Identification and partial purification of a chromatin bound calmodulin activated histone 3 kinase from calf thymus

A calcium-calmodulin (Ca2(+)-CaM) stimulated histone H3 phosphorylating activity was identified as a component of a nuclear protein complex purified from a 150 mM NaCl extract of calf thymus chromatin. This activity bound to a CaM-Sepharose affinity column in a Ca2+ dependent manner and was eluted off the column in the presence of EGTA. Equilibrium centrifugation of the EGTA eluate on a sucrose density gradient revealed that the activity is a component of a larger complex identified at 25% sucrose. This complex consisted of two major proteins, having Mr of 65 and 75 kDa. Using [125I] CaM and the gel overlay technique it was shown that the 75 kDa protein is the major CaM binding protein in the complex.     

Purification of a novel calmodulin binding protein from bovine cerebral cortex membranes

A new calmodulin (CaM) binding protein, designated P-57, has been purified to apparent homogeneity from bovine cerebral cortex membranes. In contrast to other calmodulin binding proteins, P-57 has higher affinity for calmodulin in the absence of bound Ca2+ than in its presence. The protein was purified by DEAE-Sephacel chromatography and two CaM-Sepharose affinity column steps. The first CaM-Sepharose column was run in the presence of Ca2+; the second was run in the presence of chelator in excess of Ca2+. P-57 was adsorbed by CaM-Sepharose only in the absence of bound Ca2+ and was eluted from the second column by buffers containing Ca2+. Sodium dodecyl sulfate (SDS)-polyacrylamide gels of the purified protein showed only one band at Mr 57 000. The major form of the protein on Bio-Gel A-1.5m and native polyacrylamide gradient gel electrophoresis ran with an apparent Stokes radius of 41 A. Photoaffinity labeling of P-57 with azido[125I]calmodulin yielded one cross-linked product on SDS gels with an Mr of 70 000. This interaction occurred only when excess ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid was present and was inhibited by the presence of Ca2+ in excess of chelator. It appears that P-57 has novel binding properties for calmodulin distinct from all other calmodulin binding proteins described thus far.     

Affinity chromatography of brain cyclic nucleotide phosphodiesterase using 3-(2-pyridyldithio)propionyl-substituted calmodulin linked to thiol-sepharose

[3-(2-Pyridylthio)propionyl]calmodulin (PDP-CaM), an activated thiol derivative of calmodulin (CaM), was synthesized. Preparations of this derivative containing an average of 2.8 mol of substituent/mol of protein activated purified cyclic nucleotide phosphodiesterase in a manner indistinguishable from that of native CaM. PDP-CaM was covalently coupled to free thiol-Sepharose 4B through formation of a stable mixed disulfide bond for use in affinity chromatography. The binding capacity of the disulfide-linked CaM-Sepharose for phosphodiesterase activity was proportional to substituent level up to 4 mg of CaM/mL of gel; the total capacity of the gel for binding phosphodiesterase was 4 times that of CNBr-coupled CaM-Sepharose. Quantitative recovery was achieved by desorption of both ligand and bound proteins with a reducing agent. The thiolated CaM derivative was then separated from phosphodiesterase by rapid gel filtration; the overall recovery of phosphodiesterase activity was greater than 70%. Preparations of homogeneous enzyme in good yield were obtained after a second chromatography step on CaM-Sepharose. Binding and recovery of phosphodiesterase activity were entirely reproducible, since each preparation of affinity gel was used only once. As it permits separation of interacting species in free solution, this general method may be useful with other ligands for increasing yields from affinity chromatography, particularly when dissociation of molecules in their matrix-bound conformation may be difficult to achieve.     

