Direct detection of the binding of avidin and lactoferrin fluorescent probes to heparinized surfaces

We describe the use of two heparin-binding proteins, avidin and lactoferrin, as probes for monitoring the amount of heparin immobilized to plastic surfaces. The proteins were derivatized with either fluorescent labels or europium chelates, enabling sensitive, fast, reproducible, and robust assays, and were used to measure the amount of protein bound to heparinized microplates, with particular attention to plates that have been coated with bovine serum albumin (BSA)-heparin conjugate. This direct method unequivocally shows that BSA-heparin affords an economical, convenient, and reliable method for coating both polystyrene microtiter plates and magnetic beads with heparin. We demonstrate that assays using directly labeled proteins overcome the problems of dissociation of the heparin-protein complex, which can occur during incubation and washing steps associated with antibody-based detection methods, and the loss in binding capacity caused by certain blocking regimes. We suggest that labeled avidin and lactoferrin are convenient probes for heparinized surfaces with the potential for much wider applicability than that presented here.     

The role of casein in supporting the operation of surface bound kinesin

Microtubules and associated motor proteins such as kinesin are envisioned for applications such as bioseparation and molecular sorting to powering hybrid synthetic mechanical devices. One of the challenges in realizing such systems is retaining motor functionality on device surfaces. Kinesin motors adsorbed onto glass surfaces lose their functionality or ability to interact with microtubules if not adsorbed with other supporting proteins. Casein, a milk protein, is commonly used in microtubule motility assays to preserve kinesin functionality. However, the mechanism responsible for this preservation of motor function is unknown. To study casein and kinesin interaction, a series of microtubule motility assays were performed where whole milk casein, or its α_s1 and α_s2, β or κ subunits, were introduced or omitted at various steps of the motility assay. In addition, a series of epifluorescence and total internal reflection microscopy (TIRF) experiments were conducted where fluorescently labeled casein was introduced at various steps of the motility assay to assess casein-casein and casein-glass binding dynamics. From these experiments it is concluded that casein forms a bi-layer which supports the operation of kinesin. The first tightly bound layer of casein mainly performs the function of anchoring the kinesin while the second more loosely bound layer of casein positions the head domain of the kinesin to more optimally interact with microtubules. Studies on individual casein subunits indicate that β casein was most effective in supporting kinesin functionality while κ casein was found to be least effective.     

Role of fibrinogen conformation in platelet activation

Platelet adhesion and activation induced by fibrinogen (Fbg) coating on polysaccharide layers of hyaluronic acid (Hyal) and its sulfated derivative (HyalS) were analyzed. Hyal or HyalS was coated and grafted on the glass substrate using a photolithographic method. The Fbg coating was achieved by two different routes: the immobilization of Fbg by means of covalent bond to the polysaccharide layers and the mere adsorption of Fbg to Hyal and HyalS surfaces. Platelet adhesion and activation to the surfaces were evaluated using, respectively, scanning electron microscopy (SEM) and quantifying the release of Platelet Factor 4 by ELISA. The method used for the coating of the surfaces with the Fbg influenced the platelet response. In fact, platelet adhesion and activation took place on surfaces covered by bound Fbg but not on those containing adsorbed Fbg. To explain this difference, the molecular mechanism involved in the Fbg--platelet interaction was investigated blocking platelet membrane receptors by monoclonal antibodies. Because the interaction between Fbg and the GPIIb/IIIa platelet membrane receptor was the only molecular pathway involved, Fbg conformation after the interaction (adsorption or binding) with the Hyal and the HyalS chains and the role of serum proteins adsorbed on the Fbg containing surfaces were accurately analyzed. Both adsorbed and bound Fbg prevented the adsorption of further serum proteins; consequently, a direct interaction between Fbg and platelets was supposed and the different platelet behavior was ascribed to the different conformational changes that occurred after the adsorption and the chemical binding of the Fbg to the Hyal and HyalS surfaces.     

