An extracellular retinol-binding glycoprotein in the rat eye—characterization,localization and biosynthesis

We have identified and partially purified interstitial retinol-binding protein (IRBP) from the subretinal space of the rat. It appeared to be glycosylated. Its apparent mol. wt was 270,000 by gel-filtration and 144,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Rat IRBP cross-reacted with anti-bovine IRBP sheep and rabbit sera, bound all-trans-[15-³H] retinol and was bound by concanavalin A. IRBP was not detected in the cytosols of the neural retina or retinal pigment epithelium and choroid. This distribution was confirmed by immunocytochemistry using a fluorescence-labeled second antibody. Immunospecific fluorescence was most intense in the interphotoreceptor matrix in a 6.5 μm band adjacent to the retinal pigment epithelium. It was less intense over the remainder of the rod outer segment layer and was comparatively faint over the inner segment region. Its occurrence in the interstitial space between the photoreceptors and retinal pigment epithelium coupled with the fact it bound all-trans-[15-³H] retinol supports the concept that it may be implicated in the transport of retinoids between the retina and the retinal pigment epithelium during the visual cycle. When incubated with [³H]leucine or [³H]glucosamine, isolated retinas (but not retinal pigment epithelium and choroid) secreted labeled IRBP into the medium. This suggests that the retina plays a role in regulating the amount of IRBP in the subretinal space.     

Purification and characterization of a retinol-binding glycoprotein synthesized and secreted by bovine neural retina

A retinol-binding glycoprotein ( IRBP ) was purified in milligram quantities from the extracellular matrix ( interphotoreceptor matrix) that occupies the subretinal space in bovine eyes. IRBP binds 2.2 molecules of all-trans retinol with a KD of approximately 10(-6) M. The holoprotein has lambda max at 280 nm (E1%1 cm = 10.99) and at 330 nm (E1%1 cm = 7.88). When freshly isolated from light-exposed eyes, IRBP contains up to 0.6 molecule of all-trans retinol, together with small amounts of the 11-cis and 13-cis isomers. IRBP also binds exogenous cholesterol, alpha-tocopherol, and all-trans retinoic acid, all of which are completely displaced by all trans retinol. The affinity of alpha-tocopherol for IRBP was at least several orders of magnitude less than that of all-trans retinol. IRBP contains 8.4% by weight of carbohydrate, which consists of sialic acid, neutral hexoses, and glucosamine in the molar ratio of approximately 1:3:2. No galactosamine was detected. Observations on the binding of 125I-labeled lectins to IRBP in sodium dodecyl sulfate-polyacrylamide gels before and after desialosylation suggest that at least one oligosaccharide chain is of the sialated biantennary complex type and contains fucose. The Mr of IRBP on calibrated size-exclusion columns averaged 249,000; on sodium dodecyl sulfate-polyacrylamide gels (with or without dithiothreitol) the apparent Mr was 144,000. IRBP exists in at least four isoelectric forms that bind concanavalin A and have pI values ranging from 4.4 to 4.8. Rabbit anti-bovine IRBP antiserum gave a single precipitin line against purified bovine IRBP , which showed a line of complete identity with crude bovine interphotoreceptor matrix and a line of partial identity with human interphotoreceptor matrix. The human material contains a prominent protein with lectin-binding properties similar to bovine IRBP but with a somewhat faster electrophoretic mobility. When isolated bovine neural retinas were incubated with 3H-labeled fucose, glucosamine, or leucine, a solitary labeled protein identified as IRBP was secreted into the medium. Labeled IRBP could not be detected in the medium when retinal pigment epithelium was incubated with these precursors under the same conditions. Neural retinas incubated with 3H-labeled leucine in the presence of tunicamycin secreted a form of IRBP that did not bind concanavalin A and had an Mr reduced by approximately 5,000.     

