Determination of binding parameters of cyclic AMP and its analogs to cyclic AMP-dependent protein kinase by the fluorescent probe method.

The method for determination of dissociation constants for cyclic AMP and its analogs bound to cyclic AMP-dependent protein kinase from pig brain is described. The technique for measuring the binding parameters of the ligands is based on the changes in the fluorescent spectrum of etheno cyclic AMP once it is bound to protein kinase. The dissociation constants for a number of nonfluorescent cyclic AMP analogs were determined in the competitive displacement of etheno cyclic AMP by these analogs. The number of cyclic AMP-binding sites in the pig brain protein kinase was found to be 2.2; no cooperativity was observed upon binding. The holoenzyme complex (Mr = 180,000) of the protein kinase under study was established to have the stoichiometry of R2C2 type under native conditions.     

An extended Scatchard analysis for competitively bound ligands and its application to cyclic adenosine-3',5'-monophosphate and cyclic 5'-amido-5'-deoxyadenosine-3', 5'-monophosphate binding to beef skeletal muscle protein.

A method for determining the dissociation constants of ligands and ligand analogs is described. It is based on competition binding studies in the presence of an isotope-labeled, or otherwise measurable, ligand and suitable analog concentrations.The steps used are determination of (1) the maximal amount of radioactive ligand that can be bound, (2) the slopes and intercepts from Scatchard plots at different analog concentrations and (3) the values for the dissociation constants of radioactive ligand and ligand analog from replots of the reciprocals of the slopes and intercepts obtained from the Scatchard plots. Application of the method to a cyclic AMP-binding protein from beef muscle is demonstrated, yielding dissociation constants of 2.10-9 M for cyclic (3H) AMP and cyclic AMP, and 3.10-5 M for cyclic 5'-amido-5'-deoxyadenosine-3', 5'-monophosphate.     

A kinetic study of cyclic adenosine 3':5'-monophosphate binding and mode of activation of protein kinase from Drosophila melanogaster embryos.

Cyclic AMP-dependent protein kinase and its regulatory subunit were isolated from Drosophila melanogaster embryos. The profiles of cyclic AMP binding by these proteins were significantly different. In order to explain such a difference and to find the mode of enzyme activation by cyclic AMP, a kinetic study of cyclic AMP binding was carried out. First, the association rate constant k1 and dissociation rate constant k-1 in the cyclic AMP-regulatory subunit interaction at 0 degrees C were estimated to be 2.3 X 10(6)M-1s-1 and 1.1 X 10(-3)s-1, respectively. Secondly, the three possible modes of enzyme activation by cyclic AMP were mathematically considered and could be described by a unique formula: r=APt + BQt (A + B=1) in which the parameters A, B, P, and Q are equivalent to rate constants in the sense that the rate constants are simply expressed by these parameters. Thirdly, the values of the parameters and subsequently the values of rate constants involved in the possible mechanisms were evaluated using a curve-fitting technique and compared with experimental observation. It was then found that the following mechanism was the only one which fitted the experimental observations. Namely, RC + L k3 equilibrium k-3 LRC k4 equilibrium k-4 RL + C where R, C, and L represent the regulatory and catalytic subunits and cyclic AMP as a ligand. Thus, our results indicate that in the presence of cyclic AMP the active enzyme (C) is released from a ternary intermediate which is the primary product of the cyclic AMP-holoenzyme interaction. The estimated values of the rate constants are: k3=3.5 X 10(6)M-1s-1;k-3=7.3 X 10(-1)s-1;and k4=3.8 X 10(-2)s. These estimates indicate that the reaction LRC leads to RL + C is relatively slow and limits the rate of the overall reaction. By comparing k-3 and k4, it is apparent that a large part of newly formed ternary intermediate reverts to the holoenzyme.     

Comparison of adenosine 3':5'-monophosphate-dependent protein kinases from rabbit skeletal and bovine heart muscle.

