubated with or without epinephrine Preincubation and incubation conditions as described in Table 2. Tissues were homogenized in 3 ml 20mM TrisHCI, p H 7. 5, 4 mM EDTA or 20 mM sodium acetate pH 7. and centrifuged for 5 rnin at 3000 x g, the supernatants were centrifuged once more at 500OOxg for 30 min yielding extract 1 and crack 1. The sediment of the first centrifugation was resuspended in 1. 5 ml of the tally buffer and centrifuged at 500OOxg for 30 min giving extract 2 and blastoff 2. bond activity for cyclic rSHAMP was measured in each fraction as described in the text under method A and was expressed as pmol cyclic AMP bound/l00 mg wet tissue Fraction Cyclic AMP bound in EDTA ethanoate Tris extract extract, extract, 5 yM noepinoepino epinephrine nephrine nephrine + + + + Lhrine pmo1/ speed of light mg wet tissue Extract 1 Extract 2 nip 1 Pellet 2 15. 70 1. 47 0. 76 1. 49 14. 90 1. 54 0. 83 1. 50 15. 30 1. 35 0. 80 1. 10 9. 40 0. 80 0. 44 0. 39 Table 2. Cyclic A M P levels in different extracts obtained from epinephrine- treat and untreated rat diaphragms Preincubation with 10 mM theophylline and incubation conditions in the absence or presence of 5 pM epinephrine as in Table 1. Diaphragms were homogenized in troika different solutions cold 7O/, trichloroacetic acid, Tris-HC1 pH 7. 5 or acetate p H 7. 5 as described under methods.Centrifugation was carried out for 30 rnin at 50000 x g. Soluble Tris extract, acetate extract and their similar sediments were deproteinized by 7 o/o tr ichloroacetic acid before cyclic AMP assay Incubation with epinephrine None 5wM Total cyclic AMP in Trichloroacetic 20 mM acetate acid extract pellet 57 280 20 mM Tris extract pellet 48 218 9. 5 26 extract pellet 45 242 pmo1/ snow mg wet tissue 8. 5 8. 3 As verbalisen in Table 1, epinephrine (5 pM) in the absence of theophylline increases (by a factor of 2. 5) the total cyclic AMP content of rat diaphragm extracted by trichloroacetic acid.Theophylline alone (10 mM) had a stimulating effect, repeat when both compounds were used together, the rise in cyclic AMP levels was 8- t o 9-fold, reaching 350pmol cyclic AMP/100 mg wet tissue. When cyclic AMP was assayed in either acetate or Tris extracts after deproteinization with trichloroacetic acid the determine obtained were identical t o those found when the diaphragms were directly extracted with trichloroacetic acid hence virtually no(prenominal) of the cyclic nucleotide in these extracts was associatcd with membrane-bound fract ions (Table 2). Eur. J. Biochem. 0 (1973) Location of Cyclic AMP-Binding Fractions Table 3 shows the distribution of cyclic AMP cover song activity in various fractions of three rat diaphragm homogenates measured by method A in all cases more than goo/, of this activity was recovered in the 50000 x g supernatant, virtually no cyclic AMP covering occurred in the pellets. Preincubation of the diaphragm with epinephrine did non characterise the percentage distribution of the hot nucleotide between the supernatants and the pellets, hence ensuant experiments have been performed on the soluble extracts.On the former(a) hand, in the case of epinephrine-treated diaphragms, less exogenous labelled cyclic AMP ( somewhat 50-60/0) was bound to the various fractions, indicating a decrease in the rachis capacity of the extract as compared to the untreated diaphragm. Dilution by endogenic cyclic AMP cannot explain the effect of epinephrine, since wages was made for this parameter (see Methods) the phenomenon was consistently ordered and will be get on substantiated and discussed below.The covering fire capacities of the various extracts for cyclic E3HAMP have likewise been verified in the absence of any free endogenetic cyclic AMP after removal of the latter by filtration through Sephadex G 50 (1x 37 cm) columns, previously equilibrated with 20 mM Tris-HC1 buffer, pH 7. 5 a t 4 C. I n these experiments, the detail of which w l not be reported in i l the present manuscript, the effect of epinephrine was still observed, when dorsum was measured on the main protein outpouring emerging with the void volume of the columns. When the department of corrections outlined in the clxxx Intracellular Titration of Cyclic AMP-Receptor Protein Binding Z A 0. 