Pharmacology of the biological response modifier bropirimine (PNU-54461) on experimental autoimmune encephalomyelitis (EAE) in mice.

In murine severe experimental autoimmune encephalomyelitis (EAE), an animal model for the human disease multiple sclerosis (MS), we tested the efficacy of a 5-halo-6-phenyl pyrimidinone compound, bropirimine (PNU-54461). We observed that the compound is active in suppressing EAE when administered orally, a significant pharmacological advantage compared to some current therapies for the treatment of MS. Furthermore, bropirimine was most efficacious when dosing was begun 5-10 days after injection of myelin basic protein, the protein isolated from the central nervous system and used for inducing EAE in our model. This is a period of time following the initial immunological events leading to the disease, when large-scale leukocyte infiltration into the central nervous system begins. Following oral dosing, bropirimine peaked in the blood within 3 h and was cleared to undetectable concentrations within 16-18 h. Despite the pharmacokinetics in the blood, bropirimine was fully efficacious when dosed orally every two or three days. Surprisingly, bropirimine treatment did not result in a statistically significant decrease in leukocyte infiltration into the lower spinal cord, unless the compound was dosed daily at a high concentration. We also observed the concentration and time course of alpha-interferon in blood following oral dosing of bropirimine. The kinetics of interferon in the blood are similar to, but clearly distinguishable from, the pharmacokinetics of bropirimine in the blood. It is not clear whether or not the induction of interferon plays a key role in the efficacy of bropirimine. Nevertheless, the results using bropirimine in EAE suggest that the compound may be useful for the treatment of multiple sclerosis.

Pharmacokinetic properties, induction of interferon, and efficacy of selected 5-halo-6-phenyl pyrimidinones, bropirimine analogues, in a model of severe experimental autoimmune encephalomyelitis.

We showed previously that a 5-halo-6-phenyl-pyrimidinone, bropirimine (PNU-54461), inhibited progression of severe experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis. In the work presented here, we examined the activity of a group of chemically-related bropirimine analogues. First, the pharmacokinetic properties of the bropirimine analogues were examined in normal mice following oral dosing. After equal oral doses, both PNU-56169 and PNU-63693 were found in the blood of normal mice at equal or higher concentrations than bropirimine, but PNU-54462 and PNU-56359 were present in blood only at very low concentrations. Next, we examined the bropirimine analogues for activity in our model of severe EAE. At a dose of 400 mg/kg administered orally every second day PNU-56169 nearly completely blocked EAE progression, but was ineffective at 100 mg/kg. PNU-63693 was effective in EAE at concentrations of 200 mg/kg, 100 mg/kg, 50 mg/kg, and as low as 25 mg/kg. Histopathology was examined by observing leukocyte infiltration into the lower spinal cords of the mice. Treatment with 400 mg/kg of PNU-56169 and doses of 25, 50, 100, and 200 mg/kg of PNU-63693 significantly inhibited leukocyte infiltration into the lower spinal cord of treated mice in a dose-dependent manner. Orally administered PNU-56169 and PNU-63693 also stimulated significant concentrations of IFNalpha in the serum of treated mice, which may be related to the efficacy of the compounds in EAE. However, the correlation between IFNalpha in the blood and efficacy in treating EAE was not exact. Thus, PNU-56169 and PNU-63693 were delivered to the blood following oral dosing, induced significant concentrations of IFNalpha in the blood, and were equally or more potent than PNU-54461 in inhibiting clinical signs of EAE. The results suggest that 5-halo-6-phenyl-pyrimidinones are an interesting class of compounds to investigate for development in the treatment of multiple sclerosis.

Analytical and numerical techniques for the evaluation of free radical damage in cultured cells using scanning laser microscopy.