Homogeneous preparations of adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase from rabbit skeletal (Peak I) and bovine heart muscle have been compared. Each enzyme has an S20,w value of 7.0. Each enzyme binds 2 mol of cyclic AMP per mol of enzyme and is dissociated in the presence of saturating concentrations of cyclic AMP into a demeric regulatory subunit-cyclic AMP complex and two catalytic subunits. The isolated subunits recombine, resulting in the formation of the original holoenzyme in each case. Several differences between the two enzymes were found. Different salt concentrations are necessary for elution of the respective enzyme from DEAE-cellulose. Their regulatory subunits differ with respect to their sedimentation constants and mobility on sodium dodecyl sulfate gel electrophoresis. The regulatory subunit of the heart enzyme is rapidly phosphorylated by MgATP but this does not occur with the skeletal muscle enzyme. MgATP is bound with high affinity only to the skeletal muscle enzyme. The enzymes have different apparent dissociation constants and Hill coefficients for cyclic AMP binding. With the skeletal muscle enzyme MgATP increases the dissociation constants for cyclic AMP about 10-fold and decreases the Hill coefficient, while with the heart enzyme phosphorylation decreases the cissociation constant for cyclic AMP 5- to 6-fold and increases the Hill coefficient. Different concentrations of cyclic AMP are required to dissociate the skeletal and heart muscle enzymes. The presence of MgATP increases the concentration of cyclic AMP required to dissociate the skeletal muscle enzyme but decreases the concentration necessary to dissociate the heart enzyme.     

A study of the interaction between bovine cardiac-muscle cyclic AMP-dependent protein kinase and cyclic AMP using fluorescence-polarization spectroscopy.

The C-subunit of type II cyclic AMP-dependent protein kinase from bovine heart was labelled with the fluorophore fluorescamine (FAM). The association of the dye-labelled subunit (CFAM) with the R-subunit isolated from the same source was monitored by fluorescence polarization spectroscopy. The stoichiometry of C to R in the final complex was close to 1:1. The affinity of the two subunits could be described by a dissociation constant in the nanomolar range. Holoenzyme (formed from CFAM and R) was titrated with cyclic AMP, and the changes in fluorescence anisotropy, due to dissociation of the holoenzyme, recorded. The titration curves were analysed in terms of a model which required computer simulation. Cyclic AMP-induced dissociation proceeds via one or more ternary complexes, and all four cyclic AMP-binding sites on the R-dimer are accessible in the holoenzyme. The dissociation constants describing the release of the C-subunits from the two ternary complexes containing four cyclic AMP molecules were both approx. 9 microM. The binding of two cyclic AMP molecules to protein kinase is necessary and sufficient to cause the dissociation of both C-subunits. The state of association at 'in vivo' concentrations of protein and cyclic AMP is discussed.     

Protein kinase activity in mouse mammary carcinoma.

C3H mouse mammary carcinoma contains cyclic AMP-independent (C) and dependent (RC) protein kinases and a specific cyclic AMP-binding protein (R). The specific activities of C, RC and R are markedly lower in carcinoma than the normal mammary cells. Protein kinase preparation from neoplastic cells showed markedly higher ration of $\text{C}\text{RC}$ and lower responsiveness to cyclic AMP for the activation of the enzyme than the normal cells.     

Studies on protein kinase activity and the binding of adenosine 3'5-monophosphate by membranes of hereditary spherocytosis erythrocytes.

Evidence has been presented recently of a deficiency of an endogenous membrane-associated protein kinase in erythrocytes of patients with hereditary spherocytosis (HS). We have measured endogenous protein kinase activity in erythrocyte membranes of 4 HS subjects using different membrane isolation and reaction conditions and find that the phosphorylation of the spectrin component (mean ± S.E. 17.1 ± 1.2 pmoles/10 mins per mg protein) is not significantly different to that of 4 normal controls (mean ± S.E. 20.7 ± 1.1 pmoles/10 mins per mg protein). Phosphorylation of exogenous proteins such as casein and protamine is also not deficient in HS erythrocyte membranes. Adenosine 3′5-monophosphate (cyclic AMP) binding to normal and HS erythrocyte membranes was also studied using a Millipore filtration assay. The affinity of cyclic AMP for erythrocyte membranes as determined by Hill plots of binding data from 4 HS subjects (K_D mean ± S.E. = 2.2 ± 0.2 nM) was not significantly different to 4 normal controls (K_D mean ± S.E. = 2.8 ± 0.6 nM). The rate of dissociation of bound cyclic AMP from HS membranes was also similar to control membranes. We thus cannot confirm the prediction by others that an abnormality of cyclic AMP interaction with the erythrocyte membrane underlies HS.     

