Facile preparation of the alpha-Gal-recognizing Griffonia simplicifolia I-B4 isolectin

The B4 isolectin from Griffonia simplicifolia is of great utility as a reagent for the identification of alpha-D-galactopyranosyl end groups. Its separation from isolectins containing A subunits has been greatly improved by a simple, rapid procedure using a column of N-acetylgalactosamine coupled to vinyl sulfone-activated Sepharose 4B to selectively retain the A subunit-containing isolectins. The procedure has the advantages over previous affinity procedures of speed (the isolation of B4 isolectin can be achieved in one day), simplicity, and high degree of resolution of the B4 isolectin.     

Immunochemical studies of the combining sites of the two isolectins, A4 and B4, isolated from Bandeiraea simplicifolia

The tissue distribution and the effects of starvation and streptozotocin-induced diabetes on insulin B chain-degrading neutral peptidase activity in the rat have been studied. The neutral peptidase activity in tissue extracts was determined by measuring the formation of trichloroacetic acid-soluble radioactivity from ¹²⁵I-labeled B chain of insulin in 0.1 m Tris buffer (pH 7.2). Inhibition by several different compounds (EDTA, dithiothreitol, and potassium phosphate) which are known to inhibit the purified enzyme and the effects of pH suggest that the B chain-degrading activity measured in each of 12 tissue extracts may be similar to the neutral peptidase recently purified from rat kidney (P. T. Varandani and L. A. Shroyer, 1977, Arch. Biochem. Biophys., 181, 82–93). Neutral peptidase activity was observed in all tissues examined and varied in the order kidney ? intestine > pancreas, testis > liver > thymus > heart, skeletal muscle, diaphragm > lung, spleen > fat. Neutral peptidase activity in kidney, liver, fat, and skeletal muscle from diabetic animals was significantly depressed when compared with the levels in these tissues from normal animals. Insulin treatment of diabetic animals raised the neutral peptidase activity in kidney, liver, and fat to levels equivalent to or even exceeding normal levels; however, activity in skeletal muscle persisted at depressed levels. Heart muscle neutral peptidase activity was not significantly affected in either diabetes or starvation. In the liver, starvation reduced the level of neutral peptidase activity while subsequent refeeding raised the activity to a level exceeding the control. Opposite effects were observed in kidney: starvation increased neutral peptidase activity while refeeding brought the activity back to normal levels. Only small decreases in neutral peptidase activity were observed in fat and skeletal muscle after 24 h starvation, but were not evident after 64 h starvation. The changes in neutral peptidase activity correlated well with the changes in glutathione-insulin transhydrogenase activity previously reported in liver and kidney.     

Binding of Griffonia simplicifolia 1 isolectin B4 (GS1 B4) to alpha-galactose antigens

Glycoconjugates with terminal Galalpha1-3Galbeta1-4GlcNAc sequences (alpha-galactosyl epitopes, natural xenoreactive antigens) are present on various tissues in pigs and are recognized by human anti-alphagalactosyl (alphaGal) antibodies1. Hence xenotransplantation (pig-to-human) would trigger immune reactions involving complement activation and lead to the hyperactute rejection of the graft. Xenoreactive antigens are often studied by using the lectin Griffonia simplicifolia 1 isolectin B4 (GS1 B4), which shows high affinity to galactose. We here estimate the specificity of GS1 B4 for detecting various galactosyl epitopes by measuring lectin binding to neoglycoproteins, thyroglobulin and pig skeletal muscle. Enzyme linked lectin assays confirmed that GS1 B4 was highly specific to alpha-galactosylated neoglycoproteins while the lectin did not detect a beta-galactosylated ligand. The length of the sugar chains influenced the lectin-carbohydrate interaction. A monosaccharide linked to serum albumin showed higher lectin affinity than did neoglycoproteins with di- and tri-alpha-galactosyl epitopes. When the carbohydrate was extended, as in the xenoreactive pentasaccharide (Galalpha1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc), the carbohydrate- lectin interaction was meagre. Not only the terminal, but also the subterminal sugar affected the lectin binding because the GS1 B4 affinity to Galalpha1-3Gal was much stronger than to Galalpha1-3GalNAc. In bovine and porcine thyroglobulin most alphaGal epitopes appear to be cryptic, but are unmasked by a heat denaturation. In pig skeletal muscle there was lectin reaction not only in the muscle capillaries, but also in the connective tissue and intracellularly in muscle fibres. In Western blots of isolated proteins from pig muscle at least three bands were strongly stained after incubation with lectin.     