Molecular properties of bovine interphotoreceptor retinol-binding protein

Interphotoreceptor retinol-binding protein (IRBP) is a large retinol-carrying glycoprotein, located only in the interphotoreceptor (or subretinal) space of vertebrate eyes. It has recently been purified to apparent homogeneity. The present report presents its sedimentation, spectroscopic, and binding properties. The molecular weight of bovine IRBP, determined by sedimentation equilibrium, is 133,000. The sedimentation coefficient is 5.8S. The Stokes radius, 56 A, obtained from gel-filtration chromatography, is much larger than that expected for a globular protein of the same molecular weight. These results indicate that IRBP is asymmetric (it can modeled as a prolate ellipsoid of revolution with axial ratio of about 8:1) and explain the overestimates of molecular weight obtained in previous studies based on size-exclusion methods. The molar absorption coefficients for IRBP (at 280 nm) and for bound retinol are both unaffected by ligand dissociation. Fluorescence of the holoprotein displays neither fine structure nor energy transfer from tryptophan to bound retinol. Circular dichroism suggests a secondary structure containing approximately 15% alpha-helix and approximately 20% beta-structure, unchanged by the presence of ligand. The binding of retinol creates a positive, extrinsic Cotton effect at 330 nm, proportional to the amount of retinol bound. The apparent dissociation constant for all-trans-retinol is 1.3 X 10(-6) M. This relatively loose binding implies that, if required during the visual cycle, IRBP should be able to transfer its ligand to other binding proteins in the neural retina and retinal pigment epithelium.     

Purification and Characterization of B2 Bradykinin Receptor from Rat Uterus

Abstract: A B₂ bradykinin (BK) receptor was solubilised and partially purified from rat uterine membranes by a combination of ammonium sulphate precipitation, desalting on Sephadex G-50, and hydroxyapatite and wheat germ agglutinin affinity chromatography. The partially purified BK receptor, enriched 1,500-fold, was then cross-linked to ¹²⁵l-Tyr⁰-BK using disuccinimidyl suberate and purified to homogeneity as a single protein species on two-dimensional gel electrophoresis with a molecular mass of 81 kDa. This molecular size was in agreement with the value of 80–120 kDa estimated from Sephacryl 300 size exclusion column chromatography of the B₂ receptor. The partially purified and the crude solubilised B₂ BK receptor from rat uterus showed similar affinities for BK and the BK analogues iodo-Tyr⁰-BK, D-Phe⁷-BK, and des-Arg⁹-BK, indicating that the ligand binding specificity of the receptor had been retained during the purification procedures. The biochemical properties of the solubilised B₂ BK receptor correspond to those of a hydrophobic acidic glycoprotein (isoelectric focusing gave a value of 4.5–4.7) that binds specifically to wheat germ agglutinin but has no affinity for either concanavalin A or lentil lectin, suggesting the absence of terminal mannose or glucose residues.     

Plant lectins detect age and region specific differences in cell surface carbohydrates and cell reassociation behavior of embryonic mouse cerebellar cells

When plated at high cell density in a microwell culture system, freshly dissociated embryonic mouse cerebellar cells assemble into reproducible, 3-dimensional patterns. The addition of the dimeric lectin Succinyl Concanavalin A blocks reversibly the formation of the microwell pattern, suggesting that cell surface carbohydrates affect the reassociation behavior of embryonic mouse cerebellar cells. Agglutination studes of dissociated cell populations harvested from different regions of the embryonic brain reveal that different lectins agglutinate cell populations from different embryonic brain regions. Cells from E13 cerebellum are agglutinated with Concanavalin A, wheat germ agglutinin, Ricinus communis agglutinin, mol wt 60,000, Ricinus communis agglutinin, mol wt 120,000, and Lens culinaris, but not by soybean agglutinin or a fucose-binding protein. Cells from the midbrain are agglutinated only with Concanavalin A, Ricinus communis agglutinin, mol wt 60,000 and Ricinus communis agglutinin, mol wt 120,000; those from the cerebral cortex are agglutinated only with Lens culinaris; and those from the medulla are agglutinated only with Ricinus communis agglutinin, mol wt 60,000, and Ricinus communis agglutinin, mol wt 120,000. In addition, agglutination of cerebellar cells with Concanavalin A, wheat germ agglutinin, and Ricinus communis agglutinin is diminished over the course of development from embryonic day 13 to postnatal day 7. These studies suggest regional differences in the cell surfaces of the developling brain that are further modulated during the differentiation of the tissues. On a poly(D-lysine) treated substrate in microwell cultures, cell migration is unique to the cerebellum of the 4 brain regions studied. Surfaces treated with carbohydrate-derivatized poly(D-lysine) are currently being tested for their efficacy as substrates for differential cell migration.     

