CAMP-dissociation kinetics in hormone-dependent and -independent rat mammary tumor cytosols

Cytosols from 7, 12-dimethylbenz (alpha) anthracene-induced rat mammary tumors which exhibit different hormone-responsiveness were compared with respect to their cAMP-dissociation kinetics. At 22 degree C, pH 4.5, 1 micrometer cAMP, hormone-dependent mammary tumors exhibited monophasic dissociation rates with a rate constant of k-1 = 0.06 min-1. In contrast, hormone-independent mammary tumors exhibited biphasic dissociation curves with rate constants of k-1 = 0.47 and k-2 = 0.06 min-1. The binding of cAMP was completely reversible; radio-labeled ligand was completely dissociated by 1mM nonradioactive cAMP; the binding protein could be reassociated to its original binding level after dextran-coated charcoal adsorption. The mammary cytosols exhibited specific binding for cAMP which could be displaced partially by cGMP but not by ATP, ADP, AMP, or adenosine. Receptor inactivation during the course of incubation was negligible. Both mammary tissue cytosols exhibited similar association rates at 22 degree C, pH 4.5, 1 micrometer cAMP (k+1 = 5-7 x 10(5)M-1 min-1). These data indicate that mammary tissues exhibit 2 cAMP dissociation rates. Hormone-dependent mammary tumors exhibit a dissociation constant of a high affinity binding site (k-1/k+1 = 0.07 micrometer) whereas hormone-independent mammary tumors exhibit dissociation constants of one high affinity (k-1/k+1 = 0.07 micrometer) and a second low affinity site (k-1/k+1 = 0.05 micrometer).     

Estrogen receptor in chicken oviduct: receptor dissociation kinetics and transformation

Cytosolic and nuclear estrogen receptor forms of chicken oviduct have been studied by (1) measuring hormone dissociation kinetics and by (2) sucrose density gradient analysis on high salt gradients. Estradiol dissociates from the receptor in chicken oviduct cytosol at 22 degrees C following a two-phase exponential process. The fraction of receptor with a fast dissociation rate (k = 120 X 10(-3) min-1) decreases as a function of the pre-incubation at 22 degrees C; after prolonged pre-incubation only the slowly dissociating (k = 12.3 X 10(-3) min-1) form remains. Dissociation of moxestrol, a synthetic estrogen with a higher affinity, from the cytosol receptor at 30 degrees C is similar, showing a transition of a fast dissociating form (k = 120 X 10(-3) min-1) to a slowly dissociating form (k = 7.6 X 10(-3) min-1) as a result of pre-incubation at 30 degrees C. A concomitant temperature-dependent shift of the estrogen receptor from a 4.8 S to a 6.1 S form was observed with moxestrol but not with estradiol as a ligand. Sodium molybdate (20 mM) and NaSCN (400 mM) inhibit the temperature-dependent increase in sedimentation coefficient, but molybdate allows the formation of a receptor form which shows intermediary dissociation kinetics. Estrogen receptor, precipitated with ammonium sulfate (0-35%) shows monophasic dissociation kinetics of estradiol (k = 39.5 X 10(-3) min-1) and for moxestrol (k = 10.8 X 10(-3) min-1), suggesting full receptor activation only with moxestrol as a ligand. Moxestrol-receptor complexes obtained by ammonium sulfate precipitation sediment at 0 degree C at 4.8 S. Only after subsequent incubation at 30 degrees C a shift from 4.8 S to 5.9 S is observed, suggesting that the formation of the slowly dissociating form of the receptor may precede the formation of a stable transformed receptor complex. The nuclear estrogen receptor with estradiol as a ligand shows biphasic dissociation kinetics at 22 degrees C (k = 70 X 10(-3) min-1; k = 14.0 X 10(-3) min-1). The ratio of both components (1:1) does not change after preincubation of the nuclear receptor extract at 22 degrees C. Moxestrol dissociates from the nuclear receptor at 30 degrees C monophasically with a slow rate (k = 6.1 X 10(-3) min-1), suggesting that it is extracted as an activated hormone-receptor complex.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of secretory component, a secreted glycoprotein, in the specific uptake of IgA dimer by epithelial cells.

