Cross-linking of alpha and gamma-thrombin to distinct binding sites on human platelets

The interaction of thrombin with proteins at the platelet surface was assessed by chemical cross-linking with the membrane-impermeable reagents bis(sulphosuccinimidyl)suberate and dithiobis(sulphosuccinimidyl propionate) under conditions which induced no modification of intracellular proteins and minimal cross-linking of membrane glycoproteins. The proteins covalently linked to 125I-labelled alpha and gamma-thrombin were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and crossed immunoelectrophoresis. 125I-alpha-thrombin was detected in high-molecular-mass complexes (a) at the top of a 3% acrylamide stacking gel and (b) with a Mr approximately equal to 400,000. In addition, two complexes of 240 kDa and 78 kDa were characterized. Hirudin prevented the formation of each of these complexes. The 78-kDa complex occurred spontaneously in the absence of bifunctional reagents, was only observed with active alpha-thrombin and was not dissociated by hirudin. Such characteristics are similar to those of a serpin serine-protease complex. The 240-kDa complex was formed with 0.8-100 nM alpha-thrombin, was observed after a short incubation time (30 s) and occurred with TosLysCH2Cl-inactivated alpha-thrombin. After analysis of Triton-X-100-soluble extracts of cross-linked platelets by crossed immunoelectrophoresis against a rabbit antiserum to platelets, two principal precipitates contained 125I-alpha-thrombin. These were a precipitate containing GPIIb-IIIa complexes and a precipitate in the position of GPIb. Indirect immunoprecipitation of GPIb, using a murine monoclonal antibody, confirmed it to be the major platelet component in the 240-kDa complex. Significantly, 125I-gamma-thrombin, which activates platelets with a prolonged lag phase, failed to bind to GPIb and complexes in the 240-kDa and 78-kDa molecular mass range were not observed. We conclude that several binding sites for alpha-thrombin are present at the platelet surface, and that GPIb is one of them. The studies with gamma-thrombin suggest that binding to GPIb is not obligatory for platelet activation although it could be involved in an initial step of the platelet response.     

Affinity labeling of high-affinity alpha-thrombin binding sites on the surface of hamster fibroblasts

The serine proteinase alpha-thrombin potently stimulates reinitiation of DNA synthesis in quiescent Chinese hamster fibroblasts (CCL39 line). 125I-labeled alpha-thrombin binds rapidly and specifically to CCL39 cells with high affinity (Kd approximately 4 nM). Binding at 37 degrees C was found to remain stable for 6 h or more during which time no receptor down-regulation, ligand internalization and/or degradation could be detected. The structure of alpha-thrombin receptors on CCL39 cells was identified by covalently coupling 125I-alpha-thrombin to intact cells using a homobifunctional cross-linking agent, ethylene glycol bis(succinimidyl succinate). By resolution in sodium dodecyl sulfate polyacrylamide gel electrophoresis we observed the specific labeling of a major alpha-thrombin-binding site of Mr approximately 150 000 revealed as a 125I-alpha-thrombin cross-linked complex of Mr approximately 180 000. Independent of chemical cross-linking, 125I-alpha-thrombin also formed a covalent complex with a minor, 35 000 Mr, membrane component identified as protease nexin. Two derivatives of alpha-thrombin modified at the active site are 1000-fold less than alpha-thrombin for mitogenicity. These two non-mitogenic derivatives bound to cells with similar affinity and maximal binding capacity as native alpha-thrombin, and affinity-labeled the receptor subunit of Mr 150 000. When present in large excess, during incubation of cells with alpha-thrombin, these binding antagonists were ineffective in blocking alpha-thrombin-induced DNA synthesis. These data suggest that the specific 150 000 Mr binding sites that display high affinity for alpha-thrombin do not mediate induction of the cellular mitogenic response.     