Scanning laser microscopy (SLM) was used to develop an assay to visualize the generation of intracellular reactive oxygen species (ROS) and to evaluate the effect of the lipophilic antioxidant U-87,663 on ROS formation. Cultured N18 neuroglioma cells were challenged by extracellular addition of cumene hydroperoxide, and subsequent intracellular generation of ROS was characterized by measuring the fluorescence intensity of the ROS indicator 2',7'-dichlorofluorescein (DCF). The kinetics of the reaction between ROS and the indicator DCF, or the antioxidant U-87,663, can be most accurately assessed if results from individual cell clusters are analyzed independently. It is possible and necessary to account for the these experimental and analytical properties in order to characterize the properties of the antioxidant activity precisely. We determined that the temporal increase in DCF fluorescence was consistent with the reaction of DCF with free radicals generated from cumene hydroperoxide, as was the loss of fluorescence from U-87,663. The rate constants for the free radical reactions revealed that ROS reaction with DCF is 10 times faster than with U-87,663. These differences in reaction rates combined with differences in the cellular distribution of the ROS indicator DCF, the antioxidant U-87,663, and the bulk of the ROS prevented detection of any protection of U-87,663 may offer.

The amphiphilic properties of novenamines determine their activity as inhibitors of HIV-1 RNase H.

Few inhibitors of the RNase H function associated with the HIV-1 reverse transcriptase have been discovered to date. We observed that three novenamines, U-34445, U-35122, and U-35401, are specific inhibitors of the HIV-1 RT RNase H function. All three compounds are strong amphiphiles and contain one ionizable group. Hence, a priori, in aqueous solutions the inhibitors might exist in at least four different physical states, namely protonated monomers, ionized monomers, protonated micelles, and ionized micelles. The three inhibitors all yielded anomalous dose-response curves, indicating that the four molecular species have different inhibitory potentials. In order to identify the inhibitory species, the amphiphilic properties of these compounds were studied. It was established that in alkaline solutions, around pH 8, all compounds are ionized and form micelles at concentrations above their CMC. Both the protonated and the ionized forms of these molecules form stable insoluble monomolecular layers at the air/water interface. The anomalies of the dose-response curves can be resolved by taking into account the fact that, in solution, the relative proportion of these molecules in each physical state depends on the pH and on their analytical concentration. Thus interpreted, the results indicate that RNase H is inhibited only by the ionized micellar form of these compounds and not by their monomeric form. Around their pKa (approximately pH 5), the three compounds reproducibly form uniformly sized, self-emulsified colloidal particles that may be used as an efficient drug delivery system.

Efficacy of lipid soluble, membrane-protective agents against hydrogen peroxide cytotoxicity in cardiac myocytes.

We examined the efficacy of a group of drugs that stabilize the cell membrane and can potentially prevent cytotoxicity in cultured fetal chick cardiac myocytes exposed to hydrogen peroxide (H2O2). The effects of various membrane-protective agents were determined by analysis of the kinetics of lactic dehydrogenase (LDH) release. The kinetic parameters calculated from the data include a rate constant for release of LDH (kb) and the fraction of total LDH that is released from the cells (CIIMax). The CIIMaxs derived from a range of H2O2 concentrations reveal that the mean toxic concentration of H2O2 is 1.1 mM and that the pattern of toxicity is consistent with the damage being directly proportional to the concentration of the free radicals generated from the H2O2. Maximum nontoxic concentrations of three amphiphilic membrane protective agents had no effect upon cytotoxicity from H2O2. The slightly polar lipophilic agent, Trolox C, a vitamin E derivative, was also without protective effect at a maximum nontoxic concentration. The highly lipophilic agent, probucol, had a small protective effect at 50 microM, the maximum concentration we succeeded in solubilizing in the culture medium. However, the lipophilic 21-aminosteroid U74500, delivered to the cells in an emulsion, markedly reduced cytotoxicity from H2O2. The CII Max was significantly reduced and the protection was concentration dependent over a range of concentrations from 50-400 nmol/ml. Furthermore, the inhibition by U74500 was fully consistent with a mechanism of scavenging of free radicals formed during lipid peroxidation. In support of this hypothesis, a dose of 400 nmoles/ml completely prevented an increase in lipid peroxides due to H2O2 exposure, whereas there was a sixfold increase during exposure to H2O2 in untreated myocytes. Thus, a lipid soluble 21-aminosteroid prevented lipid peroxidation and reduced cardiac myocyte injury during exposure to H2O2, probably by scavenging of free radicals formed during lipid peroxidation in the cell membrane, whereas amphiphilic agents, which probably altered the physicochemical structure of the cell membrane but did not scavenge free radicals, were not protective.

