Specificity of the sialic acid-binding lectin from the snail Cepaea hortensis.

The specificity of the sialic acid-binding lectin from the snail Cepaea hortensis, purified by affinity chromatography on fetuin-Sepharose, was studied by hemagglutination inhibition assay applying 32 sialic acid derivatives and 14 glycoproteins. 2-alpha-Methyl-9-O-acetyl-NeuAc was the most potent inhibitor, followed closely by 2-alpha-methyl-NeuAc and 2-alpha-benzyl-NeuAc. An axially orientated carboxyl group is a prerequisite for maximal lectin-sugar binding. Neither size nor polarity of the alpha-anomeric substituent significantly influenced inhibition potency. An intact sialic acid N-acetyl group is essential for optimal lectin-sugar interaction. The trihydroxypropyl side chain also is of great importance. However, a bulky hydrophobic substituent at the side chain like a 9-O-tosyl residue did not decrease binding to the lectin. The lectin did not distinguish between NeuAc alpha 2----3Gal beta 1----4Glc and NeuAc alpha 2----6Gal beta 1----4Glc. Among other sugars tested, only N-acetylglucosamine showed inhibition, although 50-fold less. The most potent glycoprotein inhibitors were those carrying O-chains only or preferentially, as ovine submaxillary mucin, bovine submaxillary mucin, and glycophorin A. Tamm-Horsfall protein was an exception being a strong inhibitor, although carrying only N-chains. Asialoglycoproteins were inactive. Glycoproteins containing the NeuAc alpha 2----3Gal sequence inhibited the lectin as well as those with NeuAc alpha 2----6GalNAc. From the results a model of the lectin's binding site for sialic acid is suggested.     

Human insulin-like growth factor binding protein-6 is O-glycosylated.

Insulin-like growth factor binding protein-6 is abundant in cerebrospinal fluid and has a marked preferential binding affinity for IGF-II over IGF-I. The present study demonstrates that IGFBP-6 is O-glycosylated but not N-glycosylated. Carbohydrate analysis revealed the presence of approximately 20-30 carbohydrate residues/molecule. Galactosamine, galactose and sialic acid were most abundant, with glucosamine and fucose present in lower concentrations. Mannose was not detected. Enzymatic deglycosylation did not alter the high affinity of IGF binding protein-6 for IGF-II (Ka 4.4 +/- 2.2 x 10(11) M-1) or its preference for IGF-II over IGF-I. Glycosylation of IGFBP-6 may affect its secretion, in vivo stability or localization, but does not affect its ligand binding properties.     

Characterization of calcium binding to adipocyte plasma membranes.

Calcium binding to adipocyte plasma membranes has been assessed by equilibrium dialysis and by membrane filtration techniques. Calcium binding was specific and saturable, displaying two distinct classes of binding sites. The affinity constants and maximum binding capacities in the presence of 0.1 M KCl were 4.5 X 10(4) M-1 and 1.8 nmol/mg of protein and 2.0 X 10(3) M-1 and 13.7 nmol/mg for the high and low affinity sites, respectively. Bound calcium was totally dissociated in the presence of excess calcium within 11.0 min in two distinct phases corresponding to the two classes of sites. Association and dissociation rate constants for the high affinity sites were 7.7 X 10(2) M-1S-1 and 9.2 X 10(-3S-1 respectively. Free energy changes at 24 degrees were +6.4 kcal mol-1 for the high affinity sites and +4.5 kcal mol-1 for the low affinity sites. The high affinity sites demonstrated a pH optimum of 7.0 whereas the binding to the low affinity sites progressively increased between pH 6.0 and 9.0. Low concentrations of MgCl2 (less than 300 muM) enhanced calcium binding slightly, whereas high concentrations of KCl and MgCl2 were noncompetitive inhibitors of calcium binding. Procaine and ruthenium red had no effect on calcium binding and lanthanum was a poor inhibitor of calcium binding. This represents the first report of calcium binding to adipocyte plasma membranes and the first kinetic analysis of calcium binding to biological membranes. The specificity of this calcium-binding system in adipocyte plasma membranes suggests its importance in cellular bioregulation.     