Photoaffinity labeling of GDP-fucose:nLcOse4Cer alpha 1----3-fucosyltransferase from human small cell lung carcinoma NCI-H69 cells with the GDP-fucose analog GDP-hexanolaminyl-4-azidosalicylic acid

An iodinatable photoactive analog of GDP-fucose, GDP-hexanolaminyl-4-azidosalicylic acid, has been prepared and applied to studies of the previously described alpha 1----3-fucosyltransferase from NCI-H69 cells (Holmes, E. H., Ostrander, G. K., and Hakomori, S. (1985) J. Biol. Chem. 260, 7619-7627). The NCI-H69 cell alpha 1----3-fucosyltransferase was obtained from a 0.2% Triton X-100-solubilized enzyme fraction after affinity purification on a GDP-hexanolamine-Sepharose column and gel filtration through a fast protein liquid chromatography Superose 12 column. Increasing concentrations of the photoaffinity reagent were found to result in loss of up to 35% of the original enzyme activity at under 100 microM final concentrations. The inactivation was photolysis dependent and could be prevented by the addition of GDP-fucose prior to photolysis. The photoprobe behaved as a competitive inhibitor with respect to GDP-fucose with a Ki of 23 microM, identical to that of GDP. Photoincorporation of 125I-labeled GDP-hexanolaminyl-4-azidosalicylic acid into the enzyme fraction labeled a slow migrating protein band in a native polyacrylamide gel which corresponded to enzyme activity. Inclusion of GDP-fucose prevented photolabeling of this band. Sodium dodecyl sulfate gel electrophoresis of the photolabeled, GDP-fucose-protected band yielded a 125I-labeled protein band that migrated at Mr 45,000, most probably corresponding to an alpha 1----3-fucosyltransferase protein subunit. These studies suggest photoaffinity labeling using nucleotide affinity ligands linked to photoactivatable, heterobifunctional cross-linking reagents may be generally applicable to photoaffinity labeling glycosyltransferase enzyme proteins.     

Molecular properties of neurotensin receptors in rat brain. Identification of subunits by covalent labeling

Neurotensin binding sites in rat brain synaptic membranes were specifically and covalently labeled by two methods. In the first, a photoreactive and highly radioactive analogue of neurotensin, 125I-labeled azidobenzoyl[Trp11]neurotensin, was synthesized and used to photoaffinity label neurotensin receptors. In the second, the reversible association between neurotensin receptors and 125I-labeled[Trp11]neurotensin, a radioactive but nonphotoreactive analogue of neurotensin, was made irreversible by means of disuccinimidyl suberate, a bifunctional cross-linking reagent. Analysis of synaptic membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that using both methods the same two protein bands with apparent molecular weights of 49,000 and 51,000 were specifically labeled. Identical results were obtained with or without reduction of the photolabeled membranes by beta-mercaptoethanol before electrophoresis. Variation of the ligand concentration did not modify the relative labeling intensities of the two bands, indicating that the high- and low-affinity neurotensin binding sites previously detected in rat brain synaptic membranes have similar molecular structures. These results indicate that neurotensin receptors in rat brain may be composed of two different protein subunits with similar molecular weight of about 50,000, that are linked together by noncovalent bonds.     

Covalent binding of thrombin to specific sites on corneal endothelial cells

Binding of 125I-labeled human alpha-thrombin to endothelial cells derived from bovine corneas was studied in tissue culture. Specific and saturable binding to the cell surface occurred at 37 degrees C but to a much smaller extent at 4 degrees C. Binding of [125I]thrombin to a specific site on these cells with formation of a 77000-dalton complex was demonstrated by NaDodSO4 (sodium dodecyl sulfate)-polyacrylamide gel electrophoresis. Binding of [125I]thrombin was blocked by a 100-fold excess of unlabeled alpha-thrombin and by the thrombin inhibitor, hirudin. There are approximately 100000 of these thrombin binding sites on the cell surface. Formation of the complex could be detected as early as 15 s, increased rapidly over the next 20-30 min, and then continued at a slower rate for the next 2.5 h. The catalytically active site of the enzyme was required for formation of the NaDodSO4-stable complex as shown by the inability of diisopropyl phosphorofluoride inactivated thrombin to form stable complexes with these cells. The complex was dissociated in NaDodSO4 with 1.0 M hydroxylamine, suggesting an acyl linkage of the enzyme to the cellular binding site. The thrombin-endothelial cell complex was distinct from the thrombin-antithrombin III complex (Mr approximately 90000) on gel electrophoresis, and its formation was not enhanced by heparin. Additional thrombin-cell complexes (Mr less than 77000) were also identified; however, they represent a small fraction of the total thrombin bound to the cells. These observations demonstrate that alpha-thrombin is capable of reacting specifically with corneal endothelial cells to form a NaDod-SO4-stable complex which requires the catalytically active enzyme.     