The binding of secretory component (SC) to epithelial cells and its role in the specific uptake of immunoglobulin A (IgA) dimer has been studied in rabbit mammary gland and liver. SC, Mr approximately 80,000, secreted by epithelial cells of the mammary gland was found associated with the cell surface of mammary cells in intact tissue. Dispersed mammary cells and plasma membrane-enriched fractions obtained from mammary glands of midpregnant rabbits bound 125I-labeled SC in a saturable time- and temperature-dependent process. The association rate followed a second order reversible reaction (k+1 approximately equal to 2.7 x 10(6) M-1 min-1 at 4 degrees C) and equilibrium was reached in about 4 h at 4 degrees C. The dissociation rate for membranes was first order (k-1 approximately equal to 1.7 x 10(-2) min-1 at 4 degrees C), whereas displacement from cells was incomplete. The apparent affinity constant was similar for membranes and cells (Ka approximately equal to 5 x 10(8) M-1) with one class of binding sites. The number of binding sites varied from one animal to another (260 to 7,000 sites/mammary cell) in relation to endogenous occupancy by SC, which was assessed by immunocytochemistry and complement-mediated cytotoxicity. Rabbit liver and heart membranes did not bind SC, and serum proteins present in rabbit milk failed to interact with mammary cells or membranes. Mammary membranes or cells and liver membranes bound 125I-labeled IgA dimer in a saturable, reversible time- and temperature-dependent process. Association and dissociation rate constants at 4 degrees C (k+1 approximately equal to 5 x 10(6) M-1 min-1 and k-1 approximately equal to 5 x 10(-3) min-1, respectively) and the apparent affinity constant (Ka approximately equal to 10(9) M-1) were similar for liver and mammary membranes; these parameters differed, however, from those reported for free SC-IgA dimer interaction. The binding capacity of membranes for IgA dimer was directly related to the amount of free SC bound to membranes. Interaction of IgA dimer with mammary or liver membranes or cells was abrogated by excess of free SC and was prevented by preincubation of membranes or cells with Fab antibody fragments directed against SC. These data indicate that the first step in the translocation process of polymeric immunoglobulins across epithelia consists of binding of SC to the surface of epithelial cells which in turn acts as a receptor for the specific uptake of IgA dimer.     

Kinetics of gonadotropin binding by receptors of the rat testis. Analysis by a nonlinear curve-fitting method.

The kinetics of the reaction between human chorionic gonadotropin (hCG) and specific gonadotropin receptors in the rat testis were determined at 24 and 37 degrees, over a wide range of hormone concentrations. Hormone concentrations were corrected for the binding activity of the (-125I)hCG tracer preparations. Analysis of the experimental data was performed with an interactive nonlinear curve fitting program, based upon the second-order chemical kinetic differential equation. The mean values for the association rate constant (k1) were 4.7 x 10-7 M-1 min-1 at 24 degrees, and 11.0 x 10-7 M-1 min-1 at 37 degrees. At both temperatures, the values of kl were independent of hormone concentration. Initial dissociation rates were consistent with first order kinetics, with dissociation rate constant (k2) of 1.7 x 10 minus -3 and 4.6 x 10 minus -3 min minus -1 at 24 and 37 degrees, respectively. When studied over longer periods at 24 degrees, the dissociation process appeared to be multiexponential. The kinetics of degradation of (-125I)hCG and receptors were determined at both temperatures, and a mathematical model was developed by modification of the second-order chemical kinetic differential equation to take these factors into account. The application of such a model to hCG kinetic binding data demonstrated that reactant degradation had little significant effect on the derivation of the association rate constant (k1), but caused significant overestimation of the dissociation rate constant (k2) values derived from association experiments. The model was also applied by computer simulation to a theoretical analysis of the effects of degradation of free hormone and receptor sites upon kinetic and steadystate binding data. By this method, the initial velocities of hormone binding were shown to be less affected by degradation than the steady-state levels of hormone-receptor complex. Also, reactant degradation in simulated steady-state experiments caused an underestimate of the apparent equilibrium association constant, but had relatively less effect on the determination of binding site concentration.     

