Half-time Of Dissociation Research Articles

Modulation of Androgen Receptor Activation Function 2 by Testosterone and Dihydrotestosterone

The androgen receptor (AR) is transcriptionally activated by high affinity binding of testosterone (T) or its 5alpha-reduced metabolite, dihydrotestosterone (DHT), a more potent androgen required for male reproductive tract development. The molecular basis for the weaker activity of T was investigated by determining T-bound ligand binding domain crystal structures of wild-type AR and a prostate cancer somatic mutant complexed with the AR FXXLF or coactivator LXXLL peptide. Nearly identical interactions of T and DHT in the AR ligand binding pocket correlate with similar rates of dissociation from an AR fragment containing the ligand binding domain. However, T induces weaker AR FXXLF and coactivator LXXLL motif interactions at activation function 2 (AF2). Less effective FXXLF motif binding to AF2 accounts for faster T dissociation from full-length AR. T can nevertheless acquire DHT-like activity through an AR helix-10 H874Y prostate cancer mutation. The Tyr-874 mutant side chain mediates a new hydrogen bonding scheme from exterior helix-10 to backbone protein core helix-4 residue Tyr-739 to rescue T-induced AR activity by improving AF2 binding of FXXLF and LXXLL motifs. Greater AR AF2 activity by improved core helix interactions is supported by the effects of melanoma antigen gene protein-11, an AR coregulator that binds the AR FXXLF motif and targets AF2 for activation. We conclude that T is a weaker androgen than DHT because of less favorable T-dependent AR FXXLF and coactivator LXXLL motif interactions at AF2.

Biochemical and Structural Characterization of the Gem GTPase

RGK proteins, encompassing Rad, Gem, Rem1, and Rem2, constitute an intriguing branch of the Ras superfamily; their expression is regulated at the transcription level, they exhibit atypical nucleotide binding motifs, and they carry both large N- and C-terminal extensions. Biochemical and structural studies are required to better understand how such proteins function. Here, we report the first structure for a RGK protein: the crystal structure of a truncated form of the human Gem protein (G domain plus the first part of the C-terminal extension) in complex with Mg.GDP at 2.1 A resolution. It reveals that the G-domain fold and Mg.GDP binding site of Gem are similar to those found for other Ras family GTPases. The first part of the C-terminal extension adopts an alpha-helical conformation that extends along the alpha5 helix and interacts with the tip of the interswitch. Biochemical studies show that the affinities of Gem for GDP and GTP are considerably lower (micromolar range) compared with H-Ras, independent of the presence or absence of N- and C-terminal extensions, whereas its GTPase activity is higher than that of H-Ras and regulated by both extensions. We show how the bulky DXWEX motif, characteristic of the switch II of RGK proteins, affects the conformation of switch I and the phosphate-binding site. Altogether, our data reveal that Gem is a bona fide GTPase that exhibits striking structural and biochemical features that should impact its regulation and cellular activities.

Stromal Cells Protect B-CLL Cells from Killing by Antigen-Specific Cytotoxic T Cells.