Evidence that rabbit muscle protein kinase has two kinetically distinct binding sites for adenosine 3' ; 5'-cyclic monophosphate.

Two distinct populations of binding sites for cyclic AMP are associated with the regulatory moity of cyclic AMP dependent protein kinase (E.C. 2.7.1.37), as judged from the kinetics of the interaction between the nucleotide and the binding protein. The two types of sites were present at the proportion 1:1. The rate of dissociation of bound cyclic AMP was more rapid for one type of site than for the other type. High ionic strength accentuated the difference in the rate of dissociation of cyclic AMP from the two sites.The two binding sites and protein kinase activity copurified during the entire procedure for preparation of protein kinase holoenzyme. The kinetic properties of each of the two sites and the proportion between them was the same in a highly purified preparation of the regulatory moiety of protein kinase and in binding protein freshly prepared in the presence of protease-inhibitor.     

Cyclic 3'.5'-nucleotide phosphodiesterase. Effect of binding protein on the hydrolysis of cyclic AMP

The rate of cyclic AMP hydrolysis by a cyclic 3′,5′-nucleotide phosphodiesterase was diminished by the presence of a cyclic AMP binding protein in the reaction mixture. The reduction was proportional to the concentration of the binding protein; and was more pronounced at 0° than at 30°, presumably because the affinity of cyclic AMP to the binding protein was greater at 0° (“apparent dissociation constant” = 3 × 10⁻⁸ M) than at 30° (“apparent dissociation constant” = 4 × 10⁻⁷ M). These experiments indicate that cyclic AMP bound to the binding protein is not susceptible to the action of phosphodiesterase. It is hydrolyzed only when dissociated from the protein, and the rate of dissociation appears to be the limiting factor. The possible physiological significance of these results is discussed.     

Cardiac adenosine 3':5'-monophosphate. Free and bound forms in the isolated rat atrium.

Adenosine 3':5'-monophosphate (cyclic AMP), a mediator of hormone action in a variety of tissues, has been measured in its free and bound forms in intact cardiac tissue. We have used a rapid high dilution technique which involves tissue homogenization, subcellular fractionation, and separation of bound from free cyclic AMP by Millopore filtration. The precision of this method is dependent upon minimization of binding and dissociation of cyclic AMP that occur during the preparation and handling of tissue homogenates. In each experiment, a tracer of cyclic [3H]AMP prebound to isolated cardiac binding protein was freed of unbound cyclic [3H]AMP by Sephadex gel filtration and added to the tissue just prior to homogenization in cold EDTA buffer. This tracer was therefore treated identically to the sample through all subsequent dilution, fractionation, and filtration procedures, and provided an acurate internal monitor for total cyclic AMP dissociation during the course of the free-bound determination. Each tissue sample was then individually corrected for dissociation. Rapid dilution to produce a 1:1000 homogenate was found to lower endogenous cyclic AMP levels sufficiently to make binding (or rebinding) during the procedure negligible (less than 5%). Spontaneously beating rat right atria (controls) contained 5.96 +/- 0.28 pmol of cyclic AMP/mg of protein (n = 19) of which 41 and 14% were bound to soluble and particulate proteins, respectively. The remaining cyclic AMP was free. Pretreatment of the tissue with 1 muM isoproterenol (30 s at 30 degrees) increased both the bound and free forms of cyclic AMP (n = 8). While free cyclic AMP increased 420% with the catecholamine, the bound forms increased 240% (soluble) and 60% (particulate). Similar results were obtained when atria (n = 6) were treated with the phosphodiesterase inhibitor, methylisobutylxanthine (0.5 mM, 10 min at 30 degrees). When both agents were used together, cyclic AMP bound to soluble proteins was elevated 4-fold over control while free cyclic AMP increased 27-fold (n = 7), indicating saturation of the soluble sites. It could be calculated that less than one-third of these sites are occupied in the unstimulated cell. These sites may represent the R subunit of cyclic AMP-dependent protein kinase. The data suggest that half-maximal binding in vivo occurs at an intracellular free cyclic AMP concentration of about 1 muM.     