51 / 0 20 40 60 duration ( m i n ) l / f r e e cyclic AMP (nM-) l / f r e e cyclic A M P (nM-) Fig. 1. The time wurse and cyclic-AMP-concentration dependence of cyclic A M P fertilisation in rat-diaphragm extrac ts (method A ) . (A) Diaphragms were incubated for 30 min in the presence of 10 mM theophylline and extracted with Tris HCI buffer (method A). Cyclic AMP fertilization was estimated in the presence of various concentrations of cyclic E3HAMP 20nM ( 0 0 ) 60nM ( ) 0 0 SO& (A-A) 100 nM ( ) -. , a t 0 C. The react,ion mixtures contained in a final volume of 2. 5 ml, 20 mM Tris-HC1 buffer, pH 7. , 10 mM MgCI,, 6. 5 mM theophylline. The reaction was initiated by the addition of 930 pg protein. At the indicated times, aliquots were pipetted, immediately diluted with cold 30 mM Tris-HC1buffer pH 7. 5,lO mM MgCl, and passed on the Millipore filters. Filters were washed with the equivalent buffer, dried and counted. Binding activity is expressed as pmol cyclic AMP bound/mg protein. (B) Data obtained from quasi(prenominal) experiments where covering fire for cyclic AMP was performed a t 0 C, for 1 h, in the presence of cyclic aHIAMP ranging from 12 nM to 110 &I. Double-reciprocal plot, according to Klotz 25 Fig. 2.Cyclic-AMP- soaking up dependence of cyclic A M P bond in rat-diaphragm extracts (method B ) . Binding assays were carried out as described under method B. unhomogeneous concentrations of cyclic 3HAMP ranging from 12nM to 200 nM were added directly to the homogenizing medium for preparing extracts from epinephrine treated (A-A) and untreated (0-0) rat diaphragms. Aliquot,s of the extracts were filtered through Millipore filters, dried and counted. Double-reciprocal plot, according to Klotz 25 present paper were use to these mental images, the results were essentially identical to those obtained with the unfiltered extracts.Specificity. Kinetics and Concentration Dependence of Exogenous Cyclic-AMP Binding in the Extracts Specificity of cyclic AMP natural covering has been assessed by dilution experiments of cyclic 3HAMP (100 nM) with unlabelled nucleotides (adenine, AMP, ATP, cyclic AMP) a t molar concentrations equalling up t o 100 times cyclic 3HAM P concentrations. I n no case, still with unlabelled cyclic AMP, the beat of money of hot material bound to proteins by either method A or B was significantly reduced (the details of these experiments are not reported).When various concentrations of cyclic 3HAMP were added to diaphragm extracts (after homogenization and centrifugation) and the concealment reaction (method A) carried out for different incubation times at 0 C (Fig. I), it appears that saturation was obtained at a concentration of 80 nM for the cyclic nucleotide which essentially coincides with previously published data 14-161 and that screening proportionality was reached a t p H 7. 5 and 0 C after less than 60 min incubation. It has also been verified that with the protein concentration used (70-150 pg in 250 pl) adhere of cyclic AMP was directly proportional to the amount of added proteins.From a reciprocal plot of cyclic AMP bond versus cyclic AMP concentration (inset of Fig. I), an unmixed Kd of 33 nM can be calculated. When similar experiments were performed by adding various concentrations of cyclic 3HAMP into the homogenizing medium (method B) and using diaphragms which have been incubated in the presence and absence of epinephrine, the double-reciprocal plots of Fig. 2 were obtained. The unmistakable Kd set calculated with this method (45 nM) are in the same range as with method A.I n addition this figure shows that epinephrine treatment of the diaphragms does not modify this Kd but decreases the amount of exogenous cyclic AMP which can be bound to the extract proteins. By comparing exogenous cyclic AMP binding value obtained with methods A and B, it appears (Table 4) that when cyclic 3HAMPwas added to the Eur. J. Biochem. 40 (1973) L. Do Khac, S. Harbon, and H. J. Clauser Table 4. Comparison of exogenous binding of cyclic SIIAMP to diaphragm extracts by method A or method B. Rat diaphragms were incubated with theophylline in the absence or presancc of 5 p M epinephrine.Ex tracts in Tris-HC1 were prepared as described under method A for subsequent binding of cyclic 3HAMP (100 nM), 1 h, a t 0 C. A second serial of extracts were prepared in the same way but in the prescnce of 100 nM cyclic 3HABIP in the homogenizing medium (method R) binding of cyclic 3HAMP was measured in a n aliquot immediately after centrifugation at 0 C ( virtually 1 h after the end of incubation). Values are expressed as pmol bound cyclic AMP/mg protein. Numerals deep down brackets indicate number of experiments Method Cvclic A P bound with M 5 pM epinephrine no epinephrine pmol/mg protein 4 f 0. 