B-cell malignancy-derived immunoglobulin (idiotype) and survivin, a member of the inhibitor of apoptosis gene family and a shared tumor-associated antigen, are expressed by B-CLL cells. Idiotype- and survivin-specific cytotoxic T cells (CTLs), capable of lysing primary autologous B-CLL cells, can be induced in patients with B-CLL. However, the leukemia cell microenvironment was shown to protect B-CLL cells from apoptosis. The protective effects of stromal cells can be reversed by CXCR4 antagonists in vitro and resensitize CLL cells to spontaneous and chemotherapy-induced apoptosis. The aim of the present study is to investigate whether stromal cell contact impairs CLL killing by CTLs raised against immunoglobulin- or survivin-derived peptides and whether the addition of CXCR4 inhibitors enhances T cell mediated cytotoxicity. To analyze the T cell response, we isolated CD8+ T cells and PBMCs from HLA-A2+ healthy donors. PBMCs were differentiated into dendritic cells (DCs) and CD40-activated B cells. CD8+ T cells were primarily stimulated with peptide-pulsed DCs and then restimulated weekly with peptide-pulsed CD40-activated B cells. Heteroclitic framework region (FR−), heteroclitic complementarity-determining region (CD−) derived peptides, and native and heteroclitic survivin-derived peptides were used for CTL induction. As expected, heteroclitic peptide modifications increased the binding affinity to HLA-A*0201 compared to the native peptide as predicted by the Parker Score (Median change of predicted half-time of dissociation to HLA class I molecules 1429 minutes) and measured by the T2 binding assay (Fluorescence Index (FI) native 0.2; FI heteroclitic 0.9). Cytotoxicity of T cells was assessed by chromium release assay and by flow cytometry against CFSE-labelled CLL cells alone and in co-culture with unlabelled stromal cells in the absence or presence of CXCR4 blocking agents. The induced CTLs efficiently lysed allogenic HLA-A2+ CLL cells (mean cytotoxicity at 30:1, 10:1, 3:1 effector-to-target (E:T) ratio: 15,5%+/−2,8; 7,5%+/−2,8; and 1,9%+/− 0,6), but not HLA-A2 negative CLL cells. Co-culture of CLL cells with the murine stromal cell line M2-10B4 resulted in protection of CLL cells from lysis by antigen-specific cytotoxic T cells in vitro, indeed suggesting a protective role of the microenvironment (mean cytotoxicity at 30:1, 10:1, 3:1 E:T ratio: 5,2%+/−4,1; 0,4%+/−1,6; 1,2%+/−2,0). In contrast to apoptosis induced by fludarabine, CXCR4 blocking agents did not reverse the protective effects of the stromal cell line on T cell mediated cytotoxicity (mean cytotoxicity 30:1, 10:1, 3:1 E:T ratio: 3,1%+/−2,4; 0,8%+/−2,5; 2,3%+/−1,6). These data indicate that the microenvironment may exert protective effects against immunotherapeutic strategies in CLL. However, the protective interaction is not entirely mediated by the CXCR4 - CXCL12 axis. Additional cell-cell interactions appear to play a role and need to be identified as therapeutic targets in order to effectively interrupt the protective effect of the microenvironment on T cell mediated cytotoxicity of B-CLL cells.

A combined immuno-informatics and structure-based modeling approach for prediction of T cell epitopes of secretory proteins of Mycobacterium tuberculosis

The role of secretory proteins of Mycobacterium tuberculosis in pathogenesis and stimulation of specific host responses is well documented. They are also shown to activate different cell types, which subsequently present mycobacterial antigens to T cells. Therefore identification of T cell epitopes from this set of proteins may serve to define candidate antigens with vaccine potential. Fifty-two secretory proteins of M. tuberculosis H37Rv were analyzed computationally for the presence of HLA class I binding nonameric peptides. All possible overlapping nonameric peptide sequences from 52 secretory proteins were generated in silico and analyzed for their ability to bind to 33 alleles belonging to A, B and C loci of HLA class I. Fifteen percent of generated peptides are predicted to bind to HLA with halftime of dissociation T 1/2 ≥100 min and 73% of the peptides predicted to bind are mono-allelic in their binding. The structural basis for recognition of nonamers by different HLA molecules was studied employing structural modeling of HLA class I–peptide complexes and there exists a good correlation between structural analysis and binding prediction. Pathogen peptides that could behave as self- or partially self-peptides in the host were eliminated using a comparative study with the human proteome, thus reducing the number of peptides for analysis. The implications of the finding for vaccine development are discussed vis-à-vis the limitations of the use of subunit vaccine and DNA vaccine.

Binding of ADAMTS13 to von Willebrand Factor

ADAMTS13, a metalloprotease, cleaves von Willebrand factor (VWF) in plasma to generate smaller, less thrombogenic fragments. The interaction of von Willebrand factor with specific ADAMTS13 domains was characterized with a binding assay employing von Willebrand factor immobilized on a plastic surface. ADAMTS13 binding was saturable and reversible. Equilibrium binding occurred within 2 h and the half-time for dissociation was approximately 4 h. Binding to von Willebrand factor was similar with either recombinant ADAMTS13 or normal plasma ADAMTS13; plasma from a patient who lacked ADAMTS13 activity showed no binding. The stoichiometry of binding was one ADAMTS13 per two von Willebrand factor monomers, and the K(d) was 14 nm. The ADAMTS13 metalloprotease and disintegrin domains did not bind VWF detectably. ADAMTS13 truncated after the first thrombospondin type 1 repeat bound VWF with a K(d) of 206 nm, whereas ADAMTS13 truncated after the spacer domain had a K(d) of 23 nm, which is comparable with that of full-length ADAMTS13. Truncation after the eighth thrombospondin type 1 repeat reduced the binding affinity by approximately 3-fold and truncation after the seventh thrombospondin type 1 repeat in addition to the CUB domains increased the affinity for von Willebrand factor by approximately 2-fold. Therefore, the spacer domain is required for ADAMTS13 binding to von Willebrand factor. The first thrombospondin repeat also affects binding, and the C-terminal thrombospondin type 1 and CUB domains of ADAMTS13 may modulate this interaction.