alpha-Conotoxins, small peptide probes of nicotinic acetylcholine receptors

alpha-Conotoxins, a family of small peptides from the venoms of the Conus marine moluscs, are selective, snake alpha-neurotoxin-competitive antagonists of the nicotinic acetylcholine receptor. A new alpha-conotoxin, SIA, has been purified, sequenced, and synthesized. Cross-linking with bivalent reagents and photoaffinity labeling of the acetylcholine receptor with alpha-conotoxin yield covalent adducts. Surprisingly, cross-linking to other subunits is considerably more efficient than to the alpha subunit. The relative efficiency of photoactivatable cross-linking to different subunits of the receptor is a function of placement of the photoactivatable group on the toxin. Since the structures of alpha-conotoxins can be solved by 2D NMR [see Pardi et al. (1989) Biochemistry 28, 5494-5508; Kobayashi et al. (1989) Biochemistry 28, 4853-4860], this family of toxins should provide a set of new ligands for probing the acetylcholine receptor with considerable precision.     

The effects of cell-released protease nexin on the measurement of thrombin binding to mouse cells

We previously showed that fibroblast-like cells release protease nexin into their growth medium. Protease nexin links to thrombin and mediates the cellular binding of thrombin via the protease nexin part of the complex to a site different from that for unlinked thrombin (1,2). To determine the effect that cell-released protease nexin had on the measurement of total cell-bound thrombin, we separately measured the cellular binding of both ¹²⁵I-thrombin and ¹²⁵I-thrombin-protease nexin complexes. Scatchard analysis of our binding data indicates that the cellular binding affinity of linked ¹²⁵I-thrombin is about 19-fold higher than that of unlinked ¹²⁵I-thrombin. We show that protease nexin acts to increase the apparent affinity of ¹²⁵I-thrombin for cellular binding sites.     

Identification of ligand binding site of phytosulfokine receptor by on-column photoaffinity labeling

Phytosulfokine (PSK), an endogenous 5-amino-acid-secreted peptide in plants, affects cellular potential for growth via binding to PSKR1, a member of the leucine-rich repeat receptor kinase (LRR-RK) family. PSK interacts with PSKR1 in a highly specific manner with a nanomolar dissociation constant. However, it is not known which residues in the PSKR1 extracellular domain constitute the ligand binding pocket. Here, we have identified the PSK binding domain of carrot PSKR1 (DcPSKR1) by photoaffinity labeling. We cross-linked the photoactivatable PSK analog [(125)I]-[N(epsilon)-(4-azidosalicyl)Lys(5)]PSK with DcPSKR1 using UV irradiation and mapped the cross-linked region using chemical and enzymatic fragmentation. We also established a novel "on-column photoaffinity labeling" methodology that allows repeated incorporation of the photoaffinity label to increase the efficiency of the photoaffinity cross-linking reactions. We purified a labeled DcPSKR1 tryptic fragment using anti-PSK antibodies and identified a peptide fragment that corresponds to the 15-amino-acid Glu(503)-Lys(517) region of DcPSKR1 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Deletion of Glu(503)-Lys(517) completely abolishes the ligand binding activity of DcPSKR1. This region is in the island domain flanked by extracellular LRRs, indicating that this domain forms a ligand binding pocket that directly interacts with PSK.     

Identification of a receptor for atrial natriuretic factor in rabbit aorta membranes by affinity cross-linking

Binding sites in rabbit aorta membranes for atrial natriuretic factor (ANF) have been specifically and covalently labeled by two methods. In the first, the photoreactive analog of ANF, 125I-azidobenzoyl-ANF, was synthesized and used to photoaffinity label ANF receptors. In the second, 125I-ANF was covalently attached to its binding site by treatment of the 125I-ANF-receptor complex with bifunctional cross-linking agents. Analysis of the labeled proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that by both methods the same three protein bands were labeled. These bands had apparent molecular masses of 60,000, 70,000, and 120,000 daltons. With the photoaffinity label, half-maximal inhibition of labeling of each of these bands was achieved when approximately 200 pM of unlabeled ANF was included in the binding assay. These results suggest that these three different polypeptides are specific components of ANF receptors in rabbit aorta membranes.     

