Isolation and partial characterization of a fragment corresponding to the dimeric form of the CH2 domain of rabbit IgG.

A fragment corresponding to the intact dimeric form of the CH2 domain of rabbit IgG, including the hinge region disulfide linkage, was obtained by plasmin digestion of crystalline Fc derived from IgG by the action of papain. Identification and assessment of purity of the fragment was established by SDS-PAGE, amino acid composition analysis, N-terminus sequence and C-terminus amino acid analysis and SDS-urea-PAGE of the reduced fragment. The fragment retains serologic reactivity with anti-Fc specific antisera. Comparison of deglycosylation by endoglycosidase F indicates a more open special relationship between the two CH2 domains in the fragment than in Fc.

Assay and characterization of IgG binding to endodermal cells of the fetal rabbit yolk sac membrane.

The transfer of passive immunity in the rabbit is mediated by the fetal yolk sac membrane (YSM) and is initiated by the specific binding of IgG to receptors on the microvillar surface of the endoderm from the YSM. This report describes the preparation of suspensions of endodermal absorptive cells of the YSM and their use in equilibrium binding experiments to characterize the nature of the binding reaction. Equilibrium binding is achieved in 4 h at 4 degrees C. The system is more rapid than, affords greater reproducibility of binding data than, and utilizes only about 1/10 the amount of YSM and ligand as the YSM disc assay system (Tsay and Schlamowitz, 1975) used previously. A Scatchard plot of the binding data over a wide range of IgG concentration was non-linear implying the presence of at least 2 binding elements. Apparent binding constant values for the stronger and weaker binding components in this population differed by about 50-fold. For the weaker binding system, binding decreased when temperature was increased indicating that the reaction was not entropy-driven (i.e., dominated by hydrophobic 'forces') and that ionic interactions might be a major factor. At low ionic strengths the measurement of specific binding was complicated by the effects of secondary ionic interactions. At physiological ionic strength the binding of IgG was species-specific.

Binding of IgG and papain-derived fragments to Fc receptors of the fetal rabbit yolk sac membrane.

Quantitative measurements of the in vitro binding of 125I-labeled rabbit IgG (IgGR), and its Fab-1, Fab-2, and Fc fragments to receptors on the fetal rabbit yolk sac membrane (YSM) were carried out by incubating equimolar solutions (1 X 10(-5) M) of IgGR and of each of its fragments with formalin-fixed discs (3.5 cm2) of the YSM. It was found that 97, 4, 2, and 96 pmoles of IgGR, Fab-1, Fab-2, and Fc, respectively, were bound per YSM disc. Since the binding characteristics of intact IgGR were fully conserved in the Fc piece, the results establish that the YSM receptor for IgG is an Fc receptor. It was also shown that whereas IgGR retained its binding ability after exposure to stress (55 degrees C, 10 min), free Fc did not. These findings, together with knowledge of the IgG structure, imply that the YSM receptor recognition unit of IgGR resides in the CH2 domain of its Fc piece.

Characterization of IgC receptors of the fetal rabbit yolk sac membrane: localization to subcellular fraction and effects of chemical agents and enzymes on binding.

Previous studies have established the existence of IgG receptors on the endodermal cells of the fetal rabbit yolk sac membrane (YSM). The present study partially characterizes these cell-associated receptors. The specific binding of rabbit IgG (IgGR) to freshly prepared cell homogenates, nuclei-free brush border preparations, and plasma membrane-rich fractions confirms that receptor function is associated with the endodermal cell, and indicates that this function is localized on its apical brush border, specifically on its plasma membrane. The protein nature of the receptor is demonstrated by the loss of specific binding capacity after treatment of formalin-fixed YSM with papain and trypsin. Evidence has also been adduced which indicates that membrane carbohydrate is not involved in receptor function. Thus, treatment of YSM with neuraminidase, beta-galactosidase, mixed glycosidases, as well as oxidation of YSM with periodate all are without effect on its capacity to bind IgGR. The interaction of IgGR with the receptor elements of formalin-fixed YSM is partially ionic in character. NaCl reversibly inhibits binding of IbGR by 60%. Divalent ions such as Ca++ are not involved in this receptor-ligand interaction since EDTA-treated YSM binds IgGR to the same extent as do controls. Receptor material on fixed YSM resists extraction by non-ionic detergents, deoxycholate, and chaotropic agents.

A comparative study of preparations of ovine lutenizing hormone by bioassay, immunodoublediffusion, radioimmunoassay, radioreceptor assay and polyacrylamide gel electrophoresis.