Purification and molecular properties of the acetylcholine receptor from Torpedo electroplax

The acetylcholine receptor of Torpedo electroplax is purified by affinity adsorption using cobra toxin (Naja naja siamensis) covalently attached to Sepharose 4B. Desorption by 10 mm benzoquinonium produces a protein that binds α-[¹²⁵I]bungarotoxin but not [³H]acetylcholine or other reversible cholinergic ligands. On the other hand, desorption by 1 m carbamylcholine produces an acetylcholine receptor protein that binds [³H]acetylcholine, [³H]decamethonium, [³H]nicotine, [¹⁴C]dimethyl-d-tubocurarine, and α-[¹²⁵I]bungarotoxin. The batch method of affinity adsorption employed gives recoveries of acetylcholine receptor (as measured by acetylcholine binding) averaging 69.2 ± 14.6%. The purity of the isolated acetylcholine receptor protein is estimated to be at best 87% as judged by disc gel electrophoresis and electrofocusing.The purified acetylcholine receptor binds 7.8 nmoles acetylcholine/mg protein based on estimation of protein concentration by a spectrophotometric method. Of these, 2.7 nmoles exhibit high affinity (K_D = 0.02 μM) and 5.1 nmoles a lower affinity (K_D = 1.97 μM. If the protein concentration used is that obtained by amino acid analysis, the total specific activity would be 10.4 nmoles acetylcholine bound per milligram protein. The subunit carrying one acetylcholine binding site is estimated to range between 83,000 and 112,000 daltons. In contrast to the membrane-bound or Lubrol-solubilized acetylcholine receptor, the purified acetylcholine receptor shows no autoinhibition with acetylcholine concentrations up to 10 μm. Binding of acetylcholine was totally inhibited by α-bungarotoxin or cobra toxin and was partially blocked by four nicotinic drugs, but not by two muscarinic ones. The amino acids of the acetylcholine receptor are analyzed and compared to those of acetylcholinesterase.     

Interaction of monoclonal antibodies with alpha-bungarotoxin and (-)-nicotine binding sites in goldfish brain. Identification of putative nicotinic acetylcholine receptor subtypes

Monoclonal antibodies raised against the nicotinic acetylcholine receptor of Electrophorus electricus electroplaque have been used as probes to characterize putative nicotinic acetylcholine receptors in goldfish brain. One monoclonal antibody (mAb), mAb 47, recognized a protein which binds both (-)-[3H]nicotine and 125I-alpha-bungarotoxin with high affinity. Another monoclonal antibody (mAb 172) recognized a protein which binds (-)-[3H]nicotine but not 125I-alpha-bungarotoxin. Both antibodies precipitated a protein(s) (biosynthetically labeled with [35S]methionine) in the absence, but not in the presence, of excess purified nicotinic acetylcholine receptor from Torpedo nobiliana. The dilution of mAb 47 that precipitated half of the maximum amount of 125I-alpha-bungarotoxin binding protein was the same as that which precipitated half of the maximum amount of (-)-[3H]nicotine binding activity. When used in combination, the two antibodies precipitated more (-)-[3H]nicotine radioactivity than either antibody alone. The (-)-[3H]nicotine and 125I-alpha-bungarotoxin binding component-mAb complexes were characterized by sucrose density centrifugation. In the presence of either mAb 172 or 47, the (-)-[3H] nicotine binding component migrated further into the gradient, but only mAb 47 shifted the 125I-alpha-bungarotoxin peak. Incubation of solubilized brain extract with alpha-bungarotoxin-coupled Sepharose reduced the amount of (-)-[3H]nicotine radioactivity precipitated by mAb 47 but not by mAb 172. These data suggest that the antibodies may recognize distinct subtypes of (-)-nicotine binding sites in goldfish brain, one subtype which binds both 125I-alpha-bungarotoxin and (-)-[3H]nicotine and a second subtype which binds only (-)-[3H] nicotine.     