Predominant contribution of surface approximation to the mechanism of heparin acceleration of the antithrombin-thrombin reaction. Elucidation from salt concentration effects

Heparin has been shown to accelerate the inactivation of alpha-thrombin by antithrombin III (AT) by promoting the initial encounter of proteinase and inhibitor in a ternary thrombin-AT-heparin complex. The aim of the present work was to evaluate the relative contributions of an AT conformational change induced by heparin and of a thrombin-heparin interaction to the promotion by heparin of the thrombin-AT interaction in this ternary complex. This was achieved by comparing the ionic and nonionic contributions to the binary and ternary complex interactions involved in ternary complex assembly at pH 7.4, 25 degrees C, and 0.1-0.35 M NaCl. Equilibrium binding and kinetic studies of the binary complex interactions as a function of salt concentration indicated a similar large ionic component for thrombin-heparin and AT-heparin interactions, but a predominantly nonionic contribution to the thrombin-AT interaction. Stopped-flow kinetic studies of ternary complex formation under conditions where heparin was always saturated with AT demonstrated that the ternary complex was assembled primarily from free thrombin and AT-heparin binary complex at all salt concentrations. Moreover, the ternary complex interaction of thrombin with AT bound to heparin exhibited a substantial ionic component similar to that of the thrombin-heparin binary complex interaction. Comparison of the ionic and nonionic components of thrombin binary and ternary complex interactions indicated that: 1) additive contributions of ionic thrombin-heparin and nonionic thrombin-AT binary complex interactions completely accounted for the binding energy of the thrombin ternary complex interaction, and 2) the heparin-induced AT conformational change made a relatively insignificant contribution to this binding energy. The results thus suggest that heparin promotes the encounter of thrombin and AT primarily by approximating the proteinase and inhibitor on the polysaccharide surface. Evidence was further obtained for alternative modes of thrombin binding to the AT-heparin complex, either with or without the active site of the enzyme complexed with AT. This finding is consistent with the ternary complex encounter of thrombin and AT being mediated by thrombin binding to nonspecific heparin sites, followed by diffusion along the heparin surface to a unique site adjacent to the bound inhibitor.     

A method for the non-covalent immobilization of heparin to surfaces

The interaction of heparan sulfate (HS) with specific proteins facilitates a wide range of fundamental biological processes including cellular proliferation and differentiation, tissue homeostasis, and viral pathogenesis. This multiplicity of function arises through sequence diversity within the HS chain. Heparin, which is very similar in structure to the sulfated regions of HS, is an excellent model for studying HS-protein interactions. The development of high-throughput enzyme-linked immunosorbent-like assays using surface-immobilized heparin has been hindered by the inability of this glycosaminoglycan to adhere to microtiter surfaces. Here we report the passive noncovalent adsorption of heparin onto microtiter wells following their treatment by plasma polymerization; there was no detectable binding of functional heparin onto untreated plates. Heparin immobilized in this way was able to interact with four different heparin-binding proteins tested, i.e., TSG-6, chemokines IL-8 and KC, and complement factor H. Heparin preparations ranging in size from high molecular weight to a defined decasaccharide could be adsorbed onto these plates in a functionally active form. Since plasma polymerization is possible for virtually any surface, this technique is likely to be of general use in the identification and characterization of heparin/HS-binding proteins in a wide range of applications.     

Localization of two binding domains for thrombospondin within fibronectin.

