Solubilised intrinsic factor receptor from pig ileum and its characteristics.

The vitamin B-12-intrinsic factor receptor was shown to be present in pig ileum and localized on the brush borders of the enterocytes, and to be solubilisable with Triton X-100. At neutral pH and in the presence of Ca2+ it bound the vitamin B-12-intrinsic factor but not the vitamin B-12-cobalophilin complex. The solubilised vitamin B-12-intrinsic factor-receptor complex consisted of two molecular species with clear Stokes radii (13.11 and 33.53 nm), sedimentation coefficients (15.1 and 45.1 S) and molecular weights (1 600 000 and 12 000 000). A third smaller macromolecule possibly also represented the receptor. Some receptor activity was present in extracts prepared with buffers lacking detergent. There was evidence that the receptor is a membrane lipoprotein from which the lipids reversibly dissociate. Free intrinsic factor also bound to the solubilized receptor and its vitamin B-12-binding site seemed not to be involved in the attachment to the receptor. A small portion of the vitamin B-12-intrinsic factor spontaneously dissociated from the receptor and nearly all dissociated in the presence of Na2-EDTA. TheStokes radius of the dissociated vitamin B-12-intrinsic factor complex was 0.17 nm smaller than before binding to the receptor. Intrinsic factor and cobalophilin were present in ileal extract and observations were made on their molecular characteristics. These proteins, polymers of the vitamin B-12-intrinsic factor complex and binding of vitamin B-12 and its protein complexes to detergent micelles may give spurious receptor-like effects which must be properly controlled.     

Studies on the solubilized porcine ileal intrinsic factor receptor and on a 340 000-dalton component binding vitamin B-12

A 340 000-dalton component “C-III” was found when Triton X-100-containing extracts of ileal mucosa were incubated with human or porcine intrinsic factor vitamin B-12 preparations. It was not formed when abnormal human intrinsic factor, unable to attach to the intrinsic factor receptor, was used. Prolonged storage promoted the transfer of vitamin B-12 to it from the vitamin B-12-intrinsic factor receptor species C-I and C-II. The component was also present in ileal extracts prepared with or without detergent and it bound vitamin B-12 directly. Immunologically and by electrofocusing it could be classified as a cobalophilin but its molecular dimensions were larger than described for cobalophilin. It thus represents a novel vitamin B-12 binding protein, possibly a macromolecular acceptor of vitamin B-12 which accepts vitamin B-12 bound via intrinsic factor to the ileal intrinsic factor receptor.In the presence of EDTA or at low pH, vitamin B-12-intrinsic factor did not bind to any of the receptor species and under the same conditions it could all be dissociated from the receptor complexes but not from C-III. The dissociated receptor was able to recombine with vitamin B-12-intrinsic factor and it appeared to bind free and vitamin B-12-bound intrinsic factor in vivo.     

Isolation of the porcine ileal intrinsic factor receptor by sequential affinity chromatography

An extract containing solubilised receptor was passed through four columns containing Sepharose to which had been covalently coupled anti-cobalophilin IgG, vitamin B-12-intrinsic factor, vitamin B-12, and free intrinsic factor, respectively. Following a wash the receptor was eluted with EDTA, then residual Triton X-100 micelles and vitamin B-12-intrinsic factor were removed by Sephadex G-200 filtration. The receptor was purified 84 000-fold, sodium dodecyl sulphate electrophoresis indicated two subunits and gel filtration of its vitamin B-12-inttrinsic factor complex resolved it into two molecular species.     

Porcine serum cobalophilin and transcobalamin. Identification, isolation and properties including electrofocusing patterns

Pooled porcine serum was found to contain cobalophilin (also called transcobalamin I) and transcobalamin (also called transcobalamin II). The two proteins were harvested by batchwise absorption with vitamin B-12 covalently coupled to Sepharose, and then separated from each other either by gel filtration or using an immunoadsorbent. Both proteins were finally isolated as single proteins using a second vitamin B-12-Sepharose chromatography step. Cobalophilin and transcobalamin complexed with vitamin B-12 had molecular weights by gel filtration of 135 000 and 38 000 and by the formula of Svedberg 104 000 and 44 000, Stokes radii 4.97 nm and 2.65 nm, and sedimentation coefficients 5.39 S and 3.75 S, respectively. Electrofocusing resolved the cobalophilin complex into three main isoproteins isoelectric at pH 3.23, 3.42 and 3.69, and transcobalamin into only the main component isoelectric at a value as low as pH 3.47. Neither protein was capable of binding to the ileal intrinsic factor receptor.     