Characterization of the Binding Interaction between ADAMTS13 and von Willebrand Factor (VWF).

VWF multimers are cleaved into smaller less thrombogenic fragments in plasma by ADAMTS13. Deficiency of ADAMTS13 activity leads to thombotic thrombocytopenic purpura (TTP), a disease characterized by thrombocytopenia, microangiopathic hemolytic anemia, fever, neurological decline, and renal insufficiency. ADAMTS13 is a member of the A Disintegrin and Metalloprotease with ThromboSpondin repeats family that has characteristic motifs including a signal sequence, a propeptide, a catalytic metalloprotease domain, a disintegrin domain, a thrombospondin-1 (TSP1) repeat, a cysteine-rich region, and a spacer domain. These domains are followed in ADAMTS13 by seven additional TSP1 repeats and two C-terminal CUB domains. Previous work has shown that ADAMTS13 truncated after the spacer domain retains proteolytic activity towards VWF, but little is known about the potential role of the additional C-terminal TSP1 and CUB domains in VWF binding or cleavage. We therefore developed an enzyme-linked immunosorbent assay (ELISA)-based system to study ADAMTS13-VWF binding. VWF was immobilized in microtiter wells and incubated with plasma or recombinant ADAMTS13 variants. Bound ADAMTS13 was detected directly by solubilization and Western blotting, or indirectly by ELISA. ADAMTS13 proteolytic activity towards VWF is enhanced by denaturation of VWF with urea or guanidine, but ADAMTS13 bound specifically to VWF without prior denaturation. EDTA increased the binding of ADAMTS13 to VWF and prevented proteolysis of the immobilized VWF. Binding was saturable and time-dependent with maximal binding in two hours. Binding was reversible with a half-time for dissociation of four hours. ADAMTS13 in normal human plasma but not in plasma from a patient with TTP bound immobilized VWF. The stoichiometry of VWF monomers to ADAMTS13 at saturation was approximately 2 (range 1–4) and the Kd of recombinant ADAMTS13 binding to VWF was 12 nM (range 5–26 nM). This Kd for binding is similar to the Km for VWF cleavage of 16 nM determined independently, and both are comparable to the estimated plasma ADAMTS13 concentration of 8 nM. The properties of C-terminally truncated ADAMTS13 constructs suggest a regulatory function for certain domains. Truncation after the 8th TSP1 domain decreased the Kd 2-fold. Further truncation after either the 6th or 7th TSP1 domain increased the Kd 2-fold relative to full-length ADAMTS13. Truncation after the spacer domain gave binding properties indistinguishable from full-length ADAMTS13. Truncation after the metalloprotease domain gave no detectable binding to VWF. Therefore, binding of ADAMTS13 to VWF requires sequences in the cysteine-rich or spacer domains, and is modulated by sequences in at least the 8th TSP1 repeat and the CUB domains.

Directed evolution of an anti-carcinoembryonic antigen scFv with a 4-day monovalent dissociation half-time at 37 degrees C.

An scFv has been engineered to bind carcinoembryonic antigen (CEA) with a dissociation half-time >4 days at 37 degrees C. Two mutations responsible for this affinity increase were isolated by screening yeast surface-displayed mutant libraries by flow cytometry. Soluble expression of the mutant scFv in a yeast secretion system was increased 100-fold by screening mutant libraries for improved yeast surface display level. This scFv will be useful as a limiting case for evaluating the significance of affinity in tumor targeting to non-internalizing antigens.