Thrombospondin is a major glycoprotein of the platelet alpha-granule and is secreted during platelet activation. Several protease-resistant domains of thrombospondin mediate its interactions with components of the extracellular matrix including fibronectin, collagen, heparin, laminin, and fibrinogen. Thrombospondin, as well as fibronectin, is composed of several discretely located biologically active domains. We have characterized the thrombospondin binding domains of plasma fibronectin and determined the binding affinities of the purified domains; fibronectin has at least two binding sites for thrombospondin. Thrombospondin bound specifically to the 29-kDa amino-terminal heparin binding domain of fibronectin as well as to the 31-kDa non-heparin binding domain located within the larger 40-kDa carboxy-terminal fibronectin domain generated by chymotrypsin proteolysis. Platelet thrombospondin interacted with plasma fibronectin in a specific and saturable manner in blot binding as well as solid-phase binding assays. These interactions were independent of divalent cations. Thrombospondin bound to the 29-kDa fibronectin heparin binding domain with a Kd of 1.35 x 10(-9) M. The Kd for the 31-kDa domain of fibronectin was 2.28 x 10(-8) M. The 40-kDa carboxy-terminal fragment bound with a Kd of 1.65 x 10(-8) M. Heparin, which binds to both proteins, inhibited thrombospondin binding to the amino-terminal domain of fibronectin by more than 70%. The heparin effect was less pronounced with the non-heparin binding carboxy-terminal domain of fibronectin. By contrast, the binding affinity of the thrombospondin 150-kDa domain, which itself lacked heparin binding, was not affected by the presence of heparin. Based on these data, we conclude that thrombospondin binds with different affinities to two distinct domains in the fibronectin molecule.     

Visualization of heparin-binding proteins by ligand blotting with 125I-heparin

A ligand-blotting procedure which allows detection of heparin-binding proteins is described. Crude commercial heparin was fractionated by chromatography on a column of human plasma low-density lipoproteins immobilized to Sepharose CL-4B. Chromatography yielded an unbound and a bound fraction of heparin, designated URH and HRH, respectively. The HRH fraction was reacted with the N-hydroxysuccinimidyl ester of 3-(p-hydroxyphenyl)propionic acid and then labeled with 125I. Proteins were separated by 3-20% pore-gradient gel electrophoresis, transferred to nitrocellulose, and then assayed for their ability to bind 125I-labeled HRH. Human plasma apolipoproteins B-100, B-48, and E of chylomicrons, very low-density lipoproteins, and low-density lipoproteins bound the 125I-labeled HRH; the radiolabeled heparin did not bind to serum albumin, ferritin, catalase, and lactate dehydrogenase. The ligand-blotting procedure should facilitate the purification of heparin-binding domains from these proteins and, moreover, may be applicable to the investigation of heparin-protein interactions in general.     

Heparin-binding proteins from seminal plasma bind to bovine spermatozoa and modulate capacitation by heparin

Bovine spermatozoa that have been exposed to seminal plasma possess more binding sites for heparin than sperm from the cauda epididymis that have not been exposed to accessory sex gland secretions. Seminal plasma exposure enables sperm, following incubation with heparin, to undergo zonae pellucidae-induced exocytosis of the acrosome. In this study, the regulatory role of seminal plasma heparin-binding proteins in capacitation of bovine spermatozoa by heparin was investigated. Plasma membranes from sperm exposed to seminal plasma in vivo or in vitro contained a series of acidic 15-17 kDa proteins not found in cauda epididymal sperm. Western blots of membrane proteins indicated that these 15-17 kDa proteins bound [125I]-heparin. Heparin-binding proteins were isolated by heparin affinity chromatography from seminal plasma from vasectomized bulls. Gel electrophoresis indicated that the heparin-binding peaks contained 14-18 kDa proteins with isoelectric variation, a basic 24 kDa protein, and a 31 kDa protein. Western blots probed with [125I]-heparin confirmed the ability of each of these proteins to bind heparin. Each of these proteins, as well as control proteins, bound to epididymal sperm. The seminal plasma proteins were peripherally associated with sperm since they were removed by hypertonic medium and did not segregate into the detergent phase of Triton X-114. Seminal plasma heparin-binding proteins potentiated zonae pellucidae-induced acrosome reactions in epididymal sperm. However, seminal plasma proteins that did not bind to the heparin affinity column were unable to stimulate zonae-sensitivity. Control proteins, including lysozyme--which binds to both heparin and sperm, were ineffective at enhancing zonae-induced acrosome reactions. These data provide evidence for a positive regulatory role of seminal plasma heparin-binding proteins in capacitation of bovine spermatozoa.     