Photoaffinity labeling of three renal cyclic 3',5'-adenosine monophosphate-binding proteins.

Evidence is presented for the presence of multiple cyclic AMP binding components in the plasma membrane and cytosol fractions of porcine renal cortex and medulla. N6-(Ethyl-2-diazomalonyl)-3',5'-adenosine monophosphate, a photoaffinity label for cyclic AMP binding sites, exhibits non-covalent binding characteristics similar to cyclic AMP in membrane and soluble fractions. Binding data for either compound to the plasma membrane fraction yields biphasic Scatchard plots while triphasic plots are obtained with the dialyzed cytosol. When covalently labeled fractions are separated on SDS-polyacrylamide gel electrophoresis, the cyclic AMP photoaffinity label is found on 49 000 and 130 000 dalton components in each kidney fraction. DEAE-cellulose and gel filtration chromatography of the labeled cortical cytosol fraction establishes that the three components suggested by the binding data correspond to two 49 000 dalton species and a 130 000 component. The 49 000 species have higher affinities for cyclic AMP than the 130 000 component (Ka(1) = 2.0 . 10(9), Ka(2) = 1.7 . 10(8), Ka(3) = 1.0 . 10(7)). The 49 000 components are associated with protein kinase activity while the 130 000 component does not exhibit protein kinase, adenosine deaminase, or cyclic nucleotide phosphodiesterase activity. Immunologic results and effects of phosphorylation and cyclic GMP on cyclic AMP binding further suggest that the 49 000 components are regulatory subunits of cyclic AMP-dependent protein kinases. Cyclic AMP binding to the 130 000 component is markedly inhibited by adenosine and adenine nucleotides, but not cyclic GMP. Thus, this component may reflect an aspect of adenosine control or metabolism which may or may not be a cyclic AMP-related cellular function.     

The use of affinity chromatography in purification of cyclic nucleotide receptor proteins.

Biospecific affinity chromatography has been used to purify specific cyclic AMP and cyclic GMP receptor proteins. Several variables are important for successful purification of the cyclic AMP receptor protein, the most critical being the length of the aliphatic spacer side arm. 8-(2-Aminoethyl)-amino-cyclic AMP coupled to the aliphatic spacer side arm. 8-(2-Aminoethyl)-amino-cyclic AMP coupled to agarose specifically retains the cyclic AMP receptor protein by interaction with the immobilized nucleotide. Binding of the cyclic AMP receptor subunit of cyclic AMP-dependent protein kinase to the immobilized nucleotide results in dissociation of the catalytic protein phosphokinase subunit which is not retained. The retained cyclic AMP receptor protein is subsequently eluted by cyclic AMP. Homogeneous cyclic AMP receptor protein prepared from rabbit skeletal muscle by affinity chromatography has been characterized. The molecular weight of the native protein as determined by analytical ultracentrifugation and polyacrylamide gel electrophoresis at varying acrylamide concentrations is 76 800 and 82 000, respectively. The protein is asymmetric with frictional and axial ratios of 1.64 and 12. SDS and urea polyacrylamide gel electrophoresis indicate that the native cyclic AMP receptor is composed of two identical subunits of 42 700 molecular weight. The native protein dimer binds 2 moles of cyclic AMP per mole of protein and is active in suppressing activity of isolated catalytic subunits of cyclic AMP-dependent protein kinase. Cyclic GMP receptor protein from bovine lung has been purified using the same affinity chromatography media. Since cyclic nucleotide binding to cyclic GMP-dependent protein kinase does not result in dissociation of regulatory receptor and catalytic phosphotransferase subunits, the cyclic GMP-dependent protein kinase holoenzyme is retained on the column and can be subsequently specifically eluted with cyclic GMP.     