Inhibition of oxidative insult in cultured cells by a novel 6-chromanol-containing antioxidant.

N18-RE-105 neuronal hybridoma cells were used in a cell culture system to evaluate the protective effects of a novel 6-chromanol-containing antioxidant, U78517F. First, the incorporation of the compound into the cells was evaluated, using a serum albumin carrier. Then the cells were exposed to peroxide-generating compounds, and the cell injury was estimated from the loss of alpha-aminoisobutyric acid (AIB) transport. We found that U78517F only protected the cells significantly when the degree of oxidative insult was below a certain limit; the measurable protection of cells by U78517F against either cumene hydroperoxide or H2O2 was limited to a narrow range of concentrations of the reactive oxygen species generator. Additionally, the protection provided by U78517F was largely localized to the cell membrane and did not extend to protection of mitochondrial function. The action of U78517 was fully consistent with a direct radical scavenging in the cells. The results indicate that the following factors must be taken into account for evaluation of antioxidants in cell culture: (a) the delivery of a compound to cells, especially when the compound is lipophilic; (b) the nature and extent of the oxidative insult used to evaluate protection; and (c) the location of the protective agent in the cells.

Kinetics and thermodynamics of emulsion delivery of lipophilic antioxidants to cells in culture.

Oil-in-water emulsions are being used increasingly for the delivery of lipophilic drugs, but the fundamental physicochemical principles governing such delivery have not been explored. We determined the kinetics and thermodynamics of delivery from emulsions to cells in culture for two lipophilic compounds, U74006 and U74500. Two fundamental properties dominate the delivery, (a) the concentration of the compound in the lipid phase of the emulsion is directly proportional to the concentration of the compound in cells at equilibrium, and (b) the rate of transfer is directly proportional to the concentration of particles in contact with the cells. Thus, the transfer is consistent with direct partitioning from the lipid phase of the emulsion to cells and occurs by the direct collision of emulsion particles with cells. The details of the mechanism of delivery differ between the two compounds. Specifically, delivery of U74006 is first-order with respect to the drug accumulating in the cells. The transfer of U74500 is best described as a sum of two simultaneous pseudo first-order processes consistent with delivery from a single donor compartment to two receiver compartments. Furthermore, two molecules of U74500 appear to be involved in each transfer event. Our results show that relatively simple principles govern the delivery of compounds from oil-in-water emulsions to cells.

Localization of damage induced by reactive oxygen species in cultured cells.

N18-RE-105 neuron-derived hybridoma cells were employed to determine the location and degree of damage induced by each of three reactive oxygen species (ROS) generators: 6-hydroxydopamine (6-OHDA), H2O2, and cumene hydroperoxide. Two readily distinguishable plasma membrane markers were used to assess cell surface damage, namely the active transport of alpha-aminoisobutyric acid (AIB) and the facilitated diffusion of glucose. In addition, staining of mitochondria with a tetrazolium dye, 3[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), was used as an intracellular marker to measure the integrity of the metabolic function of the mitochondria. The dose-response curve of inactivation of transport or of metabolic function varied with the ROS generator used and conformed to one of two patterns of toxicity: either threshold-dependent or single-hit inactivation. We determined that 6-OHDA acts simultaneously on multiple targets and steps in the cells, resulting in a very steep dose-effect curve. Similarly, damage induced by H2O2 to the AIB transporters and to mitochondria is consistent with simultaneous inactivation of multiple steps, but damage to glucose transporters conforms to single-hit inactivation of the transporter. Conversely, treatment with cumene hydroperoxide resulted in single-hit inactivation of the AIB transporter, but inactivation of the glucose transporter conformed to threshold-dependent inactivation. Thus, to evaluate quantitatively damage produced by ROS at the subcellular level, both the type of toxic agent and the target to be evaluated must be considered. Finally, the inactivation of each of the targets observed in this study for all of the ROS generators used conform to one of two simple inactivation models. Fitting the appropriate model to the data allows precise quantitative analysis of the inactivation process and provides insight into the chemistry of the inactivation process.