A simplified technique to isolate the porcine and human ileal intrinsic factor receptors and studies on their subunit structures.

The porcine intestinal intrinsic factor receptor was isolated with affinity chromatography utilizing vitamin B12-intrinsic factor-Sepharose and pH adjustments. The purification was about 70 000-fold and in sodium dodecyl sulphate electrophoresis it resolved into two carbohydrate-containing 70 000 and 130 000 dalton bands (alpha and beta subunits) indicating purity. The human receptor was similarly purified and radioiodinated for further studies. It was also composed of two subunits (90 000 and 140 000 dalton). The alpha subunits bound to anti-intrinsic factor antisera.     

Purification of rat intestinal receptor for intrinsic factor · vitamin B-12 complex by affinity chromatography

Rat intrinsic factor was bound to vitamin B-12-Sepharose to produce intrinsic factor · vitamin B-12-Sepharose. Intestinal receptor for intrinsic factor · vitamin B-12 complex was purified from rat ileal extract by affinity chromatography using the intrinsic factor · vitamin B-12-Sepharose as an affinity adsorbent with recovery of 48.5% and specific activity increased 335 fold of original sample.     

Systemic lupus erythematosus

Imerslund-Gräsbeck syndrome (IGS) or selective vitamin B₁₂ (cobalamin) malabsorption with proteinuria is a rare autosomal recessive disorder characterized by vitamin B₁₂ deficiency commonly resulting in megaloblastic anemia, which is responsive to parenteral vitamin B₁₂ therapy and appears in childhood. Other manifestations include failure to thrive and grow, infections and neurological damage. Mild proteinuria (with no signs of kidney disease) is present in about half of the patients. Anatomical anomalies in the urinary tract were observed in some Norwegian patients. Vitamin B₁₂ absorption tests show low absorption, not corrected by administration of intrinsic factor. The symptoms appear from 4 months (not immediately after birth as in transcobalamin deficiency) up to several years after birth. The syndrome was first described in Finland and Norway where the prevalence is about 1:200,000. The cause is a defect in the receptor of the vitamin B₁₂-intrinsic factor complex of the ileal enterocyte. In most cases, the molecular basis of the selective malabsorption and proteinuria involves a mutation in one of two genes, cubilin (CUBN) on chromosome 10 or amnionless (AMN) on chromosome 14. Both proteins are components of the intestinal receptor for the vitamin B₁₂-intrinsic factor complex and the receptor mediating the tubular reabsorption of protein from the primary urine. Management includes life-long vitamin B₁₂ injections, and with this regimen, the patients stay healthy for decades. However, the proteinuria persists. In diagnosing this disease, it is important to be aware that cobalamin deficiency affects enterocyte function; therefore, all tests suggesting general and cobalamin malabsorption should be repeated after abolishment of the deficiency.     

Hydrodynamic characterization of vasoactive intestinal peptide receptors extracted from rat lung membranes in Triton X-100 and n-octyl-beta-D-glucopyranoside

Rat lung membrane vasoactive intestinal peptide (VIP) receptors were covalently labeled with 125I-VIP, extracted in Triton X-100 and n-octyl-beta-D-glucopyranoside, and analyzed by gel filtration and sucrose density gradient sedimentation. The fractions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, and the identity of the 125I-VIP.receptor complex was demonstrated by its co-migration with the covalently labeled 55-kDa receptor unit identified previously. Furthermore, the radioactivity in the peak corresponding to the 125I-VIP.receptor complex was displaced in the presence of unlabeled VIP in a dose-dependent manner. The following hydrodynamic properties were determined for VIP receptors in each detergent solution: in Triton X-100, Stokes radius of 6.1 +/- 0.4 nm, sedimentation coefficient (S20,w) of 7.35 +/- 0.45 S, and partial specific volume (v) of 0.809 +/- 0.015 ml/g; in n-octyl-beta-D-glucopyranoside, Stokes radius of 5.6 +/- 0.00 nm, S20,w of 10.87 +/- 0.22 S, and partial specific volume of 0.783 +/- 0.020 ml/g. The apparent molecular weight of the 125I-VIP.receptor.detergent complex was calculated as 270,000 +/- 36,000 in Triton X-100 and 320,000 +/- 32,000 in n-octyl-beta-D-glucopyranoside. The amount of detergent bound to the receptor was estimated by using the two sets of hydrodynamic data and the significantly different partial specific volumes of the two detergents. Thus, the molecular weight of the receptor alone was calculated as 54,600 daltons, indicating that approximately 3.9 g of Triton X-100 and 4.9 g of n-octyl-beta-D-glucopyranoside were bound per g of receptor. This species contained the 55-kDa binding unit and appeared to be glycosylated as evidenced by its specific binding to wheat germ agglutinin-Sepharose. These results indicate that the rat lung VIP receptor is a glycoprotein with a single polypeptide chain of 55 kDa. The large amount of detergent bound suggests that the receptor is extensively embedded in the membrane.     