Binding characteristics of [ 3H]ucb 30889 to levetiracetam binding sites in rat brain

Levetiracetam (2 S-(2-oxo-1-pyrrolidinyl)butanamide, KEPPRA®), a novel antiepileptic drug, has been shown to bind to a specific binding site located in brain (levetiracetam binding site [Eur. J. Pharmacol. 286 (1995) 137]). However, [ 3H]levetiracetam displayed only micromolar affinity for these sites making it an unsuitable probe for further characterization. The present study describes the binding properties of an analogue of levetiracetam: [ 3H]ucb 30889, (2 S)-2-[4-(3-azidophenyl)-2-oxopyrrolidin-1-yl]butanamide. [ 3H]ucb 30889 binds reversibly to specific binding sites in rat brain. Kinetics at 4 °C were biphasic with half-times of association and dissociation of, respectively, 3 and 4 min for the fast component and 47 and 61 min for the slow component. [ 3H]ucb 30889 saturation binding curves were compatible with the labelling of a homogenous population of binding sites having a B max of 4496±790 fmol/mg protein (mean±S.D., n=5) and a K d of 62±20 nM (mean±S.D., n=5), a 20-fold increase in affinity compared to [ 3H]levetiracetam. Competition binding curves with ligands known to interact with levetiracetam binding sites and tissue distribution restricted to the brain indicated that [ 3H]ucb 30889 and [ 3H]levetiracetam bind to the same site. Although levetiracetam binding sites and GABA A (γ-aminobutyric acid) receptors share some ligands such as pentobarbital and pentylenetetrazol, experiments performed with [ 35S]TBPS ( tert-butyl-bicyclo[2.2.2]phosporothionate), a probe for the GABA A Cl − channel do not support the hypothesis that levetiracetam binding sites are part of the GABA A receptor complex. Preliminary autoradiography studies in rat brain revealed that [ 3H]ucb 30889 labels specific sites in all brain regions and that this binding is concentration-dependently displaced by levetiracetam.

Orientational Changes of Crossbridges During Single Turnover of ATP

Muscle contraction results from rotation of actin-bound myosin crossbridges. Crossbridges consist of the globular N-terminal catalytic domain and the α-helical C-terminal regulatory domain containing the essential and regulatory light chains. The essential light chain exists in two isoforms, of which the larger one has a 41-amino acid extension piece added at the N-terminus. The catalytic domain is responsible for binding to actin and for setting the stage for the main force-generating event, which is a “swing” of the regulatory domain. We measured the kinetics of the swing associated with the turnover of a single molecule of ATP. Muscle was labeled at the regulatory domain by replacing native essential or regulatory light chain with fluorescent adducts. The rotations were measured by the anisotropy of fluorescence originating from ∼400 crossbridges residing in a small volume defined by a confocal aperture of a microscope. The crossbridges were synchronized by rapid photogeneration of a stoichiometric amount of ATP. The rotations reflected dissociation from thin filaments followed by a slow reattachment. The dissociation was the same for each light chain (halftime ∼120 ms) but the rate of reattachment depended on the type of light chain. The halftimes were 920 ± 50 ms and 660 ± 100 ms for isoforms 1 and 3 of the essential light chain, respectively. The reason that the lifetimes were so long was creation of a small amount of ATP, enough only for a single turnover of crossbridges. A model was constructed that quantitized this effect. After accounting for the slowdown, the halftimes of dissociation and attachment were 34 and 200 ms, respectively.

Dissociation of the tubulin dimer is extremely slow, thermodynamically very unfavorable, and reversible in the absence of an energy source.

The finding that exchange of tubulin subunits between tubulin dimers (alpha-beta + alpha'beta' <--> alpha'beta + alphabeta') does not occur in the absence of protein cofactors and GTP hydrolysis conflicts with the assumption that pure tubulin dimer and monomer are in rapid equilibrium. This assumption underlies the many physical chemical measurements of the K(d) for dimer dissociation. To resolve this discrepancy we used surface plasmon resonance to determine the rate constant for dimer dissociation. The half-time for dissociation was approximately 9.6 h with tubulin-GTP, 2.4 h with tubulin-GDP, and 1.3 h in the absence of nucleotide. A Kd equal to 10(-11) M was calculated from the measured rate for dissociation and an estimated rate for association. Dimer dissociation was found to be reversible, and dimer formation does not require GTP hydrolysis or folding information from protein cofactors, because 0.2 microM tubulin-GDP incubated for 20 h was eluted as dimer when analyzed by size exclusion chromatography. Because 20 h corresponds to eight half-times for dissociation, only monomer would be present if dissociation were an irreversible reaction and if dimer formation required GTP or protein cofactors. Additional evidence for a 10(-11) M K(d) was obtained from gel exclusion chromatography studies of 0.02-2 nM tubulin-GDP. The slow dissociation of the tubulin dimer suggests that protein tubulin cofactors function to catalyze dimer dissociation, rather than dimer assembly. Assuming N-site-GTP dissociation is from monomer, our results agree with the 16-h half-time for N-site GTP in vitro and 33 h half-life for tubulin N-site-GTP in CHO cells.