Interaction of rabbit secretory component with rabbit IgA dimer.

Secretory component (SC), a glycoprotein with an apparent molecular weight of approximately 80,000, has been isolated from rabbit milk and found to be heterogenous in size and charge. Functionally intact IgA dimer has been dissociated from milk secretory IgA using a chaotropic agent and further purified to homogeneity. The interaction between SC and IgA dimer is a reversible time- and temperature-dependent process. At 23 degrees C, the association rate constant (2.4 x 10(5) M-1 min-1) and the dissociation rate constant (1.8 x 10(-3) min-1) have been measured independently and the affinity constant based on these rates (1.3 x 10(8) M-1) is similar to that calculated from Scatchard plots (1.9 x 10(8) M-1). One class of binding sites has been estimated from Scatchard plots in spite of the observed heterogeneity of SC. The interaction is tighter at low temperatures because the decrease in dissociation rate is greater than the decrease in association rate. The thermodynamic calculations reveal a delta G of -11.0 kcal . mol-1, a delta H of -8.9 kcal . mol-1 and a delta S of +7.0 cal. mol-1 degree-1. The pH range over which interaction occurs is rather large (5 to 8) with no significant differences in apparent Ka.     

Solubilization and partial characterization of the sex hormone-binding globulin receptor from human prostate

The sex hormone-binding globulin (SHBG) receptor was solubilized from the membranes of human prostate glands with the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid). The binding activity of the soluble receptor was measured by allowing it to bind to 125I-SHBG and precipitating the complex with polyethylene glycol-8000. The binding activity was stable for at least 4 months at -20 degrees C and had a half-life of 23 days at 4 degrees C. Like the membrane-bound receptor, Scatchard analysis revealed two sets of binding sites for the soluble one. At equilibrium (24 h), the high affinity site had an association constant (KA) of 6.8 x 10(8) M-1 and a binding capacity of 1.4 pmol/mg protein, whereas the low affinity site had a KA of 4.7 x 10(6) M-1 and a binding capacity of 43 pmol/mg protein. At 37 degrees C, the association rate constant (k1) was 8.37 x 10(5) M-1 min-1 and the dissociation rate constant (k2) was 3.43 x 10(-4) min-1. The soluble receptor was retarded on Sepharose CL-6B and had an apparent Mr = 167,000.     

Characterization of human somatotropin binding to detergent-solubilized lactogenic receptors from rat liver.

Lactogenic receptors from rat liver microsomal fraction ('microsomes') were extracted by treatment with 1% (w/v) Triton X-100. Triton X-100 exerts an inhibitory effect on both the binding reaction and the separation of the free hormone from the complex. The association and dissociation of 125I-labelled human somatotropin are time- and temperature-dependent processes. The association rate constant, k1, is 6.7 x 10(6) mol . litre-1 . min-1 at 25 decrees C, and the dissociation rate constant, k-1, is 1.1 x 10(-3) min-1 at 25 degrees C. Scatchard analysis of saturation data reveals the existence of a single class of receptors and that solubilization leads to a slight decrease in affinity and a sharp increase in binding capacity. The dissociation constant, Kd, of the solubilized preparation is 0.22 nM and the binding capacity 2900 fmol/mg of protein. Similar results were obtained from competition experiments. Binding of 125I-labelled human somatotropin to the solubilized receptors is specifically inhibited by hormones with lactogenic activity. Incubation of the solubilized preparation with trypsin resulted in an 80% decrease in binding activity. The solubilized form of the receptor has a slightly increased sensitivity to the inactivation by trypsin, heat and extremes of pH, with respect to the membrane-bound form.     