Comparative studies on intrinsic factor and cobalophilin in different parts of the gastrointestinal tract of the pig

The vitamin B₁₂ binders in the pig pyloric mucosa gastric and intestinal juice from the upper gastrointestinal tract were fractionated into only two molecular forms, classified as intrinsic factor and cobalophilin. The unsaturated vitamin B₁₂-binding power due to cobalophilin was lower in the intestinal than in the gastric juice. Electrofocusing revealed that intrinsic factor and cobalophilin in the intestinal juice contained more of the `neutral'-type isoproteins, and the suggestion is made that this is due to enzyme activity. The gastric-juice intrinsic factor contained more acidic isoproteins, which supports the hypothesis that carbohydrate is added on to the polypeptide chain of this protein before it is secreted into gastric juice. The gastric- and intestinal-juice cobalophilins, studied also by electrofocusing, differed from that of pyloric mucosa and they appeared to be of salivary origin. With regard to molecular dimensions there was no significant difference between the intrinsic factors and cobalophilins from all sources studied. All cobalophilins had molecular weights by the formula of Svedberg of approx. 92500, Stokes radii of 4.62nm and sedimentation coefficients of 5.15S. The corresponding values for the intrinsic factors were 63600, 3.57nm and 4.38S. In addition, the intrinsic factors exhibited similar avidities for binding to the solubilized ileal intrinsic-factor receptor. Also the intrinsic factors and cobalophilins, irrespective of their source, bound to the analogous specific xenoantibodies with the same avidity. The present results demonstrate that intrinsic factor remains practically unaltered during its passage through the proximal intestine and render unlikely the speculations made about the presence of an endogenous binder for intrinsic factor as well as the existence of a `pancreatic intrinsic factor'. In addition, they are compatible with the theory that the interference by undegraded cobalophilin may be the reason for the abnormal vitamin B₁₂ absorption observed in patients with pancreatic insufficiency.     

Characterization of the molybdate-stabilized glucocorticoid receptor from rat thymus.

The untransformed glucocorticoid receptor of rat thymus cytosol was characterized in the form of its complex with [1,2,4-3H]triamcinolone acetonide by ion-exchange chromatography and by gel filtration and sucrose-density-gradient ultracentrifugation at different ionic strengths. Molybdate (10 mM) was present throughout all experimental procedures and prevented receptor inactivation and degradation as well as transformation. At low ionic strength the molybdate-stabilized steroid-receptor complex was detected as a single highly asymmetric entity with a Stokes radius of 5.85 nm, a sedimentation coefficient of 9.6 S and an apparent molecular weight of 236 000. This form was converted into a smaller, even more asymmetric, form in increasing proportion as the ionic strength was increased. In the presence of 0.4 M-KCl, the smaller form had a Stokes radius of 4.95 nm, a sedimentation coefficient of 4.6 S and an apparent molecular weight of 95 500. It is concluded that the glucocorticoid-receptor complex exists at low ionic strengths as a homodimer or as a heterodimer in which only one subunit possesses a steroid-binding site, and that the process of dissociation into subunits brought about by increasing the ionic strength is a process distinct from, but possibly preceding, the transformation phenomenon responsible for conferring DNA-binding properties on the complex.     