Cooperative DNA binding and communication across the dimer interface in the TREX2 3' --> 5'-exonuclease

The activity of human TREX2-catalyzed 3' --> 5'-deoxyribonuclease has been analyzed in steady-state and single turnover kinetic assays and in equilibrium DNA binding studies. These kinetic data provide evidence for cooperative DNA binding within TREX2 and for coordinated catalysis between the TREX2 active sites supporting a model for communication between the protomers of a TREX2 dimer. Mobile loops positioned adjacent to the active sites provide the major DNA binding contribution and facilitate subsequent binding into the active sites. Mutations of three arginine residues on these loops cause decreased TREX2 activities by up to 60-fold. Steady-state kinetic assays of these arginine to alanine TREX2 variants result in increased K(m) values for DNA substrate with no effect on k(cat) values indicating contributions exclusively to DNA binding by all three of the loop arginines. TREX2 heterodimers were prepared to determine whether exonuclease activity in one protomer is communicated to the opposing protomer. Evidence for communication across the dimer interface is provided by the 7-fold lower catalytic activity measured in the TREX2(WT/H188A) heterodimer compared with the TREX2(WT) homodimer, contrasting the 2-fold lower activity measured in the TREX2(WT/R163A,R165A,R167A) heterodimer. The measured activity in TREX2(WT/H188A) heterodimer indicates that defective catalysis in one protomer reduces activity in the opposing protomer. A DNA binding analysis of TREX2 and the heterodimers indicates a cooperative binding effect within the TREX2 protomer. Finally, single turnover kinetic assays identify DNA binding as the rate-limiting step in TREX2 catalysis.     

Adenosine 3'':5''-cyclic monophosphate-dependent protein kinase in brown fat from newborn rabbits. Changes in the binding of adenosine 3'':5''-cyclic monophosphate after preincubation of the tissue with noradrenaline or incubation of the enzyme with adenosine triphosphate.

The equilibrium binding of cyclic AMP to a 150-fold purified preparation of protein kinase, when expressed as the reciprocal of bound against the reciprocal of free cyclic AMP, gave a plot consisting of two straight lines. The values of apparent Kb given by these lines were lowered by preincubating the intact tissue with noradrenaline or incubating the enzyme preparation with Mg2+ plus ATP. This effect was reversed by incubating the preparation (which contained some phosphatase impurities) with Mg2+ alone. None of these procedures affected the maximal binding of cyclic AMP. During incubation of the enzyme with Mg2+ plus ATP, the terminal phosphoryl group was incorporated into protein, over 40% being present in the kinase itself. This phosphate was removed during incubation of the preparation with Mg2+ alone. The validity of expressing cyclic AMP binding as a double-reciprocal plot is discussed, and the experimental plots are compared with those derived theoretically. The results suggest that protein kinase in brown fat is present in two forms, one with an apparent Kb for cyclic AMP or approx. 250 nM (dephosphorylation) and one with an apparent Kb of approx. 14 nM (phosphorylated). Preincubation of the tissue with noradrenaline results in phosphorylation of the kinase and an increase from 15 to 45% in the proportion of the higher-affinity form.     

An adenosine 3':5'-monophosphate-adenosine binding protein from mouse liver.