Protein binding to brush borders of enterocytes from the jejunum of the neonatal rat

The specific binding of IgG to jejunal brush borders was greatest at acidic pH, at neutral pH no specific binding occurred. Specific binding declined with age-no specific binding occurred in borders from 20-and 24-day-old animals. There was no specific binding of IgG to borders from ileal enterocytes. Human transferrin and bovine serum albumin did not bind specifically to borders. The affinity of binding (-Ka) and the receptors site numbers per border estimated for rat IgG were 18.64 X 10(6) M-1 to 3.53 X 10(6) sites; for human IgG, 25.06 X 10(6) M-1 to 3.30 X 10(6) sites; for bovine IgG, 10.48 X 10(6) M-1 to 2.11 X 10(6) sites and for sheep IgG, 7.26 X 10(6) M-1 to 2.34 X 10(6) sites.     

Characterization of a proton-driven carrier for sialic acid in the lysosomal membrane. Evidence for a group-specific transport system for acidic monosaccharides

Highly purified lysosomal membrane vesicles, obtained from rat liver lysosomes, were used to study characteristics of NeuAc transport across the lysosomal membrane. Uptake of [14C]NeuAc was found to be strongly influenced by a pH gradient across the membrane. When a proton gradient (pHin greater than pHout) was generated by impermeable buffers, NeuAc uptake above equilibrium level (overshoot) was observed. The influence of membrane diffusion potentials was ruled out by experiments where K+ and valinomycin were present. The overshoot appeared to be specifically produced by protons, since gradients of other cations (Na+ and K+) did not give stimulation. Proton-driven uptake was saturable (Kt = 0.24 mM) and mediated by a single system, as shown by linearity of the Scatchard plot. Stimulation of transport was also obtained by preincubation of vesicles with MgATP and the effect was blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, but not by the protonophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. Monocarboxylic sugars like glycuronic acids were competitive inhibitors of sialic acid transport. Transstimulation of [14C] NeuAc uptake was observed when vesicles were preloaded either with unlabeled NeuAc or with glucuronic acid. The data demonstrate that lysosomal membrane vesicles from rat liver are a suitable system for kinetic studies of solute transport events. The presence of a proton-driven carrier in the lysosomal membrane specific for sialic acid and other acidic sugars, including glucuronic acid, is shown. The possible physiological significance of these findings for the human lysosomal carrier and the patients with a sialic acid transport defect is discussed.     

Insulin receptors in rabbit erythrocyte

The existence of insulin receptors in rabbit erythrocytes was studied by evaluating the specific binding of 125I-insulin to erythrocyte membranes. The binding of 125I-insulin was pH, time and temperature dependent. Maximal binding was achieved by incubation for 20 hr at 0 degrees C. The optimum pH was 7.4. Treatment with cations and enzymes enhanced the specific binding except for with trypsin, the treatment which greatly reduced the binding. Unlabeled insulin over a wide range of concentrations competitively inhibited the binding of 125I-insulin, while the binding was little affected by structurally unrelated hormones. Scatchard plot was represented as a concave curve. Binding sites of relatively high affinity (K1 = 0.9 X 10(9) M-1) and low capacity (8.0 X 10(13)/g protein) could be distinguished from those of lower affinity (K2 = 0.8 X 10(7) M-1) and higher capacity (1.8 X 10(15)/g protein). Hill's analysis and dissociation of 125I-insulin from membranes demonstrated the characteristics of negative cooperation between receptor sites. Both incorporation of H3(32)PO4 to erythrocyte membranes and uptake of 45Ca were significantly reduced by the addition of unlabeled insulin. Unlabeled insulin produced no effect on uptake of 45Ca into trypsin-treated erythrocytes. On the basis of these results, it was suggested that rabbit erythrocytes might possess biologically significant insulin receptors located on the cell membranes.     