Solubilization of pituitary receptors for thyrotropin-releasing hormone

Receptors for thyrotropin-releasing hormone were solubilized by Triton X-100. Membrane fractions from GH₃ pituitary tumor cells were incubated with thyrotropin-releasing hormone in order to saturate specific receptor sites before the addition of detergent. The amount of protein-bound hormone solubilized by Triton X-100 was proportional to the fractional saturation of specific membrane receptors. Increasing detergent: protein ratios from 0.5 to 20 led to a progressive loss of hormone · receptor complex from membrane fractions with a concomitant increase in soluble protein-bound hormone. The soluble hormone · receptor complex was not retained by 0.22 μm filters and remained soluble after ultracentrifugation. Following incubation with high (2.5–10%) concentration of Triton X-100 and other non-ionic detergents, or following repeated detergent extraction, at least 18% of specifically bound thyrotropin-releasing hormone remained associated with particulate material. Unlike the hormone receptor complex, the free hormone receptor was inactivated by Triton X-100. A 50% loss of binding activity was obtained with 0.01% Triton X-100, corresponding to a detergent: protein ratio of 0.033.The hormone · receptor complex was included in Sepharose 6B and exhibited an apparent Stokes radius of 46 Å in buffers containing Triton X-100. The complex aggregated in detergent-free buffers. Soluble hormone receptors were separated from excess detergent and thyrotropin-releasing hormone by chromatography on DEAE-cellulose. Thyrotropin-releasing hormone dissociated from soluble receptors with a half-time of 120 min at 0°c, while the membrane hormone · receptor complex was stable for up to 5 h at 0°C.     

Requirements for cholera toxin-dependent ADP-ribosylation of the purified regulatory component of adenylate cyclase

The requirements for cholera toxin-catalyzed ADP ribosylation of the purified regulatory component of adenylate cyclase are described. In addition to the toxin, this reaction is dependent on or is facilitated by NAD, GTP, phospholipid, and a factor found associated with plasma membranes from several sources. Factor activity is heat-labile and protease-sensitive but is unaffected by treatment with N-ethylmaleimide. Gel filtration indicates that the factor behaves as a monodisperse species with a Stokes radius of 3.2 nm. The factor thus appears to be a protein that is distinct from any of the known components of adenylate cyclase. Factor activity was also detected in the cytoplasm of S49 cells. The cytoplasmic factor was smaller (Stokes radius = 2.0 nm) than the membrane-derived factor, and it was inactivated in the presence of sodium cholate. The initial rate of activation of the regulatory component of adenylate cyclase by toxin was found to be linearly related to the amount of factor present in the reaction. This has allowed the quantitation and partial purification (33-fold from detergent extracts) of the factor from turkey erythrocyte membranes.     

Cytosol oestrogen receptor of lactating mammary gland. Effect of heparin on the aggregation of the receptor and interaction of the receptor with heparin-Sepharose.

1. An oestrogen receptor is present in low-salt cytosol of the mammary gland of lactating mice as a large aggregate; it is excluded from gel matrix when filtered on a Sephadex G-200 column and sediments at 7S in sucrose gradients. After incubation of cytosol with heparin, the receptor is dissociated. On a Sephadex G-200 column, it is included in the gel matrix and eluted as a protein with mol.wt. 260000 and a Stokes radius of 6.8nm; it sediments at 6S in sucrose gradients. 2. Dissociation of the mammary-gland cytosol oestrogen receptor seems to be the result of interaction of the oestrogen-receptor complex with heparin. This receptor interacts with heparin covalently bound to Sepharose, thereafter sedimenting at 6S. By using this interaction, the cytosol receptor was purified 200-fold compared with the homogenate, with a yield of 70%. 3. The cytosol receptor that was not incubated or was incubated with heparin was much smaller during sucrose-gradient centrifugation than during gel filtration. This discrepancy can be explained by pressure-induced dissociation during high-speed centrifugation. This possibility is supported by the decrease in the sedimentation coefficient of the receptor with increased duration of centrifugation.     

Limited proteolysis of cytoplasmic and nuclear uterine estradiol receptors yields identical estradiol-binding fragments.