Kinetics of pyrophosphate induced iron release from diferric ovotransferrin

The kinetics of pyrophosphate-induced iron release from diferric ovotransferrin were studied spectrophotometrically at 37 degrees C in 0.1 M HEPES, pH 7.0. At high pyrophosphate concentrations, the kinetics are biphasic, indicating that the rates of iron release from the two, presumably noninteracting iron-binding sites of ovotransferrin are different. The pseudo-first-order rate constants for iron release from both the fast and slow sites exhibit a hyperbolic dependence on pyrophosphate concentrations. The data suggest that pyrophosphate forms complexes with the two iron-binding sites of ovotransferrin prior to iron removal. The stability constants of the complex formed with the fast site (Keqf) and slow site (Keqs) are 8.3 M-1 and 40.4 M-1, respectively. The first-order rate constants for the dissociation of ferric-pyrophosphate from the fast site (k2f) and the slow site (k2s) are 0.062 and 0.0044 min-1, respectively. Results from urea gel electrophoresis studies suggest that iron is released at a much faster rate from the N-terminal binding site of ovotransferrin. At high pyrophosphate concentration, only C-monoferric-ovotransferrin is detected during the course of iron release. At low pyrophosphate concentration, however, a detectable amount of N-monoferric-ovotransferrin is accumulated. This result is consistent with the kinetic finding that the site with a higher k2 (0.062 min-1) has a lower affinity toward pyrophosphate (Keq = 8.3 M-1) whereas the site with a lower k2 (0.0044 min-1) has a higher affinity for pyrophosphate (Keq = 40.4 M-1).     

Purification and properties of phage P22 c2 repressor.

The c2 repressor of phage P22 has been purified to homogeneity. It specifically binds to lambdaimm21 and P22 DNA. Its affinity for the presumed operator mutant P22 virB is reduced. The initial dissociation rates of the complex between c2 repressor and lambdaimm21 DNA are 0.02 min-1 at 0 degrees C, 0.08 min-1 at 20 degrees C and 0.17 min-1 at 32 degrees C. The dissociation rates of complexes formed between the c2 repressor and the lambdaimm21 operators OR, OL and OR vira were measured and compared to the corresponding rates obtained with 21 cI repressor.     

High and low affinity receptors for human interleukin for DA cells/leukemia inhibitory factor on human cells. Molecular characterization and cellular distribution.

Radioiodinated recombinant human interleukin DA (HILDA)/leukemia inhibitory factor (LIF) purified from conditioned medium of Chinese hamster ovary transfected cells enabled the identification of specific receptor sites on a variety of human cell types. Using low concentrations (up to 500 pM) of the ligand iodinated at a high specific radioactivity, high affinity receptors (equilibrium dissociation constant Kd in the range of 30-100 pM) were first demonstrated. They were expressed at low levels by human peripheral blood monocytes but not by lymphocytes, NK cells, granulocytes, and platelets. The myelomonocytic cell line THP1 as well as the T lymphoma cell line HSB2 and the lymphoblastoid B cell line DAB were also receptor-negative. In contrast, most of the non-lymphoid tumoral cell lines tested, including melanomas, neuroblastomas, and carcinomas, expressed high affinity HILDA/LIF receptors at variable levels (Bmax from 20 to 600 sites/cell). The kinetics of HILDA/LIF high affinity binding to the choriocarcinoma JAR cell line were characterized at 4 degrees C with association and dissociation rate constants of k1 = 2.2 10(9) M-1 min-1 and k-1 = 0.0084 min-1, respectively, corresponding to a steady-state dissociation constant k1/k-1 = 3.8 pM. The subsequent use of higher concentrations of HILDA/LIF labeled at a lower specific radioactivity enabled the identification of a low affinity component on several cell lines (Kd in the range of 1-4 nM; Bmax from 1,000 to 5,000 sites/cell). On JAR cells, this low affinity component was characterized by association and dissociation rate constants at 4 degrees C of k1 = 7.3 10(7) M-1 min-1 and k-1 = 0.19 min-1, respectively (k-1/k1 = 2.6 nM). Affinity cross-linking of HILDA/LIF to JAR cells showed two cross-linked species under both reducing and nonreducing conditions corresponding to receptor species of 120 and 250 kDa, respectively. Whereas both bands had similar intensities under high affinity conditions, the higher band predominated under low affinity conditions. Our data suggest that the 250-kDa chain could constitute the low affinity binding component whereas the association of both 250- and 120-Da subunits would form the high affinity structure.     

