Affinity perfusion chromatography.
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Chromatography, Affinity↗
Biomedical subjects
T R Londo
Publications and source records attributed to T R Londo.
Rotational dynamics of luteinizing hormone receptors and MHC class I antigens on murine Leydig cells.
We have examined the molecular motions of luteinizing hormone (LH) receptor and the Major Histocompatibility Complex Class I antigen on murine Leydig cells. Using time-resolved phosphorescence anisotropy methods, erythrosin (ErITC)-derivatized ovine luteinizing hormone (oLH) bound to the LH receptor appears rotationally mobile with rotational correlation times of 19.6 +/- 1.3 microseconds, 13.3 +/- 2.4 microseconds, 9.5 +/- 0.7 microseconds and 4.7 +/- 0.5 microseconds at 4 degrees C, 15 degrees C, 25 degrees C and 37 degrees C, respectively. Rotational correlation times for human chorionic gonadotropin (hCG)-occupied LH receptors were similar to those of the ErITC-oLH occupied receptor at each temperature. In addition, both oLH- and hCG-occupied LH receptors were laterally mobile in fluorescence photobleaching recovery experiments with diffusion coefficients at 29 degrees C of (5.8 +/- 0.9) x 10(-10) cm2 s-1 and (2.9 +/- 0.4) x 10(-10) cm2 s-1, respectively. We also measured the rotational correlation time of Class I antigen on murine Leydig cells using ErITC-derivatized 34-12-2S, an anti-Class I monoclonal antibody. Because there was no decay of the anisotropy function at 4 degrees C, 15 degrees C, 25 degrees C or 37 degrees C in the absence of oLH or following preincubation of Leydig cells with 1 nM oLH, it appears that Class I is rotationally immobile on the 1 ms timescale of our experiments. This result is consistent with the presence of Class I antigen in large molecular weight structures and may be the result of Class I self-aggregation. Further, treatment of cells with anti-Class I antibody had no effect on either basal or oLH-stimulated testosterone secretion. Thus, it appears that this anti-Class I antibody is not LH-mimetic on murine Leydig cells.
Animals↗
Fluorescence depletion measurements in various experimental geometries provide true emission and absorption anisotropies for the study of protein rotation.
Use of fluorescence depletion methods for measuring slow protein rotational diffusion has been limited by failure to obtain, from depletion data, well-defined anisotropy functions dependent on the distribution of either fluorophore emission or absorption transition dipoles, but not both. Such anisotropies would be directly comparable to those obtained from phosphorescence emission or triplet absorption measurements. We now describe such procedures applicable to cuvet and microscope experimental geometries, together with supporting experimental results. In cuvet measurements, the pump and probe beams are colinear and fluorescence is collected at 90 degrees to this axis. The data analysis procedure for this geometry has been suggested by Wegener (Biophys. J., 46 (1984) 795) and permits calculation of the absorption and emission anisotropies and the interdipole angle. In microscope experiments, fluorescence emission is collected along the pump/probe beam axis. For microscope measurements, a new experimental procedure permits evaluation of absorption and emission anisotropies when the interdipole angle is independently known. In either case multiple depletion measurements are required, each with different relative orientations of the probe beam polarization, pump beam polarization and emission polarizer axis. We have used these methods to calculate the time-dependent anisotropies for eosin-derivatized BSA rotation in glycerol solutions in both experimental geometries. These data correspond well with those obtained from time-resolved phosphorescence anisotropy measurements.
Chemistry, Physical↗
Biologic activity of antigen receptors artificially incorporated onto B lymphocytes.