Activation of platelets by alpha-thrombin is a receptor-mediated event. D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-thrombin, but not N alpha-tosyl-L-lysine chloromethyl ketone-thrombin, binds to the high affinity thrombin receptor

Competition binding studies have been carried out to evaluate the antagonism of TLCK-thrombin (N alpha-tosyl-L-lysine chloromethyl ketone-treated thrombin) and PPACK-thrombin (D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-treated thrombin) with alpha-thrombin using computer-assisted analysis of the binding isotherms (LIGAND). alpha-Thrombin bound to high, moderate, and low affinity sites as previously described (Harmon, J. T., and Jamieson, G. A. (1985) Biochemistry 24, 58-64). PPACK-thrombin bound to all three sites accessible to alpha-thrombin (K1, 7 nM; R1, 20 sites/platelet; K2, 3 nM; R2, 1800 sites/platelet; K3, 510 nM; R3, 84,000 sites/platelet) as well as to a separate fourth site (Kx, 0.4 nM; Rx, 20 sites/platelet) for PPACK-thrombin that was not accessible to alpha-thrombin. In contrast, TLCK-thrombin did not bind to the high affinity site for alpha-thrombin but bound to the moderate and low affinity sites for alpha-thrombin with similar affinity (K2, 2 nM; R2, 890 sites/platelet; K3, 900 nM; R3, 100,000 sites/platelet) and to another site (Ky, 0.03 nM; Ry, 10 sites/platelet) which was not accessible to alpha-thrombin. As predicted from these binding studies, TLCK-thrombin did not compete with alpha-thrombin for platelet activation at concentrations as high as 1000 nM (500-fold excess). In contrast a 300-fold excess of PPACK-thrombin (670 nM) totally inhibited platelet activation by 2 nM thrombin. These results demonstrate that the high affinity binding site for thrombin on human platelets is a classical receptor, occupancy of which is necessary for platelet activation by low concentrations of thrombin; that TLCK-thrombin does not occupy this high affinity site and hence cannot inhibit platelet activation by alpha-thrombin; and that PPACK-thrombin does compete with alpha-thrombin at the high affinity site and is an antagonist of alpha-thrombin induced activation.     

Effect of cell density on thrombin binding to a specific site on bovine vascular endothelial cells

We studied thrombin binding to proliferating and confluent endothelial cells derived from bovine vascular endothelium. [125]thrombin was incubated with nonconfluent or confluent endothelial cells and both the total amount bound and the amount linked in a 77,000-dalton thrombin- cell complex were determined. Approximately 230,000 molecules of thrombin bound per cell in nonconfluent cultures compared to 12,800 molecules per cell in confluent cultures. Approximately 67,7000 thrombin molecules were bound in an apparently covalent complex, Mr = 77,000, with each cell in sparse cultures, whereas only 4,600 thrombin molecules per cell were bound in this complex with confluent cultures. Similar studies with [125I]thrombin and endothelial cells derived from bovine cornea revealed no difference either in the total amount of thrombin bound or in the amount bound in the 77,000-dalton complex using sparse or confluent cultures. When confluent vascular endothelial cultures were wounded, additional cellular binding sites for the 77,000- dalton complex with thrombin appeared within 24 h. A 237% increase in the amount of thrombin bound to these sites was induced by a wound which resulted in a 20% decrease in cell number in the monolayer. There was no significant increase in thrombin binding to other cellular sites at 24 h. These experiments provide evidence that the first change in thrombin binding after injury is an increase in the cellular sites involved in the 77,000-dalton complex, and suggest that thrombin binding to endothelial cells may be important in the vascular response to injury.     

Platelet-derived growth factor. III. Identification of a platelet-derived growth factor receptor by affinity labeling

Two homobifunctional cross-linking reagents have been used to cross-link 125I-platelet-derived growth factor (PDGF) to a cell surface component with an approximate Mr = 164,000 that has many characteristics of a specific PDGF receptor. Excess unlabeled PDGF competed for labeling of this component, while high concentrations of fibroblast growth factor, insulin, epidermal growth factor, low density lipoprotein or acetylated low density lipoprotein had no effect. Preincubation of cells with 125I-PDGF at 37 degrees C reduced specific 125I-PDGF binding (down regulation) and produced a parallel decrease in the amount of the 164,000-dalton receptor available for labeling. The 164,000-dalton component contains at least some protein that is accessible to trypsin in the extracellular medium. A complex of comparable Mr is seen on all PDGF-responsive cell types examined, but not on a nonresponsive cell type. 125I-PDGF does not become covalently cross-linked to this component in the absence of a cross-linking reagent.     

