Molecular weight and valence of the cell-surface receptor for immunoglobulin E.

Abstract

The molecular weight of the active solubilized cell-surface receptor for immunoglobulin E (IgE) was measured in nonionic detergent. The diffusion coefficient was estimated by gel filtration, the partial specific volume was estimated from the differential sedimentation in sucrose gradients prepared from H20 and D20, and the sedimentation constant was estimated from the same centrifugation experiments. receptors were assessed using the (NH4)2SO4 assay described previously (6). Gel Filtration. Gel exclusion chromatography was performed on 92 X 1.4 cm columns of Sepharose 6B (Pharmacia, Piscataway, N.J.) equilibrated at 40 with borate buffer containing 0.5% or 1% Nonidet P-40 and 0.05% bovine serum albumin'. Column fractions of 1% bed volume were collected and flow rates were -<1 bed volume/5 hr. The exclusion volume (V0) was marked with '311-labeled tobacco mosaic virus (a gift from T. Triche, National Cancer Institute) after we observed that dextran blue appeared to interact with the solubilized receptor. A standard included volume (VG) was marked with the IgG and the RF for the unknowns and standards was calculated from their elution volumes (Ve) using the formula RF = (Ve -VO)/(VG -VO) [ 1]The RF of each standard was plotted versus 1/D20o, and the line fit by least squares was used to convert the RF values of the unknowns to their D2o0, values (15).Sucrose Density Gradient Centrifugation. Centrifugation experiments were performed with an L265B-G ultracentrifuge using an SW 40 rotor (Beckman Instrument Co., Palo Alto, Calif.). Pairs of 14.2 ml, 2% to 8% (wt/vol) linear sucrose gradients in borate buffer containing 1% Nonidet P-40 and 0.05% bovine serum albumin were prepared by pumping through mixing chambers into nitrocellulose tubes. After storage for -2 hr at 40, Al of sample in a solvent which was identical to that of the gradient except for the sucrose was layered on the gradient and the tubes were centrifuged at 40,000 rpm for 14-20 hr at 4°. Fractions of approximately 250,u (7 drops) were collected automatically. There was less than 5% variation in sample size between the second and last fraction collected and refractive index measurements on unspun gradients showed excellent linearity. For the IgE, IgE-receptor complex, and marker proteins the radioactivity of the samples could be counted directly. For the free receptor 75-140,ul portions of the fractions were diluted at least 2:1, incubated with labeled IgE, and assayed with the (NH4)2SO4 technique. The densities of the starting solvents were measured directly in a 10 or 25 ml pycnometer and the refractive index for each was determined

Measuring Aptamer Equilibria Using Gradient Micro Free Flow Electrophoresis

“…Complete mixing across a 40 μm capillary will occur for even large proteins like IgE (D = 3.3×10 −7 cm 2 /s 37) in less than 50 seconds. This leaves >4 minutes for binding to occur, which should be sufficient to reach equilibrium in a non-competitive assay considering the high affinity aptamers have for their targets.…”

Separation of rabbit mammary-gland prolactin receptors by ion-exchange chromatography, h.p.l.c.-gel filtration and ultracentrifugation

“…Triton X-100 is a mild detergent that does not inhibit protein-protein aggregation (Lang et al, 1980;Pilch & Czech, 1980). This detergent forms a high-M, micelle and binds to membrane proteins in considerable amounts (Clarke, 1975;Newman et al, 1977;Smigel & Fleischer, 1977). The difference is probably due to the amount of detergent bound to proteins and to the existence of other associated proteins in the receptor molecule.…”

Molecular versatility of antibodies

“…Therefore, our further studies were intended principally to see if the component seen on gels in the presence of denaturants might in its native form be a dimer. Our molecular weight studies could not exclude that possibility, since the higher apparent molecular weight for the binding moiety determined in the absence of harsh denaturants (36) could have resulted either from the associated detergent micelles or dimerization of the protein. In addition, we wanted to see whether the receptor was associated with other cellular components either before or after aggregation.…”