VITAMIN A RECEPTORS: RETINOL BINDING IN NEURAL RETINA AND PIGMENT EPITHELIUM

Abstract— With sucrose density gradient analysis, bovine retinal cytosol demonstrates 2S and 7S retinol-binding species; binding in pigment epithelial cytosol is predominantly to a 2S species. Binding of retinol to the 2S component in retina is unaffected by retinoic acid or retinyl palmitate whereas the ester effectively competes for 2S binding in pigment epithelium. Specific retinol binding can also be demonstrated by gel filtration on Sepharose 4B; no high molecular weight (> 100–200,000) retinol binding species are observed by this technique. Both the 2S and 7S binding species in retinal cytosol are protein in nature and differentially susceptible to proteolysis. The 2S and 7S species appear to be separate and distinct since chaotropic agents such as sodium thiocyanate or KCl and CaCl₂ do not seem to convert the 7S species into 2S subunits. Scatchard plot analysis indicates high affinity retinol binding to the 2S receptor. Computer analysis of the binding data yields K _a=3 × 10⁸, n = 1, a molecular size of 16,200 and ΔG⁰=−9.5 kcal/mol.     

Lectin affinity chromatography of cell surface proteins of novikoff tumor cells

Novikoff hepatocellular carcinoma cells were radioiodinated by a cell surface-specific method using lactoperoxid ase/¹²⁵I. The iodinated proteins were solubilized in 0.5% Nonidet P-40 and subjected to affinity chromatography on Sepharose-conjugated lectins (Ricinus communis agglutinins I or II, soybean agglutinin, concanavalin A, or wheat germ agglutinin) and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Almost all the iodinated proteins bound to one or more of the Sepharose-conjugated lectins, presumptive evidence that these peptides are glycosylated. Lectin affinity chromatography resolved defined subsets of iodinated glycoproteins and suggested that certain glycoproteins could be fractionated on the basis of heterogeneity of their heterosaccharide moieties. Incubation of the iodinated cells with neuraminidase resulted in increased binding of iodinated proteins to Sepharose-conjugated Ricinus communis agglutinins I and II and soybean agglutinin and decreased binding to Sepharose-conjugated wheat germ agglutinin. Binding of iodinated proteins to concanavalin A was unaffected by neuraminidase treatment of the cells. These studies demonstrate the utility of lectins for the multicomponent analysis of plasma membrane proteins.     

Synthesis and secretion of a novel binding protein for retinol by a cell line derived from Sertoli cells

An established cell line (TM-4) derived from murine Sertoli cells, the major supportive cell type of the testes, secretes a protein that binds retinol when grown in serum-free chemically defined medium. The protein that binds retinol is trypsin-sensitive and has an apparent Kd for retinol of 54 nM. Cholesterol, retinyl acetate, or UV-irradiated retinol at levels 100-fold in excess of retinol are poor competitors of [3H]retinol binding. Retinoic acid at a 100-fold molar excess inhibited [3H]retinol binding by 71%. In contrast, excess unlabeled retinol completely inhibits [3H]retinol binding. More than 80% of the total retinol-binding activity in confluent cultures is found in the culture medium. Prior to incubation with retinol, the protein that binds retinol has an apparent Mr of less than 150,000 by column chromatography; however, after incubation with retinol the protein that binds retinol exhibits an apparent Mr of 2 X 10(6) or greater and a sedimentation coefficient greater than 4 S. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that the major iodinatable component of the aggregated protein that binds retinol has an apparent Mr of 70,000. The secreted protein that binds retinol is not immunologically cross-reactive with either serum or cellular retinol-binding protein or transferrin. These findings suggest that Sertoli cells may secrete a protein that binds retinol. Such a protein could be involved in the transport of retinol either to the lumen of the seminiferous tubules or to the developing germ cells themselves.     