Limited tryptic hydrolysis of the estradiol cytoplasmic receptor from calf uterus has been demonstrated to yield in a high-salt buffer a stable estradiol-binding molecule with the following characteristics: sedimentation coefficient 4.0 +/- 0.1 S; Stokes radius 3.5 +/- 0.05 nm; molecular weight 60000 (for an assumed v value of 0.73 ml g-1) and frictional ratio 1.36. Nuclear KCl extracts, prepared from uteri preincubated at 37 degrees C with labeled estradiol, were analysed by Sephadex G-200 chromatography and sucrose density gradient centrifugation. The following molecular parameters were found for the estradiol-receptor complex: sedimentation coefficient 4.4 +/- 0.1 S; Stokes radius 4.12 +/- 0.02 nm; molecular weight 77000 and frictional ratio 1.47 (v = 0.73 ml g-1). Limited tryptic proteolysis of this extract gave an estradiol-binding fragment with molecular characteristics identical to the trypsin-modified cytoplasmic receptor. In addition, mild tryptic digestion of whole labeled nuclei allowed us to solubilize almost quantitatively the nuclear [3H]estradiol in a macromolecular bound form. The molecule thus obtained showed molecular parameters very similar to the 60000-dalton trypsin fragments obtained from high-salt cytoplasmic and nuclear extracts. These molecules were undistinguishable by gel electrophoresis analysis at six different acrylamide concentrations. These results in conjunction with those derived from dissociation kinetics experiments and ligand specificity studies indicate the cytosolic protein is a functional part of the nuclear receptor. Based upon these and other studies we suggest that proteolytic cleavage of the estradiol-receptor complex, which results in the removal of the estradiol-binding sites from the nuclear recognition sites of the molecule, could play a role in the inactivation of the estradiol receptor in vivo.     

Insulin binds to and promotes the phosphorylation of a Mr 210 000 component of its receptor in detergent extracts of rat liver microsomes

Insulin in the presence of Mn2+ and [gamma 32P]ATP promoted the phosphorylation of two proteins of Mr 95 000 and Mr 210 000 in detergent extracts of rat liver microsomes. The Mr 210 000 protein was identified as a component od the insulin receptor by immunoprecipitation. It also bound [125I]insulin specifically, was phosphorylated largely on a tyrosine residue and could not be cleaved to smaller subunits under extreme reducing conditions. The Mr 210 000 protein appears to be a component of a sub-population of liver membrane insulin receptors in which insulin-binding and insulin-stimulated tyrosine kinase phosphorylation site(s) reside in a single polypeptide chain.     

Differences in the association of the progesterone receptor ligated by antiprogestin RU38486 or progestin ORG 2058 to chromatin components

We assessed the hypothesis that due to variations in the conformation of the progesterone receptor induced by the antiprogestin RU38486 compared to the progestin ORG 2058, differences may result in the association of the receptor with some of the chromatin components. The physical properties of the receptor-bound chromatin fragments released by micrococcal nuclease digestion were characterized by sucrose gradient sedimentation and by gel filtration on Agarose A-1.5m or Agarose A-5m columns. The nuclear fraction was isolated from T47D cells previously exposed to 0.1 microM [3H]RU38486 or 0.1 microM [3H]ORG 2058. Micrococcal nuclease digestion solubilized two receptor forms sedimenting at 4.4 S and 6.3 S for the antiprogestin bound receptor and only one receptor at 4.4 S for the progestin ligated receptor. High-salt buffer dissociated either the antiprogestin or the progestin-bound receptor to smaller receptor forms sedimenting at 3.5 S. Chemical cross-linking with the cross-linker 2-iminothiolane of the micrococcal nuclease solubilized receptor forms resulted in 6.7-S and 4.4-S forms sedimenting on 0.4 M KCl gradients for the antiprogestin and progestin ligated receptors, respectively. Stokes radii of 7.3 nm and 6.4 nm were determined by gel filtration in 0.4 M KCl for the 6.7-S and the 4.4-S receptor forms, respectively. Using the sedimentation coefficient and the Stokes radius, molecular weights of 202,000 and 116,000 were calculated for the antiprogestin and progestin ligated receptors. We conclude that the micrococcal nuclease solubilized antiprogestin ligated receptor is associated with additional or different chromatin components compared to the progestin bound receptor.     

Sub-Optimal Vitamin B-12 Levels among ART-Na?ve HIV-Positive Individuals in an Urban Cohort in Uganda