Regulation of GH binding to specific cellular receptors in vitro--a new model of growth regulation in vivo

A new theory on the complex growth hormone (GH) mediated promotion of tissue growth has been developed on the basis of in vitro studies of growth hormone receptors on human peripheral mononuclear cells (PMC). It is hypothesized that GH acts through its tissue receptors and regulates its own receptors through two different pathways: first GH directly downregulates GH binding, second a partially GH-dependent serum factor (the so-called SM-B) enhances GH binding. Some experimental evidence for this hypothesis is presented using a new method to investigate GH receptors in circulating human blood cells: The effect of trypsin, antitrypsin, growth hormone, somatomedin-B (SM-B) and anti-SM-B-antiserum on GH binding to PMC was studied. Trypsinization of cells leads to a decrease both of specific binding and of binding affinity (affinity constant after 60 minutes of trypsinization 0.5 X 10(6) M-1 versus 1.5 X 10(6) M-1 in untreated control cells). Exposure of PMC to antitrypsin activities was followed by an increase of binding affinity and specific binding (affinity constants with 10 KIU 1.9 X 10(6) M-1, with 100 KIU 2.4 X 10(6) M-1, with 1000 KIU 3.6 X 10(6) M-1). This antitrypsin effect exceeds the binding values expected after blocking trypsin activities possibly being present in the incubation medium. In a subset of experiments the partially GH-dependent serum factor SM-B was used as the antitrypsin moiety and was shown to increase specific GH binding to PMC in a similar manner as did antitrypsin (with 1000 ng SM-B affinity constant 12.0 X 10(6) M-1, specific binding 9.7%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the binding of human growth hormone to microsomal membranes from rat liver.

The binding of 125I-labelled human growth hormone to the 100000g microsomal membrane fraction prepared from the livers of normal female rats was dependent on time, temperature, pH, membrane concentration and concentration of 125I-labelled human growth hormone. At 22 degrees C binding reached a steady state after 16h, with the mean maximal specific binding being 20% of the tracer initially added. Dissociation of 125I-labelled human growth hormone from the membranes, after addition of excess of unlabelled hormone, was relatively slow with a half-time greater than 24h. Only minor degradation of the 125I-labelled human growth hormone was observed during incubation with membranes for 16 or 25h at 22 degrees C. Similarly, no significant change in the ability of membranes to bind human growth hormone was evident after preincubation of the membranes for 16 or 25h. Specificity studies showed that up to 90% of the 125I-labelled human growth hormone bound could be displaced by 1 mug of unlabelled hormone. Ovine prolactin also showed considerable competition for the binding site. Non-primate growth-hormone preparations (ovine, bovine, porcine and rat) and non-related hormones (insulin, thyrotropin, lutropin and follitropin) all showed negligible competition. Scatchard analysis of the binding data was consistent with two classes of binding site with binding affinities of 0.64 X 10(10) +/- 0.2 X 10(10)M-1 and 0.03 X 10(10) +/- 0.007 X 10(10)M-1 and corresponding binding capacities of 98.4 +/- 10 fmol/mg of protein and 314.6 +/- 46.3 fmol/mg of protein. These studies provide data which, in general, are consistent with the criteria required for hormone-receptor interaction. However, proof of the thesis that the human-growth-hormone-binding sites in female rat liver represent physiological receptors must await the demonstration of a correlation between hormone binding and a biological response.     

