Nonactivated Complex Research Articles

Structural characterization of the catalytic γ and regulatory β subunits of phosphorylase kinase in the context of the hexadecameric enzyme complex.

In the tightly regulated glycogenolysis cascade, the breakdown of glycogen to glucose-1-phosphate, phosphorylase kinase (PhK) plays a key role in regulating the activity of glycogen phosphorylase. PhK is a 1.3 MDa hexadecamer, with four copies each of four different subunits (α, β, γ and δ), making the study of its structure challenging. Using hydrogen-deuterium exchange, we have analyzed the regulatory β subunit and the catalytic γ subunit in the context of the intact non-activated PhK complex to study the structure of these subunits and identify regions of surface exposure. Our data suggest that within the non-activated complex the γ subunit assumes an activated conformation and are consistent with a previous docking model of the β subunit within the cryoelectron microscopy envelope of PhK.

Yttrium half-sandwich complexes bearing the 2-(N,N-dimethylamino)ethyl-tetramethylcyclopentadienyl ligand

The synthesis of bis(dimethylsilyl)amide and dimethyl half-sandwich complexes of yttrium bearing the 2-(N,N-dimethylamino)ethyl-tetramethylcyclopentadienyl ligand revealed unusual Si‒H activation reactions and ate complex formation, respectively. Protonolysis of complex Y[N(SiHMe2)2]3(thf)2 with cyclopentadiene C5Me4HCH2CH2NMe2 (HCpNMe2) at elevated temperature yielded complex CpNMe2Y[η2-SiMe2(NSiHMe2)2](thf) featuring a four-membered metallacycle. The bis(amido) ligand [η2-SiMe2(NSiHMe2)2]2− is shown to form via separation of H2SiMe2. In contrast, the salt metathesis reaction of YCl3(thf)3 with LiN(SiHMe2)2 and LiCpNMe2 in a 1/2/1 ratio at ambient temperature generated the non-activated complex CpNMe2Y[N(SiHMe2)2]2, exhibiting a distinct ligand activation at elevated temperatures, as evidenced by a comprehensive NMR spectroscopic study. Application of a sequential salt metathesis protocol involving YCl3(thf)3, LiCpNMe2, and LiMe led to the isolation of ate complexes [CpNMe2YCl2]2[LiCl(thf)2] and [CpNMe2YMe2(LiMe)]2. Aforementioned half-sandwich complexes were all characterized by X-ray structure analyses and the respective silylamido and methyl derivatives are demonstrated to produce the half-sandwich yttrium bis(tetramethylaluminate) complex [CpNMe2AlMe3Y(AlMe4)2] in the presence of excess trimethylaluminium, by NMR spectroscopic studies.

The structure of the complex between rubisco and its natural substrate ribulose 1,5-bisphosphate

The three-dimensional structure of the complex of ribulose 1,5-bisphos phate carboxylase/oxygenase (rubisco; EC 4.1.1.39) from spinach with its natural substrate ribulose 1,5-bisphosphate (RuBP) has been determined both under activating and non-activating conditions by X-ray crystallography to a resolution of 2.1 Å and 2.4 Å, respectively. Under activating conditions, the use of calcium instead of magnesium as the activator metal ion enabled us to trap the substrate in a stable complex for crystallographic analysis. Comparison of the structure of the activated and the non-activated RuBP complexes shows a tighter binding for the substrate in the non-activated form of the enzyme, in line with previous solution studies. In the non-activated complex, the substrate triggers isolation of the active site by inducing movements of flexible loop regions of the catalytic subunits. In contrast, in the activated complex the active site remains partly open, probably awaiting the binding of the gaseous substrate. By inspection of the structures and by comparison with other complexes of the enzyme we were able to identify a network of hydrogen bonds that stabilise a closed active site structure during crucial steps in the reaction. The present structure underlines the central role of the carbamylated lysine 201 in both activation and catalysis, and completes available structural information for our proposal on the mechanism of the enzyme.