Expression of externally-disposed heparin/heparan sulfate binding sites by uterine epithelial cells

A class of high-affinity binding sites that preferentially bind heparin/heparan sulfate have been identified on the external surfaces of mouse uterine epithelial cells cultured in vitro. [3H]Heparin binding to these surfaces was time-dependent, saturable, and was blocked specifically by the inclusion of unlabeled heparin or endogenous heparan sulfate in the incubation medium. A variety of other glycosaminoglycans did not compete for these binding sites. The presence of sulfate on heparin influenced, but was not essential for, recognition of the polysaccharide by the cell surface binding sites. [3H]-Heparin bound to the cell surface was displaceable by unlabeled heparin, but not chondroitin sulfate. Treatment of intact cells on ice with trypsin markedly reduced [3H]heparin binding, indicating that a large fraction of the surface binding sites were associated with proteins. Scatchard analyses revealed a class of externally disposed binding sites for heparin/heparan sulfate exhibiting an apparent Kd of approximately 50 nM and present at a level of 1.3 x 10(6) sites per cell. Approximately 9-14% of the binding sites were detectable at the apical surface of cells cultured under polarized conditions in vitro. Detachment of cells from the substratum with EDTA stimulated [3H]heparin binding to cell surfaces. These observations suggested that most of the binding sites were basally distributed and were not primarily associated with the extracellular matrix. Collectively, these observations indicate that specific interactions with heparin/heparan sulfate containing molecules can take place at both the apical and basal cell surfaces of uterine epithelial cells. This may have important consequences with regard to embryo-uterine and epithelial-basal lamina interactions.     

Inhibition of platelet-surface-bound proteins during coagulation under flow II: Antithrombin and heparin

Blood coagulation is a self-repair process regulated by activated platelet surfaces, clotting factors, and inhibitors. Antithrombin (AT) is one such inhibitor that impedes coagulation by targeting and inactivating several key coagulation enzymes. The effect of AT is greatly enhanced in the presence of heparin, a common anticoagulant drug. When heparin binds to AT, it either bridges with the target enzyme or induces allosteric changes in AT leading to more favorable binding with the target enzyme. AT inhibition of fluid-phase enzymes caused little suppression of thrombin generation in our previous mathematical models of blood coagulation under flow. This is because in that model, flow itself was a greater inhibitor of the fluid-phase enzymes than AT. From clinical observations, it is clear that AT and heparin should have strong inhibitory effects on thrombin generation, and thus we hypothesized that AT could be inhibiting enzymes bound to activated platelet surfaces that are not subject to being washed away by flow. We extended our mathematical model to include the relevant reactions of AT inhibition at the activated platelet surfaces as well as those for unfractionated heparin and a low molecular weight heparin. Our results show that AT alone is only an effective inhibitor at low tissue factor densities, but in the presence of heparin, it can greatly alter, and in some cases shut down, thrombin generation. Additionally, we studied each target enzyme separately and found that inactivation of no single enzyme could substantially suppress thrombin generation.     

Identification of a heparin-releasable lipoprotein lipase binding protein from endothelial cells.

Triglycerides in circulating plasma lipoproteins are hydrolyzed by lipoprotein lipase (LPL) which is thought to bind to proteoglycans on the luminal endothelial cell surface. Previous studies from this laboratory using LPL-Sepharose affinity chromatography identified a 220-kDa LPL binding proteoglycan. Using ligand blotting with 125I-LPL, we now report a 116-kDa LPL binding protein in plasma membrane preparations of endothelial cells. 125I-LPL binding to this protein was abolished by addition of unlabeled LPL. When the cell surface of endothelial cells was labeled with biotin, a 116-kDa protein was biotinylated. Furthermore, the biotinylated 116-kDa protein bound to LPL-Sepharose and eluted with 0.4 M NaCl suggesting that the 116-kDa LPL binding protein is present on the cell surface. When detergent extracts of endothelial cells were applied to LPL-Sepharose in the presence of 0.15 M NaCl, the 116-kDa, but not the 220-kDa, protein still bound to LPL-Sepharose. The 116-kDa protein was not labeled with 35SO4 and eluted from DEAE-cellulose prior to proteoglycans, suggesting that it is not a proteoglycan. However, a 116-kDa endothelial cell surface protein was metabolically labeled with [35S]methionine. This protein was dissociated from the cell surface by incubating cells with heparin (50 units/ml)-containing buffer. After heparin treatment of endothelial cells, LPL binding to and internalization by the cells decreased greater than 70% compared to control cells. These results suggest that endothelial cells synthesize a heparin-releasable, high affinity 116-kDa LPL binding protein. We postulate that this protein is associated with proteoglycans on luminal endothelial surfaces and mediates LPL binding, internalization, and recycling. We name this protein hrp (heparin-releasable protein)-116.     