Lectin-Induced Inhibition of Nerve Growth Factor Binding by Receptors of Sympathetic Ganglia

Abstract: Nerve growth factor (NGF) initiates a pleiotypic response in numerous tissues derived from the neural crest by binding to specific plasma membrane receptors. In sympathetic ganglia this receptor has been characterized as a highly asymmetric, minimally hydrophobic, intrinsic membrane protein with a molecular weight of 135,000 (Costrini et al., 1979b). To further characterize this moiety we assessed the effects of lectins on ¹²⁵I-NGF specific binding to preparations of particulate and nonionic detergent-extracted micro-somal receptors of rabbit superior cervical ganglia (SCG). Concanavalin A (Con A) and wheat germ agglutinin (WGA), but not soybean agglutinin or Ulex europaeus I, induced a concentration-related, carbohydrate-specific decrease in ¹²⁵T-NGF binding. Following Con A exposure, ¹²⁵I-NGF specific binding to particulate SCG receptors was maximally reduced to 23% of control values. WGA similarly reduced NGF binding to particulate microsomal receptors to 37% of control values. Scatchard analysis of growth factor binding following Con A exposure indicated that this lectin effect was principally due to a sixfold reduction in maximum receptor affinity. Lectin-associated impairment of NGF binding was also demonstrated by using a Triton X-100 solubilized receptor preparation. These results provide evidence that the high-affinity-state NGF receptor of SCG is a glycoprotein containing N -acetylglucosamine and α-D-mannopyranoside residues. These residues are probably located in close proximity to the growth factor binding region of the NGF receptor.     

Identification of Strychnine Binding Sites in the Rat Retina

Abstract: [³H]Strychnine specifically binds to membrane fractions isolated from rat retinae. The binding is saturable, with an apparent dissociation constant, K _D, of 14.3 × 10⁻⁹ M and 205 fmol bound/mg protein. Specific binding is time-dependent and proportional to protein concentration. Glycine and taurine are equally potent inhibitors of [³H]strychnine binding ( K _i= 4 × 10⁻⁵ M); no other amino acids endogenously present in the retina inhibited [³H]strychnine binding.     

Lectins as differentiation markers of human gliomas.

The lectins Concanavalin A (Con A), Ricinus communis agglutinin (RCA-I), Peanut agglutinin (PNA) and Wheat germ agglutinin (WGA) as well as the immunomarkers for glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) were used in a series of 21 glial tumors (4 pylocytic astrocytomas, 5 grade II astrocytomas, 3 anaplastic astrocytomas, 4 glioblastomas and 5 oligodendrogliomas). ConA binds to all tumoral astrocytes in low grade astrocytomas, as well as to well differentiated tumoral astrocytes in anaplastic astrocytomas and glioblastomas. RCA-I has a similar behaviour. PNA, and to a lesser degree WGA, binds selectively to the oligodendroglial plasma membrane in well differentiated oligodendrogliomas. The results suggest that these lectins are markers of differentiation in gliomas rather than of malignancy.     