Nuclease sensitivity of estradiol-charged estrogen receptor binding sites in nuclei isolated from normal and neoplastic rat mammary tissues

The interaction of partially purified calf uterine estradiol-charged estrogen receptor ([3H]ER) with rat nuclei was studied in vitro. We previously observed a significantly greater number of [3H]ER binding sites (at saturation) in nuclei of R3230AC mammary tumors from intact vs ovariectomized (ovex) rats with no difference in the affinity of [3H]ER binding for these nuclei. We now report on the nuclease sensitivity of [3H]ER binding sites in nuclei from these tumors and from normal rat tissues. Digestion of tumor nuclei with deoxyribonuclease I (DNase I) prior to incubation with [3H]ER in vitro resulted in a progressive loss of [3H]ER binding capacity, which was not accompanied by alterations in the affinity of [3H]ER for the nuclei (Kd = 1-3 nM). A significantly lower concentration (P less than 0.005) of DNase I eliminated 50% of the [3H]ER binding sites in nuclei of tumors from intact hosts (8 unit.min/ml) compared to tumors from ovex hosts (22 unit.min/ml). These results indicate that DNA regions capable of binding ER are more susceptible to DNase I digestion in tumors from intact rats than those from ovex hosts, suggesting that the endogenous hormonal milieu is responsible, at least in part, for maintenance of nuclease-sensitive DNA conformations in this hormone-responsive mammary tumor. The amount of DNase I required to eliminate 50% of [3H]ER binding to nuclei from lactating mammary gland, liver, and kidney ranged from 14 to 56 unit.min/ml. Therefore, accessibility of [3H]ER binding sites to nuclease digestion in normal rat tissue is generally less than that of R3230AC tumors.     

Calmodulin binding to the Fas death domain. Regulation by Fas activation

Fas (APO-1/CD95) is a cell surface receptor that initiates apoptotic pathways, and its cytoplasmic domain interacts with various molecules suggesting that Fas signaling is complex and regulated by multiple proteins. Calmodulin (CaM) is an intracellular Ca(2+)-binding protein, and it mediates many of the effects of Ca2+. Here, we demonstrate that CaM binds to Fas directly and identify the CaM-binding site on the cytoplasmic death domain (DD) of Fas. Fas binds to CaM-Sepharose and is co-immunoprecipitated with CaM. Other death receptors, such as tumor necrosis factor receptor, DR4, and DR5 do not bind to CaM. The interaction between Fas and CaM is Ca(2+)-dependent. Deletion mapping analysis with various GST-fused Fas cytoplasmic domain fragments revealed that the fragment containing helices 1, 2, and 3 of the Fas DD has the CaM-binding ability. Sequence analysis of this fragment predicted a potential CaM-binding site in helix 2 and connected loops. A valine 254 to asparagine mutation in this region, which is analogous to the identified mutant allele of Fas in lpr mice that have a deficiency in Fas-mediated apoptosis, showed reduced CaM binding. Computer modeling of the interaction between CaM and helix 2 of the Fas DD predicted that amino acids, which are important for Fas-CaM binding, and point mutations of these amino acids caused reduced Fas-CaM binding. The interaction between Fas and CaM is increased approximately 2-fold early upon Fas activation (at 30 min) and is decreased to approximately 50% of control at 2 h. These findings suggest a novel function of CaM in Fas-mediated apoptosis.     

Biochemical characterization of the BETA-adrenergic receptor of the frog erythrocyte

Summary The beta-adrenergic receptor which is coupled to adenylate cyclase in the frog erythrocycte plasma membrane provides a convenient model system for probing the molecular characteristics of an adenylate cyclase coupled hormone receptor. Direct radioligand binding studies with beta-adrenergic agonists and antagonists such as [³H]hydroxybenzylisoproterenol and [³H]dihydroalprenolol have shed new light on the biochemical properties of the receptor as well as on its mode of interaction with other components of the adenylate cyclase system. Agonist binding to the receptor induces a high affinity state of the receptor which can be selectively reverted to a low agonist affinity state by guanyl nucleotides. This agonist-induced high affinity state of the receptor appears to correspond to a receptor moiety which has larger apparent molecular weight and which is probably a complex of the beta-adrenergic receptor and nucleotide regulatory binding protein. Antagonists do not appear capable of inducing or stabilizing the formation of this high affinity receptor-nucleotide site complex.The beta-adrenergic receptors have been solubilized using the plant glycoside digitonin as the detergent and have been highly purified by biospecific affinity chromatography on an alprenolol-agarose affinity support. These highly purified receptor preparations retain all of the binding characteristics observed in the unpurified soluble receptor preparations.Remarkably, antibodies raised in rabbits against affinity chromatography purified preparations of the receptor, themselves bind beta-adrenergic ligands with typical beta-adrenergic specificity. Such antibodies which possess binding sites similar to those of physiological receptors provide useful model systems for further probing the molecular characteristics of beta-adrenergic binding sites.     