Malnutrition is common among HIV-infected individuals and is often accompanied by low serum levels of micronutrients. Vitamin B-12 deficiency has been associated with various factors including faster HIV disease progression and CD4 depletion in resource-rich settings. To describe prevalence and factors associated with sub-optimal vitamin B-12 levels among HIV-infected antiretroviral therapy (ART) naïve adults in a resource-poor setting, we performed a cross-sectional study with a retrospective chart review among individuals attending either the Mulago-Mbarara teaching hospitals’ Joint AIDS Program (MJAP) or the Infectious Diseases Institute (IDI) clinics, in Kampala, Uganda. Logistic regression was used to determine factors associated with sub-optimal vitamin B-12. The mean vitamin B-12 level was 384 pg/ml, normal range (200–900). Sub-optimal vitamin B-12 levels (<300 pg/ml) were found in 75/204 (36.8%). Twenty-one of 204 (10.3%) had vitamin B-12 deficiency (<200 pg/ml) while 54/204 (26.5%) had marginal depletion (200–300 pg/ml). Irritable mood was observed more among individuals with sub-optimal vitamin B-12 levels (OR 2.5, 95% CI; 1.1–5.6, P = 0.03). Increasing MCV was associated with decreasing serum B-12 category; 86.9 fl (±5.1) vs. 83 fl (±8.4) vs. 82 fl (±8.4) for B-12 deficiency, marginal and normal B-12 categories respectively (test for trend, P = 0.017). Compared to normal B-12, individuals with vitamin B-12 deficiency had a longer known duration of HIV infection: 42.2 months (±27.1) vs. 29.4 months (±23.8; P = 0.02). Participants eligible for ART (CD4<350 cells/µl) with sub-optimal B-12 had a higher mean rate of CD4 decline compared to counterparts with normal B-12; 118 (±145) vs. 22 (±115) cells/µl/year, P = 0.01 respectively. The prevalence of a sub-optimal vitamin B-12 was high in this HIV-infected, ART-naïve adult clinic population in urban Uganda. We recommend prospective studies to further clarify the causal relationships of sub-optimal vitamin B-12, and explore the role of vitamin B-12 supplementation in immune recovery.     

Water-soluble acetylcholine receptor from Torpedo californica. Solubilization,purification and characterization

The nicotinic acetylcholine receptor from electrogenic tissue of Torpedo californica was solubilized by tryptic digestion of membrane fragments obtained from autolysed tissue, without use of detergent. The water-soluble acetylcholine receptor was purified by affinity chromatography on a cobra-toxin-Sepharose resin. The purified receptor bound 4000–6000 pmol per mg protein of α-[¹²⁵]bungarotoxin, and toxin-binding was specifically inhibited by cholinergic ligands. Gel filtration revealed a single molecular species of Stokes radius $\text{125 ± 10}$ Å and on sucrose gradient centrifugation one major peak was observed of 20–22 S. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and β-mercaptoethanol revealed two major polypeptides of mol. wt. 30 000 and 48 000.     

Solubilisation and Molecular Characterisation of Muscarinic Acetylcholine Receptors

Abstract

Stable, soluble preparations of rat brain muscarinic receptors can be prepared by extracting membranes with digitonin, or with combinations of sodium cholate and sodium chloride. The stability of the cholate/NaCl extract is enhanced by the addition of egg phosphatidylcholine, which, at the same time, suppresses the considerable dispersity apparent in the hydrodynamic behaviour of the solubilised receptor. The Stokes radius of the brain muscarinic receptor in cholate/NaCl/lecithin extracts is 6.7 nm, with very similar values in other detergents, including digitonin and sodium dodecyl sulphate. Its sedimentation coefficient is 3.78s, and its molecular weight approximately 110,000 after correction for detergent binding. The isoelectric point of the digitonin - solubilised receptor is approximately 4.5.     

Binding assay and physicochemical characteristics of solubilized intrinsic factor receptor in ileal mucosal homogenates using phenyl-Sepharose to separate the saturated receptor from free intrinsic factor.

A radioisotopic assay was set to determine the physicochemical properties of the solubilized intrinsic factor receptor in pig mucosal extracts. In this assay, phenyl-Sepharose was used to separate the receptor-intrinsic factor-labelled cobalamin complex from the free saturated intrinsic factor. The association constant (at pH 7.4) of the receptor-intrinsic factor complex was estimated at 3.4 +/- 0.3 nM-1. Adsorption of the apo-receptor to phenyl-Sepharose allowed its binding site to be made accessible to intrinsic factor with an association constant in order of 6 nM-1. The receptor binding activity obtained with five mucosal extracts was closely correlated with that obtained by gel filtration of the intrinsic factor-receptor complex (r = 0.99). The radioisotope assay was used to detect the unsaturated receptor (apo-receptor) in sucrose density ultracentrifugation and in superose 6 gel filtration. The sedimentation coefficient was 9.5 s. The apo-receptor was eluted in three peaks in gel filtration, corresponding to the formation of oligomers. The peak of the monomer was increased in presence of EDTA. Its molecular mass was estimated at 270 kDa and its Stokes radius at 5.9 nm. It was concluded that calcium is involved in the oligomerisation of the apo-receptor.