The presence of a soluble interphotoreceptor retinol-binding protein (IRBP) in the retinal interphotoreceptor space

A new, gentle technique has been developed for washing of the retinal interphotoreceptor space (IPS) to obtain soluble components of the extracellular matrix (ECM). Using this method, we have determined that the major soluble coustituent of monkey IPS is a 146,000 M_r glycoprotein, which binds [³H]retinol, sediments on sucrose gradients at 7S and has an R_f of 0.42 on native gel electrophoresis. Using size-exclusion high performance liquid chromatography, the apparent molecular weight of the native protein was calculated to be 250,000 daltons. In contrast to previous studies, no 15,000-dalton cellular retinol-binding protein (CRBP) or 33,000-dalton cellular retinaldehydebinding protein (CRALBP) was observed in the IPS wash, indicating that these proteins are probably not involved in retinol transport between retina and pigment epithelium (PE). In the supernatant fraction of retinal homogenates that contains soluble intracellular proteins as well as extracellular constituents, the 146,000 M_r protein was closely associated with a 93,000 M_r protein that could be separated on SDS-gel electrophoresis; the 93,000 M_r protein was not found in the IPS wash. The 146,000 M_r interphotoreceptor retinol-binding protein (IRBP) may function in extracellular retinol transport in the IPS.     

Retinoid-binding proteins and retinol esterification in cultured retinal pigment epithelium cells

The esterification of all-trans retinol and the occurrence of cytosolic retinoid-binding proteins was investigated in cultured bovine retinal pigment epithelium (RPE) cells. ³H-labeled all-trans retinyl ester (mainly palmitate) was formed at an initial rate of 0.1 nmol·mg protein⁻¹·min⁻¹ when ³H-labeled all-trans retinol was incubated with the 100,000 g pellet obtained from a homogenate of freshly-harvested cells. No esterification could be detected under the same conditions after 14 days in culture in defined medium (DM) or in medium containing 20% fetal bovine serum (CM). No enhancement or restoration of esterifying capacity was observed when the assay mixture was supplemented with palmitoyl CoA. As determined by specific, saturable binding of ³H-labeled all-trans retinol and ³H-labeled 11-cis retinal to proteins with mol. wts 16,000 and 33,000 dalton on calibrated Bio-Sil TSK 250 size-exclusion columns, the cytosol of freshly-harvested RPE cells contained cellular retinol-binding protein (CRBP) and cellular retinal-binding protein (CRAlBP). By comparison with the quantity of ³H-labeled all-trans retinol bound under identical conditions to pure dog liver CRBP, it was estimated that fresh RPE cells contained 102 ± 3 ng CRBP·μg cytosol protein⁻¹. In cultured and subcultured cells, CRBP was present at much lower levels (down to one-tenth of the initial amounts) and CRAlBP could not be detected. Since binding of ³H-labeled all-trans retinoic acid to a protein with molecular weight of 17,000 dalton was not observed in the cytosols of fresh or cultured cells, it was concluded that cellular retinoic acid binding protein (CRABP) was either present at very low levels or absent altogether. An unidentified peak of specific ³H-labeled all-trans-retinoic acid binding at mol. wt 61,000 dalton was prominent in subcultured cells. These results show that in RPE cells in culture the expression of differentiated phenotype with respect to retinoid utilization undergoes significant modification. It is postulated that changes in the composition of the extracellular matrix (e.g. absence of interstitial retinol-binding protein, IRBP) may be involved.     

Development of Synaptic Glycoproteins: Effect of Postnatal Age on the Synthesis and Concentration of Synaptic Membrane and Synaptic Junctional Fucosyl and Sialyl Glycoproteins