Recycling of the asialoglycoprotein receptor in isolated rat hepatocytes. Dissociation of internalized ligand from receptor occurs in two kinetically and thermally distinguishable compartments

We have examined the rate of dissociation of internalized 125I-asialo-orosomucoid-receptor complexes in freshly isolated rat hepatocytes. Cell suspensions were washed with ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid at 0 degrees C to remove surface-bound ligand and then assessed for the retention of radioactive glycoprotein in the presence of digitonin, which permeabilized the cells and released the internal soluble contents. In cells which initially contained only surface-bound ligand, about 50% of the internalized ligand dissociated from receptor very rapidly (t1/2 less than or equal to 2.5 min, k greater than or equal to 0.28 min-1), at 37 degrees C, whereas the other 50% dissociated more slowly with apparent first order kinetics (t1/2 = 50 min, k = 0.014 min-1). This equal distribution of internalized ligand into two compartments, from which dissociation occurred with very different kinetics, did not depend on the extent of surface receptor occupancy and also occurred under non-steady state conditions of continuous exposure to ligand. Ligand entering both the rapid and slow dissociation compartments was eventually degraded with apparent first order kinetics (k = 0.0047 min-1), suggesting that the intracellular routing of ligand to lysosomes after dissociation from either compartment was via the same pathway. The fast and slow dissociation of receptor-ligand complexes were also distinguished by different temperature sensitivities; the slow dissociation process ceased below 18 degrees C, whereas the fast dissociation process still proceeded. The equal partition of internalized complexes into the two kinetic compartments did not change as a function of temperature but did change as cells continued to endocytose asialo-orosomucoid at 37 degrees C. As the internal receptor pool approached a steady state level of occupancy, there was an increase in the average time for receptor recycling and an increase in the fraction of incoming receptor-ligand complexes which dissociated rapidly (approximately 75%). In addition, under steady state conditions, the rate of the slow dissociation process increased (k = 0.026 min-1, t1/2 = 27 min).     

Activation of Torpedo acetylcholine receptors expressed in mouse fibroblasts. Single channel current kinetics reveal distinct agonist binding affinities.

The experiments described examine single channel currents recorded through Torpedo acetylcholine receptor channels stably expressed by a mouse fibroblast cell line. Closed-duration histograms were constructed from currents elicited by 0.5-300 microM acetylcholine (ACh). The concentration dependence of closed durations is well described by a four-state linear scheme with the addition of open-channel block by ACh. Analysis of closed durations measured at low concentrations gives estimates of the rate of opening of doubly liganded receptors, beta, the rate of dissociation of ACh from doubly liganded receptors, k-2, and the rate of channel closing, alpha. The rate of ACh dissociation from singly liganded receptors, k-1, is then deduced from closed-duration histograms obtained at intermediate ACh concentrations. With k-1, k-2 and beta determined, the rates of ACh association, k+1 and k+2, are estimated from fitting closed-duration histograms obtained over a range of high ACh concentrations. A complete set of rate constants is presented for three experimental conditions: (a) Ca2(+)-free extracellular solution containing 1 mM free Mg2+ at 22 degrees C, (b) Ca2(+)-free solution at 12 degrees C, and (c) extracellular Ca2+ and Mg2+, both at 0.5 mM, at 22 degrees C. For all three conditions the dissociation constant for the first agonist binding site is approximately 100-fold lower than that for the second site. The different affinities are due primarily to different dissociation rates. Both the association and dissociation rates depend strongly on temperature. At 22 degrees C ACh associates at diffusion-limited rates, whereas at 12 degrees C association is 30- to 60-fold slower. Also slowed at 12 degrees C are beta (4-fold), k-2 (3-fold), k-1 (25-fold), and alpha (15-fold). In contrast to the activation rate constants, those for ACh-induced block decrease only twofold between 22 and 12 degrees C. Changing from a Ca2(+)-free to a Ca2(+)-containing extracellular solution does not affect k+1 and k+2, but increases beta (twofold) and decreases k-2, k-1, and alpha (all twofold). Spectral analysis of single channel currents supports the parameter estimates obtained from fitting the open- and closed-duration histograms, and improves resolution of brief channel blockages produced by ACh.     

