The influence of various steroids on the binding of methyltrienolone (R1881) to human placenta: evidence for a second steroid-binding site which stabilizes the R1881 binding site

Specificity studies of the binding of R1881 to crude placental homogenate gave surprising results in that certain steroids increased the binding of [3H]R1881 rather than displacing it. While data for 'competing', i.e. displacing, steroids were similar to those reported by other authors, there have been no previous reports of increased binding due to added steroids. This increased binding was due mainly to an increase in capacity (about 10-fold). These data suggest that the placental steroid-binding protein is unusual in that there is a second steroid-binding site whose occupancy increases the stability of the protein, thereby increasing its capacity to bind R1881.     

Inhibitory effect of fatty acids on the binding of androgen receptor and R1881

Unsaturated long chain fatty acids are known to inhibit the binding between estrogen and estrogen receptor, or progesterone and progesterone receptor in rat uterus. The effects of long chain fatty acids on the binding between androgen receptor of castrated rat prostate and 3H-R1881 were studied. The binding was not affected by saturated fatty acids such as palmitic acid (16:0) or stearic acid (18:0). But unsaturated fatty acids such as oleic acid (18:1), arachidonic acid (20:4) and docosahexaenoic acid (22:6) inhibited the binding between androgen receptor and 3H-R1881. The inhibitory effect of arachidonic acid was dose dependent. Scatchard analysis showed that the addition of arachidonic acid markedly decrease the number of binding sites of androgen receptor. But the dissociation constant was not affected. The inhibitory effect of arachidonic was not a competitive one.     

The promoter of the tgt/sec operon in Escherichia coli is preceded by an upstream activation sequence that contains a high affinity FIS binding site.

The tgt/sec operon in E. coli consists of five genes: queA, tgt, ORF12, secD, and secF. QueA and Tgt participate in the biosynthesis of the hypermodified t-RNA nucleoside Queuosine, whereas SecD and SecF are involved in protein secretion. Examination of the promoter region of the operon showed structural similarity to promoter regions of the rrn-operons. An upstream activation sequence (UAS) containing a potential binding site for the factor of inversion stimulation (FIS) was found. Gel retardation assays and DNaseI footprinting indicated, that FIS binds specifically and with high affinity to a site centred at position -58. Binding of FIS caused bending of the DNA, as deduced from circular permutation analysis. Various 5' deletion mutants of the promoter region were constructed and fused to a lacZ reporter gene to determine the influence of the UAS element on the promoter strength. An approximately two-fold activation of the promoter by the UAS element was observed.     

Evidence that the antiestrogen binding site is a histamine or histamine-like receptor

N,N-diethyl-2-[(4 phenylmethyl)-phenoxy]-ethanamine X HCl (DPPE), a compound selective for the antiestrogen binding site, is structurally similar to the aminoethyl ether group of antihistamines. Our studies now reveal that H1-, but not H2-antagonists, also compete for this site in the order: DPPE = hydroxyzine = perchlorperazine greater than phenyltoloxamine greater than pyrilamine greater than diphenhydramine. The affinity of these compounds for the antiestrogen binding site correlates with their in vitro cytotoxicity against MCF-7 and EVSA-T human breast cancer cells. Tamoxifen, DPPE and hydroxyzine also bind to H1 receptors present in digitonin-solubilized rat liver microsomes, but with less affinity than pyrilamine, which is selective for this site; the ratio of H1 to antiestrogen binding sites in this preparation is 4:1. The data suggest that the antiestrogen binding site may be, in whole or in part, a receptor for histamine different from H1 and H2.     

Assay of androgen binding sites by exchange with methyltrienolone (R 1881).

Methyltrienolone (R 1881 - 17beta-hydroxy-17alpha-methyl-estra-4,9,11-trien-3-one) binds specifically to androgen receptor in rat prostate cytosol where, unlike androstanolone, it is not metabolized. By exchanging bound endogenous hormone in rat prostate cytosol with labelled R 1881, it is possible to measure total (free anc occupied) binding sites. This assay method has also been applied to the measurement of androgen receptor sites in human benign prostatic hypertrophy where R 1881 has the added advantage of not being bound by any contaminating plasma protein (sex hormone binding protein).     