22 (9) 4. 80 5 0. 2 (5) 181 6 t e . ? 4 Q Q E A B 2 f 0. 13 (9) 3 f 0. 19 (5) 0 I I I 30 60 90 * Time (rnin) homogenization medium (extract B) prouder binding determine were obtained both with epinephrine-treated and untreated diaphragms, than with method A. This demonstrates that some additional binding of endogenetic cyclic AMP occurred during the homogenization and fractionation p rocedures, which tends to decrease the amount of un set-aside(p) binding sites available for exogenous cyclic 3HAMP. so method B has been currently used to measure exogenous cyclic AMP binding, since the values obtained with this method seem to think over intracellular conditions more accurately.Fig. 3. Time human body of cyclic 3HAMP binding in extracts from rat diaphragms incubated in the absence or presence of theophylline orland epinephrine. Half rat diaphragms were preincubated in the absence (m, A ) or in the presence ( 0 , 0 ) of 10 m31 theophylline for 30 min at 37 C. Epinephrine (5 pM) was added ( A , 0 )and incubation continued for 5min. Tissue was homogenized in 1. 5 ml Tris-HC1 buffer containing 200 nnf cyclic 3HAMP and centrifuged at 5000xg for 10 min at 0 C.Binding of cyclic 3HAMP was measured in aliquots of the supernatant at the times indicated, through Millipore filtration, t = 0 corresponds to the onset of the extraction. Results are expressed as pmol cyclic AMP bound/ mg protein (without correction for cyclic AMP deepen) Effect of Theophylline and Epinephrine Treatment on the Binding of Exogenous Cyclic 3HAMP by Diaphragm Extracts Fig. 3 shows the results of a common experiment in which diaphragms have been incubated in the absence or presence of theophylline and epinephrine. Homogenization has been performed according to method B, the centrifugation time of the homogenate kept to a inimum (10 min), and the binding capacity for cyclic 3HAMP determined a t different times. As may have been expected, this cyclic 3HAMP binding (which measures the residual binding capacities of the extracts) was, in the course of the whole titration period, in return related t o the amount of endogenetic cyclic AMP present in the relevant extracts (see Table 1). Hence the agents which increase the intracellular cyclic AMP level appear to decrease the amount of binding sites available for exogenous cyclic 3HAMP, probably through an increase of endogenic cy clic AMP binding to the receptors.I n order to titrate endogenous binding of cyclic AMP accurately, experiments were designed to estiEm. J. Biochem. 40 (1973) mate the total binding capacities of the extracts through complete alternate of endogenously bound cyclic AMP with cyclic 3HAMP, and also to estimate the actual amount of exchange occurring in the extracts between endogenous bound unlabelled cyclic AMP and exogenous cyclic 3HAMP during the titration period. A precise knowledge of these dickens parameters is required for the determination of the binding sites occupied by endogenous cyclic AMP at the moment where the tissues are homogenized.Cyclic-AM P Exchange and Determination of Maximal Binding Capacities Total cyclic AMP exchange has been measured under the conditions defined by Wilchek et al. 19 for parotid gland and skeletal muscle extracts from both treated and untreated diaphragms were f i s t incubated at 0 C with cyclic 3HAMP (100 nM) under binding conditions of met hod A and then allowed t o exchange with 1 pM unlabelled cyclic AMP at 20 C in the presence of 100p. M ATP and 10mM MgCl,. Fig. 4 shows that almost complete exchange of the bound labelled nucleotide occurred at heart 30 min, 182Intracellular Titration of Cyclic AMP-Receptor Protein Binding 0 10 20 30 40 50 60 Time (min) 70 80 90 Fig. 4. Exchange of bound cyclic SHIAMP. Extracts were prepared from epinephrine-treated ( + o ) and untreated (0-0) rat diaphragms. Binding of cyclic 3HAMP was carried out a t 0 C in a volume of 2. 5 ml with 500 pg proteins, and 100 nM cyclic r3HAMP in Tris-HC1 buffer, MgCl, and theophylline a t the concentrations described for the standard binding assay. After 1-h incubation, 1 pM unlabelled cyclic AMP and 100 pM ATP were added and the mixture allowed to stand at 20C.At the different times indicated in the figure, aliquots corresponding t o 50 pg protein were pipetted, promptly diluted with 20 mM Tris-HC1 buffer, 2. 