Multipoint Attachment Research Articles

Activation of blood coagulation at heparin‐coated surfaces

It is hypothesized that immobilized heparin exerts a dual role in blood coagulation. On the one hand, the heparinized surface is because of its dense negative charge, thought to initiate the intrinsic pathway of blood coagulation. On the other hand, heparin is known as a potent anticoagulant drug. However, it remains to be seen how much contact-phase activation of factor XI contributes to thrombin formation and how this process is counterbalanced by which of the anti-protease activities of immobilized heparin. In the present study we examined the generation of factors XIa, IXa, and Xa, and thrombin in recalcified normal and antithrombin-depleted plasma exposed to polyacrylamide-graft polyurethane (PU) sheets modified by multipoint attachment of two different heparin species. One of them, HAH, contained the specific antithrombin binding sequence and the other one, NAH, had a low affinity for antithrombin and had no anticoagulant activity. Our data demonstrate that in contrast to PU, PU-NAH and PU-HAH are strong mediators of factor XIa and factor IXa formation in normal and antithrombin-deficient plasma. Interestingly, compared to PU-HAH and PU-NAH, thrombin formation was only slightly diminished in antithrombin-deficient plasma exposed to PU. In contrast, thrombin formation was dramatically delayed and diminished in normal plasma exposed to PU-HAH. These findings indicate that very low amounts of factor XIa apparently suffice to induce significant amounts of thrombin. In this sense, heparinized surfaces are highly thrombogenic, but our data also indicate that this activity is effectively counterbalanced by the anti-thrombin activity of the immobilized anti-coagulant species of heparin. © 1997 John Wiley & Sons, Inc. J Biomed Mater Res, 37, 108–113, 1997.

Gustatory responses of non-human primates to dipeptide derivatives or analogues, sweet in man

Dipeptide derivatives or analogues, sweet in man, can be divided into three classes according to their gustatory responses in primates: (i) dipeptides which are sweet to all primates (prosimians, New World simians and Old World simians), such as alitame or l-aspartyl- d-alanine propyl ester (class I); (ii) dipeptides which are sweet to prosimians and Old World simians, but not to New World simians, such as l-aspartyl-( R)-α-methylphenethylamine or l-aspartyl- l-( O- tert-butyl)serine methyl ester (class II); and, (iii) compounds which are sweet only to Old World simians, but not to prosimians and New World simians, such as aspartame (class III). Analysis of these results by means of the multipoint attachment (MPA) theory of sweetness reception (Nofre & Tinti, 1996) leads to the conclusion that the seven basic recognition sites of the sweetness receptor, as inferred from the MPA theory, are (i) in prosimians: Asp-1 or Glu-1, Lys-2, Asp-3 or Glu-3, Thr-4, Ala-5 or Ser-5, Thr-6, Thr-7; (ii) in New World simians: Asp-1 or Glu-1, Lys-2, Asp-3 or Glu-3, Thr-4, Ala-5 or Ser-5, Ala-6 or Ser-6, Thr-7; and, (iii) in Old World simians: Asp-1 or Glu-1, Lys-2, Asp-3 or Glu-3, Thr-4, Thr-5, Thr-6, Thr-7.