The kinetics of mebendazole binding to Haemonchus contortus tubulin.

The kinetics of the binding of mebendazole (MBZ) to tubulin from the third-stage (L3) larvae of the parasitic nematode, Haemonchus contortus, have been characterized. In partially purified preparations, the association of [3H]MBZ to nematode tubulin was rapid, k1 = (2.6 +/- 0.3) x 10(5) M-1 min-1, but dissociation was slow, k-1 = (1.58 +/- 0.02) x 10(-3) min-1. The affinity constant (K(a)) for the interaction, determined by the ratio k1/k-1, was (1.6 +/- 0.2) x 10(8) M-1. Similar results were obtained with crude cytosolic fractions. In equilibrium studies, performed with partially purified nematode tubulin under similar conditions, a K(a) of (5.3 +/- 1.6) x 10(6) M-1 was obtained. The best estimate for the K(a) of the MBZ-nematode tubulin interaction is considered to be the 'kinetic' value determined from the ratio of rate constants. The slow dissociation of MBZ from nematode tubulin, which contrasts with the rapid dissociation of MBZ from mammalian tubulin, supports the hypothesis that the selective toxicity of the benzimidazole anthelmintics results from a difference between the affinities of mammalian and nematode tubulins for these drugs.     

Mannitol and glucose movement across the Golgi membrane of lactating-rat mammary gland.

1. Purified Golgi-membrane vesicles of lactating-rat mammary gland were penetrated by glucose. 3-O-methylglucose, mannose, fructose, sorbitol and mannitol, but not by lactose or sucrose. 2. The kinetics of mannitol uptake and release were followed at 2-6 degrees C with the aid of fine filters (0.45 micrometers pore size) to separate the vesicles from the medium. 3. Mannitol efflux exhibited apparent first-order kinetics with k approximately 1 min-1. Neither saturability, nor inhibition by excess sorbitol or glucose, could be observed. 4. Mannitol efflux at 18 degrees C was about seven times faster than at 1 degrees C, and rates at higher temperatures were too fast to be measured. The rate of glucose efflux at 2-6 degrees C exceeded that of mannitol severalfold. 5. These findings imply a channel or carrier of definite, but limited, specificity straddling the Golgi membrane and able to supply glucose for lactose synthesis.     

Cholic acid uptake and isolated rat hepatocytes.

Cholic acid uptake was studied in isolated rat hepatocytes using a centrifugal filtration technique to allow rapid sampling. Hepatocytes were found to adsorb as well as to transport cholic acid. The adsorption was characterized by a capacity of 24 nmol X mg cell protein-1 and an association constant of 0.59 X 103 M-1. Cholic acid uptake was linear with respect to concentration at or below 10 degree C, suggesting a unsaturable uptake process which was considered to represent simple diffusion and is quantitated by a diffusion coefficient of 1.76 pmol cholic acid X min-1 X mg protein-1 X muM-1. Above 10 degrees C the uptake curve was biphasic. After subtracting the unsaturable component from uptake rates at higher temperatures, a curve showing saturable kinetics resulted. The apparent Km and V values at 37 degrees C were calculated to be 31muM and 0.8 nmol X min-1 X mg protein-1 respectively. This saturable uptake process was temperature-dependent with an activation energy of 13 kcal X mol-1 (5.44 X 104 J X mol-1) and was inhibited by oligomycin and KCN. Countertransport was demonstrated with cholic, taurocholic and chenodeoxycholic acids. The results suggest that cholic acid is transported by an energy-dependent carrier-mediated process in addition to simple diffusion by hepatocytes, and that the postulated carrier has affinity for other bile acids.     