Evidence that both exosites on thrombin participate in its high affinity interaction with fibrin

Exosite 1 on thrombin mediates low affinity binding to sites on the NH2 termini of the alpha- and beta-chains of fibrin. A subpopulation of fibrin molecules (gammaA/gamma'-fibrin) has an alternate COOH terminus of the normal gamma-chain (gammaA/gammaA-fibrin) that binds thrombin with high affinity. To determine the roles of exosites 1 and 2 in the high affinity interaction of thrombin with gammaA/gamma'-fibrin, binding studies were done with thrombin variants and exosite 1- or 2-directed ligands. alpha-Thrombin bound gammaA/gamma'-fibrin via high and low affinity binding sites. A peptide analog of the COOH terminus of the gamma'-chain that binds alpha-thrombin via exosite 2 blocked the high affinity binding of alpha-thrombin to gammaA/gamma'-fibrin, suggesting that the interaction of alpha-thrombin with the gamma'-chain is exosite 2-mediated. In support of this concept, (a) gamma-thrombin, which lacks a functional exosite 1, bound to gammaA/gamma'-fibrin, but not to gammaA/gammaA-fibrin; (b) thrombin R93A/R97A/R101A, an exosite 2-defective variant, bound only to gammaA/gamma'-fibrin via low affinity sites; and (c) exosite 2-directed ligands reduced alpha-thrombin binding to gammaA/gamma'-fibrin. However, several lines of evidence indicate that exosite 1 contributes to the high affinity interaction of thrombin with gammaA/gamma'-fibrin. First, the affinity of gamma-thrombin for gammaA/gamma'-fibrin was lower than that of alpha-thrombin. Second, removal of a low affinity binding site on the beta-chain of gammaA/gamma'-fibrin reduced its affinity for alpha-thrombin. Third, exosite 1-directed ligands reduced alpha-thrombin binding to gammaA/gamma'-fibrin. Taken together, these data suggest that, although exosite 2 mediates the interaction of thrombin with the gamma'-chain of gammaA/gamma'-fibrin, simultaneous ligation of exosite 1 by low affinity binding sites is essential for the high affinity interaction of thrombin with gammaA/gamma'-fibrin.     

Monoclonal antibody to the thrombin receptor stimulates DNA synthesis in combination with gamma-thrombin or phorbol myristate acetate

Studies with various thrombin derivatives have shown that initiation of cell proliferation by thrombin requires two separate types of signals: one, generated by high affinity interaction of thrombin or DIP-thrombin (alpha-thrombin inactivated at ser 205 of the B chain by diisopropylphosphofluoridate) with receptors and the other, by thrombin's enzymic activity. To further study the role of high affinity thrombin receptors in initiation, we immunized mice with whole human fibroblasts and selected antibodies that blocked the binding of 125I- thrombin to high affinity receptors on hamster fibroblasts. One of these antibodies, TR-9, inhibits from 80 to 100% of 125I-thrombin binding, exhibits an immunofluorescent pattern indistinguishable from that of thrombin bound to receptors on these cells, and selectively binds solubilized thrombin receptors. By itself, TR-9 did not initiate DNA synthesis nor did it block thrombin initiation, but TR-9 addition to cells in the presence of alpha-thrombin, gamma-thrombin (0.5 microgram/ml), or PMA stimulated thymidine incorporation up to threefold over controls. In all cases, maximal stimulation was observed at concentrations of TR-9, ranging from 1 to 4 nM corresponding to concentrations required to inhibit from 30 to 100% of 125I-thrombin binding. These results demonstrate that the binding of the monoclonal antibody to the alpha-thrombin receptor can mimic the effects of thrombin's high affinity interaction with this receptor in stimulating cell proliferation.     