Local delivery of basic fibroblast growth factor (bFGF) using adsorbed silyl-heparin,benzyl-bis(dimethylsilylmethyl)oxycarbamoyl-heparin

A growth factor delivery system was developed that is based on the use of silyl-heparin, a chemically modified analogue of heparin. The silyl-heparin was adsorbed onto surfaces by hydrophobic interaction via the prosthetic unit and can then be used as a solid-phase adsorbent for bFGF. All the coating steps were performed by adsorption, a process that allowed preparation of surfaces by immersion or "dip-coating". In this study a series of silyl-heparins were synthesized and each of the analogues found to function similar to unmodified heparin relative to their binding of antithrombin III and also the binding of bFGF. The silyl-heparins were found to be adsorbed onto a wide variety of substrates including polystyrene and lactide:glycolide copolymer. Enzyme-linked immumosorbant assay (ELISA) was used to establish that bFGF was readily bound to surface adsorbed silyl-heparin, and that the amount bound was directly related to amount offered for binding. Once adsorbed the silyl-heparin/FGF was able to induce capillary tube formation of endothelial cells and to increase the growth of endothelial cells. When coated onto suture material and implanted in muscle, the FGF/silyl-heparin coating caused an increased density of mesenchymal cells in the area of the implant. This coating method could prove to be useful in a number of tissue engineering applications for the local delivery of FGF and other growth factors.     

Acylation of gelatin-agarose and the enhancement by heparin of fibronectin binding

The enhancement of the binding of plasma fibronectin to collagen or gelatin by heparin was previously thought to be due primarily to interaction of heparin with fibronectin. We observed, however, that the elution of purified human plasma fibronectin from heparin-treated gelatin-agarose required the same high urea concentrations regardless of whether heparin treatment preceded or followed fibronectin adsorption. Acylation of gelatin-agarose with acetic anhydride or succinic anhydride had little effect upon fibronectin binding, yet the heparin enhancement of fibronectin binding was abolished by either acylation reaction. When heparin binding to gelatin-agarose was investigated with dansyl heparin, gelatin-agarose bound substantial quantities of labeled heparin which could be readily dissociated from the matrix with 2 M NaCl. Acetylated gelatin-agarose did not bind detectable amounts of dansyl heparin. We interpret these results as evidence that the stronger binding of fibronectin to gelatin-agarose in the presence of heparin is due to heparin itself binding to gelatin, thus allowing fibronectin to bind simultaneously to both immobilized ligands through appropriate domains of the glycoprotein.     

Kinetic and Structural Studies on the Interactions of Heparin and Proteins of Human Seminal Plasma using Surface Plasmon Resonance