Evidence that insulin receptor from human placenta has a high affinity for only one molecule of insulin

Insulin receptor, partially purified from human placenta by chromatography on wheat germ agglutinin, was shown, by means of double probe labeling, to bind only one molecule of insulin with a high affinity. In the double probe labeling protocol used, 125I-insulin (probe 1) was affinity cross-linked to its receptor in the presence of an excess of unlabeled N epsilon B29-biotinylinsulin (probe 2). The ability of succinylavidin to bind to receptor-linked probe 2 and alter the electrophoretic mobility of the cross-linked complex (during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate) was used to determine the amount of receptor which was cross-linked to both probes relative to that which was cross-linked to only probe 1. The fraction of receptor bound to two molecules of insulin prior to cross-linking was estimated from the cross-linking efficiency and the yield of receptor cross-linked to both probes relative to the yield of receptor cross-linked only to probe 1. The low fraction of receptor bound to both probes in the presence of high concentrations of probe 2 indicated that the affinity of the receptor for a second molecule of insulin was approximately 100 times less than that for the first and that in the range of insulin concentrations (less than 20 nM) usually used to determine the stoichiometry for the interaction between receptor and insulin, more than 80% of the receptor molecules should be bound to only one molecule of insulin. This knowledge of how insulin receptor interacts with insulin was shown to be important for proper determination of receptor purity, interpretation of curvilinear Scatchard plots, and interpretation of the insulin-enhanced rate of dissociation of receptor-bound insulin.     

Evidence that the platinum-reactive methionyl residue of the alpha 2-macroglobulin receptor recognition site is not in the carboxyl-terminal receptor binding domain

Digestion of human alpha 2-macroglobulin-methylamine (alpha 2M-CH3NH2) with papain prior to gel filtration resulted in the resolution of three distinct peaks. The material in peak I (Mr approximately 600,000) and peak II (Mr approximately 55,000) did not have any receptor binding ability as determined by in vivo clearance studies and in vitro competitive binding studies using mouse peritoneal macrophages. In contrast, the material in peak III (Mr approximately 20,000) bound to macrophage alpha 2-macroglobulin (alpha 2M) receptors with a Kd of 250 nM. This represents a 500-fold decrease in affinity relative to undigested alpha 2M-CH3NH2. Sequence analysis demonstrated that this material constituted the carboxyl-terminal fragment (COOH-terminal fragment) of alpha 2M. alpha 2M is known to possess a methionyl residue which is susceptible to modification by cis-dichlorodiammineplatinum (II) (cis-DDP) with the result being a loss of receptor binding ability by alpha 2M. For this reason, experiments were performed to determine if the platinum-reactive methionyl residue is located in the COOH-terminal receptor binding fragment of alpha 2M. The results of this investigation demonstrate that cis-DDP is not reactive with either the isolated COOH-terminal fragment or the COOH-terminal fragment isolated from alpha 2M-CH3NH2 which had been pretreated with cis-DDP. In addition, the COOH-terminal fragment did not bind to monoclonal antibody 7H11D6, a monoclonal antibody which binds to the platinum-reactive epitope of the alpha 2M-CH3NH2 receptor recognition site. In contrast, the 55-kDa fragment of alpha 2M bound approximately 1 mol platinum/mol of 55-kDa fragment and also bound to monoclonal antibody 7H11D6. Since the COOH-terminal fragment retains some receptor binding ability, the results of this investigation demonstrate that this fragment is not the complete receptor recognition site and suggest that a platinum-reactive methionyl residue located in the 55-kDa fragment of alpha 2M is another component of this site.     

Acetylcholine receptor binding site contains a disulfide cross-link between adjacent half-cystinyl residues