Staphylococcal enterotoxin B modulates V beta 8+ TcR-associated T-cell memory against conventional antigen.

Primary in vivo challenge with the superantigen staphylococcal enterotoxin B (SEB) induces polyclonal proliferation of an unusually large proportion of circulating T-cells that bear the V beta 8-T-cell receptor (TcR) domain. Early and vigorous proliferation of V beta 8+ T-cells precedes their selective deletion, leaving the host unresponsive upon rechallenge with the native immunogen SEB. Nonetheless, this induction of anergy is incompletely understood. Recently we demonstrated that more cells than just V beta 8+ T-cells undergo clonal proliferation after challenge with SEB (Cell. Immunol. 154, 440, 1994). These findings suggested that non-V beta 8+ T-cells may have a role in the induction of superantigen-induced anergy. To further investigate this, we enumerated CD4+ and CD8+ T-cells in lymph nodes and spleens from Balb/c mice at various times after primary and secondary challenge with either a high or a low dose of SEB. Using these kinetic data we investigated whether challenge with SEB would modulate antigen-specific V beta 8-associated T-memory responses. To this end, the V beta 8+ T-cell-associated responses induced by SEB were compared with the V beta 8+ TcR-associated memory responses induced by the nominal antigen sperm whale myoglobin (SWM). Results indicated that challenge of SWM-primed mice with SEB abrogated the V beta 8-associated SWM-specific T-cell memory for an extended but transient period of time. Moreover, prechallenge with SEB blocked the establishment of de novo V beta + T-cell-mediated immunity. These findings suggest that administration of low and controlled doses of microbial superantigen could provide long-term suppression of antigen-specific cell-mediated immunity.

Tirilazad mesylate protects stored erythrocytes against osmotic fragility.

The hypoosmotic lysis curve of freshly collected human erythrocytes is consistent with a single Gaussian error function with a mean of 46.5 +/- 0.25 mM NaCl and a standard deviation of 5.0 +/- 0.4 mM NaCl. After extended storage of RBCs under standard blood bank conditions the lysis curve conforms to the sum of two error functions instead of a possible shift in the mean and a broadening of a single error function. Thus, two distinct sub-populations with different fragilities are present instead of a single, broadly distributed population. One population is identical to the freshly collected erythrocytes, whereas the other population consists of osmotically fragile cells. The rate of generation of the new, osmotically fragile, population of cells was used to probe the hypothesis that lipid peroxidation is responsible for the induction of membrane fragility. If it is so, then the antioxidant, tirilazad mesylate (U-74,006f), should protect against this degradation of stored erythrocytes. We found that tirilazad mesylate, at 17 microM (1.5 mol% with respect to membrane lecithin), retards significantly the formation of the osmotically fragile RBCs. Concomitantly, the concentration of free hemoglobin which accumulates during storage is markedly reduced by the drug. Since the presence of the drug also decreases the amount of F2-isoprostanes formed during the storage period, an antioxidant mechanism must be operative. These results demonstrate that tirilazad mesylate significantly decreases the number of fragile erythrocytes formed during storage in the blood bank.

Early activation and cell trafficking induced by staphylococcal enterotoxin B: effects of high- versus low-dose challenge on induction of anergy.

