Opioids trigger alpha 5 beta 1 integrin-mediated monocyte adhesion

Inflammatory reactions involve a network of chemical and molecular signals that initiate and maintain host response. In inflamed tissue, immune system cells generate opioid peptides that contribute to potent analgesia by acting on specific peripheral sensory neurons. In this study, we show that opioids also modulate immune cell function in vitro and in vivo. By binding to its specific receptor, the opioid receptor-specific ligand DPDPE triggers monocyte adhesion. Integrins have a key role in this process, as adhesion is abrogated in cells treated with specific neutralizing anti-alpha5beta1 integrin mAb. We found that DPDPE-triggered monocyte adhesion requires PI3Kgamma activation and involves Src kinases, the guanine nucleotide exchange factor Vav-1, and the small GTPase Rac1. DPDPE also induces adhesion of pertussis toxin-treated cells, indicating involvement of G proteins other than Gi. These data show that opioids have important implications in regulating leukocyte trafficking, adding a new function to their known effects as immune response modulators.     

Urokinase receptors are required for alpha 5 beta 1 integrin-mediated signaling in tumor cells

Up-regulation of urokinase receptors is common during tumor progression and thought to promote invasion and metastasis. Urokinase receptors bind urokinase and a set of beta1 integrins, but it remains unclear to what degree urokinase receptor/integrin binding is important to beta1 integrin signaling. Using site-directed mutagenesis, single amino acid mutants of the urokinase receptor were identified that fail to associate with either alpha3beta1 (D262A) or alpha5beta1 (H249A) but associate normally with urokinase. To study the effects of these mutations on beta1 integrin function, endogenous urokinase receptors were first stably silenced in tumor cell lines HT1080 and H1299, and then wild type or mutant receptors were expressed. Knockdown of urokinase receptors resulted in markedly reduced fibronectin and alpha5beta1-dependent ERK activation and metalloproteinase MMP-9 expression. Re-expression of wild type or D262A mutant receptors but not the alpha5beta1 binding-deficient H249A mutant reconstituted fibronectin responses. Because urokinase receptor.alpha5beta1 complexes bind in the fibronectin heparin-binding domain (Type III 12-14) whereas alpha5beta1 primarily binds in the RGD-containing domain (Type III 7-10), signaling pathways leading to ERK and MMP-9 responses were dissected. Binding to III 7-10 led to Src/focal adhesion kinase activation, whereas binding to III 7-14 caused Rac 1 activation. Tumor cells engaging fibronectin required both Type III 7-10- and 12-14-initiated signals to activate ERK and up-regulate MMP-9. Thus urokinase receptor binding to alpha5beta1 is required for maximal responses to fibronectin and tumor cell invasion, and this operates through an enhanced Src/Rac/ERK signaling pathway.     

Differences in phosphatase modulation of alpha4beta1 and alpha5beta1 integrin-mediated adhesion and migration of B16F1 cells.

It is well established that a biphasic relationship exists between the adhesive strength of beta1 integrins and their ability to mediate cell movement. Thus, cell movement increases progressively with adhesive strength, but beyond a certain point of optimal interaction, cell movement is reduced with further increases in adhesive function. The interplay between the various kinase and phosphatase activities provides the balance in beta1 integrin-mediated cell adhesion and migration. In the present study, the significance of protein tyrosine phosphatases (PTP) and ser/thr protein phosphatases (PP) in alpha4beta1 and alpha5beta1 integrin-mediated mouse melanoma B16F1 cell anchorage and migration on fibronectin was characterized using phosphatase inhibitors. At low fibronectin concentration, alpha5beta1 functioned as the predominant receptor for cell movement; a role for alpha4beta1 in B16F1 cell migration increased progressively with fibronectin concentration. Treatment of B16F1 cells with PTP inhibitors, sodium orthovanadate (Na3VO4) and phenylarsine oxide (PAO), or PP-1/2A inhibitor, okadaic acid (OA), abolished cell movement. Inhibition of cell movement by PAO and OA was associated by a reduction in the adhesive strength of alpha4beta1 and alpha5beta1. In contrast, treatment of B16F1 cells with Na3VO4 resulted in selective stimulation of the adhesive function of alpha5beta1, but not alpha4beta1. Therefore, our results demonstrate that (i) both PTP and PP-1/2A have roles in cell movement, (ii) modulation of cell movement by PTP and PP-1/2A may involve either a stimulation or reduction of beta1 integrin adhesive strength, and (iii) distinct phosphatase-mediated signaling pathways for differential regulation of the various beta1 integrins exist.     