A conserved feature of all nicotinic receptors is the presence of a readily reducible disulfide bond adjacent to the acetylcholine binding site. Previously we showed that in intact receptor from Torpedo californica electric tissue reduction of this disulfide followed by affinity alkylation with 4-(N-maleimido)benzyltri[3H] methylammonium iodide specifically and uniquely labels the alpha subunit residues Cys-192 and Cys-193. To identify all of the half-cystinyl residues contributing to the binding site disulfide(s), we have now reduced receptor under mild conditions and alkylated with a mixture of 4-(N-maleimido)benzyltri[3H]methylammonium iodide and N-[1-14C]ethylmaleimide and find that Cys-192 and Cys-193 are labeled exclusively. Furthermore, from unreduced receptor we have isolated two cyanogen bromide peptides of alpha, one containing Cys-192 and Cys-193, and the other containing Cys-128 and Cys-142 (which are the other potential contributors to the binding site disulfide(s]. These isolated peptides incorporate iodo[1-14C]acetamide only following reduction by dithiothreitol. Our results demonstrate that: 1) the binding site disulfide is between Cys-192 and Cys-193; 2) Cys-128 is disulfide-cross-linked to Cys-142; and 3) under conditions that reduce Cys-192 and Cys-193 completely, Cys-128 and Cys-142 remain cross-linked. At the acetylcholine binding site, agonists induce a local conformational change that stabilizes the binding site disulfide against reduction. We suggest that a transition between two stable conformations of the vicinal disulfide, both involving a nonplanar cis peptide bond between Cys-192 and Cys-193, is associated with receptor activation by agonists.     

Estrogen and androgen receptor binding affinity of 10 beta-chloro-estrenen derivatives

10 beta-Chloroestradien-3-one and its derivatives with chlorine substitution in ring A have been prepared. Efficient synthetic methods for 2-chloro- and 4-chloroestradiol are described. The binding affinity of these chlorinated estrogens to the uterine estrogen receptor was measured by a competitive binding assay using [3H]estradiol as ligand. 4-Chloroestradiol showed high binding affinity for the receptor (110% of that of estradiol). 2-Chloroestradiol, 10 beta-chloroestradien-3-one and 4,10 beta-dichloroestradien-3-one had moderate binding affinity. The structures of 10 beta-chloroestradien-3-one and androst-1,4-dien-3-one are very similar and can almost be superimposed. However, their binding affinities to the estrogen and androgen receptor were different. Androst-1,4-dien-3-one displayed no measurable affinity for the estrogen receptor and measurable affinity for the androgen receptor whereas 10 beta-chloroestradien-3-one had very low affinity for the androgen receptor.     

Human synaptotagmin-II is not a high affinity receptor for botulinum neurotoxin B and G: increased therapeutic dosage and immunogenicity

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release by hydrolysing SNARE proteins essential for exocytosis. The synaptic vesicle protein synaptotagmin-II of rat and mouse acts as neuronal high affinity receptor for BoNT/B and BoNT/G. Here, we show that human synaptotagmin-II is not a high affinity receptor for BoNT/B and G due to a phenylalanine to leucine mutation in its luminal domain present only in humans and chimpanzees. It eliminates one of three major interactions between synaptotagmin-II and BoNT/B and hereby explains the disparity in potency of BoNT/B in humans and mice as well as the 40-fold higher dosage of rimabotulinumtoxinB versus onabotulinumtoxinA.     

Evidence that the acetylcholine binding site is not formed by the sequence alpha 127-143 of the acetylcholine receptor

The sequence alpha 127-143 of the alpha subunit of the acetylcholine receptor has been proposed to contain several important features: (1) the acetylcholine binding site, (2) the only N-glycosylation site of the alpha subunit, at asparagine-alpha 141, and (3) two cysteine residues, at alpha 128 and alpha 142, that may participate in a disulfide bond known to be near the binding site. We tested these hypotheses by using antisera to receptor and its subunits and monoclonal antibodies to the synthetic peptide alpha 127-143 cyclized by a disulfide bond between alpha 128 and alpha 142. Antisera to receptor and its alpha subunit were able to immunoprecipitate the iodinated peptide, and this reaction was inhibited by soluble receptor, but not by membrane-bound receptor. alpha-Bungarotoxin did not inhibit antiserum binding to solubilized receptor. Similarly, cholinergic ligands had little or no effect on binding to immobilized receptors of anti-peptide monoclonal antibodies. In addition, these monoclonal antibodies, when bound to the receptor, did not affect toxin binding kinetics. By contrast, preincubation with concanavalin A did inhibit monoclonal antibody binding. Reduction of the receptor significantly decreased the binding of three of the monoclonal antibodies, but subsequent alkylation with N-ethylmaleimide or the affinity labeling reagent bromoacetylcholine had no additional effect on binding. A dithiothreitol concentration about 100-fold higher that the one needed to reduce the disulfide near the acetylcholine binding site was necessary to inhibit monoclonal antibody binding. We conclude that the sequence alpha 127-143 is not fully exposed on the surface when the receptor is in the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apolipoprotein E mediates binding of normal very low density lipoprotein to heparin but is not required for high affinity receptor binding