The in vivo challenge with exogenous superantigen, staphylococcal enterotoxin B (SEB), selectively induces vigorous polyclonal proliferation of T cells bearing the V beta 8+ TcR domain, whereafter the responsive cells become anergic. We used kinetic analyses to compare the effects of primary (1 degree) and secondary (2 degrees) challenge with a high and a low dose of SEB and the conventional antigen, sperm whale myoglobin, to determine the differential effects of in vivo challenge with a superantigen compared with a conventional antigen. We demonstrate that SEB induces very early activation-associated intralymphatic proliferation and trafficking of more T cells than can be accounted for by V beta 8+ T cells alone. Overall, this study indicates that challenge with SEB causes an apparent loss of CD4+ T-helper cell function and provides an essential foundation for the understanding of the mechanisms of peripheral tolerance induction and T cell "memory."

Immunological determinants of nerve growth factor involved in p140trk (Trk) receptor binding.

Monoclonal anti-NGF antibodies that specifically inhibit the biological activity of mouse beta-NGF were used to study the structural determinants involved in the interaction of NGF with its receptors gp75LNGFR and Trk. None of the three antibodies--N60, M15, and 27/21--showed any reactivity toward denatured NGF. Three experimental methods--radioimmunoassay (RIA), enzyme-linked immunoassay (ELISA), and slot blots--detected no significant cross reactivity between the antibodies and BDNF or NT-3. RIA showed that M15 and N60 recognize the same or an overlapping antigenic site, but 27/21 recognizes a different epitope. Only 27/21, and not N60 or M15, immunoprecipitated beta-NGF crosslinked to LNGFR receptor. Thus, the epitope recognized by 27/21 does not overlap the LNGFR receptor binding site. N60, M15, and 27/21 all block binding of NGF to Trk in a manner consistent with competitive inhibition. Purified Fab fragments of N60 and M15 gave similar results to the intact antibodies. The other subunits present in the 7S complex of NGF, i.e. the alpha and gamma subunits, competitively inhibited binding of antibodies to beta-NGF. Only the gamma subunit inhibited phosphorylation of Trk and biological activity of beta-NGF. These findings suggest that the M15, N60, and 27/21 antibodies bind to a specific site on the surface of NGF where they competitively inhibit binding to the Trk NGF receptor. The region encompassing the N-terminus, the C-terminus, and the loop on the surface of beta-NGF containing residues 60-80 is proposed as important for binding to the Trk receptor.

Competitive irreversible inhibition of dopamine uptake by 6-hydroxydopamine.

We examined the effects of 6-hydroxydopamine (6-OHDA) treatment on the human neuroblastoma cell line SK-N-SH-SY5Y (SY5Y) and the rat pheochromocytoma cell line, PC12. Structural and metabolic integrity was tested by measuring the ability of cells to transport the non-metabolizable amino acid analogue [3H]-alpha-aminoisobutyric acid (AIB). We determined that treatment with 6-OHDA at concentrations of 49 microM and 62 microM inhibited 50% of the AIB uptake in SY5Y and PC12 cells, respectively. Inhibition of AIB uptake was prevented by the addition of catalase, but was not influenced by the addition of 1 mM dopamine. This indicated that cell damage resulted from the generation of H2O2 and was independent of the catecholamine uptake system. Effects directly on the catecholamine uptake system were observed by measuring the uptake of 3H-dopamine. In contrast to the effects on amino acid uptake, dopamine uptake was significantly inhibited by 6-OHDA treatment, and this inhibition was not prevented by the addition of catalase. The results indicate a Ki of 430 microM for inhibition of dopamine uptake by 6-OHDA treatment of PC12 cells. The results are consistent with a competitive irreversible inhibition of the dopamine uptake sites by 6-OHDA or one of its metabolites. Thus, the lack of a catecholamine uptake-dependent cellular toxicity appears to result from the direct inactivation of catecholamine uptake sites. Similarly, the inhibition of dopamine uptake in vivo by 6-OHDA may be explained, at least in part, by direct inactivation of dopamine uptake sites rather than exclusively by intracellular transport and action of 6-OHDA.