Heterogeneous histochemical reaction pattern of the lectin Bandeiraea (Griffonia) simplicifolia with blood vessels of human full-term placenta

Bandeiraea simplicifolia lectin (BS-I) stains vascular endothelium in various species. In humans, less than 10% of the specimens studied exhibit a reaction with BS-I. In the present histochemical study, the reactivity of BS-I with placental blood vessels and its correlation with the blood group from mother and newborn child was investigated. Acetone-fixed cryosections of representative tissue segments of human full-term placenta and umbilical cord were stained with BS-I. The staining pattern of tissues from patients with different blood groups was identical, although the reaction of BS-I in the placenta was heterogeneous. BS-I did not react with the umbilical cord. Vascular smooth muscle cells at the insertion site of the umbilical cord into the chorionic plate, and endothelium deeper in the chorionic plate, became progressively stained. The endothelial cells and tunica muscularis of smaller arteries and veins in stem villi lost their reactivity in parallel with decreasing vessel size. Arterioles and venules reacted heterogeneously. Capillaries, trophoblastic basement membranes, especially epithelial plates, and sometimes the syncytiotrophoblast were labelled in several terminal villi. The data indicate that 1) the placenta binds BS-I to fetal endothelium independent of the blood group, 2) cell-surface antigens on placental endothelial cells are expressed heterogeneously and 3) cell-surface glycans are constituted in an organ-specific manner on human endothelial cells.     

Cell type-specific lectin staining of the tracheobronchial epithelium of the rat: quantitative studies with Griffonia simplicifolia I isolectin B4.

We compared lectin staining patterns to cell population densities, as determined by morphological criteria in rat airways. Eight lectins were studied: Griffonia simplicifolia I isolectin B4 (GSI-B4), Arachis hypogaea (PNA), Wisteria floribunda (WFA), Glycine maximus (SBA), Dolichos biflorus (DBA), Helix pomatia (HPA), Ulex europaeus (UEA-1), and Maclura pomifera (MPA). Two of the lectins strongly stained morphologically distinct cell subpopulations. GSI-B4 stained basal cells, and MPA stained non-ciliated bronchiolar (Clara) cells. The specificity and sensitivity of GSI-B4 as a marker for basal cells was examined. In the trachea, 35% of all cells were GSI-B4 positive; 84% of these were basal cells, 7% were unidentified cells, 5% were serous/mucous cells, and 4% were ciliated, brush, or inflammatory cells. Comparison to cell population density data strongly suggested that all basal cells were GSI-B4 positive. The segmental bronchus was a transitional area; GSI-B4 positive basal cells were present in the region closest to the lobar bronchus but were absent in the distal region; instead, MPA-positive Clara cells appeared. When dissociated tracheal cells were obtained by pronase digestion, 43% were GSI-B4 positive. These results show that GSI-B4 is a sensitive and relatively specific marker for basal cells in the rat trachea which can be used to study dissociated epithelial cells.     

Isolectins I-A and I-B of Griffonia (Bandeiraea) simplicifolia. Crystal structure of metal-free GS I-B(4) and molecular basis for metal binding and monosaccharide specificity.

Seeds from the African legume shrub Griffonia simplicifolia contain several lectins. Among them the tetrameric lectin GS I-B(4) has strict specificity for terminal alpha Gal residues, whereas the closely related lectin GS I-A(4) can also bind to alpha GalNAc. These two lectins are commonly used as markers in histology or for research in xenotransplantation. To elucidate the basis for the fine difference in specificity, the amino acid sequences of both lectins have been determined and show 89% identity. The crystal structure of GS I-B(4), determined at 2.5-A resolution, reveals a new quaternary structure that has never been observed in other legume lectins. An unexpected loss of both Ca(2+) and Mn(2+) ions, which are necessary for carbohydrate binding in legume lectins, may be related to a particular amino acid sequence Pro-Glu-Pro in the metal binding loop. Comparison with demetallized concanavalin A reveals a different process for the loss of metal ions and for the subsequent loss of carbohydrate binding activity. The GS I-A x alpha GalNAc and GS I-B x alpha Gal complexes were constructed using homology modeling and docking approaches. The unusual presence of an aromatic amino acid at position 47 (Tyr in I-A and Trp in I-B) explains the strong preference for alpha-anomeric sugars in both isolectins. Alteration at one amino acid position, Ala(106) in I-A versus Glu(106) in I-B, is the basis for the observed specificities toward alpha GalNAc and alpha Gal.     