A cyclic AMP-adenosine binding protein from mouse liver has been purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis in the absence and presence of sodium dodecyl sulfate and by analytical ultracentrifugation. The binding protein had a Stokes radium of 48 A based on gel chromatography. Both the purified binding protein and the binding activity in fresh cytosol sedimented as 9 S on sucrose gradient centrifugation. The homogeneous protein had a sedimentation coefficient (S20, w) of 8.8 x 10-13 s, as calculated from sedimentation velocity experiments. By use of the Stokes radius and S20, w', the molecular weight was calculated to be 180,000. The protein was composed of polypeptides having the same molecular weight of 45,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and thus appeared to consist of four subunits of equal size. The isoelectric point, pI = 5.7. The binding capacity for cyclic AMP increased by preincubating the receptor protein in the presence of Mg2+ ATP. This process, tentatively termed activation, was studied in some detail and was shown not be be be accompanied by dissociation, aggregation, or phosphorylation of the binding protein. Cyclic AMP was bound to the protein with an apparent dissociation constant (Kd) of 1.5 x 10-7 M. The binding of cyclic AMP was competitively inhibited by adenosine, AMP, ADP, and ATP whose inhibition constants were 8 x 10-7 M, 1.2X 10-6 M, 1.5 X 10-6 M, and higher than 5 x 10-6 M respectively. A hyperbolic Scatchard plot was obtained for the binding of adenosine to the activated binding protein, indicating more than one site for adenosine. The binding of adenosine to the site with the highest affinity (Kd=2 x 10-7 M) for this nucleoside was not suppressed by excess cyclic AMP and was thus different from the aforementioned cyclic AMP binding site. Cyclic GMP, GMP, guanosine, cyclic IMP, IMP, and inosine did not inhibit the binding of either cyclic AMP or adenosine. The binding protein had no cyclic AMP phosphodiesterase, adenosine deaminase, phosphofructokinase, or protein kinase activities, nor does it inhibit the catalytic subunit of the cyclic AMP-dependent protein kinase.     

Purification and some properties of rabbit skeletal muscle fructose 6-phosphate kinase inhibitor.

The loss of activity of rabbit skeletal muscle FPK on storage and its restoration by ATP, AMP and cyclic AMP has prompted us to look for an inhibitory unit of the enzyme. We have purified this inhibitory factor from the crude muscle extract and isolated from crystalline FPK; both proteins have the same Mw of about 68,000 (SDS). Carboxymethylation revealed species of lower molecular weight. It is suggested that two different kinds of FPK exist, one composed only of "active" subunits and another composed only of "inactive" (inhibitor) subunits. States of intermediate activity exist, created by dissociation, reassociation and exchange of subunits, because the inhibitor and FPK share several subunits. A model is proposed where one or several inhibitors of molecular mass 68,000 replace the corresponding number of active subunits of 93,000 daltons, the structure of the native molecule remaining tetrameric. It is shown that cyclic AMP exerts its activation function on FPK only in the presence of the inhibitory protein, probably by displacing the exchange of the subunit in favor of the active tetrameric species of 360,000. Ammonium chloride plays probably an opposite role in this exchange. The inhibitor coverts the Michaelian behavior with respect to F-6-P into a cooperative response (sigmoidal shape of the curve) characterized by a Hill coefficient of 2. The Michaelian response with respect to ATP is preserved, the corresponding constant being only slightly affected. In the presence of subsaturating concentration of inhibitor, mixed species are detected. As a first approximation one can propose that a reversible equilibrium exists between free and complex FPK subunits. The dissociation constant of this equilibrium being equal to 4 X 10(-8) M in moles of FPK protomers.     

Cyclic AMP binding proteins in early embryos of Drosophila melanogaster.

A variety of effects of cyclic AMP on cellular and subcellular phenomena suggest that there may be other modes of action of cyclic AMP then activation of protein kinase. It is also known that developing embryos contain cyclic AMP and its related enzymes. In order to explore the role of cyclic AMP in embryogenesis, a survey of proteins capable of binding cyclic AMP in the embryonic supernatant of Drosophila melanogaster was carried out. As the result, two cyclic AMP-binding proteins were found and characterized. The one (L) is, as expected, associated with protein kinase and has a dissociation constant of about 10(-9) M. Its molecular weight of 21 000 daltons is extremely small when compared with similar proteins in other organisms. The other (H), whose function is yet to be found, has a molecular weight of about 200 000 daltons and has a dissociation constant of about 10-7 M. Some laxity in binding specificity of the latter protein among adenosine nucleotides was observed, but cyclic AMP is the strongest ligand among them.     

An adenosine 3':5'-monophosphate-adenosine binding protein from mouse liver. Factors affecting the activation of the binding protein by adenosine 5'-triphosphate.

