These studies assessed the acute effects of a single dose of prostaglandin F2 alpha-1-isopropylester (PGF2 alpha-IE) on the IOP and AHF, as well as on conventional and uveoscleral outflow, in cynomolgus monkeys. The AHF was determined by a dilution method, using radioactive albumin as a marker. Arterial blood samples were collected and analyzed for radioactivity to determine the flow of aqueous humor to blood, corresponding to the conventional outflow. The uveoscleral outflow was calculated as the difference between the measured AHF and the conventional outflow. Topical application of PGF2 alpha-IE (1 microgram free acid equivalents) to eyes of cynomolgus monkeys caused a small initial increase in IOP followed by a gradual decrease, which was greatest (2.9 +/- 0.6 mmHg below the control eye) at about three hours after the PG application. The mean AHF during 4 hours was slightly higher in the experimental eyes than in the control eyes. The mean uveoscleral outflow during 4 hours was significantly higher in the experimental eyes (0.98 +/- 0.12 microliters.min-1) than in the control eyes (0.61 +/- 0.10 microliters.min-1), while the conventional outflow was significantly lower. The ability of PGF2 alpha-IE to increase the uveoscleral outflow was also demonstrated by autoradiography. These results suggest that PGs exert at least part of their ocular hypotensive effect by increasing the uveoscleral outflow. It remains to be established whether this effect is caused by relaxation of the ciliary muscle or whether other mechanisms, such as structural and metabolic changes, are involved.

To compare the receptor patterns for mitogenic and non-mitogenic substances, surface glycoproteins of human lymphocytes were labelled with the lactoperioxidase-catalysed iodination technique and with a galactose oxidase-tritiated sodium borohydride technique. Labelled cells were detergent-solubilized, and the lysates were allowed to react with insolubilized purified mitogenic lectins, phytohaemagglutinin, leucoagglutinin and an insolubilized non-mitogenic lectin, oxidized leucoagglutinin. Lectin-reactive proteins were eluted with sodium dodecyl sulphate (SDS) buffer. Cell membrane components reactive with anti-lymphocyte globulin (ALG) were retrieved by indirect immunoprecipitation with protein-A-bearing staphylococcus Cowan I strain (SaCI). Lectin- and ALG-reactive proteins were analysed by SDS polyacrylamide gel electrophoresis. Iodinated glycoproteins regularly showed four major components with molecular weights of 120,000, 70,000, 60,000 and 43,000 daltons, respectively, on 7% gels. An additional broad peak in the molecular weight range 20,000--35,000 daltons was found on 10% gels. Tritiated glycoproteins also showed four major components with MW 120,000, 70,000, 60,000 AND 42,000, RESPECTIVely, which reacted with lectin and ALG. In addition, ALG reacted with some glycoproteins with MW between 150,000 and 230,000 daltons. On 10% gels additional lectin- and ALG-binding glycoproteins with MW around 30,000 daltons were found. The similarity in structures bound by mitogenic and non-mitogenic substances indicates that lymphocyte activation may depend on some property conferred by the mitogen.

The concanavalin-A-binding cell surface glycoproteins from normal and certain leukaemic human lymphocytes were radiolabelled and then solubilized with detergent, isolated by affinity chromatography on Con A insolubilized on agarose beads, and subsequently analysed by SDS-polyacrylamide gel electrophoresis. Leukaemic T cells from patients with Sezary syndrome were found to express major concanavalin-A-binding glycoproteins on their outer surface similar to those of normal T lymphocytes. Leukaemic B cells from patients with chronic lymphocytic leukaemia expressed Con-A-binding proteins similar to those of B-cell lines. HLA antigens were predominant among the major Con-A-binding proteins on the surface of the normal and the malignant T cells studied. Human Ia-like antigens, HLA antigens, and the cell surface immunoglobulins IgD and IgM represented the major Con-A-binding proteins on the B cells studied. beta 2-microglobulin was found associated with HLA antigens on both leukaemic and non-leukaemic T and B cells. The presence of additional Con-A-binding proteins expressed on the surface of the different cell types studied is discussed along with some physical characteristics of the human Ia-like antigens isolated.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTIsolation and characterization of lectin binding proteins from murine lymphoid cellsSten F. Nilsson and Myron J. WaxdalCite this: Biochemistry 1978, 17, 5, 903–910Publication Date (Print):March 7, 1978Publication History Published online1 May 2002Published inissue 7 March 1978https://doi.org/10.1021/bi00598a025RIGHTS & PERMISSIONSArticle Views23Altmetric-Citations21LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (930 KB) Get e-Alerts Get e-Alerts

Cell surface molecules of rat normal lymphoid cells were selectively labelled by lactoperoxidase catalysed iodination or by a galactose oxidase tritiated sodium borohydride technique, subsequently detergent solubilized, isolated by indirect immunoprecipitation and analysed on SDS‐polyacrylamide gel electrophoresis. Four polypeptide chains were isolated by using the alloantiserum DA anti‐Lewis. The molecular weights of the antigens were calculated as 41,000, 33,000, 27,000 and 12,000. Based on functional in vitro characteristics of the antiserum used and on the physiochemical properties as well as genetics of inheritance and tissue distribution, the polypeptide chains were identified as being subunits of Ag‐B and Ia antigens. Two types of immunoglobulin heavy chains exhibiting the molecular weights 70,000 and 64,000 were isolated from unfractionated normal spleen cells by use of a polyvalent rabbit anti‐rat immunoglobulin serum and tentatively identified as μ and δ chain. Using the same anti‐immunoglobulin serum, no molecules could be precipitated from the lysates of Lewis thymocytes or peripheral T cells.