5 mM MgC1, p H 7 5 and filtered throug h Millipore filters. The filters . were washed with the same buffer, dried and counted. Results are expressed as pmol/mg protein 0 30 60 90 120 Time (rnin) 180 240 Total binding capacities of the proteins could thus be measured by incubating the extracts first with 100 nM unlabelled cyclic AMP a t 0 C and carrying on the exchange reaction in the presence of 1 pM cyclic I13HAMP at 20 C for 1-2 h the values obtained fairishd 8. -9. 5 pmol cyclic 3HAMP/mg soluble protein, both with epinephrine-treated and untreated diaphragms. These results were corroborate by direct assay of bound cyclic AMP the extracts have been richly modify with unlabelled 1pM cyclic AMP and filtered as described. After washing the Millipore filters, bound cyclic AMP was extracted by cold 7 O/, trichloroacetic acid and the cyclic nucleotide was directly assayed according to Gilman 16. The average value was 9. 8 f 0. 4 pmol cyclic AMP bound per mg protein, which is of the same order of magnitude as the amoun t of bound cyclic 3HAMP calculated above.Previously published data are in close agreement with these values. Walton and GarFen 15 reported supreme binding capacities of 9. 8 pmol/mg protein for adrenal extracts, whereas Gilman l6 found a total binding of 12pmol/mg protein in muscle extracts. The values for maximum cyclic AMP binding are truly low as compared t o the total endogenous cyclic AMP present in the extract (46 pmol/mg protein with the theophylline-treated diaphragm and clxx pmol/mg protein with the epinephrine theophylline-treated diaphragm).It must be added that the binding proteins, saturated with cyclic AMP or not, were almost completely bear on the Millipore filters, and that endogenous cyclic AMP, not Fig. 5. T i m e course of cyclic A M P exchange under binding (0 C) and exchange (20 C conditions. Extracts were prepared from epinephrine treated (0,A ) and untreated ( 0 , A) r a t diaphragms. Binding of cyclic AMP was performed as described in Fig. 2 in the presen ce of 100 nM cyclic AMP for 60 min at 0 C. A t the end of the binding reaction 1 pM cyclic 3HAMP was added t. the different extracts, in the absence (A, A ) or presence ( 0 , 0 ) of l00p. M ATP. The reaction mixtures were maintained a t 0 C for 2 h and then at 20 C (arrow) for 2 more hours. At the different times indicated on the figure, aliquots corresponding t o 70 pg protein were pipetted and treated as in Fig. 4. Results are expressed as cyclic rHAMP bound in pmol/mg protein. bound to these fractions, was quantitatively recovered in the Millipore filtrates after trichloroacetic acid extraction. The consummation t o which this free cyclic AMP may or not be bound to other proteins is presently not known.Cyclic-AMP Exchange under Binding Conditions The extent of cyclic AMP exchange under binding conditions (0 C, 1 h, 100 nM cyclic AMP) must be controlled if corrections for simultaneous exchange have to be applied t o binding data extracts of rat diaphragms treated with theophyllin e and theophylline epinephrine were first saturated with 1 O O n M unlabelled cyclic AMP (binding conditions) and then exchanged with 1 pM cyclic 3HAMP but a t 0 C. After 2 h, the temperature was brocaded to 20 C and completion ofthe exchange measured after 1-2 h further incubation.Fig. 5 shows that a t 0 C, within 1h incubation time, which are the conditions described above for the binding assay, about 200/, of total sites were exchangeable. Under these conditions, ATP and Mg ions slightly increase the exchange velocity. I n addition, this figure confirms that a t 20 C total exchange capacities were identical for epinephrine-treated and untreated diaphragms hence initial + + Em. J. Biochem. 40 (1973) L. Do Khac, S. Harbon, and H. J. Clauser 183 Table 5. relationship between intracellular cyclic A M P levels and cyclic AM P binding in extracts from diaphragm incubated under various conditions Diaphragms were incubated with or without 10 mM theophylline for 30 min at 37 C, 5 pM epin ephrine was added where indicated and incubation continued for varying times. From each incubation, half a diaphragm was extracted by trichloroacetic acid for cyclic AMP estimation. The other half was homogenized with Tris-HC1buffer lOOnM cyclic 3HAMP(method B) for exogenous cyclic AMP binding after 1 h a t 0 C maximal binding capacities were determined in the same extracts a t 20 C in the presence of 1 pM cyclic 3HAMP under conditions described for cyclic A P exchange.R. esults are expressed as pmol cyclic AMP/mg M protein. endogenic binding values were calculated as the difference between maximal binding capacities ( A )and exogenousbinding ( B ) and corrected for the 200/, exchange + Incubation conditions Theophylline 10 mM Epinephrine 5t*M Time Cyclic AMP Total level Maximal binding Exogenous capacity binding (a) (b) Endogenous binding (a-b) corrected min pmol/mg protein + + + + + + 0 2 10 30 5 5 20. 