Sweetness reception in man: the multipoint attachment theory

The multipoint attachment (MPA) theory assumes the presence, in the human sweetness receptor (probably a seven-pass transmembrane receptor), of at least eight fundamental recognition sites, namely, the B-, AH-, XH-, G1-, G2-, G3-, G4- and D-recognition sites. The B-recognition site is assumed to be a Lys residue; the AH- and XH-recognition sites, Asp (or Glu) residues; the G1-, G2-, G3- and G4-recognition sites, Thr residues; and the D-recognition site, a Ser (or Thr) residue. These eight recognition sites are able to interact with eight sweetener interaction sites, namely, the B, AH, XH, G1, G2, G3, G4 and D interaction sites. The number of interaction sites in a given sweetener may be equal to or lower than eight. Apart from D, the interaction sites are made up of two elementary interaction sites (interaction points or subsites). These interaction points (namely, the B1, B2, AH1, AH2, XH1, XH2, G1, E1, G2, E2, G3, E3, G4, E4 and D points) operate with the receptor through three types of elementary interactions, namely, ionic, H-bonding and steric interactions. B (B1, B2) is an anionic group (CO 2 −) or an H-bond acceptor atom (Cl, Br, O); AH1, AH2, XH1, XH2 are H-bond donor groups (NH +, NH, OH); E1, E2, E3, E4 are H-bond acceptor atoms (N, O, Cl, Br); G1, G2, G3, G4 are steric interaction subsites (CH 3, CH 2, CH, halogens); and D is an H-bond acceptor group (CN). Several examples are given to illustrate the theory. For example, according to the MPA theory, D-glucose is a B1, B2, AH1, AH2, XH1, XH2-type sweetener, D-fructose an E1, E2, E3, E4-type sweetener, and sucrose a B1, B2, AH1, AH2, XH1, XH2, G1, E1, G2, E2, G3, E3, G4, E4-type sweetener.

Study of the stabilization of pure lipases: comparison of two different lipase-microgel derivatives

The immobilization/stabilization of pure and very labile lipases was studied. Two types of lipase-microgels derivatives, which may be used in aqueous and/or organic media were designed and optimized. The first type consisting in the covalent linkage of the protein to the surface of a previously formed microgel. The second type was obtained in a reverse micellar system of AOT. The lipase was microencapsulated into the acrylic microgels formed after a polymerization process carried out in the micellar droplets. In this case, a crosslinking agent was simultaneously used to enhance the protein rigidity. Due to the distinct lipase localization the two microgel derivatives differ in their activities and stabilities: the microgel with the lipase at its surface had a similar activity and stability as the native lipase, while an important reduction of the conformational mobility of the protein was found when the lipase was microencapsulated, and it gave rise to a high stabilization factor. Thus, a new immobilization method which stabilizes by 352 times at 45°C the pure isolipase B from Candida cylindracea is described. These results were also better than those of the crude lipase stabilization by multipoint attachment to agarose gels.

Antithrombin activity of surface‐bound heparin studied under flow conditions

Polyacrylamide-grafted polyetherurethane sheets were modified by end-point and multipoint attachment of heparin. The surface-bound heparin was firmly attached. No release of heparin activity could be detected when the surface was rinsed at a wall shear rate of 2000 s-1. Uptake of antithrombin and thrombin inactivation were investigated under well-defined flow conditions by the use of a spinning device with an attached disk-shaped heparinized surface. It is demonstrated that the rate of thrombin inactivation at the antithrombin-heparin surface equals the maximal rate of transport of thrombin toward the surface when the surface coverage of antithrombin exceeds 10 pmol/cm2. This result indicates that a higher intrinsic catalytic efficiency of a surface does not necessarily result in a higher antithrombin activity. We varied the heparin content of the surfaces between 0 and 35 micrograms/cm2 by increasing the number of functional groups to which heparin could be covalently attached. The uptake of antithrombin increased with the heparin content of the surface, but the stoichiometry decreased from 2 to 0.5 pmol antithrombin/micrograms heparin. Apparently, antithrombin could not bind to heparins buried in the poly(acrylamide) layer. The rate of thrombin inactivation at surfaces with low heparin content (2 micrograms/cm2) fells below the transport limit of thrombin and became proportional with the heparin content of the surface. Although the contribution of surface-bound heparin to the neutralization of fluid-phase thrombin was found to be negligible compared with the effect of fluid-phase antithrombin at physiologic relevant concentrations, these heparinized surfaces markedly delayed the onset of thrombin generation in platelet-rich plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective oxidation: stabilisation by multipoint attachment of ferredoxin NADP + reductase, an interesting cofactor recycling enzyme