The major glycoproteins which bind concanavalin A have been isolated and identified from murine spleen cells, thymocytes,and purified thymus-derived (T) lymphocytes, and from the spleen cells of congenitally athymic (nude) mice. The cells were radiolabeled by lactoperoxidase catalyzed 125I iodination or by culturing the cells in media containing [3H]leucine or [3H]fucose. The cell membrane was solubilized with Nonidet P-40 and the concanavalin A binding proteins were isolated by affinity chromatography and analyzed according to their mobility on polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The major proteins from various lymphocyte preparations were identified by immunoprecipitation with specific antisera. The molecules coded by the histocompatibility-2 complex acted as concanavalin A binding proteins H-2K and H-2D were isolated from T lymphocytes, thymocytes, and bone marrow derived (B) lymphocytes. The Ia antigens were identified from B lymphocytes and tentatively identified from T lymphocytes. In addition to these H-2 complex proteins, immunoglobulin M and D on B lymphocytes also bound concanavalin A binding. All these glycoproteins have previously been identified as cell surface molecules. The presence of certain minor unidentified concanavalin A binding proteins on lymphoid cells is indicated.

Thyroxine-binding globulin was isolated from human plasma by ammonium sulfate fractionation, chromatographic separations on diethylaminoethyl-Sephadex, gel chromatography, and two different electrophoretic procedures. The highly purified was homogeneous when subjected to polyacrylamide gel electrophoresis, ultracentrifugation analyses, and immunochemical determinations. The weight average molecular weight as determined by sedimentation equilibrium ultracentrifugations was 54,000 and by sedimentation diffusion data 55,000. Amino acid analyses indicated a minimum of 110 amino acid residues per molecule. By determination of the minimum in the curve for the fraction of maximum deviation from the amino acid analyses it was found that the minimum molecular weight for the polypeptide was 12,200. Carbohydrate analyses demonstrated the presence f equimolar amounts of amnnose, galactose, and glucosamine, and the carbohydrate portion constituted 7.5% of the total weight. The amino acid analyses suggested that thyroxine-binding globulin is composed of 4 subunits. Molecular weight determinations by gel chromatography in 6 M guanidine hydrochloride indicated the presence of three species of globulin with apparent molecular weights 52,000, 25,000, and 13,500, respectively. Prolonged storage in guanidine hydrochloride promoted a more than 60% yield of the monomeric species. Moreover, a half-molecule of thyroxine-binding globulin was isolated and shown to consist of two polypeptide chains of similar molecular weight...

Vitamin A in human plasma is transported by its specific carrier protein, the retinol-binding protein. Under physiological conditions the protein forms a stable protein-protein complex with the tetrameric plasma protein, the thyroxine-binding prealbumin. Human prealbumin was shown to interact with the fluorescent probes 1,8-anilinonaphthalene sulfonate (ANS) and 2-p-toluidinylnaphthalene-6-sulfonate (TNS). ANS bound to the protein at two independent sites with the apparent association constant 3 X 10(5) M-1, whereas TNS interacted with a single site with the binding constant 5 X 10(k) M-1. The fluorescent yield of protein-bound ANS was 0.95, a more than 200-fold enhancement compared with that of ANS in aqueous solutions. TNS enhanced its quantum yield nearly 500-fold to 0.37. On addition of thyroid hormones the fluorescent probes could be quantitatively displaced from the protein. This finding suggested that triiodthyronine, thyroxine, and the probes bound to a common site in prealbumin, which is likely to have a strongly hydrophobic character. The association constants for the interaction between prealbumin and the thyroid hormones could be calculated by using the hormones as competitive inhibitors in the TNS-prealbumin titrations. The data from the competition experiments together with those obtained from equilibrium dialysis revealed one major hormone binding site on the protein. The calculated association constant were 9 X 10(6) M-1 and 1 X 10(8) M-1 for triiodothyronine and thyroxine, respectively. Prealbumin monomers were bound to Sepharose by covalent attachment, and their properties were examined. Evidence was obtained demonstrating that the retinol-binding protein could interact with a single subunit of prealbumin. The estimated apparent association constant for the interaction of the protein and the matrix-bound monomeric prealbumin was 3 X 10(4) M-1, approximately 250-fold lower than that measured for protein and matrix-bound tetrameric prealbumin. The data, however, strongly suggest that there are four retinol-binding protein sites per prealbumin molecule. Using the technique of sedimentation equilibrium ultracentrifugation the prealbumin subunit self-association has been studied. The energy of the interaction for the prealbumin subunits is very high, and various concentrations of guanidine hydrochloride has to be used to perturb the equilibrium. All experiments indicated that prealbumin dissociates directly into monomers without the presence of intermediate forms. Thyroxine perturbed the chemical equilibrium of the prealbumin monomer-tetramer system by strengthening the interaction between the subunits.