5 52 43 38 46 clxx f 4. 7 & 0. 47 f2 f 10. 7 9. 6 f 0. 9 9. 4 f 0. 1 9. 2 0 9. 40 8. 9 5 0. 73 8. 9 & 0. 85 5. 35 f0. 40 4. 50 f 0. 133 4. 40 4. 70 4. 46 f 0. 20 2. 7 f0. 224 5. 31 6. 13 6 5. 5 5. 53 7. 77 differences in residual binding capacities reflect variations in the degree of saturation of the receptor proteins by endogenous cyclic AMP, rather than modifications of their maximal binding capacity. 1 Titration o Endogenous Cyclic-AMP Binding in Rat f Diaphragm. set up of Theophylline and Epinephrine Since total binding capacities of the receptor proteins in the extracts and the amount of exogenous cyclic 3HAMP bound by these extracts after homogenization may be estimated, it appears thinkable to calculate endogenous cyclic AMP bound in the intact harmoniums, correcting for a 2001, exchange during the titration period.Table 5 summarizes the results of a series of experiments where diaphragms have been incubated under conditions which modify endogenous levels of cyclic AMP in every case, half of the diaphragm was extracted with cold trichloroaceti c acid (see Methods) for the assay of intracellular cyclic AMP levels the second half was extracted according to method B for the estimation of exogenous cyclic 3HAMP binding and of total cyclic AMP binding capacities. The endogenous cyclic AMP bound was calculated from the latter observational data.This table definitely establishes that the average values obtained for the intracellular binding of endogenous cyclic AMP in the intact organ seem to correlate with its cyclic AMP levels. A reciprocal plot of intracellular binding versus intracellular cyclic AMP concentrations (Fig. 6) shows that this correlation fits unproblematic saturation kinetics very accurately. I n the unstimulated diaphragm (no theophylline nor epinephrine added to the incubation medium) about 50/, of the available binding sites are occupied by endogenous cyclic AMP this Eur. J. Biochem. 40 (1973) -0. 002 I 0. 002 l/Free cyclic AMP (nM-) 0 0. 004 . Fig. 6. Reciprocal plot of intracellular cyclic A M P levels an d cyclic A M P binding in rat-diaphragm extracts. Data arc obtained from experiments performed as described in Table 5 and replotted according t o the Klotz equation. The interpose on the y axis yields a n estimate of the number of binding sites and the x intercept provides a n estimation of the intracellular seeming dissociation constant. Statistical analysis of the data were performed according to Cleland 26 using a Wang electronic computing machine alue increases to almost goo/,, when the diaphragms have been fully stimulated with both theophylline and epinephrine. Various treatments with one of the agonists alone cause endogenous bindings ranging between these 2 extreme values. The spare Kd value for intracellular binding according to this plot was estimated to 330 nM f 50, as compared to the apparent Kd (33-45 nM) when binding was assayed in the extracts (Fig. l and 2). Hence a difference of about one order of magnitude appears to obtain between the Kd values calculated wi thin the cell and the 84 Intracellular Titration of Cyclic AMP-Receptor Protein Binding same constant measured with diaphragm homogenates. The double-reciprocal plot may also be used to calculate the intracellular maximal binding capacities, from its intercept with the set up axis. A value of 8. 9 pmol/mg protein was found which coincides with the values measured in the extracts by total cyclic 3HAMP exchange. This dissonance between the intracellular Kd and the Kd measured in vitro in a variety of tissue extracts including diaphragm may a t first sight seem surprising.It has however repeatedly been pointed out that cyclic AMP concentration even in the unstimulated cell was far in excess of the concentration which should result in almost maximal stimulation of protein kinases and compartmentalization of the nucleotide within the cell has usually been postulated to explain this contradiction in terms 8,9,20. The present work shows that despite these high intracellular