Inactivation of oestrogen receptor in vitro by nuclear dephosphorylation.

1. Nuclei of the calf uterus are endowed with an activity inactivating crude oestrogen-receptor complex. This activity has been partially purified. It shows a very high affinity for the oestrogen-receptor complex (Km = 0.8 X 10(-9) mol of specific [3H]oestradiol-17 beta-binding sites/l) as well as for the oestrogen-free receptor (Km = 1.5 X 10(-9) mol of specific [3H]oestradiol-17 beta binding sites/l). 2. The nuclear receptor-inactivating activity is enhanced by dithiothreitol and inhibited by several phosphatase inhibitors as well as by 4-nitrophenyl phosphate, as well known phosphatase substrate. This inhibition shows that a dephosphorylation process is required for the receptor inactivation. 3. The purified nuclear activity also inactivates pure receptor and phosphatase inhibitors prevent this inactivation. From these observations it appears that receptor inactivation is due to a nuclear phosphatase directly acting on the oestrogen receptor. 4. The nuclear localization of the receptor-inactivating activity, its high affinity for specific oestrogen binding sites and, as previously reported, its presence only in oestrogen target tissues suggest that this activity is the same as that involved in the nuclear loss of the receptor observed in intact cells.     

Role of calmodulin methionine residues in mediating productive association with cardiac ryanodine receptors

Calmodulin (CaM) binds to the cardiac ryanodine receptor Ca2+ release channel (RyR2) with high affinity and may act as a regulatory channel subunit. Here we determine the role of CaM Met residues in the productive association of CaM with RyR2, as assessed via determinations of [3H]ryanodine and [35S]CaM binding to cardiac muscle sarcoplasmic reticulum (SR) vesicles. Oxidation of all nine CaM Met residues abolished the productive association of CaM with RyR2. Substitution of the COOH-terminal Mets of CaM with Leu decreased the extent of CaM inhibition of cardiac SR (CSR) vesicle [3H]ryanodine binding. In contrast, replacing the NH2-terminal Met of CaM with Leu increased the concentration of CaM required to inhibit CSR [3H]ryanodine binding but did not alter the extent of inhibition. Site-specific substitution of individual CaM Met residues with Gln demonstrated that Met124 was required for both high-affinity CaM binding to RyR2 and for maximal CaM inhibition. These results thus identify a Met residue critical for the productive association of CaM with RyR2 channels.     

The control of the interaction of sex hormone-binding globulin with its receptor by steroid hormones

Sex hormone-binding globulins (SHBG) is a plasma glycoprotein that binds certain steroids. It, in turn, binds to a specific receptor on cell membranes. This work was undertaken to investigate the role of steroids in the interaction of SHBG with its receptor. Because the probe for the interaction of SHBG with its receptor is 125I-SHBG, we first showed that 125I-SHBG binds [3H]dihydrotestosterone (DHT) at 4 degrees C and 37 degrees C with KD values similar to those published previously for pure radioinert SHBG. 125I-SHBG could be prevented from binding to its receptor by a variety of steroids whose relative inhibitory activity (dihydrotestosterone much greater than 2-methoxyestradiol greater than testosterone greater than estradiol much greater than methyltrienolone greater than cortisol) was almost identical to their relative ability to bind to SHBG. Because significant binding of [3H]DHT to the SHBG receptor could not be demonstrated, steroid inhibition of SHBG binding must be noncompetitive. If steroids bound to SHBG prevent binding to the SHBG receptor, then liganded SHBG should have a higher apparent KD for its receptor than unliganded SHBG. This is the case. The KD was 0.86 +/- 0.25 nM for the high affinity receptor site using liganded SHBG and 0.19 +/- 0.024 nM for unliganded SHBG. Thus, only liganded SHBG assumes a conformation that prohibits interaction with the SHBG receptor. However, when unliganded SHBG was prebound to its receptor, it retained its ability to bind [3H] DHT. The model that emerges from these observations is as follows. Unliganded SHBG can bind either steroids or receptor in a reversible reaction; SHBG bound to a steroid cannot bind to the receptor, but unliganded SHBG that first binds to the receptor can subsequently bind steroids.     