Carbohydrate binding specificity of a beetle (Allomyrina dichotoma) lectin

Carbohydrate binding specificity of a lectin, allo A, isolated from a beetle (Allomyrina dichotoma), was investigated by means of lectin affinity chromatography. Sialylated complex-type and hybrid-type oligosaccharides/glycopeptides, and sialyllactose were retained by the column, whereas desialylated ones were retarded but not retained by the column. The association constants of allo A for biantennary oligosaccharides from human serum transferrin, determined by frontal analysis, were 8.0 X 10(5) M-1, 4.5 X 10(5) M-1, and 2.5 X 10(5) M-1 for disialo-, monosialo-, and asialo-oligosaccharides, respectively. Removal of the beta-galactose residues markedly reduced the association constant to 3.5 X 10(3) M-1. Furthermore, allo A was found to have no affinity for mucin-type glycopeptides carrying the sialylated Gal beta 1----3 GalNAc sugar sequence (Ka: 3.5 X 10(3) M-1). The results of this study indicated that allo A strongly binds to the trisaccharide structure, NeuAc alpha 2-3(6)Gal-beta 1-4GlcNAc, and that its binding potency is affected by the inner core structures of oligosaccharides and glycopeptides, because the presence of a bisecting N-acetyl-glucosamine residue and an alpha-fucose residue linked to the innermost N-acetylglucosamine residue reduced the association constants for oligosaccharides and glycopeptides.     

Residue Y161 of influenza virus hemagglutinin is involved in viral recognition of sialylated complexes from different hosts

Influenza A virus glycoprotein hemagglutinin (HA) binds to host cell surface sialic acid (SA)-terminated sugars in glycoproteins to initiate viral entry. It is thought that avian influenza viruses preferentially bind to N-acetylneuraminic acid α3 (NeuAcα3) sugars, while human influenza viruses exhibit a preference for NeuAcα6-containing sugars. Thus, species-specific SA(s) is one of the determinants in viral host tropism. The SA binding pocket of the HA1 subunit has been extensively studied, and a number of residues important for receptor binding have been identified. In this study, we examined the potential roles of seven highly conserved HA surface-located amino acid residues in receptor binding and viral entry using an H5 subtype. Among them, mutant Y161A showed cell-type-dependent viral entry without obvious defects in HA protein expression or viral incorporation. This mutant also displayed dramatically different ability in agglutinating different animal erythrocytes. Oligosaccharide binding analysis showed that substituting alanine at Y161 of HA changed the SA binding preference from NeuAc to N-glycolylneuraminic acid (NeuGc). Rescued mutant Y161A viruses demonstrated a 5- to 10-fold growth defect, but they were robust in viral replication and plaque forming ability. Our results demonstrate that Y161 is a critical residue involved in recognition of different SA species. This residue may play a role in determining influenza virus host tropism.     

Classification and localization of hemoglobin binding sites on the red blood cell membrane.

The binding of hemoglobin to the red cell membrane was characterized over a wide range of free hemoglobin concentrations by measurement of membrane bound and supernatant hemoglobin. Scatchard analysis of the binding data revealed two classes of sites: high affinity sites with a binding constant of 1 X 10(8) M-1 and 1.2 X 10(6) sites per cell, and a second, low affinity class of sites with a binding constant of 6 X 10(6)M-1 and 6 X 10(6) sites per cell. The low affinity sites are shown to be nonspecific and appear to be a result of the ghost preparation. The high affinity sites are shown to be specific to the inner surface of the red cell membrane. The competition of hemoglobin and glyceraldehyde-3-phosphate dehydrogenase suggests band III proteins as a potential binding site for hemoglobin.     

Human alpha-fetoprotein. Fluorescence studies on binding and proximity relationships for fatty acids and bilirubin.

The binding of bilirubin and the polyene fatty acids cis-parinaric acid and cis-eleostearic acid to human alpha-fetoprotein was studied using fluorescence quenching and fluorescence enhancement techniques. alpha-Fetoprotein has three fatty acid binding sites of decreasing affinity (association constants 2.1 x 10(7) M-1 9.1 X 10(5) M-1, and 1.4 x 10(5) M-1) and one relatively strong and one relatively weak bilirubin binding site (association constants 1.1 x 10(7) M-1 and 1.8 x 10(5) M-1). These association constants are slightly weaker than the corresponding association constants for binding to human albumin. Competition experiments failed to show preferential binding of polyunsaturated fatty acids. Fluorescence quenching was used to determine 11 ligand-ligand and ligand-tryptophanyl residue distances. Each of these 11 calculated distances (ranging from 19 A to 32 A) was within 5 A of the corresponding distances measured previously for human albumin (Berde, C.B., Hudson, B.S., Simoni, R.D., and Sklar, L.A. 1979, J. Biol. Chem. 254, 391-400). Thus, in addition to previously described sequence homology, immunologic cross-reactivity, and other similarities, human albumin and human alpha-fetoprotein have functional and geometric homologies.     