Temperature-dependent effects of high pressure on the bioluminescence of firefly luciferase

This study measured the effect of high pressure on the enzyme kinetics of firefly luciferase. When firefly luciferase is mixed with luciferin and ATP, a transient flash of light is produced, followed by a weak light, lasting hours. The first stage reaction produces an enzyme-luciferin-AMP complex and pyrophosphate. Addition of pyrophosphate to the reaction mixture decelerated the reaction rate, and the initial flash was prolonged to a plateau, showing a quasi-equilibrium state. The effects of temperature and pressure were analyzed at the plateau. The temperature scan showed that the maximum light intensity was observed at about 22.5 degrees C. When pressurized below the temperature optimum, pressure decreased the light intensity, while increasing it above the temperature optimum. According to the theory of absolute reaction rate, the following values were obtained for the bioluminescent reaction: delta V++ = 823.7 - 2.8 T cm3/mol and delta V = -280.47 + 0.94T cm3/mol, where T is the absolute temperature, delta V++ and delta V are, respectively, activation volume and the volume change due to thermal unfolding. The optimal temperature for the maximum light output occurs because the reaction rate increases with the temperature elevation at low temperature range, but the thermal unfolding of the enzyme decelerates the reaction velocity when the temperature exceeds a critical value. The intensity of luminescence is modified by the influence of pressure on both delta V++ and delta V. So long as the volume of the activated complex (V++) exceeds the average volume of the nonactivated complex (VN), pressure will slow down the reaction. At the point where the volumes become equal, there is no change in the rate under pressure. When the volume of the activated complex is less than that of the reactants, pressure will speed up the rate. This study showed that firefly luciferase is not exceptional to other enzymes in responding to high pressure.

Cyclic, nonequilibrium models of glucocorticoid antagonism: Role of activation, nuclear binding and receptor recycling

Quantitative models that have been proposed to date to explain mechanisms of glucocorticoid antagonism have generally been of the equilibrium type, involving hypothetical allosteric equilibria between active and inactive states of the receptor or the steroid-receptor complex. We describe here the agonist-antagonist relationships predicted by a nonequilibrium cyclic model that we have recently devised to account for the kinetic behavior of glucocorticoid-receptor complexes in intact rat thymus cells. This model simulates quantitatively most kinetic and steady state results that have been obtained so far. It postulates the existence of only well-established receptor species, and its kinetic parameters can in principle be determined by receptor measurements with intact cells. To calculate the steady state agonist-antagonist properties it is assumed that biological activity is proportional to the total amount of nuclear-bound complex, whether formed by agonist or antagonist. The agonist activity of a steroid is determined by the steady state ratio of nuclear-bound to total complexes it forms. This ratio varies from 0 for a pure antagonist to 1 for a pure agonist. It turns out to be independent of agonist and antagonist concentrations, and a function only of the rate constants for the reactions of the complexes formed by a steroid. Analysis of the dependence of the ratio on each rate constant shows quantitatively how each reaction in the cyclic model—activation of the nonactivated complex, nuclear binding of the activated complexes, and dissociation and recycling of activated and nuclear-bound complexes—affects antagonist properties. Steady state interactions of agonists with antagonists are found to be determined by equations that are identical to those for competition in simple equilibrium systems. Predicted dose-response relations agree qualitatively with experimentally observed relations. They are similar to those predicted by two-state allosteric models, although the cyclic model has no allosteric mechanisms, and is based on quite different assumptions. Present limitations of the model arise particularly from lack of information about the mechanisms by which nuclear-bound complexes generate biological activity; for lack of such information the model includes no steps to account for substances that have low agonist activity despite forming nuclear-bound complexes.

Activation of cytosolic glucocorticoid-receptor complexes in intact WEHI-7 cells does not dephosphorylate the steroid-binding protein.