Indirect Measurement of Bioavailable Testosterone with the Bayer Immuno 1 System

Testosterone is the principal circulating androgen in men. A major fraction of testosterone is specifically bound with high affinity and low capacity to sex-hormone-binding globulin (SHBG), whereas most of the remaining testosterone is bound with low affinity to albumin (ALB), leaving only 1–2% to circulate as “free” testosterone (FT) not bound to protein in serum (1). Because the SHBG–testosterone complex dissociates very slowly in vitro (dissociation half-time, 20 s), SHBG-bound testosterone (SHBG-T) is not considered to be available for biologic action in tissues. According to the original “free hormone hypothesis”, only FT is able to enter target cells. Tait and Burstein (2), however, suggested that because testosterone is only loosely bound to ALB, this fraction is also available for tissue uptake (dissociation half-time, 1 s). The concept of bioavailable testosterone (BAT), also known as “non-SHBG-bound testosterone” [or both FT and ALB-bound testosterone (ALB-T)], was supported by in vitro kinetic calculations (3), but others (4)(5) have challenged the assumptions used for these calculations. Indeed, if SHBG facilitates entry of testosterone into tissues, as reported by Pardridge (6), then SHBG-T would also be “available” for tissue uptake and action. Controversy remains about the best biochemical measurement for testosterone function in humans. The initial clinical diagnostic evaluation for men with symptoms of hypogonadism involves measurement of the serum total testosterone. In situations such as hyper- and hypothyroidism, obesity, and the use of antiepileptic drugs, the correlation between clinical symptoms and the serum total testosterone concentration has been reported to be poor. This may be attributable in part to the inactivity of the SHBG-T fraction and to variation in the plasma concentration of SHBG in these situations. The fact that plasma SHBG changes during life and in several pathologic situations is well established (7)(8). It is currently common practice to …

Binding characteristics of cetirizine and levocetirizine to human H(1) histamine receptors: contribution of Lys(191) and Thr(194).

Competition experiments with [(3)H]mepyramine showed that cetirizine and its enantiomers, levocetirizine and (S)-cetirizine, bound with high affinity and stereoselectivity to human H(1) histamine receptors (K(i) values of 6, 3, and 100 nM, respectively). Cetirizine and levocetirizine were 600-fold more selective for H(1) receptors compared with a panel of receptors and channels. Binding results indicated that the interaction between cetirizine, its enantiomers, and histamine is compatible with a competitive behavior, in contrast with the noncompetitive profile of cetirizine and levocetirizine observed in isolated organs. Binding kinetics provided a suitable explanation for this observation, because levocetirizine dissociated from H(1) receptors with a half-time of 142 min; that of (S)-cetirizine was only 6 min, implying that the former could act as a pseudo-irreversible antagonist in functional studies. The carboxylic function of levocetirizine seemed responsible for its long dissociation time. Indeed, hydroxyl or methyl ester analogs dissociated more rapidly from H(1) receptors, with half-times of 31 min and 7 min, respectively. The importance of the carboxylic function of levocetirizine for the interaction with the H(1) receptor was further supported by the results from the mutation of Lys(191) to Ala(191). This mutation decreased the dissociation half-time of levocetirizine from 142 to 13 min and reduced its affinity from 3 to 12 nM, whereas the affinity and dissociation kinetics of hydroxyl and methyl ester analogs were hardly affected. The mutation of Thr(194) reduced the binding stereoselectivity by selectively enhancing the affinity of the distomer.

Rapid dynamics of the microtubule binding of ensconsin in vivo.