Basolateral membrane lipid dynamics alter Na-K ATPase activity in rabbit small intestine.

The role of basolateral membrane fluidity in regulating Na-K ATPase activity along the crypt-villus axis in rabbit distal small intestine was assessed. Basolateral membranes were prepared from isolated villus and crypt enterocytes at 24- to 28-fold enhancement. Villus basolateral membranes were significantly (p < 0.001) more fluid than crypt basolateral membranes as measured by 1,6-diphenyl-1,3,5-hexatriene. No difference was seen between the two groups as measured by either 2-(9-anthroyloxy)-stearic fatty acid or 16-(9-anthroyloxy)-palmitic acid. Fluidity alterations were accompanied by an increased phospholipid content in villus membranes, which resulted in a decreased cholesterol:phospholipid ratio and an increased lipid:protein molar ratio. Na-K ATPase activity was significantly (p < 0.01) greater in villus basolateral membranes than in crypt membranes, and demonstrated a greater sensitivity to ouabain inhibition. Ouabain inhibition curves calculated from villus data fit well (p < 0.001) with a two binding site model, with a high affinity (Ki 16 nM) and a low affinity (Ki 4.2 microM) ouabain binding site. In crypt basolateral membranes, only a low affinity site was apparent (Ki 3.0 microM). Fluidizing crypt basolateral membranes in vitro with benzyl alcohol to levels seen in villus basolateral membranes resulted in the appearance of a high affinity ouabain binding site (Ki 110 nM) and an increased sensitivity of Na-K ATPase to ouabain inhibition. The fluidization of villus basolateral membranes eliminated the binding associated with the high affinity site. Treatment with methanol, as a control, did not alter Na-K ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of Mg2+ on the Ca2+-binding properties of non-activated phosphorylase kinase.

The calcium binding properties of non-activated phosphorylase kinase at pH 6.8 have been studied by the gel filtration technique at calcium concentrations from 50 nM to 50 muM. Taking into account the subunit structure alpha4beta4gamma4 the enzyme binds 12 mol Ca2+ per mol with an association constant of 6.0 X 10(7) M-1, 4 mol with an association constant of 1.7 X 10(6) M-1 and 36 mol with a binding constant of 3.9 X 10(4) M-1 at low ionic strength. In buffer of high ionic strength, i.e. 180 mM NH4Cl or 60 mM (NH4)2SO4, only a single set of eight binding sites with a binding constant of 5.5 X 10(7) M-1 is left. In a buffer containing 155 mM NH4Cl and 10 mM MgCl2, the calcium affinity of these sites is reduced to a KCa of 3.0 X 10(6) M-1, indicating competition between Ca2+ and Mg2+. From these measurements, the binding constant of Mg2+ for these sites is calculated to be 1.7 X 10(3) M-1 is left. In a buffer containing 155 mM NH4Cl and 10 mM MgCl2, the calcium affinity of these sites is reduced to a KCa of 3.0 X 10(6) M-1, indicating competition between Ca2+ and Mg2+. from these measurements, the binding constant of Mg2+ for these sites is calculated to be 1.7 X 10(3) M-1. Additionally, 10 mM Mg2+ induces a set of four new Ca2+ binding sites which show positive cooperativity. Their half-saturation constant under the conditions described is 3.5 X 10(5) M-1, and they, too, exhibit competition between Ca2+ and Mg2+. Since this set of sites is induced by Mg2+ a third group of binding sites for the latter metal must be postulated.     