Benzodiazepine receptors along the nephron: [3H]PK 11195 binding in rat tubules

Binding of [3H]PK 11195, an isoquinoline carboxamide derivative, was measured in microdissected tubule segments of rat nephron. High specific binding capacities (1.1-1.8 fmol X mm-1) were found in the thick ascending limb of the Henle's loop and in the collecting tubule, whereas specific binding could not be detected in the proximal tubule. In the medullary collecting tubule, the association and dissociation rate constants at 4 degrees C were k1 = 3.0 X 10(6) M-1 X min-1 and k-1 = 0.021 min -1; the ratio k-1/k1 = 7.0 nM was in agreement with the estimated equilibrium dissociation constant (Kd = 2.4 nM). [3H]PK 11195 binding sites from medullary ascending limb and medullary collecting tubule revealed the following sequence of specificity: PK 11195 = Ro 5-4864 much greater than clonazepam, indicating that tubule binding sites might be the peripheral benzodiazepine receptors of the rat kidney.     

Kinetics and concentration dependence of reversible cAMP-induced modification of the surface cAMP receptor in Dictyostelium

We have previously reported that extracellular cAMP induced a reversible shift, from apparent Mr = 40,000 to 43,000, in the electrophoretic mobility of a polypeptide identified by photoaffinity labeling with [32P]8-N3-cAMP as the cAMP receptor of Dictyostelium (Klein, P., Theibert, A., Fontana, D., and Devreotes, P. (1985) J. Biol. Chem. 260, 1757-1764). In this report, we examine the kinetics and concentration dependence of this stimulus-induced receptor modification. Prior to stimulation, 90% of the receptors migrated as the higher mobility form (Mr = 40,000) and 10% as the lower mobility form (Mr = 43,000). Following 15 min of persistent stimulation with 1 microM cAMP, the per cent of receptors migrating as the lower mobility form rose to 80%. This transition occurred with a half-time of 2.5 min. Removal of the stimulus initiated a return to the basal state which occurred with a half-time of about 6 min at 22 degrees C. No reversal occurred at 0 degrees C. Addition and removal of a 50 nM cAMP stimulus induced transitions with similar kinetics, but the final plateau value reached was only 40% lower mobility form. The stimulus concentration which induced 50% of the maximal transition from higher to lower mobility forms at steady state was 27 nM, similar to the KD for [3H]cAMP binding. Scatchard analysis of [3H]cAMP binding indicated that, although a 20% down-regulation occurs during cAMP stimulation, there is no significant difference in the affinities of the higher and lower mobility forms of the receptor. The unoccupied higher and lower mobility forms of the receptor, designated R and D, are considered to be in rapid equilibrium with liganded forms, designated RL and DL. The rate constants for interconversion of the receptor forms R (Formula: see text) D and RL (Formula: see text) DL were calculated from the kinetic data: k1 = 0.012, k-1 = 0.104, k2 = 0.222, and k-2 = 0.055. The interconversion steps are not at equilibrium, suggesting that an energy expenditure occurs during the receptor modification. The pattern of modulation of the cAMP-induced receptor modification suggests that it may be the biochemical mechanism of adaptation.     