Carbohydrate-carbohydrate binding of ganglioside to integrin alpha(5) modulates alpha(5)beta(1) function

Gangliosides GT1b and GD3, components of keratinocyte membranes, inhibit keratinocyte adhesion to fibronectin. Although ganglioside sialylation is known to be important, the mechanism of inhibition is unknown. Using purified insect recombinant alpha(5) and beta(1) proteins and alpha(5)beta(1) integrin from lysed keratinocyte-derived SCC12 cells, we have shown that GT1b and GD3 inhibit the binding of alpha(5)beta(1) to fibronectin. Co-immunoprecipitation of GT1b and alpha(5)beta(1) from SCC12 cells and direct binding of GT1b and GD3 to affinity-purified alpha(5)beta(1) from SCC12 cells and insect recombinant alpha(5)beta(1), particularly the alpha(5) subunit, further suggest interaction between ganglioside and alpha(5)beta(1). The carbohydrate moieties of integrin appear to be critical since gangliosides are unable to bind deglycosylated forms of alpha(5)beta(1) from SCC12 and insect cells or poorly glycosylated recombinant alpha(5)beta(1) from Escherichia coli cells. The GT1b-alpha(5)beta(1) interaction is inhibited by concanavalin A, suggesting that GT1b binds to mannose structures in alpha(5)beta(1). The preferential binding of GT1b to high mannose rather than reduced mannose ovalbumin further implicates the binding of GT1b to mannose structures. These data provide evidence that highly sialylated gangliosides regulate alpha(5)beta(1)-mediated adhesion of epithelial cells to fibronectin through carbohydrate-carbohydrate interactions between GT1b and the alpha(5) subunit of alpha(5)beta(1) integrin.     

Structural requirements of echistatin for the recognition of alpha(v)beta(3) and alpha(5)beta(1) integrins

There are key differences between the amino acid residues of the RGD loops and the C termini of echistatin, a potent antagonist of alpha(IIb)beta(3), alpha(v)beta(3) and alpha(5)beta(1), and eristostatin, a similar disintegrin selectively inhibiting alpha(IIb)beta(3). In order to identify echistatin motifs required for selective recognition of alpha(v)beta(3) and alpha(5)beta(1) integrins, we expressed recombinant echistatin, eristostatin, and 15 hybrid molecules. We tested them for their ability to inhibit adhesion of different cell lines to fibronectin and von Willebrand factor and to express ligand-induced binding site epitope. The results showed that Asp(27) and Met(28) support recognition of both alpha(v)beta(3) and alpha(5)beta(1). Replacement of Met(28) with Asn completely abolished echistatin's ability to recognize each of the integrins, while replacement of Met(28) with Leu selectively decreased echistatin's ability to recognize alpha(5)beta(1) only. Eristostatin in which C-terminal WNG sequence was substituted with HKGPAT exhibited new activity with alpha(5)beta(1), which was 10-20-fold higher than that of wild type eristostatin. A hypothesis is proposed that the C terminus of echistatin interacts with separate sites on beta(1) and beta(3) integrin molecules.     