Interaction of ADP and magnesium with the active site of myosin subfragment-1 observed by reactivity changes of the critical thiols and by direct binding methods at low and high ionic strength

Comprehensive binding studies using direct and indirect methods yield stoichiometry and affinities for the binding of Mg X ADP and uncomplexed ADP to the active site of myosin subfragment-1. Additionally, the binding parameters for Mg2+ in the ternary complex protein X Mg X ADP are presented for the first time. The indirect method makes use of reactivity changes of the critical thiol-1 and thiol-2 groups, which occur upon the binding of the ligand at the active site. The affinity constants derived by this method are corroborated by two independent direct methods, equilibrium dialysis and centrifugation transport. For Mg2+, ADP and Mg X ADP just one mole of ligand binds/mole subfragment-1. The affinity of Mg X ADP at low ionic strength is 2.1 X 10(6) M-1 and only five-times lower in the absence of Mg2+. In the ternary complex Mg2+ has a low affinity of 4.1 X 10(4) M-1. At high ionic strength the uncomplexed ADP binds with a 43-times-lower affinity than Mg X ADP, whose affinity is 6.9 X 10(5) M-1. In this case Mg2+ interacts in the ternary complex with the higher affinity of 3.2 X 10(5) M-1, implying that at high salt concentration it plays a more prominent role in anchoring ADP at the active site.     

Protease inhibitors from the parasitic worm Parascaris equorum

Two proteic inhibitors (I and II) of serine proteases have been purified from the parasitic worm Parascaris equorum by affinity chromatography on immobilized trypsin followed by preparative electrophoresis. They have an apparent relative molecular mass of 9000 and 7000 as determined by gel filtration, a slightly acid isoelectric point (5.5 and 6.1) and a similar amino acid composition. Both inhibitors lack serine, methionine and tyrosine. They bind bovine trypsin extremely strongly with an association constant, Ka, larger than 10(9) M-1, and form a 1:1 complex with this protease. The Ka values for the binding to bovine chymotrypsin are approximately 3.3 X 10(8) M-1 (inhibitor I) and approximately 2 X 10(6) M-1 (inhibitor II). Inhibitor I interacts also with porcine elastase (Ka approximately 5 X 10(7) M-1), while inhibitor II is inactive towards this enzyme.     

Effect of deglycosylation on the binding and immunoreactivity of human thyroxine-binding globulin.

Thyroxine-binding globulin (TBG), prepared from human serum by an improved purification method, was treated with a mixture of neuraminidase, beta-galactosidase, alpha-mannosidase, and beta-N-aectylglucosaminidase, which resulted in the removal of approximately 86% of saccharides. Purification by thyroxine-Sepharose affinity chromatography gave a homogeneous protein as shown by equilibrium sedimentation and sodium dodecylsulfate-polyacrylamide gel electrophoresis. Amino acid and NH2-terminal sequence analysis indicated that the protein moiety was intact. Deglycosylation had no effect on the stoichiometry of the binding of L-thyroxine as shown by tryptophanyl fluorescence quenching and equilibrium dialysis at pH 8.6 and 25 degrees C. However, the affinity constant for L-thyroxine was reduced from 1.6 X 10(9) M-1 to 0.58 X 10(9) M-1. Analysis of radioimmunoassay data revealed that deglycosylation resulted in a slight decrease of the affinity constant for anti-TBG antibody from 3.9 X 10(10) M-1 to 1.8 X 10(10) M-1. These results suggest that the polypeptide moiety, rather than the heterosaccharides, contains the antigenic determinants. Removal of the majority of the heterosaccharides of TBG has only a minor effect on its immunoreactivity and on the binding of thyroid hormone.     