Three preparations of ovine LH were compared for biological potency and by several in vitro parameters. All were found to be heterogenous by immunodoublediffusion and by electrophoresis in polyacrylamide gels. They all also showed similarities and/or differences with respect to their characteristics in immunodoublediffusion, radioimmunoassay, radioreceptor assay, gel electrophoresis and in dye-binding capacity, but in ways that preclude establishing a meaningful correlation between biopotency and the in vitro parameters or even among the in vitro parameters themselves. The implications of these findings for the use of these in vitro parameters for screening and assessing biological potencies of LH preparations and for inferring chemical and/or structural similarities between LH preparations are discussed. Aspects of polymorphism of LH, observed by electrophoresis on polyacrylamide gels, are also discussed.

Membrane receptors in the specific transfer of immunoglobulins from mother to young.

Transfer of immunity from mother to young takes place prenatally, postnacally, or both depending on the animal species. Where prenatal transfer occurs the fetal tissues across which the immunoglobulins pass are the yolk sac or the hemochorioplacenta. Postnatal transfer is effected via the gut of the newborn. Transfer mechanisms are discussed and evaluated as is the evidence from both in vivo and in vitro studies that support the view that membrane receptors specific for IgG and its Fc fragment mediate the transfer process. This is followed by a brief discussion of the properties of the receptors.

Comparison of the binding affinities of rabbit IgG fractions to the rabbit fetal yolk sac membrane: use of 22Na to facilitate quantitation of 125I-IgG binding.

Rabbit IgG was purified, Fr-1-(G-200)2, and then separated by DEAE-cellulose column chromatography into three major fractions, Fr-I, -II, and -III. Binding affinities of the 125I-labeled IgG and its fractions to the rabbit fetal yolk sac membrane were studied. At a concentration of 2 mg/ml, rabbit IgG is bound to the extent of 9 mug/cm2 membrane, whereas the values for fractions Fr-I, -II, and -III are 13, 7, and 5 mug/cm2, respectively. The maximal amount of IgG bound appears to be approximately the same, i.e., 23 mug of IgG/cm2 membrane, for Fr-1-(G-200)2 and its three fractions. In contrast, bovine IgG does not bind to the membrane over the range of concentration tested. Binding constants for Fr-1-(G-200)2, fraction Fr-I, -II, and -III are estimated to be 5.4, 8.6, 4.0 and 2.0 times 10(4) M-1, respectively. The binding affinities of IgG fractions to the yolk sac membrane correlate with the chemical and physicochemical properties of the fractions. Also detailed in the text is the use of 22Na to facilitate quantitation of specific binding of the 125I-IgG to the membrane under equilibrium conditions.

Preparation of IgG-binding membrane vesicles from the microvillar brush border of fetal rabbit yolk sac.

A one step procedure is described for the production of membrane vesicles from the endodermal microvillar brush border of the fetal rabbit yolk sac splanchnopleur. The vesicles, examined by light and electron microscopy, were shown to consist of biomolecular leaflet unit membrane, coated to varying extents with glycocalyx. By fluorescence microscopy, the homologous immunoglobulin, FITC-IgGR, has been shown to bind to the glycocalyx-coated vesicles as well as the glycocalyx-coated brush border of the intact yolk sac, whereas, the heterologous bovine immunoglobulin, FITC-IgGB, fails to bind under comparable conditions. These observations demonstrate the specificity of the receptors for the homologous IgG.

Preparation and characterization of rabbit IgG fractions.

Rabbit IgG has been purified and further fractionated by deae-cellulose column chromatography to yield three fractions, Fr-I,-II and -III-1 that span a broad range in the heterogeneous molecular population of IgG. Immunodiffusion established that these fractions are free from contamination by IgA, IgM, transferrin, and hemopexin. The three fractions were assayed for neutral hexose and sialic acid; Fr-III-1 and Fr-I yielded the highest and lowest values for these components, resepctively. The order of elution from DEAE-cellulose and the electrophoretic mobilities of the fractions correlate with their content of sialic acid. Digestion of Fr-I with papain is much more rapid and complete than that of Fr-III-1. Treatment with neuraminidase to remove sialic acid from each fraction does not affect the comparative susceptibility to digestion by papain. The fractions can also be distinguished by the relative amounts of H-L half molecules and H chains formed under identical conditions of reduction with cysteine and by differences in their ability to bind with IgG receptors on the fetal rabbit yolk sac membrane. The results are discussed in the context of the structures of IgG in the heterogeneous population of molecules and the possible relation to the oligosaccharide moieties of the IgG molecules in that population.