Design,synthesis and evaluation of photoactivatable derivatives of microtubule (MT)-active [1,2,4]triazolo[1,5-a]pyrimidines

The [1,2,4]triazolo[1,5-a]pyrimidines comprise a promising class of non-naturally occurring microtubule (MT)-active compounds. Prior studies revealed that different triazolopyrimidine substitutions can yield molecules that either promote MT stabilization or disrupt MT integrity. These differences can have important ramifications in the therapeutic applications of triazolopyrimidines and suggest that different analogues may exhibit different binding modes within the same site or possibly interact with tubulin/MTs at alternative binding sites. To help discern these possibilities, a series of photoactivatable triazolopyrimidine congeners was designed, synthesized and evaluated in cellular assays with the goal of identifying candidate probes for photoaffinity labeling experiments. These studies led to the identification of different derivatives that incorporate a diazirine ring in the amine substituent at position 7 of the triazolopyrimidine heterocycle, resulting in molecules that either promote stabilization of MTs or disrupt MT integrity. These photoactivatable candidate probes hold promise to investigate the mode of action of MT-active triazolopyrimidines.     

Disulfides of the lutropin receptor

Affinity cross-linking of the lutropin receptor with 125I-human choriogonadotropin (hCG) on porcine granulosa cells produced four distinct homone-receptor complexes under reducing conditions. They contain 18-, 24-, 28-, and 34-kDa components (Ji, I., Bock, J. H., and Ji, T. H. (1985) J. Biol. Chem. 260, 12815-12821). Photoaffinity labeling and cross-linking produced 136-, 102-, and 74-kDa hCG-receptor complexes under reducing conditions and the 136-kDa complex under nonreducing conditions. In addition, the unreduced 102-kDa complex was seen in photoaffinity labeling but not in cross-linking. When the unreduced 136-kDa complex was reduced, the 102- and 74-kDa complexes were generated, indicating release of the 34- and the 28-kDa components in two steps. When the unreduced 102-kDa complex was reduced, the 74-kDa complex was produced, indicating the release of a 28-kDa component. The 74-kDa complex could not be reduced but was cleaved by alkaline treatment to produce the hCG alpha beta dimer. The results indicate that the 24-kDa component is released from the 74-kDa complex, since the apparent mass of the hCG alpha beta dimer on gels is 50 kDa. The 24-kDa component appears to be the initial site for photoaffinity labeling or cross-linking and to be disulfide linked to the 28-kDa component which is in turn disulfide linked to the 34-kDa component. These intercomponent disulfides exist in some receptors but not all. Formation of the disulfide-linked 136-kDa band required the presence of a sulfhydryl-blocking agent, N-ethylmaleimide. In particular, the 34-kDa component was vulnerable to reduction. There was no significant evidence of disulfides between the hormone and any of the receptor components.     

Identification of a high-affinity binding protein for N-acetylchitooligosaccharide elicitor in the plasma membrane of suspension-cultured rice cells by affinity labeling

A high-affinity binding protein for the N-acetylchito-oligosaccharide elicitor of phytoalexin biosynthesis was identified by photoaffinity labeling and affinity cross-linking in the plasma membrane of suspension-cultured rice cells. Both a [¹²⁵I]-labeled photolabile 2-(4-azidophenyl)ethylamino conjugate ([¹²⁵I]-GN₈-AzPEA) and a [¹²⁵I]-labeled 2- (4-aminophenyl)ethylamino conjugate ([¹²⁵]-GN₈-APEA) of N-acetylchito-octaose were synthesized. The two conjugates were separately incubated with the plasma membrane prepared by aqueous two-phase partitioning, and covalently cross-linked to the elicitor binding site by irradiation with UV light or treatment with the cross-linking agent glutaraldehyde, respectively. Autoradiography of the SDS-PAGE gel of the solubilized membrane proteins revealed the labeling of a single 75 kDa band in both cases. The incorporation of the radiolabeled ligands into the 75 kDa protein showed a saturable mode of binding, with half-maximal incorporation at 45 and 52 nM for photoaffinity labeling and affinity cross-linking, respectively. The labeling of the 75 kDa protein was inhibited by N-acetylchito-oligasaccharides in a size-dependent manner, and N-acetylchito-octaose (GlcNAc)₈ showed a half-maximal inhibition at concentrations of the order of 10 nM. However, neither chito-octaose (GlcN)₈, cellopentaose nor α-1,4 linked N-acetylgalactosamine octamer (GalNAc)₈ at concentrations as high as 25 μM inhibited the labeling of the 75 kDa protein. These results are in good agreement with the sensitivity and the specificity of the ‘high-affinity binding site’ previously identified by binding assays, as well as with the activities of these oligosaccharides in the induction of phytoalexin biosynthesis and other cellular responses. These results suggest that the 75 kDa protein identified by the affinity labeling represents a functional receptor for this elicitor.     