Characterization of ultra-high affinity monoclonal antibodies with a dimeric, symmetrical antigen: inhibition of the receptor recognition site of nerve growth factor.

We generated a family of ultra-high affinity monoclonal antibodies (MAb) which inhibit competitively the binding of nerve growth factor (NGF) to its receptor. Preliminary experiments indicated that the dissociation constants (Kd) of some of the MAb:NGF complexes were substantially less than 0.1 nM. Conventional methods, such as ELISA and radioimmunoassays (RIA), were not sufficiently sensitive to measure the Kds of these MAb. Therefore, experimental conditions were developed to determine binding constants for these very high affinity MAb. The experiments establish that the Kds for our anti-NGF MAb range from 2.6 nM to 39 fM. Additionally, the inhibition of NGF binding to NGF-receptor by MAb is fully consistent with a purely competitive model but is not consistent with a model allowing the formation of a ternary complex of NGF, MAb, and NGF-receptor. One MAb, M4, immunoprecipitates NGF indicating interaction between each protomer of the NGF dimer and individual MAb molecules. We also evaluated the effects of mild denaturing conditions on the binding and biological activity of NGF and on recognition by the MAbs. Guanidine HCl or heat treatment of NGF resulted in only small, but significant, changes in binding or biological activity, in parallel with changes in recognition by the MAbs. However, binding, biological activity, and recognition by six of seven MAbs were completely eliminated by beta-mercaptoethanol reduction. Thus, our results are consistent with the MAbs interacting with the receptor recognition site on the surface of the NGF molecule. The high affinity MAbs will serve as sensitive probes of structural elements of NGF responsible for binding and biological activity.

Effects of dopamine agonists on dopamine secretion from PC12 cells: lack of functional autoreceptor activity.

In an effort to determine if PC12 cells have functional dopamine autoreceptors we found that carbachol-stimulated release of dopamine from undifferentiated PC12 cells was inhibited by the dopamine autoreceptor agonists apomorphine and U-68553B. Studies were conducted to determine the mechanism of this effect. The inhibition of dopamine release by apomorphine or U-68553B did not appear to result from effects on dopamine metabolism. When cells were exposed to the dopamine agonists for 2 minutes no changes in dopa, DOPAC or dopamine were observed. Over this same time interval, apomorphine and U68553B at 10 microM inhibited carbachol-stimulated dopamine release by 45.6% and 57.4% respectively. However, these drugs failed to inhibit the potassium dependent release of dopamine from cells suggesting no direct involvement with ion fluxes. Furthermore, haloperidol did not block the inhibitory effects of U-68553B on PC12 cells. This would appear to preclude activation of a dopamine autoreceptor as a possible mechanism. Kinetic analysis revealed that U-68553B is most likely a non-competitive inhibitor (Ki = 2 microM) of the nicotinic acetylcholine receptor. These data do not provide evidence for functional dopamine autoreceptors on undifferentiated PC12 cells but rather indicate that dopamine agonists may alter dopamine release by alternative mechanisms.

Cell surface molecules involved in early events in T-cell mitogenic stimulation by staphylococcal enterotoxins.