Binding properties of beta-adrenergic receptors in early human fetal lung

The characteristics of beta-adrenergic receptors in human fetal lung were examined using the beta-adrenoceptor antagonist 3H-dihydroalprenolol /DHA/. Steady-state binding was reached by 15 min at 25 degrees C, and the association and dissociation rate constants were 0.0422 nM-1 min-1 and 0.0874 min-1, respectively. From saturation experiments, Bmax of 82.0 +/- 38 fmol/mg protein and KD = 1.85 +/- 0.92 nM were calculated. Inhibition of 3H-DHA binding by beta-1 /metoprolol/ and beta-2 /zinterol, IPS-339, fenoterol/ selective drugs resulted in biphasic displacement curves with slope factors less than 1.0. Analysis of these curves revealed a beta-1: beta-2 ratio of 40:60 in human fetal lung. The presence of 3H-DHA binding sites in early human lung may have a developmental importance which is not yet understood.     

Kinetics and thermodynamics of ouabain binding by intact turkey erythrocytes: effects of external sodium ion, potassium ion, and temperature

The kinetics of association and dissociation for the ouabain-Na+,K+- dependent ATPase complex have been studied in intact turkey erythrocytes as a function of external Na+ concentration, K+ concentration, and temperature. At free ligand concentrations substantially exceeding the concentration of available binding sites, the association reaction exhibits pseudo-first-order kinetics with an association rate constant (k1) that is conveniently determined over a wide range of temperatures (5-37 degrees C). The dissociation reaction exhibits strict first-order kinetics with a dissociation rate constant (k-1) that has the unusual property, in the turkey cell, of being sufficiently great to permit its direct determination even at temperatures as low as 5 degrees C. Values for the equilibrium binding constant for the ouabain-ATPase complex (KA) predicted from the ratio of the association and dissociation rate constants agree closely with independently measured values of KA determined directly under conditions of equilibrium binding. KA is a sensitive function of the composition of the external ionic environment, rising with increasing Na+ concentration and falling with increasing K+ concentration. These changes in KA are shown to be quantitatively attributable to changes in the rate constant k1, k-1 in contrast being unaffected at any given temperature by even very large changes in Na+ or K+ concentration. Arrhenius plots of k1 and k-1 both yield straight lines over the entire temperature range corresponding to activation energies for association and dissociation of 29.5 and 24.2 kcal/mol, respectively. These observations have made it possible to calculate the following standard values for the ouabain binding reaction in the presence of 150 mM Na+: delta G degree = -9.8 kcal/mol; delta H degree = +5.3 kcal/mol; delta S degree = +48.7 cal/degree/mol. The large positive value of delta S degree presumably reflects a highly ordered configuration of the ouabain-free ATPase molecule that is lost upon ouabain binding and that "drives" the reaction despite the positive value of delta H degree.     

Kinetic identification of a two-state glucagon receptor system in isolated hepatocytes. Interconversion of homogeneous receptors

A detailed kinetic study was performed to investigate the interaction of glucagon with receptors on freshly isolated hepatocytes. Competition binding assay results fit a mathematical expression for a single site noncooperative model of binding. Glucagon was shown to bind with first-order kinetics at six-hormone concentrations (0.02-0.50 nM) at 0 and 37 degrees C. The observed pseudo-first-order rate constants are directly proportional to the hormone concentration at 0 degree C, but display a downward deviation from linearity at 37 degrees C. Dissociation of glucagon exhibited biexponential character at 37 degrees C which was not seen at 0 degree C. The biphasic dissociation at 37 degrees C was resolved into rapid (t1/2 = 1.9 min) and slow (t1/2 = 27.7 min) components. The distribution of the total bound hormone between the rapidly and slowly dissociating complexes was not dependent upon the extent of receptor occupancy. The absolute quantity of rapidly dissociating hormone-receptor complexes was constant at all times examined; however, the fraction of slowly dissociating hormone-receptor complexes was found to increase with increasing incubation time. The results indicate that a homogeneous population of hepatic receptors undergoes a time-dependent, temperature-dependent conversion from one state to another in a two-stage sequential manner.