Solubilization and biochemical characterization of the high affinity [3H]ryanodine receptor from rabbit brain membranes

A high affinity [3H]ryanodine receptor has been solubilized from rabbit brain membranes and biochemically characterized. [3H]Ryanodine binding to rabbit brain membranes is specific and saturable, with a Kd of 1.3 nM. [3H]Ryanodine binding is enriched in membranes from the hippocampus but is significantly lower in membranes from the brain stem and spinal cord. Approximately 60% of [3H]ryanodine-labeled receptor is solubilized from brain membranes using 2.5% CHAPS and 10 mg/ml phosphatidylcholine containing 1 M NaCl. The solubilized brain [3H]ryanodine receptor sediments through sucrose gradients like the skeletal receptor as a large (approximately 30 S) complex. Solubilized receptor is specifically immunoprecipitated by sheep polyclonal antibodies against purified skeletal muscle ryanodine receptor coupled to protein A-Sepharose. [3H]Ryanodine-labeled receptor binds to heparin-agarose, and a protein of approximately 400,000 Da, which is cross-reactive with two polyclonal antibodies raised against the skeletal muscle ryanodine receptor, elutes from the column and is enriched in peak [3H]ryanodine binding fractions. These results suggest that the approximately 400,000-Da protein is the brain form of the high affinity ryanodine receptor and that it shares several properties with the skeletal ryanodine receptor including a large oligomeric structure composed of approximately 400,000-Da subunits.     

125I] 3-quinuclidinyl 4-iodobenzilate: a high affinity, high specific activity radioligand for the M1 and M2-acetylcholine receptors

We have prepared a radioiodinated ligand which binds with high affinity to the muscarinic acetylcholine receptor (m-AChR). A derivative of 3-quinuclidinyl benzilate, [125I] labeled (R) 1-aza-bicyclo(2.2.2)oct-3-yl (R,S)-alpha-hydroxy-alpha-(4-[125I]iodophenyl)phenyl acetate (4- IQNB ) exhibits an affinity for the m-AChR from corpus striatum higher than that of (R) [3H] QNB. Additionally, [125I] 4- IQNB exhibits receptor selectivity for the M1 receptor since the affinity for the receptor from dog and rat heart is lower than that using dog or rat corpus striatum.     

Binding of glucocorticoid receptors to mammary chromatin acceptor sites

We have recently characterized the interaction of mouse mammary estrogen receptors (ER) with mammary chromatin acceptor sites and demonstrated that ER from estrogen resistant lactating mammary glands do not bind to chromatin. In this study we have characterized the chromatin binding of the glucocorticoid receptor from mouse mammary glands isolated from nulliparous and lactating mice in order to better understand the relationship between receptor binding to chromatin and steroidogenic sensitivity of the tissue. Mammary chromatin was linked covalently to cellulose and deproteinized sequentially by 0-8 M Gdn-HCl. Binding to intact chromatin as well as to chromatin deproteinized by Gdn-HCl was determined using partially purified [3H]dexamethasone labelled glucocorticoid-receptor complexes (GR) obtained by fractionation on DEAE-cellulose columns. The binding of [3H]GR from mammary glands of nulliparous mice to chromatin fractions from the same tissue revealed maximal binding activity (acceptor sites) on chromatin previously extracted with 5-6 M Gdn-HCl. Binding of [3H]GR was of high affinity (Kd = 0.2 nM) and saturable. A simultaneous comparison of the chromatin binding patterns for [3H]ER and [3H]GR isolated from mammary glands of nulliparous mice revealed that the chromatin subfractions obtained with 4-6 M Gdn-HCl extraction contained acceptor sites for both [3H]ER and [3H]GR; however, while the [3H]ER bound to a 4.5 M and a 5.5 M site, the [3]GR bound a 5 M and a 6 M site. Competition experiments supported the steroid receptor specificity of the chromatin acceptor sites. Thus, the 4-6 M chromatin fractions contain distinct acceptor sites for the glucocorticoid receptor and for the estrogen receptor. In addition our studies reveal that the binding patterns of [3H]GR isolated from mammary glands of nulliparous and lactating mice to their homologous chromatin is essentially similar. Thus, in contrast to estrogen receptors, glucocorticoid receptors from lactating mammary glands are able to effectively bind to chromatin acceptor sites which supports our previous suggestion that the estrogenic insensitivity of lactating mouse mammary glands may at least be in part due to the impeded interaction of ER with chromatin acceptor sites.     