We describe a method for incorporating monoclonal antibody molecules onto viable murine lymphocytes and summarize the biologic activity of these artificial receptors on B cells. Mouse spleen cells incubated overnight with palmitate conjugates of a monoclonal anti-DNP IgA (protein 315) in the presence of deoxycholic acid incorporate about 50,000 antibody molecules per cell. When concentrations of deoxycholate and palmitoyl-protein 315 are carefully controlled, this labeling procedure does not affect the viability or the normal functions of the receptor-decorated cells. The incorporated antibody specifically binds DNP-antigens, although it appears to be unable to communicate directly with internal cellular components. Yet when these receptor-decorated, unprimed cells are challenged with any one of several DNP-antigens, up to 42,000 per 10(6) B cells differentiate into Ig-secreting cells. This response is about 23-fold greater than that induced in normal cell cultures and is of the same magnitude as that induced by the polyclonal B cell activator LPS. This, in addition to the observation that only about 3.6% of receptor-decorated B cells responding to DNP-conjugated polymerized flagellin (DNP-POL) produce hapten-specific antibody, demonstrates that these antigens cause polyclonal B cell differentiation. Normal spleen cells in the presence of DNP-POL and irradiated spleen cells bearing the artificial receptors do not exhibit the polyclonal antibody response. Also, the response of receptor-decorated B cell is blocked by high but nontoxic concentrations of the nonimmunogenic hapten DNP-lysine. These observations demonstrate that the polyclonal B cell response in this system requires the binding of antigen to artificial receptors on functionally viable cells. The polyclonal B cell response to a thymus-dependent antigen DNP-conjugated bovine gamma-globulin (DNP-BGG) requires the presence of the carrier-primed T cells. On the other hand, T cell depletion by anti-Thy-1.2 monoclonal antibody and complement causes only a slight reduction in the number of receptor-decorated B cells that respond to the relatively thymus-independent antigen DNP-POL. This type of phenomenon is also seen with natural antigen-specific B cells. Thus, polyclonal activation of receptor-decorated B cells exhibits the same gross helper cell requirements as antigenic activation of natural antigen-specific B cells. The results of this study are discussed in the context of the role of membrane-bound surface Ig in antigen-dependent B cell activation.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Lateral diffusion of antigen receptors artificially incorporated onto B lymphocytes.
In the companion paper, we have shown that palmitate conjugates of a monoclonal anti-DNP IgA (protein 315) incorporated onto B lymphocytes can bind DNP antigens and that this binding causes polyclonal B cell activation. In this study we use fluorescence photobleaching recovery (FPR) techniques to examine the lateral diffusion and mobile fractions of antigen-receptor complexes on receptor-decorated B cells as functions of antigen concentration and epitope density. Antigens used in this study are DNP conjugates of polymerized flagellin (DNP-POL) and linear dextran of 2 X 10(6) m.w. (DNP-DEX). The diffusion coefficient observed for antigen bound to artificial receptors decreases monotonically with increased antigen dose and epitope density. When the artificial receptor-bearing cells are labeled with either relatively high concentrations of medium epitope density antigen or high epitope density antigen, a large fraction of antigen-receptor complexes become immobile in the time scale of the experiment. We attribute this behavior to extensive receptor cross-linking by antigen. In parallel with these FPR experiments, we examined the effects of antigen concentration and epitope density on the polyclonal humoral response of receptor-decorated B cells. We found that the response is a function of both antigen concentration and epitope density similar to that seen in natural B cells. The combined results of these experiments show that cell activation results when the diffusion coefficient of the antigen-receptor complex ranges between 10 X 10(-11) cm2 sec-1 and 5 X 10(-11) cm2 sec-1. These values represent threefold and sixfold decreases from the diffusion coefficient of antigen-free receptors, respectively. However, when either a high antigen concentration or epitope density causes a large fraction of antigen-receptor complexes to become immobile, B cells become unresponsive not only to the bound antigen, but also to LPS. Results obtained in this study are very similar to those obtained in a study performed with natural antigen-specific B cells. Therefore, for the responding population of receptor-decorated B cells, it is possible that antigens activate and paralyze these B cells by mechanisms similar to those by which antigens regulate normal B cell responses.
Animals↗