In order to determine the ratio of phosphates to hormone-binding sites on nonactivated (non-DNA-binding) glucocorticoid receptors in WEHI-7 mouse thymoma cells, we have extracted these receptors from cells grown to a steady state with 32P, labeled them with a saturating concentration of [3H]dexamethasone 21-mesylate, purified them using a monoclonal antibody, and analyzed them by polyacrylamide gel electrophoresis under denaturing and reducing conditions. The complexes contained approximately 5 mol of phosphate/mol of bound steroid. Only half of the phosphates were associated with the approximately 100-kDa protein which is labeled with [3H]dexamethasone 21-mesylate. The remaining phosphates were associated with the approximately 90-kDa non-steroid-binding component of the nonactivated complex. Dual label studies, using [35S]methionine to measure receptor protein and 32P to measure receptor phosphates, have enabled us to determine the phosphate content, relative to receptor protein, of both nonactivated and activated cytosolic complexes generated in intact WEHI-7 cells exposed to triamcinolone acetonide at 37 degrees C. The total amount of phosphate associated with the activated complex is roughly half of that associated with the nonactivated complex, the decrease being accounted for by dissociation of the approximately 90-kDa phosphoprotein which accompanies activation. However, the ratio of 32P to 35S counts associated with the approximately 100-kDa steroid-binding protein is the same for the activated and nonactivated complexes. These results indicate that there is no net change in the phosphorylation of the approximately 100-kDa steroid-binding component of the cytosolic glucocorticoid-receptor complex upon activation in the intact cell.

Molybdate-stabilized nonactivated glucocorticoid-receptor complexes contain a 90-kDa non-steroid-binding phosphoprotein that is lost on activation.

We have used a monoclonal antibody to purify glucocorticoid-receptor complexes from WEHI-7 mouse thymoma cells. Molybdate-stabilized, nonactivated complexes were found to contain two distinct proteins which could be separated by polyacrylamide gel electrophoresis under denaturing and reducing conditions. One of the proteins, 100 kDa, was labeled when cytosol was incubated with the affinity ligand [3H]dexamethasone 21-mesylate. The second protein, 90 kDa, was not labeled. Several lines of evidence, including Western blot analysis of purified nonactivated complexes, indicate that only the 100-kDa protein is directly recognized by the antibody. The 90-kDa protein appears to be purified as a component of the nonactivated complex due to noncovalent association with the 100-kDa protein. Both the 100-kDa and 90-kDa components of the nonactivated complex become labeled with 35S when cells are grown in medium containing [35S]methionine. Using cells labeled in this manner, we have shown that activated (i.e. DNA-binding) cytosolic complexes, formed by warming either in intact cells or under cell-free conditions, contain only the 100-kDa protein. Complexes extracted from nuclei of warmed cells similarly contain only the 100-kDa protein. These results indicate that the 100-kDa and 90-kDa components of nonactivated complexes separate upon activation. Purification of nonactivated complexes from cells grown in medium containing [32P]orthophosphoric acid indicates that both the 100-kDa and 90-kDa components are phosphoproteins which can be labeled with 32P. Therefore, resolution of the two proteins will be essential in order to determine whether the receptor is dephosphorylated on activation.

Degradation without apparent change in size of molybdate-stabilized nonactivated glucocorticoid-receptor complexes in rat thymus cytosol.

We have investigated the stability of the [3H]dexamethasone 21-mesylate-labeled nonactivated glucocorticoid-receptor complex in rat thymus cytosol containing 20 mM sodium molybdate. Cytosol complexes were analyzed under nondenaturing conditions by gel filtration chromatography in the presence of molybdate and under denaturing conditions by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. When analyzed under nondenaturing conditions, complexes from fresh cytosol and from cytosol left for 2 h at 3 degrees C eluted from gel filtration as a single peak of radioactivity with a Stokes radius of approximately 7.7 nm, suggesting that no proteolysis of the complexes had occurred in either cytosol. When analyzed under denaturing conditions, however, whereas the fresh cytosol gave a receptor band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at Mr approximately 90,000 (corresponding to the intact complex), the cytosol that had been left for 2 h at 3 degrees C gave only a fragment (Mr approximately 50,000). This fragment, just as the intact complex, could be thermally activated to a DNA-binding form. Proteolysis of the receptor could be blocked by preparing the cytosol in the presence of EGTA, leupeptin, or a heat-stable factor present in the cytosol of rat liver and WEHI-7 mouse thymoma cells. From these results we conclude: (i) 20 mM molybdate does not protect the nonactivated glucocorticoid-receptor complex present in rat thymus cytosol against proteolysis under conditions which are commonly used for cell-free labeling of the receptor, and (ii) the demonstration of a Stokes radius of approximately 8 nm for the nonactivated glucocorticoid-receptor complex is not sufficient to indicate that the receptor complex is present in its intact form.