Protease nexin. Properties and a modified purification procedure

The present paper describes chemical and functional properties of protease nexin, a serine protease inhibitor released from cultured human fibroblasts. It is shown that protease nexin is actually synthesized by fibroblasts and represents about 1% of their secreted protein. Analysis of the amino acid composition of purified protease nexin indicates that it is evolutionarily related to antithrombin III and heparin cofactor II. Protease nexin contains approximately 6% carbohydrate, with 2.3% amino sugar, 1.1% neutral sugar, and 3.0% sialic acid. The Mr calculated from equilibrium sedimentation analysis is 43,000. Protease nexin is a broad specificity inhibitor of trypsin-like serine proteases. It reacts rapidly with trypsin (kassoc = 4.2 +/- 0.4 X 10(6) M-1 s-1), thrombin (kassoc = 6.0 +/- 1.3 X 10(5) M-1 s-1), urokinase (kassoc = 1.5 +/- 0.1 X 10(5) M-1 s-1), and plasmin (kassoc = 1.3 +/- 0.1 X 10(5) M-1 s-1), and slowly inhibits Factor Xa and the gamma subunit of nerve growth factor but does not inhibit chymotrypsin-like proteases or leukocyte elastase. In the presence of heparin, protease nexin inhibits thrombin at a nearly diffusion-controlled rate. Two heparin affinity classes of protease nexin can be detected. The present characterization pertains to the fraction of protease nexin having the higher affinity for heparin. The low affinity material, which is the minor fraction, is lost during purification.     

Characterization of prolactin receptors in pig mammary gland.

Prolactin receptors present in the particulate fraction of lactating pig mammary gland were solubilized by 7.5mM-3-[(3-cholamidopropyl)dimethylammonio]-1-propane-su lph onic acid (Chaps) and purified by affinity chromatography on prolactin coupled to Affi-Gel 10. Nearly 30% of the particulate receptors were solubilized by the detergent and over a 1000-fold purification from homogenates was achieved. A water-soluble fraction rich in receptors was observed during the preparation of membranes, although this fraction has not yet been purified. Prolactin binding to the receptors was a time-dependent, reversible and saturable reaction in particulate, Chaps-solubilized and purified receptors. In all forms, receptors showed the same specificity to peptide hormones. Prolactin and human growth hormone bound to the same receptors, whereas bovine growth hormone, follicle-stimulating hormone, luteinizing hormone, thyroid-stimulating hormone and insulin failed to bind. After solubilization, the dissociation constant (Kd) for prolactin was decreased 5-fold from 9.8 X 10(-11) M in the particulate receptors to 1.8 X 10(-11) M in solubilized and purified receptors, being due principally to an increase in the association rate constant from 1.0 X 10(9)M-1 X h-1 to (3.9-4.6) X 10(9)M-1 X h-1, respectively, with the dissociation rate constant remaining unchanged at (1.1-1.3) X 10(-2)h-1. Isoelectric focusing of the prolactin-receptor complex revealed two peaks, one at a pI of 5.5-5.6 and the other at 5.2-5.3. Microsomal receptors were covalently cross-linked to 125I-labelled ovine prolactin with ethylene glycol bis(succinimidyl succinate) and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Autoradiography of the gel revealed a major subunit of Mr 28 000-35 000 and a minor one of Mr 67 000-69 000. Anti-(prolactin receptor) antibodies raised against rabbit mammary gland prolactin receptors were equally effective in inhibiting prolactin binding to particulate, solubilized and affinity-purified receptors, suggesting that purified prolactin receptors have a structure indistinguishable immunologically from particulate receptors and rabbit mammary gland prolactin receptors. The present demonstration shows that particulate prolactin receptors from a domestic animal can be solubilized and purified without losing the original properties of high affinity and binding specificity for hormones.     

A high affinity thyroid hormone binding protein in the cytosol of embryonic rat brain cells in primary cultures

A thyroid hormone binding protein(s) has been characterized in the cytosol of fetal rat brain cells in primary cultures. This protein is closely related to the one described in brain supernatants with respect to its electrophoretic mobility, binding kinetic parameters and estimated molecular weight (65 000 daltons). However, in contrast to the brain cytosolic binding protein, two classes of affinity sites for triiodothyronine (T3) and thyroxine (T4) have been demonstrated: a high affinity site (KA = 1.2-3.7(3) X 10(9) M-1 for T3 and KA = 3.7-5 X 10(8) M-1 for T4) and a low affinity site (KA = 0.8-1.4 X 10(8) M-1 for T3 and 1.6-2.9 X 10(7) M-1 for T4). The results are discussed with respect to their cellular significance.