Multiple binding sites for phencyclidine on the nicotinic acetylcholine receptor from Torpedo ocellata electric organ

Binding of [3H]-phencyclidine ( [3H]-PCP) to acetylcholine-receptor enriched membrane from Torpedo ocellata electric organ was studied over a ligand concentration range of 1 to 200 microM. The results indicate that [3H]-PCP is bound to two classes of sites: high affinity (Kd = 6-9 microM) and low affinity (Kd = 85 microM) binding sites. In the absence of cholinergic drugs the ratio of high affinity [3H]-PCP binding sites to 125I-alpha-bungarotoxin (alpha-Bgt) binding sites is 0.37, and that of low affinity [3H]-PCP binding sites to 125I-alpha-Bgt is 1.06. Low affinity [3H]-PCP binding can be completely inhibited by alpha-bungarotoxin (alpha-Bgt), carbamylcholine and d-tubocurarine. This inhibition, together with the one to one stoichiometry with 125I-alpha-Bgt, suggests that the sites to which [3H]-PCP binds with low affinity are the acetylcholine (AcCho) binding sites. In the presence of 1 microM alpha-Bgt which blocks binding of [3H]-PCP to the AcCho binding sites, the ratio of high affinity [3H]-PCP sites to 125I-alpha-Bgt sites is 0.5, indicating the existence of one high affinity PCP site per receptor molecule, The toxin, however, decreases the apparent affinity of [3H]-PCP towards the AcCho receptor as well as the potency of tetracaine or dibucaine in inhibiting [3H]-PCP binding to that receptor. In the latter case the effect involves changes from a biphasic to a simple inhibition curve. The results suggest that non-competitive blockers to the AcCho receptors may affect their own sites as well, and that they do this also by binding to the AcCho binding sites. This is also inferred from the accelerated dissociation of [3H]-PCP from its high affinity binding sites by unlabeled PCP in the concentration range of 10(-3) to 10(-4) M, at which the drug occupies AcCho binding sites as well.     

Testis-specific calmodulin-dependent phosphodiesterase. A distinct high affinity cAMP isoenzyme immunologically related to brain calmodulin-dependent cGMP phosphodiesterase

A cell-specific isozyme of calmodulin (CaM)-dependent phosphodiesterase that exhibits micromolar affinity for cAMP has been purified 900-fold from mouse testis by DEAE chromatography, gel filtration, affinity chromatography with CaM-Sepharose 4B, and isoelectric focusing. The highly purified enzyme is stimulated 5-6-fold by CaM in the presence of Ca2+ and hydrolyzes both cAMP and cGMP with anomalous substrate dependence, i.e. high and low affinity components (Km 2 and 20 microM) are observed either in the presence or absence of CaM. Each of the substrates acts as a noncompetitive inhibitor of the other, suggesting the presence of two distinct catalytic sites on the enzyme. Hydrodynamic studies suggest that the testis phosphodiesterase is an asymmetric monomer of 68-70 kDa that forms a dimer after interaction with Ca2+ and CaM; the tetrameric complex exhibits an apparent molecular size of 180 kDa. These enzymatic and biophysical properties differ in many respects from those of the brain isozyme, suggesting that they are different proteins. Nevertheless, common epitopes do exist, since the testis enzyme interacted with rabbit antibodies raised against bovine brain CaM-dependent phosphodiesterase. The major peptide of 68 kDa was strongly reactive on immunoblots, and was distinguished unambiguously from the 60-kDa species from mouse brain. A comparison of the immunoreactive fragments produced by limited proteolysis with staphylococcal V-8 protease indicated several similarities in the domains of these polypeptides. Thus, although differing in several important physical and biochemical parameters, the testis enzyme appears immunologically related to CaM-dependent phosphodiesterase from brain. On the basis of these data, we conclude that common elements of the structural genes for these isozymes have been conserved, whereas certain biological properties, including substrate specificity, have diverged substantially.     