Griffonia simplicifolia I-B4 isolectin receptors in the microcirculatory vessels of the gastrocnemius muscle and heart: Heterogeneity among families of anuran amphibians

• 1.1. A fluorescent derivative of the Griffonia simplicifolia I-B₄ isolectin was used to examine historically the distribution of terminal methyl-α-galactosyl residues in the microcirculatory vessels of the gastrocnemius muscle and the ventricles from four families of anuran amphibians.
• 2.2. The isolectin preferentially bound to capillaries in the gastrocnemius muscles from members of the families Ranidae, Bufonidae and Pipidae, but not from the family Hylidae.
• 3.3. Histological and ultrastructural analyses revealed a primitive sinusoidal endothelial system in the anuran heart, with a less extensive expression of the GSI-B₄ receptors than in skeletal muscle.
• 4.4. These results suggest phylogenetic differences among families of anuran amphibians with regard to the distribution of GSI-B₄ receptors in skeletal and cardiac muscle.

     

Study of the transformation of amoeboid microglial cells into microglia labelled with the isolectin Griffonia simplicifolia in postnatal rats.

The transformation of amoeboid microglial cells into ramified microglial cells in the brain of postnatal rats has been studied by labeling the cells with the isolectin Griffonia simplicifolia (GSA1-B4). The latter served as a specific membrane marker of the cell type. Thus, at the light-microscopic level, the amoeboid microglial cells in 1- to 5-day-old rats were intensely stained with GSA1-B4. All the stained cells appeared round. In 10-day-old rats, while most of the stained cells were round, some had assumed an oval appearance. In older rats, i.e. 15-22 days, all the stained cells became flattened or fusiform with long cytoplasmic processes. The present electron-microscopic study confirmed the above features but also added the fact that the reaction for GSA1-B4 was localized at the plasma membrane in the amoeboid microglial cells in all the age groups studied. The reaction for the isolectin was also detected in some vacuoles in the cytoplasm of the round cells. It was concluded from this study that the round amoeboid microglial cells differentiate to become the ramified microglia with age. In the course of this transformation, they retained specific membrane receptors for the isolectin which distinguished them from other glial cell types.     

Distribution of Bandeiraea simplicifolia lectin binding sites in the genital organs of female and male mice

Summary Fluorescein isothiocyanate labelled type I lectin from Bandeiraea simplicifolia (BSA-I) known for its specific binding to -d-galactopyranosyl and 2-acetamido-2-deoxy-d-galactose groups, has been used to map the distribution of the lectin specific binding sites in the genital organs of female and male mice. In non-pregnant female mice, strong lectin reactivity was restricted to the epithelium of the distal oviduct, the cervix and vagina. In pregnant mice strong BSA-I reactivity was also noted in the epithelium of uterine glands from the time of implantation on day 5 onward. In the testis BSA-I bound selectively to sperm but did not react with other cells in the seminiferous tubules. In the proximal caput epididymis BSA-I reacted with the epithelial cells, the underlying basement membranes and the intraluminal sperm. The intraluminal contents of the seminal vesicles reacted strongly with the lectin. Our data thus show a widespread but selective distribution of BSA-I lectin binding sites in the male and female genital organs and altered lectin binding in the uterus during pregnancy.     

Distribution of Bandeiraea simplicifolia lectin binding sites in the genital organs of female and male mice

Fluorescein isothiocyanate labelled type I lectin from Bandeiraea simplicifolia (BSA-I) known for its specific binding to alpha-D-galactopyranosyl and 2-acetamido-2-deoxy-D-galactose groups, has been used to map the distribution of the lectin specific binding sites in the genital organs of female and male mice. In non-pregnant female mice, strong lectin reactivity was restricted to the epithelium of the distal oviduct, the cervix and vagina. In pregnant mice strong BSA-I reactivity was also noted in the epithelium of uterine glands from the time of implantation on day 5 onward. In the testis BSA-I bound selectively to sperm but did not react with other cells in the seminiferous tubules. In the proximal caput epididymis BSA-I reacted with the epithelial cells, the underlying basement membranes and the intraluminal sperm. The intraluminal contents of the seminal vesicles reacted strongly with the lectin. Our data thus show a widespread but selective distribution of BSA-I lectin binding sites in the male and female genital organs and altered lectin binding in the uterus during pregnancy.     