Heparin is naturally occurring polysaccharides which interacts with seminal plasma proteins and regulate multiple steps in fertilization process. Qualitative and quantitative information regarding the affinity for heparin-seminal plasma proteins interactions is not generally well documented and there are no reports of a comprehensive analysis of these interactions in human seminal plasma. Such information should improve our understanding of how GAGs especially heparin present in the reproductive tract regulate fertilization. In this study, we use SPR to study interactions of heparin with various seminal plasma heparin-binding proteins (HBPs). HBPs like lactoferrin (LF), fibronectin fragment (FNIII), semenogelinI (SGI) and prostate specific antigen (PSA) all bind heparin with different binding kinetics and affinities. Kinetic data suggests that FNIII binds heparin with a high affinity (KD=3.2 nM), while PSA binds heparin with a micromolar affinity (KD=11.1 μM). Preincubation of SGI with heparin inhibits the binding of SGI to immobilized PSA in a dosedependent manner, while FNIII incubated with heparin binds with an increased affinity to PSA. Solution-competition studies show that the minimum size of a heparin oligosaccharide capable of binding with PSA is greater than a tetrasaccharide, with LF and SGI is larger than a hexasaccharide and for FNIII is larger than an octasaccharide.     

Relationship between fertilizing ability of frozen human spermatozoa and capacity for heparin binding and nuclear decondensation.

Nuclear decondensation of spermatozoa induced by heparin, reduced glutathione (GSH) or a mixture of heparin and GSH was studied using frozen-thawed human spermatozoa. The percentages of decondensed spermatozoa in controls and after treatment for 60 min with 30 mumol heparin l-1, 5 mmol GSH l-1, or heparin-GSH mixture were 1.5, 22.1, 4.3 and 37.6%, respectively. Most of the decondensed spermatozoa were eliminated by Percoll gradient centrifugation of samples previously treated with heparin or heparin-GSH mixture. However, comparable numbers of motile spermatozoa were recovered in the control and in each treated sample, demonstrating that a major proportion of motile spermatozoa was resistant to heparin (or heparin-GSH) effects on nuclear decondensation of spermatozoa. Fertilization of hamster oocytes was attempted using spermatozoa recovered in the 90% Percoll fraction and resistant to heparin-GSH decondensing mixture. Although insemination used a constant number of motile spermatozoa, fertilization rates were higher after treatments with heparin and GSH alone than in control or heparin-GSH-treated samples. In addition the number of spermatozoa that attached to the oocyte plasma membrane was a sixth or a half for sperm pretreated with heparin-GSH or heparin alone, respectively compared with untreated values. However, there was no evidence for induced acrosomal reaction by heparin and GSH, at least at the concentrations used. Qualitative analyses of heparin-binding sites were performed on untreated spermatozoa recovered in the 90% Percoll fraction by incubating spermatozoa in the presence of heparin covalently linked to albumin and coupled to colloidal gold (5 nm). Among this population of spermatozoa, 40.5% bound heparin-gold and labelling was mainly observed on the sperm head surface (88% of labelled spermatozoa) with (59.5%) or without (28.5%) tail labelling. Only a small proportion (23%) of spermatozoa that attached to the oocyte plasma membrane bound heparin-gold conjugate and only weak labelling was observed on the sperm head. Moreover, the proportion of spermatozoa that bound heparin-gold conjugate decreased (r = -0.77, P less than 0.0001) in relation to increasing concentrations of motile spermatozoa in the sample.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lectin binding to newt epidermis: fluorescent localization and effects on motility

The ability of seven lectins to bind to newt epidermal cells and influence their motility was examined. Of the seven fluoresceinated lectins applied to frozen sections containing intact newt skin and migrating epidermis (wound epithelium), only Con A (concanavalin A), WGA (wheat germ agglutinin), and PNA (peanut agglutinin) produced detectable epidermal fluorescence. Con A and WGA each heavily labeled all layers of intact epidermis, but PNA bound only to the more superficial layers. In contrast to a single population of labeled cells in migrating epidermal sheets after treatment with Con A, there were both labeled and unlabeled cells after exposure to either WGA or PNA. The wound bed was labeled by both Con A and WGA, but not by PNA. DBA (Dolichos bifloris agglutinin), RCA I (Ricinus communis agglutinin), and UEA (Ulex europaeus agglutinin), did not produce significant fluorescence with either migrating or intact epidermis. In general, inhibitory effects on epidermal motility correlated with the binding studies. Thus, Con A, WGA, and PNA, the lectins which clearly bound to the epidermis, all produced a concentration-dependent depression in the rate of epidermal wound closure. RCA was somewhat paradoxical in that it was moderately inhibitory despite showing essentially no binding. The effects of SBA and UEA were equivocal. DBA had no effect. These results indicate that the inhibition of motility produced by Con A that we have described previously is not peculiar to this mannose-binding lectin, but is shared by at least one lectin with an affinity for D-GlcNAc (WGA), and one with an affinity for B-D-Gal(1-3)-D-GalNAc (PNA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein-heparin interactions measured by BIAcore 2000 are affected by the method of heparin immobilization