Identification and purification of the calcium‐regulated Ca2+‐ATPase from the endoplasmic reticulum of a higher plant mechanoreceptor organ

Erythrosin b, a potent inhibitor of the Ca²⁺‐ATPases and the Ca²⁺‐release channel (BCC1) in mechanosensitive tissue of Bryonia dioica Jacq., effectively suppresses a tendril's reaction to touch, suggesting that Ca²⁺‐transporters are involved in signal transduction in this organ. The Ca²⁺‐ATPase located in the endoplasmic reticulum (ER) represents a multiregulated enzyme that is stimulated by calmodulin (CaM), KCl and lysophospholipids. Limited proteolysis of ER‐membranes by trypsin results in an irreversible activation of the Ca²⁺‐ATPase and loss of the CaM sensitivity, presumably through removal of an autoinhibitory domain where CaM binds. Mild trypsination mimics the effects of CaM on V_max and the affinity for Ca²⁺ and ATP. Irrespective of a trypsin treatment, the enzyme can be additionally stimulated by KCl and lysolipids, indicating that the sites of interaction for these effectors are not located in the domain removed by the protease. CaM‐stimulated ATPase activity was purified from microsomal and ER fractions using a combination of CaM‐affinity and anion‐exchange chromatography. The isolated polypeptide was enzymatically active, showed a calcium‐dependent mobility‐shift in SDS‐PAGE from 109 kDa in the absence of Ca²⁺ to 104 kDa in the presence of 10 m M CaCl₂ and could be radiolabeled with [³⁵S]‐CaM. The characteristics of the purified enzyme remained closely similar to those of the ER‐bound Ca²⁺‐transporting activity, including the enzymatic data, CaM stimulation, and the sensitivity towards a range of inhibitors.     

Two Saturable Recognition Sites for (-)[125I]Iodo-N6-(4-Hydroxyphenyl-Isopropyl)-Adenosine Binding on Purified Cardiac Sarcolemma

Abstract

Analysis of (-)[¹²⁵]iodo-N⁶-(4-hydroxyphenylisopropyl)-adenosine ([¹²⁵I]HPIA) binding to purified sarcolemmal preparations of guinea pig and bovine hearts revealed two classes of binding sites when unlabeled iodo-HPIA (100 μmol/1) was used as non-specific binding marker. In the presence of 1 mmol/1 theophylline, however, only the high affinity component was detected. Adenosine receptor agonists caused biphasic displacement of [¹²⁵I]HPIA binding, with a high affinity potency rank order typical of interaction with A₁-adenosine receptors. Biphasic competition curves were also observed with 8-phenyltheophylline and isobutylmethylxanthine, whereas the theophylline curve was monophasic up to 1 mmol/1. In brain membranes, specific binding of [¹²⁵I]HPIA as well as of [³H]PIA was further reduced when unlabeled iodo-HPIA replaces theophylline as the non-specific binding marker. These results suggest the presence of two [¹²⁵I]HPIA binding sites on cardiac sarcolemma and brain membranes, but receptor function can only be ascribed to the high affinity sites. The low affinity site probably represents an artefact, which is often observed when non-specific binding is defined with the unlabeled counterpart or a structurally related ligand of the radioligand used.     