Prealbumin was isolated from human plasma by ammonium sulfate fractionation, chromatographic separations on diethylaminoethyl-Sephadex, and gel filtrations. The highly purified protein was subjected to quantitative amino acid analyses, which showed a minimum of 140 amino acid residues per prealbumin molecule. By determination of the minimum in the curves for the fraction of maximum deviation it was found that the minimum molecular weight for prealbumin was 15,500. Molecular weight determinations by sedimentation equilibrium ultracentrifugations gave a value of 62,500 ± 2,200. These results indicate that prealbumin is composed of four subunits. Molecular weight determinations by gel chromatography in 6 m guanidine hydrochloride gave values of about 16,000 for prealbumin. By this method it was shown that the subunits are held together by noncovalent bonds. A trypsin digest of prealbumin was examined by peptide-mapping techniques and the number of peptides (15 to 19) detected was in good agreement with the total number of lysine and arginine residues calculated from the amino acid composition of a prealbumin tetramer consisting of four identical chains. The NH2-terminal sequence of the protein was shown to be uniquely Gly-Pro. Prealbumin, reduced and 14C-carboxymethylated, was subjected to tryptic digestion, and the radioactivity was used to trace and isolate the cysteine-containing peptides. Only one peptide contained radioactivity, and analysis of this peptide revealed the unique sequence Gly-Pro-Ser-Met-Val-Cys(Cm)-Lys. These data strengthen the view that preal-bumin is composed of identical subunits. The mode of dissociation of the prealbumin tetramer was investigated by sedimentation equilibrium ultracentrifugation in various concentrations of guanidine hydrochloride. Determinations of local weight and number average molecular weights were consistent with three species being involved in chemical equilibrium, i.e. monomers, dimers, and tetramers of the prealbumin subunits.

The human retinol binding protein, which in plasma is bound to thyroxine binding prealbumin, has been isolated in free form from urine of patients with tubular proteinuria and from normal serum. The isolation procedure for urinary retionl binding protein involved gel chromatography and affinity chromatography on a column of prealbumin coupled to Sepharose, whereas the isolation of serum retinol binding protein required three fractionation steps; DEAE‐Sephadex chromatography, and affinity chromatography.The retinol binding protein from urine and serum was obtained in a yield of 31% and 42% respectively. The relatively low yield of the urinary protein was explained by the fact that two forms of retinol binding protein were present in the urine, one of which did not bind to prealbumin. The purity of the retinol binding protein isolated from urine and serum was established by the following criteria: immunoelectrophoresis, polyacrylamide gel electrophoresis, amino acid analyses, and sedimentation equilibrium ultracentrifugations.The use of affinity chromatography offers a simple and rapid procedure for the isolation of moderate quantities of highly purified retinol binding protein from urine and serum.

The binding of thyroxine, triiodothyronine, and diiodothyronine to prealbumin, and to the prealbumin-retinol-binding protein (RBP) complex has been investigated by measurements of the quenched tryptophan fluorescence and by equilibrium rate dialysis. Fluorescence quenching revealed a single site of high affinity for prealbumin and the prealbumin-RBP complex. The binding constants under physiological conditions for the combination of prealbumin with a single molecule of thyroxine, triiodothyronine, and diiodothyronine were 1.1 x 107 m-1, 1.3 x 106 m-1, and 6.2 x 104 m-1, respectively. At pH 7.4, the binding of thyroxine to prealbumin was less sensitive to temperature changes within the range 2–40° than the binding between triiodothyronine and prealbumin. The calculated thermodynamic constants were compatible with the difference in ionization of thyroxine and triiodothyronine at pH 7.4. The phenolic hydroxyl group of thyroxine, bound to prealbumin, is highly ionized at this pH, whereas the phenolic hydroxyl group of triiodothyronine is essentially un-ionized. Variations of pH exerted a marked influence on the association constants for the thyroid hormones and prealbumin. Thus an increase in the binding strength under conditions promoting ionization of the phenolic hydroxyls indicated that this group participates in the binding with prealbumin. Reduced and carboxymethylated prealbumin did not show any binding to thyroxine but retained completely its affinity for the retinol-binding protein. The binding of thyroxine and triiodothyronine to prealbumin and to the prealbumin-RBP complex were also investigated over a wide range of ligand to protein ratios by means of equilibrium rate dialysis. Identical results were obtained for both hormones irrespective of whether prealbumin was free or in complex with RBP. A single site of high affinity, and three sites of weaker affinity were revealed. The differences in binding characteristics between the sites of high and low affinities would be most easily explained by assuming a negative homotropic effect of the thyroid hormone binding.