An improved staining method for rat microglial cells using the lectin from Griffonia simplicifolia (GSA I-B4)

A simple method for the lectin histochemical visualization of rat microglial cells is described. Advantages include ease of fixation of brain tissue using paraformaldehyde, and rapidity of tissue processing by vibratome sectioning. Furthermore, in addition to providing good structural preservation, the method achieves improved lectin binding, resulting in complete labeling of all microglial cells and in superior visualization of cellular processes. The lectin histochemical technique for rat microglia has the potential to be adapted to any mammalian species, and should prove valuable for neuroscientists interested in studying this glial cell type.     

The xenograft antigen bound to Griffonia simplicifolia lectin 1-B(4). X-ray crystal structure of the complex and molecular dynamics characterization of the binding site.

The shortage of organs for transplantation into human patients continues to be a driving force behind research into the use of tissues from non-human donors, particularly pig. The primary barrier to such xenotransplantation is the reaction between natural antibodies present in humans and Old World monkeys and the Gal alpha(1-3)Gal epitope (xenograft antigen, xenoantigen) found on the cell surfaces of the donor organ. This hyperacute immune response leads ultimately to graft rejection. Because of its high specificity for the xenograft antigen, isolectin 1-B(4) from Griffonia simplicifolia (GS-1-B(4)) has been used as an immunodiagnostic reagent. Furthermore, haptens that inhibit natural antibodies also inhibit GS-1-B(4) from binding to the xenoantigen. Here we report the first x-ray crystal structure of the xenograft antigen bound to a protein (GS-1-B(4)). The three-dimensional structure was determined from orthorhombic crystals at a resolution of 2.3 A. To probe the influence of binding on ligand properties, we report also the results of molecular dynamics (MD) simulations on this complex as well as on the free ligand. The MD simulations were performed with the AMBER force-field for proteins augmented with the GLYCAM parameters for glycosides and glycoproteins. The simulations were performed for up to 10 ns in the presence of explicit solvent. Through comparison with MD simulations performed for the free ligand, it has been determined that GS-1-B(4) recognizes the lowest energy conformation of the disaccharide. In addition, the x-ray and modeling data provide clear explanations for the reported specificities of the GS-1-B(4) lectin. It is anticipated that a further understanding of the interactions involving the xenograft antigen will help in the development of therapeutic agents for application in the prevention of hyperacute xenograft rejection.     

Carbohydrate-binding properties of an immobilized alpha-D-galactopyranosyl-binding protein (lectin) from the seeds of Bandeiraea simplicifolia.

The alpha-D-galactopyranosyl-binding lectin from Bandeiraea simplicifolia has been coupled to cyanogen bromide-activated Sepharose 4B. Using this immobilized system, we have been able to study the interaction of the lectin with model carbohydrate-protein conjugates and polysaccharides, and to reaffirm this protein's carbohydrate-binding specificity. The opportunity for the isolation of biopolymers containing alpha-D-galactopyranosyl end-groups is demonstrated by the single-step purification of a new galactomannan from the seeds of Cassia alata.     

A structural comparison of the A and B subunits of Griffonia simplicifolia I isolectins

A structural comparison between the A and B subunits of the five tetrameric Griffonia simplicifolia I isolectins (A4, A3B, A2B2, AB3, B4) was undertaken to determine the extent of homology between the subunits. The first 25 N-terminal amino acids of both A and B subunits were determined following the enzymatic removal of N-terminal pyroglutamate blocking groups with pyroglutamate aminopeptidase. Although 21 amino acids were common to both subunits, there were four unique amino acids in the N-terminal sequence of A and B. Residues 8, 9, 17, and 19 were asparagine, leucine, lysine, and asparagine in subunit A and threonine, phenylalanine, glutamic acid, and serine in subunit B. The last six C-terminal amino acids, released by digestion with carboxypeptidase Y, were the same for both subunits: Arg-(Phe, Val)-Leu-Thr-Ser-COOH. Subunit B, which contains one methionyl residue, was cleaved by cyanogen bromide into two fragments, a large (Mr = 31,000) and a small (Mr = 2700) polypeptide. Failure of the small fragment to undergo manual Edman degradation indicated an N-terminal blocking group, presumably pyroglutamate. Both subunits were digested with trypsin and the tryptic peptides were analyzed using reverse-phase HPLC. Tryptic glycopeptides were identified by labeling the carbohydrate moiety of the A and B subunit using sodium [3H] borohydride. Cysteine-containing tryptic peptides were similarly identified by using [1-14C]iodoacetamide. Approximately 30% of the tryptic peptides were common to both subunits. Thus, although the N- and C-terminal regions of A and B are similar, the subunits each possess unique sequences.     