Ferredoxin-NADP + reductase (FNR, EC 1.18.1.2) is an enzyme that is able to catalyse the oxidation of NADPH + H +. A strategy to prepare industrial derivatives of this enzyme for use as an ‘NADP’ regenerating enzyme in oxidizing reactions is presented. The strategy is based on a strictly controlled process of multipoint covalent attachment between the enzyme, via its amino groups, and a pre-existing solid activated with a monolayer of simple aldehyde groups linked by a space-arm of moderate length to the surface of the support. Controlling the variables which may have an influence in the multi-interaction process, we have prepared a number of enzyme derivatives with very different activity/stability properties. The ‘optimum derivative’ was found to be much more stable than its corresponding soluble enzyme under all the denaturation conditions assayed (high temperatures, extreme pH, organic solvents, etc.). Because of the excellent properties of this enzyme derivative, we can regenerate NADP + by using molecular oxygen directly as the oxidizing agent under a wide range of conditions. Coupling this oxidative system to other NADP-dependent redox enzymes, we should be able to develop a very specific and selective oxidative procedure under very mild oxidizing conditions.

Competitive immunosorbent assays for biotin using bifunctional unilamellar vesicles.

Competitive immunosorbent assays for the model antigen biotin were performed using both unilamellar vesicles with covalently attached biotin and horseradish peroxidase (HBVs) and commercially available biotin-labeled horseradish peroxidase (B-HRP) as the enzyme-labeled antigen. The assays were performed using anti-biotin antibody (ABA) surface densities ranging from one-tenth to full monolayer coverage. It was found that assays using HBVs strongly depended on the antibody surface density, while assays using B-HRP were relatively insensitive to the antibody surface density. The HBV assay dependence on the ABA surface density was most likely due to multiple point attachment of vesicles to the surface. The lowest detectable antigen concentration (least detectable dose) for vesicles (approximately 10(-9) M) was an order of magnitude lower than the value found for B-HRP (approximately 10(-8) M). The sensitivity (slope of the response vs biotin concentration curve) of assays with B-HRP was comparable to the sensitivity of assays with HBVs at low antibody surface density, probably due to less extensive multipoint attachment. It was also found that assays could be performed with vesicles at antibody surface densities that were at least 5 times lower, in terms of the bulk antibody concentrations used to coat the wells, than antibody surface densities at which B-HRP gave comparable signals (approximately 1 delta A/min).

Ligand-induced accelerated dissociation of (+)-cis-diltiazem from L-type Ca2+ channels is simply explained by competition for individual attachment points.

We have studied the kinetics of (+)-[3H]cis-diltiazem binding to L-type Ca2+ channels. The association reaction was of the second order with a rate constant of k1 = 5.3 x 10(-5) nM-1 min-1. A dissociation rate of the first order (k-1 = 0.0022 min-1) and the monophasic equilibrium binding curve (KD = 42 nM) seemed to reflect the simple case of one ligand binding to one site. However, when dissociation was monitored after isotopic rather than volume dilution, the dissociation rate constant increased with the concentration of added non-radioactive (+)-cis-diltiazem. Half-logarithmic plots were linear with rate constants of 0.0058, 0.011, 0.015, and 0.020 min-1 when 3, 10, 30, and 50 microM (+)-cis-diltiazem was added, respectively. We quantitatively analyzed our experimental data in terms of three different binding models, which can account for ligand-induced accelerated dissociation: 1) the classical allosteric model of pre-existing conformational states, 2) a general reaction scheme for two sites with negative cooperativity, and 3) competition of ligands for single attachment points within one binding site. Computer fitting of the experimental data to simplified reaction schemes derived from these models revealed that all three can be used to fit our data. However, only the multipoint attachment model does not require additional structural assumptions, not even an additional binding site, and is supported by structure-activity relationship analysis of diltiazem analogues. We conclude that ligand-induced accelerated dissociation should be a widely observed phenomenon that does not prove a non-competitive binding mechanism. Instead, it can be explained with the thermodynamics of non-covalent binding, where high affinity results from the cooperative interaction of a ligand molecule with multiple attachment points.