Surface plasmon resonance (SPR) biosensors such as the BIAcore 2000 are a useful tool for the analysis of protein-heparin interactions. Generally, biotinylated heparin is captured on a streptavidin-coated surface to create heparinized surfaces for subsequent binding analyses. In this study we investigated three commonly used techniques for the biotinylation of heparin, namely coupling through either carboxylate groups or unsubstituted amines along the heparin chain, or through the reducing terminus of the heparin chain. Biotinylated heparin derivatives were immobilized on streptavidin sensor chips and several heparin-binding proteins were examined. Of the surfaces investigated, heparin attached through the reducing terminus had the highest binding capacity, and in some cases had a higher affinity for the proteins tested. Heparin immobilized via intrachain bare amines had intermediate binding capacity and affinity, and heparin immobilized through the carboxylate groups of uronic acids had the lowest capacity for the proteins tested. These results suggest that immobilizing heparin to a surface via intrachain modifications of the heparin molecule can affect the binding of particular heparin-binding proteins.     

Visualization of Histamine Binding to Nuclei

The binding of histamine to cultured microvascular endothelial cells and glycol methacrylate embedded ovarian tissue sections has been localized using fluorescein-albumin-histamine conjugate. Histamine conjugate was bound to the plasma membranes and nuclei of luteal, endothelial, and ovarian stromal cells. An apparent increase in the binding of histamine to nuclei was observed in the presence of cimetidine but the plasma membrane staining was still evident. Unlike cimetidine, pyrilamine completely inhibited the binding of histamine to the plasma membrane. Instead, in the presence of pyrilamine, histamine bound exclusively to the nuclei of endothelial, germinal epithelial, granulosa, and stromal cells. However, the nuclei of terminally differentiated luteal cells and oocytes were not labeled. The functional significance of these nuclear histamine binding sites remains to be determined.     

Separation, characterization and identification of boar seminal plasma proteins

Methods used for the isolation, separation and characterization of boar seminal plasma proteins are discussed, as well as techniques applied to study their binding properties. Attention is paid to interactions of these proteins with different types of saccharides and glycoconjugates, with membrane phospholipids, and to interactions between proteins. Boar seminal plasma contains different types of proteins: spermadhesins of the AQN and AWN families; DQH and PSP proteins belong to the most abundant. Some of these proteins are bound to the sperm surface during ejaculation and thus protein-coating layers of sperm are formed. Sperms coated with proteins participate in different types of interactions occurring in the course of the reproduction process, e.g. formation of the oviductal sperm reservoir, sperm capacitation, oocyte recognition and sperm binding to the oocyte.     

The interaction between heparin and antithrombin III: a comparison of two different heparin-dye conjugates

The interactions of antithrombin III with two heparin-dye conjugates have been compared using their fluorescence anisotropy. The first, heparin labelled with 5-isothiocyanatofluorescein, where the dye was mostly bound to unsulphated glucosamine residues, exhibited binding which was characteristic of heparin with a low affinity for antithrombin III. The second, heparin labelled with a reactive naphthalene dye (DENMT), showed similar binding character. However, when the heparin was treated with an amino group blocking agent prior to labelling with DENMT, the resultant heparin-dye conjugate showed binding behaviour, the strength of which was consistent with heparin molecules having both high and low affinity for antithrombin III. Heparin molecules with a high affinity for antithrombin III did not possess free amino groups. The implications of these findings are discussed with regard to the reliability of the data obtained using heparin-fluorescein conjugates.