Calcium modulates protease resistance and carbohydrate binding of a plant defense legume lectin,Griffonia simplicifolia lectin II (GSII)

Site-directed mutagenesis previously identified the residues responsible for the biological activity of the plant defense legume lectin, Griffonia simplicifolia lectin II (GSII) [Proc. Natl. Acad. Sci. USA 95, (1998) 15123-15128]. However, these results were inconclusive as to whether these residues function as direct defense determinants through carbohydrate binding, or whether substantial changes of the protein structure had occurred in mutated proteins, with this structural disruption actually causing the loss of biochemical and biological functions. Evidence shown here supports the former explanation: circular dichroism and fluorescence spectra showed that mutations at carbohydrate-binding residues of GSII do not render it dysfunctional because of substantial secondary or tertiary structure modifications; and trypsin treatment confirmed that rGSII structural integrity is retained in these mutants. Reduced biochemical stability was observed through papain digestion and urea denaturation in mutant versions that had lost carbohydrate-binding ability, and this was correlated with lower Ca(2+) content. Accordingly, the re-addition of Ca(2+) to demetalized proteins could recover resistance to papain in the carbohydrate-binding mutant, but not in the non-binding mutant. Thus, both carbohydrate binding (presumably to targets in the insect gut) and biochemical stability to proteolytic degradation in situ indeed contribute to anti-insect activity, and these activities are Ca(2+)-dependent.     

Comparative analysis by frontal affinity chromatography of oligosaccharide specificity of GlcNAc-binding lectins, Griffonia simplicifolia lectin-II (GSL-II) and Boletopsis leucomelas lectin (BLL)

Lectin-based structural glycomics requires a search for useful lectins and their biochemical characterization to profile complex features of glycans. In this paper, two GlcNAc-binding lectins are reported with their detailed oligosaccharide specificity. One is a classic plant lectin, Griffonia simplicifolia lectin-II (GSL-II), and the other is a novel fungal lectin, Boletopsis leucomelas lectin (BLL). Their sugar-binding specificity was analyzed by frontal affinity chromatography using 146 glycans (125 pyridylaminated and 21 p-nitrophenyl saccharides). As a result, it was found that both GSL-II and BLL showed significant affinity toward complex-type N-glycans, which are either partially or completely agalactosylated. However, their branch-specific features differed significantly: GSL-II strongly bound to agalacto-type, tri- or tetra-antennary N-glycans with its primary recognition of a GlcNAc residue transferred by GlcNAc-transferase IV, while BLL preferred N-glycans with fewer branches. In fact, the presence of a GlcNAc residue transferred by GlcNAc-transferase V abolishes the binding of BLL. Thus, GSL-II and BLL forms a pair of complementally probes to profile a series of agalacto-type N-glycans.     

An HPLC method for the direct assay of the serotonin precursor, 5-hydroxytrophan,in seeds of Griffonia simplicifolia

5-Hydroxytryptophan (1) is a naturally occurring amino acid found in significant levels in seeds of Griffonia simplicifolia and used in the treatment of the numerous effects of serotonin deficiency syndrome. An HPLC method has been developed for the direct assay of 1 in seeds of G. simplicifolia which overcomes the problems associated with previous techniques. By optimising the solvent extraction procedures and the HPLC conditions, levels of 1 could be estimated following a single-step seed extraction. The chromatographic conditions, solvent system and the extraction technique developed make this method relatively simple, fast and efficient. Using the described methods, the highest ever levels of 1 (namely, 20.83% on a fresh weight basis) have been determined in seeds of G. simplicifolia obtained in Ghana.     