Effect of cross-linking on glucocorticoid receptor activation

Chick thymus cytosol glucocorticoid receptor complexed with [ 3H]-triamcinolone acetonide was heat-activated after treatment with diimidates of varying chain length. Diimidates with maximal effective reagent length shorter than 0.5 nm influenced DNA-cellulose binding only slightly, whereas sebacic diimidate (maximal effective reagent length 1.21 nm) blocked activation completely. Coupled activation — deactivation process induced in the presence of calcium was again inhibited only by the longer diimidates. Sebacic diimidate treated complex had a molecular mass similar to that of the molybdate stabilized nonactivated complex, as judged by gel permeation chromatography. It is suggested that prevention of size reduction by cross-linking blocks receptor activation as well.

Characterization of nonactivated and activated glucocorticoid-receptor complexes from intact rat thymus cells.

In cells exposed to glucocorticoids at 37 degrees C activated glucocorticoid-receptor complexes (complexes with affinity for nuclei and DNA) are formed after nonactivated complexes. Activation thus appears to be an obligatory physiological process. To investigate this process we have characterized cytoplasmic complexes formed in rat thymocytes at 0 and 37 degrees C. Complexes in cytosols stabilized with molybdate were analyzed by sucrose gradient centrifugation and by chromatography on DNA-cellulose, DEAE-cellulose, and agarose gels. Two major complexes were observed: the nonactivated complex, eluted from DEAE at approximately 200 mM KCl, was formed at 0 and 37 degrees C, gave S20,w = 9.2 S, Stokes radius = 8.3 nm, and calculated Mr = 330,000; the activated complex, eluted from DEAE at approximately 50 mM KCl, appeared only at 37 degrees C, gave S20,w = 4.8 S, Stokes radius = 5.0 nm, and Mr = 100,000. A third, minor complex, probably mero-receptor, which appeared mainly at 37 degrees C, bound to neither DNA nor DEAE, and gave S20,w = 2.9 S, Stokes radius = 2.3 nm, and Mr = 27,000. With three small columns in series (DNA-cellulose, DEAE-cellulose and hydroxylapatite), the three complexes can be separated in 5-10 min. By this method we have examined the stability of complexes under our conditions. We conclude that in intact thymus cells glucocorticoid-receptor complexes occur principally in two forms, nonactivated and activated, and that activation is accompanied by a large reduction in size. The origin of the mero-receptor complex remains uncertain.

Aqueous two-phase partition studies of the glucocorticoid receptor activation and pH-dependent changes of rat liver glucocorticoid-receptor complex in partly purified preparations

1. 1. The activation of the glucocorticoid receptor of rat liver cytosol was studied by the use of partitioning in an aqueous dextran-poly(ethylene glycol) two-phase system. 1.1. a, Incubation under conditions generally found to stimulate the activation results in a time-dependent increase of the partition coefficient of the glucocorticoid-receptor complex in the two-phase system. 1.2. b, This increase was found to result from the fact that the activated form of the complex has a higher partition coefficient that the nonactivated one, since the two forms are eluted from DNA-Sepharose columns as clearly separated peaks with different partition coefficients, and a redistribution of complex between the peak occurs without any changes of the partition coefficient of each single peak. 1.3. c, The partition coefficient of a partly purified nonactivated complex was found to follow the same time course towards a higher value under ‘activating’ incubation conditions as the complex in whole cytosol. 2. 2. The dependence of the partition coefficient of the glucocorticoid-receptor complex on the pH of the two-phase system revealed a pH-dependence of the state of the complex. Experiments with partly purified preparations of the complex showed that this change was either a conformational change or an aggregation of high affinity of the complex with itself or another DNA-binding substance.