We tested the mitogenic response to staphylococcal enterotoxin (SE) type A and SE type B in spleen cells from five strains of mice and found consistent and significant differences among the strains. We chose to study the mitogenic responses of two of these strains, C58BL/6J and BALB/cJ, in greater detail. We investigated the effects of specific monoclonal antibodies to cell surface determinants on SE-induced mitogenesis. Monoclonal antibodies against Ia (class II major histocompatibility complex) determinants blocked SE-induced mitogenesis. Both I-A and I-E molecules can participate in the stimulation, and in BALB/cJ mice which express both types of class II molecules both must be blocked to prevent mitogenesis. Mitogenesis was not inhibited by monoclonal antibodies specific for class I major histocompatibility complex antigens or monoclonal antibodies specific for Mac-1, Lyt-1, or Lyt-2 cell surface proteins. Monoclonal antibodies specific for the T-cell surface antigens L3T4 and T3 also substantially inhibited SE-induced mitogenesis. This implicates participation of the T-cell antigen receptor complex in stimulation induced by the SEs. Elimination of L3T4+ helper-inducer T cells abolished the mitogenic response of spleen cells to SE. Reconstitution of L3T4-depleted spleen cells with L3T4+ T cells showed that the level of the mitogenic response was directly proportional to the number of L3T4+ cells added. Elimination of Lyt-2+ cells resulted in a 50% decrease in the response to SEs. These results indicate that L3T4+ T cells are required for the mitogenic response to SE, but both L3T4+ and Lyt 2+ T cells participate in SE-induced mitogenesis. Our results suggest that both Ia and the T-cell antigenic receptor complex are involved in SE-induced mitogenesis.

Change in state of nerve growth factor receptor. Modulation of receptor affinity by wheat germ agglutinin.

The binding of 125I-labeled nerve growth factor (NGF) to human melanoma cell (A875) membranes, detergent-soluble membrane extracts, and membrane extracts reconstituted into phospholipid vesicles was significantly increased when binding was carried out in the presence of wheat germ agglutinin (WGA). In the absence of WGA, all 125I-NGF binding was rapidly eliminated by trypsin treatment or rapidly dissociated in the presence of a high concentration of unlabeled NGF. However, in the presence of WGA, up to 75% of 125I-NGF bound was resistant to trypsin digestion and was only slowly dissociated by a high concentration of unlabeled NGF. The effects of WGA can be blocked or reversed by N-acetylglucosamine. Both WGA and NGF rapidly associate with soluble extracts and reconstituted vesicles and, at the concentrations used here, reach binding equilibrium within 2 min. The conversion to slowly dissociating, trypsin-resistant binding, however, was not complete for at least 10 min. Both WGA and NGF are required for maximum accumulation of trypsin-resistant, slowly dissociating binding. The order of addition of NGF and WGA has no effect on the rate of conversion of NGF-receptor, and the conversion occurs after both NGF and WGA are present. The amount of conversion is dependent on the incubation temperature, and significantly greater conversion occurs at 37 than at 0 degrees C. The generation of the trypsin-resistant, slowly dissociating state of NGF-receptor is consistent with a time- and temperature-dependent conformational change in NGF-receptor which occurs after interaction of both NGF and WGA with the receptor or closely associated structures.

Purification of the receptor for nerve growth factor from A875 melanoma cells by affinity chromatography.

The receptor for nerve growth factor (NGF) has been purified to near homogeneity from octylglucoside extracts of A875 melanoma cell membranes by the use of repetitive affinity chromatography on NGF-Sepharose. Elution of purified receptor (NGF receptor) was accomplished with 0.15 M NaCl, pH 11.0, containing phosphatidylcholine and octylglucoside. Chromatography on two columns of NGF-Sepharose yielded a 1500-fold purification of the receptor, as assessed by 125I-NGF binding, and permitted recovery of 9% of the total binding activity in the soluble extract. Scatchard analysis of equilibrium binding of 125I-NGF provided similar Kd values for NGF receptors in soluble extracts of A875 membranes (2.2 nM) and with purified NGF receptor (3.1 nM). Examination of NGF receptor after electrophoresis on sodium dodecyl sulfate-polyacrylamide gels revealed the presence of two major peptides, of Mr = 85,000 and Mr = 200,000. Affinity labeling experiments, done with 125I-NGF and A875 cells, soluble extracts of A875 cell membranes, and purified receptor, show that both of these components of the NGF receptor can be specifically cross-linked to 125I-NGF.