Preparation of an affinity chromatography matrix for the selective purification of the dopamine D2 receptor from bovine striatal membranes

A ligand affinity matrix has been developed and utilized to purify the dopamine D2 receptor approx. 2100 fold from bovine striatal membranes. 3-[2-Aminoethyl]-8-[3-(4-fluorobenzoyl)propyl]-4-oxo-1-phenyl-1,3,8- triazaspiro[4.5]decan-4-one (AES) was synthesized and used to prepare the affinity matrix by coupling to epoxy-activated Sepharose 6B (AES-Sepharose). AES (Ki approximately 1.7 nM) is similar in potency to the parent compound, spiperone (Ki approximately 0.8 nM), in competing for [3H]spiperone-binding activity. AES has no significant potency in competing for the dopamine D1 receptor as assessed by competition for [3H]SCH23390 binding (Ki greater than 1 microM). Covalent photoaffinity labeling of the dopamine D2 receptor in bovine striatal membranes with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS) was prevented by AES at nanomolar concentrations. The dopamine D2 receptor was solubilized from bovine striatal membranes using 0.25% cholate in the presence of high ionic strength, followed by precipitation and subsequent treatment with 0.5% digitonin. Nearly 100% of the [3H]spiperone-binding activity in the cholate-digitonin solubilized preparation was absorbed at a receptor-to-resin ratio of 2:1 (v/v). Dopamine D2 receptor was eluted from the affinity resin using a competing dopaminergic antagonist molecule, haloperidol. Recovery of dopamine D2 receptor activity from the affinity matrix was approx. 9% of the activity adsorbed to the resin. The [3H]spiperone-binding activity in AES-Sepharose affinity purified preparations is saturable and of high affinity (0.2 nM). Affinity-purified preparations maintain the ligand-binding characteristics of a dopamine D2 receptor as assessed by agonist and antagonist competition for [3H]spiperone binding.     

Interaction of the catecholamine release-inhibitory peptide catestatin (human chromogranin A(352-372)) with the chromaffin cell surface and Torpedo electroplax: implications for nicotinic cholinergic antagonism

The catecholamine release-inhibitory chromogranin A fragment catestatin (chromogranin A(344-364)) exhibits non-competitive antagonism of nicotinic cholinergic signaling in chromaffin cells. A previous homology model of catestatin's likely structure suggested a mode of interaction of the peptide with the nicotinic receptor, but direct evidence has been lacking. Here we found that [125I]-catestatin binds to the surface of intact PC12 and bovine chromaffin cells with high affinity (K(D)=15.2+/-1.53 nM) and specificity (lack of displacement by another [N-terminal] fragment of chromogranin A). Nicotinic agonist (carbamylcholine) did not displace [125I]-catestatin from chromaffin cells, nor did catestatin displace the nicotinic agonist [3H]-epibatidine; these observations indicate a catestatin binding site separate from the agonist binding pocket on the nicotinic receptor, a finding consistent with catestatin's non-competitive nicotinic mechanism. [125I]-catestatin could be displaced from chromaffin cells by substance P (IC(50) approximately 5 microM), though at far lower potency than displacement by catestatin itself (IC(50) approximately 350-380 nM), suggesting that catestatin and substance P occupy an identical or overlapping non-competitive site on the nicotinic receptor, at different affinities (catestatin > substance P). Small, non-peptide non-competitive nicotinic antagonists (hexamethonium or clonidine) did not diminish [125I]-catestatin binding, suggesting distinct non-competitive binding sites on the nicotinic receptor for peptide and non-peptide antagonists. Similar binding and inhibitory profiles for [125I]-catestatin were observed on chromaffin cells as well as nicotinic receptor-enriched Torpedo membranes. Covalent cross-linking of [125I]-catestatin to Torpedo membranes suggested specific contacts of [125I]-catestatin with the delta, gamma, and beta subunits of the nicotinic receptor, a finding consistent with prior homology modeling of the interaction of catestatin with the extracellular face of the nicotinic heteropentamer. We conclude that catestatin occludes the nicotinic cation pore by interacting with multiple nicotinic subunits at the pore vestibule. Such binding provides a physical explanation for non-competitive antagonism of the peptide at the nicotinic receptor.