The relationship between the cholesteryl ester content of normal human very low density lipoprotein (VLDL) and its ability to bind to apolipoprotein E (apoE), heparin, and the low density lipoprotein (LDL) receptor have been compared. Plasma VLDL were separated by heparin affinity chromatography into two fractions: one with apoE and one without. Both fractions had the same cholesteryl ester content relative to apolipoprotein B (apoB). LDL, on the other hand, had a greater cholesteryl ester content. VLDL were modified by lipolysis to express the ability to bind apoE (Ishikawa, Y., Fielding, C. J., and Fielding, P. E. (1988) J. Biol. Chem. 263, 2744-2749). Lipolyzed VLDL with or without apoE were compared for their ability to bind to heparin or the up-regulated fibroblast LDL receptor. Lipolyzed VLDL bound with the same affinity to the receptor whether or not the particles contained apoE. ApoB, not apoE, appears then to be the important ligand for normal VLDL. On the other hand, modified VLDL without apoE, even though binding to the LDL receptor, did not bind to heparin. These data suggest that apoE mediates heparin binding in normal VLDL, that apoB mediates receptor binding, and that the cholesteryl ester content of VLDL is not a factor in the induction of the ability to bind apoE.     

Arabidopsis thaliana GLX2-1 contains a dinuclear metal binding site, but is not a glyoxalase 2

In an effort to probe the structure and function of a predicted mitochondrial glyoxalase 2, GLX2-1, from Arabidopsis thaliana, GLX2-1 was cloned, overexpressed, purified and characterized using metal analyses, kinetics, and UV-visible, EPR, and (1)H-NMR spectroscopies. The purified enzyme was purple and contained substoichiometric amounts of iron and zinc; however, metal-binding studies reveal that GLX2-1 can bind nearly two equivalents of either iron or zinc and that the most stable analogue of GLX2-1 is the iron-containing form. UV-visible spectra of the purified enzyme suggest the presence of Fe(II) in the protein, but the Fe(II) can be oxidized over time or by the addition of metal ions to the protein. EPR spectra revealed the presence of an anti-ferromagnetically-coupled Fe(III)Fe(II) centre and the presence of a protein-bound high-spin Fe(III) centre, perhaps as part of a FeZn centre. No paramagnetically shifted peaks were observed in (1)H-NMR spectra of the GLX2-1 analogues, suggesting low amounts of the paramagnetic, anti-ferromagnetically coupled centre. Steady-state kinetic studies with several thiolester substrates indicate that GLX2-1 is not a GLX2. In contrast with all of the other GLX2 proteins characterized, GLX2-1 contains an arginine in place of one of the metal-binding histidine residues at position 246. In order to evaluate further whether Arg(246) binds metal, the R246L mutant was prepared. The metal binding results are very similar to those of native GLX2-1, suggesting that a different amino acid is recruited as a metal-binding ligand. These results demonstrate that Arabidopsis GLX2-1 is a novel member of the metallo-beta-lactamase superfamily.     

The T-cell receptor zeta chain contains a GTP/GDP binding site.

In a search for nucleotide binding proteins associated with the T-cell receptor (TCR)-CD3 complex, a novel labeling technique involving introduction of [alpha-32P]GTP or [alpha-32P]ATP into permeabilized cells followed by in situ periodate oxidation was developed. To test the method we first demonstrated that p21ras and other classical GTP binding proteins could be labeled in a GTP-specific manner. In human T lymphocytes the TCR zeta chain was found to be specifically labeled by GTPoxi but not by ATPoxi or CTPoxi. Labeling kinetics and competition experiments demonstrated that zeta had a capacity to bind GTP and GDP but not GMP or ATP. Proteolytic cleavage experiments identified lysine 128 as the GTP crosslinking site. This result was confirmed by studies using oligonucleotide-directed mutagenesis. Lysine residues 128, 135 and 149 were each replaced by arginine and glycine 134 by valine and mutated proteins were expressed in CHO cells. Labeling of mutants K128R and G134V was abrogated whereas mutant proteins K135R and K148R could still be specifically crosslinked to GTP. We conclude that Lys128 and Gly134 are part of a GTP/GDP binding site suggesting that zeta is a unique GTP/GDP binding structure.     