Stabilization of heterodimeric enzyme by multipoint covalent immobilization: Penicillin G acylase from Kluyvera citrophila

We have developed a strategy for immobilization-stabilization of penicillin G acylase (PGA) from Kluyvera citrophila by controlled multipoint covalent attachment to agarose-aldehyde gels. This enzyme is composed by two dissimilar subunits noncovalently bound. Thus, in this article we establish clear correlations between enzyme stabilization and the multipoint immobilization and/or between enzyme stabilization and the involvement of the two subunits in the attachment of them to the support. We have demonstrated that important thermal stabilizations of derivatives were only obtained through a very intense enzyme-support multipoint attachment involving the whole enzyme molecule. In this way, we have prepared derivatives preserving more than 90% of catalytic activity and being more than 1000-fold more stable than soluble and one-point attached enzyme. In addition, the involvement of the two subunits in the covalent attachment to the support has proved to be essential to develop interesting strategies for reactivation of inactivated enzyme molecules [e.g., by refolding of immobilized PGA after previous unfolding with urea and sodium dodecyl sulfate (SDS)].

Affinity thermoprecipitatin: Contribution of the efficiency of ligand–protein interaction and access of the ligand

Conjugates to two thermoprecipitating polymers, poly(N-vinyl caprolactam) and poly(N-isopropylacrylmide), with soybean trypsin inhibitor, Cibacron Blue 3GA, Cu-iminodiacetic acid, and p-aminobenzamidine were synthesized. The interaction of these conjugates with trypsin and lactate dehydrogenase was studied. Coupling of the ligand to a polymer resulted in a 100-1000-fold decrease in enzyme-affinity. Rough theoretical estimates revealed that a successful affinity precipitation required that the binding of a target protein and a ligand coupled to a polymer have binding constants on the order of 10(-7)-10(-8) M. Such strong affinity of low molecular weight ligands that can provide binding constants of 10(-9)-10(-11) M or alternatively multipoint attachment of the target protein molecule. The ligand in the ligand-polymer conjugate is still accessible to the protein after thermoprecipitation, and the latter can bind with the particle of the dispersion of thermoprecipitated ligand-polymer precipitate may result in stripping of enzyme molecules from the surface of the particles.

Stabilization of Micrococcal Endonuclease by Immobilization on Agarose Gels Highly Activated with CNBr

The attachment of enzymes, through their amino groups, to CNBr activated agarose gels has been tested as an immobilization stabilization system. By using this system, the development of a strategy to immobilize enzymes through multipoint covalent attachment has been studied. We have prepared different staphylococcal nuclease-Agarose derivatives by using Sepharose 2B gels previously activated with CNBr. Activity and stability of the derivatives obtained were very dependent on the degree of activation of the support. The most stable derivatives, prepared with the most activated supports, were 700 fold more stable than the soluble enzyme in irreversible thermal inactivation experiments, at 40d`C. In contrast, a significant loss of catalytic activity (kcat decreases down to 40%) was associated with the increase in stability. Colorimetric titration of amine groups in the stabilized derivatives suggested that enzyme-support multipoint attachment was the main reason for the observed stabilizing effect.Index Entr...

Effect of polymer matrix on penicillin acylase immobilization on copolymers of butyl acrylate and ethylene glycol dimethacrylate

Copolymers of butyl acrylate and ethylene glycol dimethacrylate with various degree of crosslinking and aminolysis were used for penicillin acylase immobilization. The amount and activity of the bound protein decreased with the decrease of crosslinking when copolymers having nearly constant degrees of aminolysis were compared. The increase of the carrier amino group content increased the ability to bind the protein (by glutaraldehyde method) without a proportional rise of enzyme activity. The comparative studies of immobilization effect and performance of the derivatives showed 40% crosslinked EGDMA, having an amino group content of 0.67 mmol g-3, to be the carrier of choice. This selected carrier was tested for active protein immobilization by use of crude acylase preparation. It was shown that up to a protein concentration of 9 mg cm-3 in the immobilization mixture the active protein was preferentially bound to the polymer matrix. It was indicated also that the derivative stability was mostly affected by multipoint attachment of the enzyme to the carrier surface.