Microtubule-associated proteins (MAPs) are proteins that reversibly bind to and regulate microtubule dynamics and functions in vivo. We examined the dynamics of binding of a MAP called ensconsin (E-MAP-115) to microtubules in vivo. We used 5xGFP-EMTB, a construct in which the microtubule-binding domain of ensconsin (EMTB) is fused to five copies of green fluorescent protein (GFP), as a reporter molecule amenable to the use of fluorescent speckle microscopy. Fluorescent speckle microscopy (FSM) sequences and kymograph analyses showed rapid dynamics of speckles comprised of 5xGFP-EMTB in untreated cells. By contrast, in detergent-lysed cytoskeletons, speckles were not dynamic. Since detergent-lysed cytoskeletons differ from living cells in that they lack both ATP and dynamic microtubules, we used azide treatment to substantially reduce the level of ATP in living cells and we used Taxol to halt microtubule dynamics. Both treatments slowed the dynamics of 5xGFP-EMTB speckles observed by FSM. We also used fluorescence recovery after photobleaching (FRAP) to quantify the half-time of binding and dissociation of the 5xGFP-EMTB chimera and to compare this half-time to that of the full-length MAP molecule. In untreated cells, the t(g) of either 5xGFP-EMTB or full-length GFP-ensconsin was similarly rapid (approximately 4 seconds), while in ATP-reduced and Taxol-treated cells, t(g) was increased to 210 seconds and 40 seconds, respectively. In detergent-extracted cells no recovery was seen. Consistent with the rapid dynamics of 5xGFP-EMTB measured with fluorescent speckle microscopy and FRAP, we estimated that the affinity of the MAP for microtubules is approximately 40 microM in untreated living cells, compared with approximately 1 microM in vitro. However, K(D,app) was not significantly changed in the presence of azide and was increased to 110 microM in the presence of Taxol. To test whether changes in the phosphorylation state of cellular proteins might be responsible for altering the dynamics of ensconsin binding, we used FSM to monitor staurosporine-treated cells. Staurosporine treatment substantially halted dynamics of 5xGFP-EMTB speckles along MTs. Our results show that ensconsin is highly dynamic in its association with microtubules, and its microtubule association can be altered by in vivo phosphorylation events.

Androgen-induced NH2- and COOH-terminal Interaction Inhibits p160 coactivator recruitment by activation function 2.

The androgen receptor undergoes an androgen-specific NH(2)- and COOH-terminal interaction between NH(2)-terminal motif FXXLF and activation function 2 in the ligand binding domain. We demonstrated previously that activation function 2 forms overlapping binding sites for the androgen receptor FXXLF motif and the LXXLL motifs of p160 coactivators. Here we investigate the influence of the NH(2)- and COOH-terminal interaction on androgen receptor function. Specificity and relative potency of the motif interactions were evaluated by ligand dissociation rate and the stability of chimeras of transcriptional intermediary factor 2 with full-length and truncated androgen or glucocorticoid receptor. The results indicate that the androgen receptor activation function 2 interacts specifically and with greater avidity with the single FXXLF motif than with the LXXLL motif region of p160 coactivators, whereas this region of the glucocorticoid receptor interacts preferentially with the LXXLL motifs. Expression of the LXXLL motifs as a fusion protein with the glucocorticoid receptor resulted in loss of agonist-induced receptor destabilization and increased half-time of ligand dissociation. The NH(2)- and COOH-terminal interaction inhibited binding and activation by transcriptional intermediary factor 2. We conclude that the androgen receptor NH(2)- and COOH-terminal interaction reduces the dissociation rate of bound androgen, stabilizes the receptor, and inhibits p160 coactivator recruitment by activation function 2.

Recombinant Expression and Purification of Human Androgen Receptor in a Baculovirus System

A full-length human androgen receptor (hAR) cDNA was used to produce recombinant baculovirus. Spodoptera frugiperda (Sf9) cells infected with this virus expressed protein with an N-terminal hexahistidine tag (His6-hAR) in soluble and insoluble forms. The soluble cytosolic His6-hAR demonstrated similar association and dissociation half-times for mibolerone, similar binding affinity for mibolerone, and similar steroid specificity as bona fide AR. Under native conditions, the soluble cytosolic His6-hAR was purified to apparent homogeneity in the presence of dihydrotestosterone, using metal ion affinity chromatography. The insoluble pellet fraction was solubilized with strong denaturant 6 M guanidine HCl, and His6-hAR was purified from it in the presence of 6 M guanidine HCl. Both the solubilized crude pellet fraction and the solubilized/purified His6-hAR could be renatured to bind mibolerone. The baculovirus system will therefore provide an efficient means for producing hAR for ligand-binding assays, as well as purifying hAR for detailed molecular analyses.