Abstract: Synaptic plasma membranes (SPM) and synaptic junctions (SJ) were isolated from the cortices of rats varying in age between 5 and 28 days. Gel electrophoresis of SPM and SJ indicated a marked increase in the concentration of the "PSD protein" (M. W. 52,000) with development. The biosynthesis of glycoproteins was measured following the intracranial injection of [³H]fucose or [³H] N '-acetylmannosamine. The incorporation of [³H]fucose into synaptic fractions decreased two- to threefold between 10 and 28 days whereas little change in the incorporation of [³H] N '-acetylmannosamine occurred over the same period. Gel electrophoretic analyses of labeled synaptic membranes indicated major increases in the relative incorporation of radiolabeled precursors into glycoproteins with apparent molecular weights of 74,000, 65,000, 50,000, and 40,000 with increasing age. Identification of fucosyl and sialyl glycoproteins following reaction with ¹²⁵I-fucose-binding protein or labeling of sialic acid with NaIO₄NaB[³H₄] demonstrated similar increases in the concentrations of these glycoproteins. Synaptic junctions contained three major glycoproteins with apparent molecular weights of 180,000, 130,000 and 110,000. The reaction of these glycoproteins with ¹²⁵I-fucose-binding protein increased one- to twofold between 10 and 28 days but little variation in their relative distribution or synthesis occurred over this period. The reaction of synaptic junctional glycoproteins GP 180 and GP 110 with ¹²⁵I-wheat germ ag-glutinin increased between 10 and 28 days. The results indicate that the molecular composition of the synapse continues to evolve after the initial synaptic contact has been formed.     

Synthesis and proof-of-function of a [14C]-labelled form of the plant iron chelator nicotianamine using recombinant nicotianamine synthase from barley

The amino acid nicotianamine (NA) is essential for micronutrient metabolism in plants. Lack of NA results in a chlorotic phenotype and oxidative stress, since NA is a chelator of iron and other metal nutrients. To investigate the precise cellular function of NA in micronutrient transport and homeostasis, a protocol for the production of [¹⁴C]-labelled NA was developed. Recombinant NA synthase was used to generate [¹⁴C]-NA from [¹⁴C]- S -adenosylmethionine. After purification by solid-phase ion exchange about 66% yield was achieved. The identity of the [¹⁴C]-NA with chemically synthesized NA was demonstrated by several independent methods, including two TLC systems, two HPLC systems and immuno-detection. Moreover, biological function was shown by complementation of the Lycopersicon esculentum mutant chloronerva that is free of NA due to a defect in NA synthase. Proof-of-function for the produced [¹⁴C]-NA as a suitable tool for transport studies was provided monitoring the distribution of [¹⁴C]-NA after feeding to tomato and Ricinus communis seedlings.     

An extracellular retinol-binding glycoprotein in the eyes of mutant rats with retinal dystrophy: development, localization, and biosynthesis

Interstitial retinol-binding protein (IRBP) is a soluble glycoprotein in the interphotoreceptor matrix of bovine, human, monkey, and rat eyes. It may transport retinol between the retinal pigment epithelium and the neural retina. In light-reared Royal College of Surgeons (RCS) and RCS retinal dystrophy gene (rdy)+ rats, the amount of IRBP in the interphotoreceptor matrix increased in corresponding proportion to the amount of total rhodopsin through postnatal day 22 (P22). In the RCS-rdy+ rats, the amount increased slightly after P23. However, in the RCS rats there was a rapid fall in the quantity of IRBP as the photoreceptors degenerated between P23 and P29. No IRBP was detected by immunocytochemistry in rats at P28. The amount of rhodopsin fell more slowly. Although retinas from young RCS and RCS-rdy+ rats were able to synthesize and secrete IRBP, this ability was lost in retinas from older RCS rats (P51, P88) but not their congenic controls. The photoreceptor cells have degenerated at these ages in the RCS animals, and may therefore be the retinal cells responsible for IRBP synthesis. The putative function of IRBP in the extracellular transport of retinoids during the visual cycle is consistent with a defect in retinol transport in the RCS rat reported by others.     