A cyclic AMP-adenosine binding protein, whose binding sites are activated by preincubation in the presence of Mg⁺-ATP, has been purified to apparent homogeneity from mouse liver (P.M. Ueland and S.O. Døskeland, 1977, J. Biol. Chem.,252, 677–686). The degree of activation of both the cyclic AMP binding site and a high-affinity site for adenosine depends on the concentration of ATP during the preincubation. The velocity and the degree of activation are dependent on the temperature and the presence of Mg²⁺ and K⁺. The NH₄⁺ ion can be substituted for K⁺, whereas Na⁺ is inefficient. Low pH promotes the conversion from the inactive to the active form. The apparent affinity for adenosine to the high-affinity site for this adenine derivative and the affinity for cyclic AMP to the site specific for this nucleotide are independent of the degree of activation as judged from the slope of Scatchard plots. The activation of the cyclic AMP binding site by ATP (6 mm) was determined at pH 7 in the presence of 10 μm cyclic AMP, AMP, ADP, or adenosine. Adenosine specifically inhibits the activation and does not promote the inactivation of the binding protein. The possibility that the apparent inhibition of activation was effected by interference with cyclic AMP binding by adenosine was ruled out.     

Reversible, positive cooperative interaction of 11 beta-chloromethyl-[3H]estradiol-17 beta with the calf uterine estrogen receptor

The binding of 11 beta-chloromethyl-[3H]estradiol-17 beta [3H]CME2) with the calf uterine estrogen receptor was investigated. The equilibrium binding analysis indicated a positive cooperative interaction yielding curvilinear Scatchard plots and Hill coefficients of 1.4-1.5. This positive cooperative interaction of [3H]CME2 was indistinguishable from the typical cooperative interaction of [3H]estradiol with the receptor. The apparent relative association constant and the relative binding affinity of CME2 for the estrogen receptor measured by competitive binding assay were 146 and 184%, respectively. The dissociation kinetics of [3H]CME2 from the receptor was biphasic, composed of a fast dissociating component (15%, t1/2 = 4 min at 0 degrees C; 9%, t1/2 = 4 min at 28 degrees C) and a slow dissociating component (85%, t1/2 greater than 50 h at 0 degrees C; 91%, t1/2 greater than 50 h at 28 degrees C). The dissociation kinetics of [3H]estradiol was also biphasic: the t1/2 of the fast dissociating component was 4 min at 0 and 28 degrees C and approximately 200 min for the slow dissociating component at both temperatures. The fraction of the slow [3H]estradiol dissociating component increased from 56 to 92% upon warming. Ethanol extraction and trichloroacetic acid treatment proved that the binding of [3H]CME2 is fully reversible. The unusual dissociation kinetics and the binding mechanism of CME2 are discussed.     

Binding proteins for adenosine 3′:5′-cyclic monophosphate in bovine adrenal cortex

Five peaks of cyclic AMP-binding activity could be resolved by DEAE-cellulose chromatography of bovine adrenal-cortex cytosol. Two of the binding peaks co-chromatographed with the catalytic activities of cyclic AMP-dependent protein kinases (ATP-protein phosphotransferase, EC 2.7.1.37) of type I or type II respectively. A third binding protein was eluted between the two kinases, and appeared to be the free regulatory moiety of protein kinase I. Two of the binding proteins for cyclic AMP, sedimenting at 9S in sucrose gradients, could also bind adenosine. They bound cyclic AMP with an apparent equilibrium dissociation constant (K(d)) of about 0.1mum, and showed an increased binding capacity for cyclic AMP after preincubation in the presence of K(+), Mg(2+) and ATP. The two binding proteins differed in their apparent affinities for adenosine. The isolated regulatory moiety of protein kinase I had a very high affinity for cyclic AMP (K(d)<0.1nm). At low ionic strength or in the presence of MgATP, the high-affinity binding of cyclic AMP to the regulatory subunit of protein kinase I was decreased by the catalytic subunit. At high ionic strength and in the absence of MgATP the high-affinity binding to the regulatory subunit was not affected by the presence of catalytic subunit. Under all experimental conditions tested, dissociation of protein kinase I was accompanied by an increased affinity for cyclic AMP. To gain some insight into the mechanism by which cyclic AMP activates protein kinase, the interaction between basic proteins, salt and the cyclic nucleotide in activating the kinase was studied.