An ultrasensitive competitive binding assay for the detection of toxins affecting protein phosphatases

An ultrasensitive assay is described for microcystin-LR and other substances (microcystins, nodularin, okadaic acid, calyculin A, tautomycin) which block the active site of protein phosphatases (PP) 1 and 2A. The assay is based on competition between the unknown sample and [ 125I]microcystin-YR for binding to the catalytic subunit of PP2A. The PP2A-bound [ 125I]microcystin-YR was stable (half-time of dissociation=1.8 h), allowing non-bound [ 125I]microcystin-YR to be removed by Sephadex G-50 size-exclusion chromatography. Compared to current assays based on inhibition of protein phosphatase activity, the present assay was more robust against interference (from fluoride, ATP, histone, and casein), and had an even better sensitivity. The detection limit was below 50 pM (2.5 fmol) for nodularin and microcystin-LR, and below 200 pM (10 fmol) for okadaic acid. The method was used successfully to detect extremely low concentrations of either microcystin or nodularin in drinking water or seawater, and okadaic acid in shellfish extract.

Binding specificity of a class II-restricted hepatitis B epitope by DR molecules from responder and nonresponder vaccine recipients

A small but significant proportion of people who receive the hepatitis B vaccine do not produce anti-hepatitis B antibodies, a phenomenon associated with certain human leukocyte antigen (HLA) class II haplotypes. We were interested in determining whether natural allelic differences between two HLA-DR4 molecules associated with responder versus nonresponder subtypes differed with respect to binding of an immunodominant hepatitis B surface antigen (HBsAg) peptide as measured using a resonant mirror biosensor. In contrast to our original hypothesis, we found a ten-fold difference in the affinity in favor of the nonresponder DRB1∗0401 allele, with a K D of 6.89 × 10 −8 M versus a K D of 6.71 × 10 −7 M for the responder DRB1∗0404 allele. Half-times of dissociation were 1.3 min and 7.7 min, respectively, although association rate constants for both HLA class II molecules were similar (approximately 10 4 M −1s −1). Of particular interest was the observation of different on-rates during the association phase, suggesting that stoichiometry of binding was not 1:1 or that different structural forms of the HLA-peptide complex exist. Our observations indicate that whereas HBsAg peptide binding to HLA class II molecules is influenced by HLA polymorphism, the nonresponse to hepatitis B vaccine associated with this HLA-DR4 subtype is not a result of failure of processed HBsAg to bind HLA class II molecules.

Binding and Dissociation of Cytochrome c to and from Membranes Containing Acidic Phospholipids

Membrane association and detachment of cytochrome c (cyt c) in millisecond to second time domain were investigated by stopped-flow fluorescence spectroscopy monitoring the efficiency of energy transfer from a pyrene-fatty acid containing phospholipid derivative, 1-palmitoyl-2-[10-(pyren-1-yl)-decanoyl]-sn-glycero-3-phosphoglyce rol (PPDPG, mole fraction X = 0.01) to the heme of the cyt c. Large unilamellar liposomes composed of egg phosphatidylcholine (eggPC) with varying content of the acidic phospholipid phosphatidylglycerol (eggPG) were employed. Unexpectedly, the rate of binding of cyt c to membranes was attenuated upon increasing the mole fraction of the acidic phospholipid (XPG). For example, at 50 microM phospholipid and 5 microM cyt c, when XPG was increased from 0.20 to 0.40 the half-time for the single-exponential decay in fluorescence increased from 4.7 to 8.6 ms. A similar observation was made for the membrane binding of another cationic protein, histone H1. We suggest that the formation of cooperative hydrogen-bonded networks by deprotonated and protonated PG in the vesicle surface retards the binding of cyt c to the liposome surface. However, once formed, the complex of cyt c with acidic phospholipids is stabilized by increasing XPG. Accordingly, significantly prolonged half-times of dissociation of cyt c from liposomes by NaCl, ATP, and different cationic proteins are measured upon increasing XPG. Differences between the latter cationic membrane binding ligands most likely reflect the varying relative contributions of hydrophobicity and Coulombic forces to their attachment to liposomes. Our data on the release and binding of cyt c to liposomes as a function of XPG and in the presence of ATP also provide the first direct experimental evidence for multiple lipid binding sites in cyt c.

Characterization of [125I]-PD164333, an ETA selective non-peptide radiolabelled antagonist, in normal and diseased human tissues.