The human alpha 2-macroglobulin receptor contains high affinity calcium binding sites important for receptor conformation and ligand recognition.

The receptor for alpha 2-macroglobulin-proteinase complexes (alpha 2MR) was purified recently, and its binding of ligand was shown to depend on calcium ions (Moestrup, S. K., and Gliemann, J. (1989) J. Biol. Chem. 264, 15574-15577). This paper shows that the 440-kDa human placental alpha 2MR is a cysteine-rich glycoprotein with high affinity calcium binding sites important for receptor conformation; and the relationship between Ca2+ concentration and receptor function is presented. Autoradiography showed 45Ca2+ binding to the 440-kDa alpha 2MR blotted onto nitrocellulose from a sodium dodecyl sulfate-polyacrylamide gel. alpha 2MR immobilized on nitrocellulose in the absence of sodium dodecyl sulfate bound 45Ca2+ in the presence of 5 mM Mg2+, and 2-3 microM unlabeled Ca2+ was required to displace half of the bound 45Ca2+. The calcium concentration dependence showed upward concave Scatchard plots, and the number of binding sites was estimated to be approximately eight/alpha 2MR molecule. Binding of calcium did not change in the pH range 6.5-8.0 but decreased at lower pH values. Addition of Ca2+ to the medium was necessary for receptor binding of the alpha 2-macroglobulin-trypsin complex, and half of the maximal binding capacity was obtained with about 16 micrograms Ca2+ at pH 7.8. The requirement for calcium was increased at lower pH values, and half of the maximal 125I-alpha 2M-trypsin binding was obtained with about 30-40 microM Ca2+ at pH 7.0. Monoclonal antibodies were produced against alpha 2MR, and one of them distinguished between the Ca2(+)-occupied and nonoccupied forms. Like Ca2+, Sr2+ and Ba2+ elicited ligand binding affinity and competed for binding with 45Ca2+ in the order Ca2+ greater than Sr2+ greater than Ba2+. In conclusion, calcium ions bind specifically to alpha 2MR with high affinity, and it is likely that several sites on the alpha 2MR molecule have to be occupied to elicit the conformation recognizing the ligand.     

Immunochemical evidence that the FITC-labeling site on Na+,K+-ATPase is not the ATP binding site

The covalent labeling of the alpha subunit of lamb kidney Na+,K+-ATPase by fluorescein 5'-isothiocyanate at Lys-501 has generally been assumed to occur at the ATP binding site. We have found that the peptide sequence 496HLLVMKGAPER506 serves as the antigenic determinant for monoclonal antibody M8-P1-A3. This antibody binds to both native and FITC-labeled enzyme and while this epitope undergoes ligand-induced changes these changes are not involved in either enzyme function or the E1 in equilibrium E2 conformational changes monitored by FITC-fluorescence intensity.     

Riboflavin binding protein contains a type II copper binding site

Riboflavin binding protein, purified from egg white, binds copper(II) under dialysis conditions in an approximately 1:1 molar ratio. Results further indicate a small, but not negligible, amount of copper is present in the protein as purified from egg white. Electron paramagnetic resonance indicates a single type II copper site present in the protein. These results suggest the possibility of a previously unknown function of riboflavin binding protein in the storage or transport of copper.     

Bovine mitochondrial ribosomes possess a high affinity binding site for guanine nucleotides

Mammalian mitochondrial ribosomes possess a binding site for guanine nucleotides. GTP binds in unit stoichiometry and with high affinity (Kd = 15.3 +/- 2.8 nM) to the small subunit of bovine mitochondrial ribosomes. This binding activity survives high salt washes, indicating that the nucleotide binds to an integral site within this subunit. GDP also binds to the small subunit with high affinity (Kd = 17 +/- 5.8 nm) and in unit stoichiometry. The GTP binding activity can be competed with GDP but not appreciably by other nucleotides, indicating that both GTP and GDP bind specifically and to the same site. The non-hydrolyzable analogs of GTP, guanylyl-5'-imidophosphate, and guanylyl-(beta,gamma-methylene)- diphosphonate also bind to the small subunit, but with reduced affinity. These results indicate that mammalian mitochondrial ribosomes, unlike other ribosomes, are able to interact directly with guanosine triphosphate, suggesting that the bound GTP may be involved in a novel regulatory mechanism in mitochondrial protein synthesis.