Avidin-induced lysis of biotinylated erythrocytes by homologous complement via the alternative pathway depends on avidin's ability of multipoint binding with biotinylated membrane

It was reported that avidin and streptavidin induce lysis of prebiotinylated red blood cells via the alternative pathway of both homologous and heterologous complement. Both of these proteins have four biotin-binding sites, providing a polyvalent interaction with biotinylated components of the erythrocyte membrane. We have compared the effects of mono- and multipoint avidin attachment on the sensitivity of biotinylated erythrocytes to lysis by the complement system. In the presence of anti-avidin antibody, avidin-bearing biotinylated erythrocytes were rapidly lysed by heterologous serum. This lysis was independent from the mode of avidin attachment, implying that complement activation by the classical pathway triggered by interaction between Cl and avidin-bound antibody on the erythrocyte surface is independent from the avidin's ability of polyvalent (multipoint) binding with biotinylated membrane components. In the absence of anti-avidin antibody, biotinylated erythrocytes bearing polyvalently attached avidin were lysed by homologous complmment better than cells bearing avidin, which possesses reduced ability for multipoint binding with biotinylated erythrocyte. Two independent approaches to reduce avidin's ability of multipoint binding were used: decreased in surface density of biotin on the erythrocyte membrane and blockage of biotin-binding sites of avidin. Both methods result in reduced lysis of avidin-bearing erythrocytes as compared with erythrocytes bearing an equal amount of polyvalent-bound avidin. Thus the activation of homologous complement via the alternative pathway depends on avidin's ability to ‘cross-link’ to the biotinylated components of the erythrocyte membrane.

Immobilization‐stabilization of α‐chymotrypsin by covalent attachment to aldehyde‐agarose gels

We have developed a strategy for immobilization-stabilization of alpha-chymotrypsin by multipoint covalent attachment of the enzyme, through its amino groups, to agarosealdehyde gels. We have studied the role of the main variables that control the intensity of these enzyme-support multi-interaction processes (surface density of aldehyde groups in the activated gel, contact time between the immobilized enzyme and the activated support prior to borohydride reduction of the derivatives, etc.). In this way, we have prepared a number of very different chymotrypsinagarose derivatives. Our best derivatives, with the most intense multipoint attachment, were more stable than one-point attached derivatives and were more than 60,000-fold more stable than soluble enzyme in the absence of autolysis phenomena. In spite of the dramatic stabilization, the catalytic activity of these derivatives is little changed (they only lose 35% of intrinsic activity after this intense enzyme-support multi-interaction process). In addition, we have also demonstrated the very high capacity of 6% aldehyde-agarose gels to immobilize pure chymotrypsin (40 mg enzyme/mL catalyst). Furthermore, we have been able to establish a clear correlation between enzyme-support multipoint covalent attachment, stabilization against very different denaturing agents (heat, urea, organic cosolvents), and insensitivity of those immobilized chymotrypsin molecules to some activating agents.

Avidin attachment to biotinylated erythrocytes induces homologous lysis via the alternative pathway of complement

Noncovalent attachment of avidin to the membrane of prebiotinylated red blood cells (RBCs) induces lysis via the alternative pathway of complement (APC). Lysis is not species-dependent; RBCs from humans, rabbits, rats, and sheep were lysed with both autologous and all heterologous sera. Both biotinylated and native cells were not lysed. Lysis was observed at an avidin surface density of about 10(5) molecules per cell. Acylation of avidin prevents lysis and decreases the positive charge of the avidin. Lysis depends on the length of the cross-linking agent used for the biotin attachment to the membrane. An increase in the length of the cross-linking agent was accompanied by an enhancement of the lysis and the agglutination titer of biotinylated RBCs in a solution of avidin. It is suggested that avidin attachment induces some transformations of the cell membrane that lead to the conversion from "APC nonactivator" cells to "APC activator" cells. The interaction of avidin with membrane APC-restrictors (decay-accelerating factors, type 1 receptor for complement, homologous restriction factor, and others), the charge of avidin, and its cross-linking ability in lysis are discussed. It is proposed that membrane rearrangement induced by multipoint avidin attachment to biotinylated membrane is the main reason for avidin-induced elimination of APC restriction.