A histochemical comparison of human corneal stromal glycoconjugates with eight other species. Distinct species-dictated differences in binding sites of Griffonia simplicifolia I

Frozen sections of human, calf, rabbit, rat, cat, dog, goat, lamb, and hog corneas were stained with various lectins using an avidin-biotin-peroxidase complex to study glycoconjugates of stromal matrix. Staining of the stromal matrix and keratocytes with an alpha-galactose-specific lectin, Griffonia simplicifolia I (GSA-I) was species-dependent. The stromal matrices of cat, dog, and hog corneas invariably reacted intensely with this lectin, whereas those of the human, calf, rabbit, rat, and lamb did not react. A positive reaction with GSA-I could be abolished in each instance by preincubation of the sections with alpha-galactosidase. The stromal matrices and keratocytes of all nine species reacted positively with wheat germ agglutinin, concanavalin A, and Ricinus communis agglutinin but did not react with soybean agglutinin. Results of this study may help select an appropriate animal model for further investigate human corneal stromal glycoconjugates.     

Fluorescein-Conjugated Bandeiraea simplicifolia Lectin as a Marker of Endodermal, Yolk Sac, and Trophoblastic Differentiation in the Mouse Embryo

Abstract. The Bandeiraea simplicifolia lectin I (BSA-I) conjugated to fluorescein isothiocyanate was used as a histochemical reagent to study the mouse embryos from fertilization to early somitogenesis. No lectin binding could be detected on the embryonic cells in the preimplantation embryo. Lectin labeled intensely the zona pellucida. In the implanting embryos lectin binding was detected along the subtrophectodermal and Reichert's membrane, in the cytoplasm of the parietal and visceral endoderm, and the trophoblastic giant cells, but not in the ectodermal cells. Studies on explanted blastocyts cultured in vitro disclosed that the cytoplasmic BSA-I binding sites in trophoblastic cells develop gradually. In the 9-day somitic embryo BSA-I reacted with epithelial cells of the yolk sac, but not with the mesenchymal cells. A continuity between the lectin-reactive endoderm and the foregut epithelium could be demonstrated. These data indicated that BSA-I lectin can be used as a histochemical probe for endodermal (yolk sac) and trophoblastic differentiation in the peri-implantational mouse embryo.     

Differential binding of the lectins Griffonia simplicifolia I and Lycopersicon esculentum to microvascular endothelium: organ-specific localization and partial glycoprotein characterization

The lectins Griffonia simplicifolia I and Lycopersicon esculentum were used to assess the presence of endothelium-specific glycoproteins in the microvasculature of the rat myocardium, diaphragm and superficial cerebral cortex. Organs fixed by intravascular perfusion were processed to obtain semithin (0.5 micron) and thin (less than 0.1 micron) frozen sections that were reacted with biotinylated lectin followed by streptavidin conjugated to Texas Red, for semithin sections, or by streptavidin conjugated to 5-nm colloidal gold particles, for thin sections. Lycopersicon esculentum lectin exclusively labeled the endothelium of all small vessels in all three microvascular beds; it did not bind to components of either the parenchyma or the extracellular matrix. Griffonia simplicifolia I lectin exclusively labeled the endothelium of the entire microvasculature in the myocardium and diaphragm, but marked primarily pericytes in the cerebral microvasculature. It did not label any parenchymal or interstitial organ component. At the electron microscope level, the lectin Griffonia simplicifolia I labeling was associated with the plasmalemma proper and especially with plasmalemmal vesicles and their introits, and Lycopersicon esculentum lectin bound primarily to the luminal plasmalemma in the microvascular beds of the myocardium and diaphragm. In the cerebral cortex, labeling of the microvasculature was clearly different: Griffonia simplicifolia I bound primarily to pericytes and vascular smooth muscle cells whereas Lycopersicon esculentum labeled only the microvascular endothelium. Lysates prepared from the myocardium, diaphragm and cerebral cortex were processed through Griffonia simplicifolia I lectin affinity separation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the fraction obtained. A number of putative endothelium-specific glycoproteins was detected and found to differ qualitatively and quantitatively from organ to organ. The most prominent polypeptide, approximately 97 kDa, was present in substantial amounts in the myocardium and diaphragm, but in considerably lower concentration in the cerebral cortex. The reverse applied for a approximately 55 kDa protein. The preferential distribution of the approximately 97 kDa protein parallels differences in Griffonia simplicifolia I lectin binding by fluorescence and electron microscopy on sections of the corresponding organs. The results provide further evidence for the existence of endothelial glycoproteins specific for different microvascular beds and possibly connected with local functional differentiations.