“Non-activated” form of the progesterone receptor from chick oviduct: Characterization

Summary A non-activated form of the progesterone receptor from chick oviduct cytosol can be stabilized and prepared in the presence of sodium molybdate. This form of the receptor sediments at 8–9 S on 0.15 M KCl salt-containing sucrose gradients. Its Stokes radius, as determined on Ultrogel AcA-22, is 7.9 nm. The data permit calculation of a molecular weight for the non-activated receptor form of ∼ 290,000 d and a frictional ratio of 1.7. The non-activated receptor is eluted from DEAE-cellulose as a single peak, at an ionic strength of 0.1. Studies of dissociation kinetics of the hormone-receptor complex demonstrate that the non-activated complex dissociates 2.5 times faster than the activated complex (t 1/2 at 0°: 19 and 48 h, respectively).

Activated and non-activated glucocorticoid-receptor complexes in rat thymus cells: Kinetics of formation and relation to steroid structure

Previously we showed that when glucocorticoids initially enter rat thymus cells incubated at 37°C. non-activated hormone-receptor complexes are formed within 15 s and are then rapidly replaced by activated complexes. Activated and non-activated forms were identified in cytosols from the cells by DEAE-cellulose chromatography, where they are eluted with progressively higher salt concentrations in what are referred to as Peaks I and II. On DNA-cellulose columns dexamethasone-receptor complexes in cytosols that on DEAE-cellulose give mainly Peak II (the non-activated complex, referred to as Complex II) are not bound significantly: they are eluted at the lowest salt concentration along with free steroid, from which they can be separated by binding to hydroxyapatite. This DNA-cellulose peak is referred to as Peak a. Complexes in cytosols that give only Peak I on DEAE, however, on DNA separate into two components. One of these (Complex Ia) is not bound significantly and appears in Peak a. The other (Complex Ib, presumably the true activated complex) appears in a later Peak b. These three complexes have been measured by first adsorbing Ib with DNA-cellulose, then separating Complexes Ia and II. which are not adsorbed on DNA. by means of a DEAE-cellulose column. When [ 3H]-dexamethasone is added to cells at 37°C. II is formed within 15 s and is rapidly replaced by Ib, in agreement with our earlier results. Complex Ia accounts for 15–20% of total complexes at all times, and is also present after 120 min at 0°C. Various steroids have been compared on DNA-cellulose after 30 min at 37°C. when the amount of Complex II is negligible. The ratio of Ib to Ia is highest for dexamethasone and triamcinolone acetonide, intermediate for cortisol and corticosterone and lowest for cortexolone (which seems to form only Ia), roughly in proportion to intrinsic glucocorticoid activity. Complex Ia may thus be a third normal form of extranuclear glucocorticoid-rcceptor complex, but the possibility remains that it is an artifact formed after cells are broken. The relation of Complexes Ia, Ib and II to glucocorticoid activity is discussed.

The activated hepatic glucocorticoid-receptor complex: A simple one-step method for its separation from nonactivated complex

Protamine sulfate was found to precipitate completely the nonactivated [ 3H]-dexamethasone-receptor complex of rat liver. This observation was then used as the basis of a method to separate activated from nonactivated complex. Thus, addition of 10 mg/ml of protamine sulfate to the rat hepatic cytosol [ 3H]dexamethasone-receptor complex, incubated at 0–4°C for 2 hr, resulted in the complete precipitation of [ 3H]dexamethasone-receptor complex. The remaining supernatant obtained on centrifugation at 800 g was unable to bind either to nuclei or to DNA-cellulose. An increase in temperature to 25°C or the addition of 10 m m CaCl 2 to the cytosol resulted in the appearance of activated [ 3H]dexamethasone-receptor complex in the supernatant obtained by addition of protamine sulfate. This was determined by characteristic binding to nuclei or DNA cellulose and by p I. Protamine sulfate could not affect the separation of activated [ 3H]dexamethasone-receptor complex at salt concentrations above 100 m m NaCl. This procedure therefore had to be carried out under conditions of relatively low ionic strength. Finally, a one-step rapid method is described for the separation of activated [ 3H]dexamethasone-receptor complex from nonactivated receptor complex. The homogeneous population of activated complex thus obtained should have considerable applicability in studies of the mechanisms of in vitro glucocorticoid-receptor activation.