Avidin Attachment to Biotinylated Erythrocytes Induces Homologous Lysis Via the Alternative Pathway of Complement

AbstractNoncovalent attachment of avidin to the membrane of prebiotinylated red blood cells (RBCs) induces lysis via the alternative pathway of complement (APC). Lysis is not species-dependent; RBCs from humans, rabbits, rats, and sheep were lysed with both autologous and all heterologous sera. Both biotinylated and native cells were not lysed. Lysis was observed at an avidin surface density of about 105 molecules per cell. Acylation of avidin prevents lysis and decreases the positive charge of the avidin. Lysis depends on the length of the cross-linking agent used for the biotin attachment to the membrane. An increase in the length of the cross-linking agent was accompanied by an enhancement of the lysis and the agglutination titer of biotinylated RBCs in a solution of avidin. It is suggested that avidin attachment induces some transformations of the cell membrane that lead to the conversion from “APC nonactivator” cells to “APC activator” cells. The interaction of avidin with membrane APC-restrictors (decay-accelerating factors, type 1 receptor for complement, homologous restriction factor, and others), the charge of avidin, and its cross-linking ability in lysis are discussed. It is proposed that membrane rearrangement induced by multipoint avidin attachment to biotinylated membrane is the main reason for avidin-induced elimination of APC restriction.

G-protein-coupled receptor kinases

Rhodopsin kinase and the β-adrenergic receptor kinase (βARK) catalyse the phosphorylation of the activated forms of the G-protein-coupled receptors, rhodopsin and the β 2-adrenergic receptor (β 2AR), respectively. The interaction between receptor and kinase is independent of second messengers and appears to involve a multipoint attachment of kinase and substrate with the specificity being restricted by both the primary amino acid sequence and conformation of the substrate. Kinetic, functional and sequence information reveals that rhodopsin kinase and βARK are closely related, suggesting they may be members of a family of G-protein-coupled receptors kinases.

Multipoint attachment to a support protects enzyme from inactivation by organic solvents: α‐Chymotrypsin in aqueous solutions of alcohols and diols

Inactivation of alpha-chymotrypsin in aqueous solutions of alcohols and diols proceeds both reversibly and irreversibly. Reversible loss of the specific enzyme activity results from conformational changes (unfolding) of the enzyme detected by fluorescence spectroscopy. Multipoint covalent attachment to the matrix of polyacryl-amide gel by copolymerization method stabilizes alpha-chymotrypsin from denaturation by alcohols, the stabilizing effect increasing with the number of bonds between the protein and the support. Immobilization protects the enzyme also from irreversible inactivation by organic solvents resulting from bimolecular aggregation and autolysis.

Strategy for Stabilizing Enzymes Part One: Increasing Stability of Enzymes via their Multi-Point Interaction with a Support

This review states that the covalent multi-point attachment of enzymes to a support is the most general approach to stabilize them against different denaturing conditions, namely against their inactivation caused by protein unfolding. It is suggested that the change in the wavelength of the maximum emission in fluorescence spectra of a protein, resulting from its denaturation, can be used to evaluate a priori the effectiveness of stabilization. The copolymerization method of enzyme immobilization, as the most promising approach to stabilizing enzymes, is discussed in detail.

Molecular Recognition by Saccharides. Asymmetric Complexation between Bilirubin and Nucleosides

Abstract Asymmetric complexation between bilirubin (BR) and nucleosides has been studied in water (pH 10.8) by UV-vis absorption and circular dichroism (CD) spectroscopy. The complexes of BR with ribo- and deoxyribonucleosides show the plus to minus bisignated CD signals, which indicate that BR in these complexes selectively takes the (R)-helix conformation. Thymine and uracil arabinosides exhibit the oppositely signed CD spectra. A multi-point attachment model may explain the asymmetric complexation.