concentration s of cyclic AMP, protein kinases could indeed not be fully activated, since under the same conditions, the receptor proteins appear not to be fully saturated with cyclic AMP. think RemarksAs might have been expected from Equation (1) (if this reaction truly reflects intracellular conditions) a rise in cyclic AMP should be mateed by an increase in the amount of cyclic AMP bound to receptor protein in the cell. The results reported show this indeed to be the case in the isolated rat diaphragm when this tissue is stimulated by various agents which increase the level of cyclic AMP the amount of protein receptors endogenously saturated by cyclic AMP (R cyclic AMP) rises, as indicated in our experiments by a decrease in their ability to bind exogenously added cyclic 3HAMP after tissue extraction.Maximal binding capacities for cyclic AMP do not seem to be affected under any circumstance. A parallel approach t o the study of this problem has been undertaken by Corbin et al. 12 and Soderli ng et al. 13 who investigated in fatty tissue under various stimulatory conditions, the state of activation of the catalytic subunit (C) by assaying the cyclic AMP dependence of the protein kinase in tissues extracts. These authors demonstrated that under well-defined xperimental conditions, in that location was a quantitative relationship between the intracellular level of cyclic AMP and the amount of the active C unit which could be separated from the complex protein kinase RC. even so in their experiments high concentrations of NaCl had to be added to the extracts, since in its absence R and C tended to reassociate almost immediately, indicating that cyclic AMP is no monthlong bound to its receptor protein (R). The situation in various other tissue xtracts has been found to be analogous, except with skeletal muscle, where preliminary results obtained by the authors led them to suggest that the protein kinase subunits do not readily reassociate. This seems also to be the case for the diaphragm, since under the conditions of the present work, it has been possible to titrate for R * cyclic AMP in the in the buff extracts even in the absence of high salt concentrations acccurate estimations of intracelM a r binding of cyclic AMP have been obtained and correlated with the absolute amounts of the nucleotide present in the stimulated and unstimulated cell.The binding seems t o obey simple saturation kinetics but the apparent Kd of this binding is about10 times higher as compared with the crude extracts. These results may be explained by cyclic AMP compartmentalization within the cell in this case, however, the simple saturation kinetics would indicate that the various pools of the cyclic nucleotide attain equilibrium very rapidly.Or else, if cyclic AMP within the cell is not compartmentalized, and if the reaction described by Equation (1) may be applied, without any modification, to intracellular equilibria, a decrease in the apparent Kd could be merely a c onsequence of the dilution (about 10-fold) of the protein components during extraction of the tissue, while cyclic AMP concentrations are maintained by the addition of exogenous cyclic 3HAMP.However these two hypotheses are certainly oversimplified, since they do not take into account factors like the intracellular concentration of the heat-stable kinase inhibitor 21,22, ATP or Mg2+ 19,23, which are known to affect cyclic AMP binding either in crude extracts or with purified protein kinase preparations. It seems impossible to decide at present which of these interpretations is most likely to reflect true intracellular conditions. It is noteworthy that the apparent Kd estimated is close to the intracehlar cyclic AMP concentration of the nstimulated tissue, a fact which should account for maximal sensitivity of the regulatory mechanisms under physiological conditions. Hormonal controls at the level of cyclic AMP-receptor protein interaction have hitherto never been described the data reported above provide a suitable means for investigating such problems. The authors are very much indebted to Mrs Ginette Delarbre for her excellent skillful assistance and to Mrs Marie-ThBrBse Crosnier for preparing the manuscript. The present work has been performed give thanks to two official grants of the C. N. R. S. Paris, France ERA No 33 and ATP No 429. 914), to a grant obtained from the D. G. R. S. T. 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