1 We have synthesized a new low molecular weight, non-peptide radioligand, [125I]-PD164333, an analogue of the orally active butenolide antagonists of the endothelin ETA receptor. 2 Analysis of saturation binding assays demonstrated that [125I]-PD164333 bound with high affinity to a single population of receptors (n > or = 3 individuals +/- s.e.mean) in human aorta (KD=0.26+/-0.08 nM; Bmax=8.8+/-3.95 fmol mg(-1) protein), left ventricle from the heart (KD=0.16+/-0.02 nM; Bmax=34.2+/-3.02 fmol mg(-1) protein) and kidney (KD=1.24+/-0.16 nM; Bmax=125.3+/-35.07 fmol mg(-1) protein). In each case Hill slopes were close to unity. 3 In kinetic experiments, the binding of [125I]-PD164333 to ETA receptors in sections of heart was time-dependent and rapid at 23 degrees C. The data were fitted to a one site model, with an association rate constant (K1 of 2.66+/-0.213x10(8) M(-1) min(-1), and a half-time for association of 11 min. The binding was reversible at 23 degrees C: analysis of the data indicated [125I]-PD164333 dissociated from a single site, with a dissociation rate constant of 0.0031+/-0.0004 min(-1), a half-time for dissociation of 216 min and a KD calculated from these kinetic data of 0.01 nM. 4 Unlabelled PD164333 inhibited the binding of [125I]-ET-1 to left ventricle (which expresses both subtypes) in a biphasic manner with a KDETA of 0.99+/-0.32 nM and KDETB of 2.41+/-0.22 microM, giving a selectivity of 2500 fold. ETA-selective ligands competed monophasically for [125I]-PD164333 binding in left ventricle, a one site fit was preferred to a two site model giving similar nanomolar affinities: BQ123, KD=3.93+/-0.18 nM; FR139317 KD=3.53+/-0.69 nM. In contrast, the ETB selective agonists, BQ3020 and sarafotoxin S6c (1 microM) did not inhibit binding. 5 In human isolated saphenous vein, unlabelled PD164333 was a functional antagonist, producing parallel rightward shifts of the endothelin-1 (ET-1) concentration-response curve (pA2=8.84) and a slope of unity. 6 In the human brain, autoradiography revealed high levels of [125I]-PD164333 binding to the pial arteries of the cerebral cortex and to the numerous smaller intercerebral vessels penetrating the underlying grey and white matter. Conduit and resistance vessels contributing to the control of blood pressure from the heart, kidney, lungs and adrenal also displayed high densities of binding. In diseased vessels, binding of [125I]-PD164333 was confined to the medial layer of both coronary arteries with advanced atherosclerotic lesions or occluded saphenous vein grafts. In contrast, little or no binding was detected in the proliferated smooth muscle of the intimal layer or occluded lesion. 7 These results show [125I]-PD164333 is a specific, high affinity, reversible non-peptide radioligand for human ETA receptors, which will facilitate the further characterization of this subtype, in vitro and in vivo.

Insect tolerance to a neurotoxic polypeptide: pharmacokinetic and pharmacodynamic aspects

Androctonus australis insect toxin (AaIT) is an insect-selective neurotoxic polypeptide from scorpion venom used to probe insect Na+ channels and to design insecticidal recombinant baculoviruses. When injected into susceptible insects (such as flies or cockroaches), nanogram doses of the toxin induce a rapid paralysis within seconds. More tolerant insects respond to microgram doses by developing either a slow progressive paralysis, as in lepidopterous larvae, or a rapid but reversible paralysis, as in Trachyderma philistina, a tenebrionid beetle. Using toxicity and binding assays, microscopy and chromatography, we show that the tolerance of insects to AaIT occurs at both the pharmacokinetic and pharmacodynamic levels. Pharmacokinetic effects occur in Trachyderma philistina in which the toxin undergoes a progressive process of degradation and elimination from the hemolymph, resulting in the loss of 95&shy;97 % of toxin activity 6 h after injection. The pharmacodynamic aspect was demonstrated in studies of the kinetics of binding dissociation of [125I]AaIT from neuronal membranes of susceptible and tolerant insects. Stable binding is shown in susceptible insects such as cockroaches and locusts, which have a dissociation half-time of approximately 9 and 5 min, respectively. This contrasts strongly with the fast half-time of dissociation of 7 s for Spodoptera littoralis larvae and 9 s for Trachyderma philistina, which are both relatively tolerant to AaIT. These differences in binding kinetics may reflect a structural and functional diversity of Na+ channels in different insects that is responsible for their diverse susceptibility to neurotoxic polypeptides.