Synthesis and use of bifunctional chloromethylalkanedione derivatives of variable chain length for cross-linking thiol groups in oligomeric proteins. Specific cross-linking in glyceraldehyde 3-phosphate dehydrogenase

Bischloromethylpentanedione, bischloromethylhexanedione, bischloromethyloctanedione and bischloromethyldecanedione were synthesized from their corresponding dicarboxylic acids via the bis-acyl chloride and the bisdiazomethylketone derivatives. These compounds proved to be highly specific cross-linking reagents for rabbit skeletal-muscle glyceraldehyde 3-phosphate dehydrogenase. Incubation of the enzyme with cross-linking reagents resulted in both a time- and concentration-dependent formation of covalently linked oligomeric structures. The major cross-linked product detected by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was the dimer (mol. wt. 72000). Sepharose 6B chromatography of the cross-linked enzyme showed that it still existed as the tetramer. Cross-linking was dependent on the native structure of the enzyme, since it was abolished on denaturation of the enzyme. The actual covalently linked product depends on the conditions of modification and the chain length of the reagent. The maximum yield of dimer (70-80%) was obtained with bischloromethylhexanedione, and the yield decreased with either shorter- or longer-chain compounds. The calculated distance between the two reactive points in bischloromethylhexanedione is 1.21-1.45nm. Bischloromethylhexanedione modified at least two thiol groups per monomer. Modification of the active-site thiol, cysteine-149, was not essential for cross-linking, since glyceraldehyde 3-phosphate dehydrogenase carboxymethylated on cysteine-149 still reacted to form the dimer. The rate of chemical cross-linking was markedly decreased by increasing the NAD(+) occupancy of the enzyme active sites. These experiments are discussed in terms of the asymmetry of the enzyme structure in solution.     

Thrombin-induced increase in albumin permeability across the endothelium

We studied the effect of thrombin on albumin permeability across the endothelial monolayer in vitro. Bovine pulmonary artery endothelial cells were grown on micropore membranes. Morphologic analysis confirmed the presence of a confluent monolayer with interendothelial junctions. Albumin permeability was measured by the clearance of 125I-albumin across the endothelial monolayer. The control 125I-albumin clearance was 0.273 +/- 0.02 microliter/min. The native enzyme, alpha-thrombin (10(-6) to 10(-10) M), added to the luminal side of the endothelium produced concentration-dependent increases in albumin clearance (maximum clearance of 0.586 +/- 0.08 microliter/min at 10(-6) M). Gamma (gamma) thrombin (10(-6) M and 10(-8) M), which lacks the fibrinogen recognition site, also produced a concentration-dependent increase in albumin clearance similar to that observed with alpha-thrombin. Moreover, the two proteolytically inactive forms of the native enzyme, i-Pr2 P-alpha-thrombin and D-Phe-Pro-Arg-CH2-alpha-thrombin, increased the 125I-albumin clearance (0.610 +/- 0.09 microliter/min and 0.609 +/- 0.02 microliter/min for i-Pr2 P-alpha-thrombin and D-Phe-Pro-Arg-CH2-alpha-thrombin at 10(-6) M, respectively). Since the modified forms of thrombin lack the fibrinogen recognition and active serine protease sites, the results indicate that neither site is required for increased albumin permeability. The increase in albumin clearance with alpha-thrombin was not secondary to endothelial cell lysis because lactate dehydrogenase concentration in the medium following thrombin was not significantly different from baseline values. There was also no morphological evidence of cell lysis. Moreover, the increase in 125I-albumin clearance induced by alpha-thrombin was reversible by washing thrombin from the endothelium. The basis for the increased albumin permeability following the addition of alpha-thrombin appears to be a reversible change in endothelial cell shape with formation of intercellular gaps.