Lectin-Reactive Components in White Matter Membranes from Normal and Multiple Sclerosis Brains

Abstract: Polypeptides derived from human white matter membranes reacted with the radioiodinated lectins concanavalin A, Lens culinaris phytohemagglutinin, Ricinus communis agglutinin and wheat germ agglutinin after electrophoresis in polyacrylamide pore gradient gels. The molecular weights of these lectin-reactive bands were estimated by comparison with radioiodinated protein standards by using the linear relationship between log of the molecular weight and log of the gel concentration reached by the protein after electrophoresis in a polyacrylamide gradient gel. The molecular weight estimates for components reactive with concanavalin A were 176,800, 141,200, 72,800, 52,800, 44,700, 40,000, 24,800 and 23,900. The molecular weights of the bands reactive with both wheat germ agglutinin and Lens culinaris phytohemagglutinin were 138,000, 113,500, 92,100, 52,800, 44,700, 24,800 and 23,900. Wheat germ agglutinin was bound also to a band with a molecular weight of 72,800. Ricinus communis agglutinin bound to bands with estimated molecular weights of 138,000, 72,800, 52,800, 44,700, 24,800 and 23,900. The electrophoretic pattern of lectin-reactive polypeptides derived from normal-appearing white matter of multiple sclerosis brains was not qualitatively different from the lectin-binding pattern of control brain membrane polypeptides.     

The binding of lectins to components of plasma membranes from porcine submaxillary lymph node lymphocytes

By sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis the plasma membranes from porcine lymphocytes contain at least 30--35 glycopolypeptides and one or more glycolipids to which one or more of 12 purified lectins bind. The specificities of binding generally followed the same pattern as those of the reaction of the lectin with intact pig lymphocytes. Some lectins (e.g., the isolectin pair, Agaricus bisporus lectins A and B and a group consisting of the Lens culinaris A and B isolectins and the closely related Pisum sativum lectins) bind to almost identical populations of plasma membrane components and compete with each other for all their binding sites. Others (e.g., Concanavalin A and the Lens culinaris-Pisum sativum group and a group consisting of phytohemagglutinin-L, Ricinus communis lectin-60 and Ricinus communis lectin-120 bind in a cross reactive manner to some common binding moieties but, in addition, to certain nonshared ones. Still others (e.g., soybean agglutinin, peanut agglutinin and wheat germ agglutinin) do not share any common binding moieties with the other lectins. The amount of lectin binding and the number of membrane components to which a lectin binds is directly related to the Ka of binding of the lectin to the intact lymphocyte. Those with high Ka (Cocanavalin A Lens culinaris lectins, Pisum sativum lectins, phytohemagglutinin-L), bind to 20-30 different components giving very complex binding patterns while those with lower Ka (Agaricus bisporus lectins, wheat germ agglutinin, peanut agglutinin, and soybean agglutinin) bind to 8--13 components with easily distinguishable patterns. Soybean agglutinin binds almost exclusively to a glycolipid fraction while for the others one or more glycopolypeptides served as the major lectin-binding molecule. The Ricinus lectins, two lymphocyte toxins, bind to essentially every plasma membrane component to which the mitogen phytohemagglutinin-L binds, in fact competing for most of those plasma membrane moieties which bind phytohemagglutinin-L.     

Synergistic effects of lectins in the interaction of thrombin with human platelets

Several lectins have been studied for their effects on the interaction of thrombin with human platelets. Wheat germ agglutinin, concanavalin A and Ricinus communis lectin increased the number of high affinity sites for diisopropylphosphothrombin on washed platelets from 3000 to about 12 000 but the binding affinities were unchanged (Kd approx 4 nM). Two other lectins, Lens culinaris and Bandieria simplicifolia, were without effect. (2) Using formalinized platelets to avoid possible complications of the platelet release reaction, wheat germ agglutinin showed a marked increase (5-fold) in the binding of active thrombin, peanut agglutinin had no effect while Ricinus communis and :Bandieria simplicifolia showed marginal increases (2-fold). Thrombin binding was decreased to about one quarter with Lens culinaris, Phaseolus vulgaris and concanavalin A. (3) Wheat germ agglutinin caused a synergistic increase of platelet aggregation at low concentrations of thrombin (12.5 mU/ml) and ADP (1 microM), both in the absence and presence of added fibrinogen, but had no effect on ristocetin-induced aggregation.