TGF-beta 1 modulated the expression of alpha 5 beta 1 integrin and integrin-mediated signaling in human hepatocarcinoma cells

Integrins are a family of cell surface adhesion molecules which mediate cell adhesion and initiate signaling pathways that regulate cell spreading, migration, differentiation, and proliferation. TGF-beta is a multifunctional factor that induces a wide variety of cellular processes. In this study, we show that, TGF-beta 1 treatment enhanced the amount of alpha 5 beta 1 integrin on cell surface, the mRNA level of alpha 5 subunit, and subsequently stimulated cell adhesion onto a fibronectin (Fn) and laminin (Ln) matrix in SMMC-7721 cells. TGF-beta 1 could also promote cell migration. Furthermore, our results showed that TGF-beta1 treatment stimulated the tyrosine phosphorylation level of FAK, which can be activated by the ligation and clustering of integrins. PTEN can directly dephosphorylate FAK, and the results that TGF-beta 1 could down-regulate PTEN at protein level suggested that TGF-beta 1 might stimulate FAK phosphorylation through increasing integrin signaling and reducing dephosphorylation of FAK. These studies indicated that TGF-beta 1 and integrin-mediated signaling act synergistically to enhance cell adhesion and migration and affect downstream signaling molecules of hepatocarcinoma cells.     

Induction of alpha v beta 3 integrin-mediated attachment to extracellular matrix in beta 1 integrin (CD29)-negative B cell lines.

beta 1 integrin containing complexes have been implicated as the primary adhesion structures in many lymphocyte extracellular matrix (ECM) interactions. However, many B lymphocytes lack surface expression of the beta 1 subunit, implying that this subpopulation of lymphoid cells must employ alternate adhesion structures if they are to maintain an interactive capacity with ECM. An examination of the adherence properties of the beta 1 integrin-negative B cell line JY indicated that these cells exhibit little or no basal adherence to any of the ECM components examined. However, these cells could be induced to adhere to the ECM components fibronectin, laminin, and vitronectin following treatment with PMA. Blocking studies with monoclonal antibodies indicated the alpha v beta 3 integrin complex was involved in the attachment to each of these ligands. However, the adherence to fibronectin displayed a complex pattern of inhibition suggesting the involvement of other ECM receptors. The utilization of the alpha v beta 3 complex was not unique to the JY cell line. Other B cell lines were observed to employ alpha v beta 3, and these lines similarly lacked expression of beta 1 integrin. These results indicate that alpha v beta 3 can act as a lymphoid ECM-adhesion structure which may provide an alternative means for lymphocytes to interact with ECM. Furthermore, these studies provide evidence for the presence of lymphoid-associated alpha v beta 3 integrins with regulatable activity, which contrasts with the constitutive adhesive potential of these complexes when present on other cell types.     

Determinants of ligand binding specificity of the alpha(1)beta(1) and alpha(2)beta(1) integrins.

The alpha(1)beta(1) and alpha(2)beta(1) integrins are cell surface collagen receptors. Cells expressing the alpha(1)beta(1) integrin preferentially adhere to collagen IV, whereas cells expressing the alpha(2)beta(1) integrin preferentially adhere to collagen I. Recombinant alpha(1) and alpha(2) integrin I domains exhibit the same collagen type preferences as the intact integrins. In addition, the alpha(2) integrin I domain binds echovirus 1; the alpha(1) I domain does not. To identify the structural components of the I domains responsible for the varying ligand specificities, we have engineered several alpha(1)/alpha(2) integrin I domain chimeras and evaluated their virus and collagen binding activities. Initially, large secondary structural components of the alpha(2) I domain were replaced with corresponding regions of the alpha(1) I domain. Following analysis in echovirus 1 and collagen binding assays, chimeras with successively smaller regions of alpha(1) I were constructed and analyzed. The chimeras were analyzed by ELISA with several different alpha(2) integrin monoclonal antibodies to assess their proper folding. Three different regions of the alpha(1) I domain, when present in the alpha(2) I domain, conferred enhanced collagen IV binding activity upon the alpha(2) I domain. These include the alpha3 and alpha5 helices and a portion of the alpha6 helix. Echovirus 1 binding was lost in a chimera containing the alphaC-alpha6 loop; higher resolution mapping identified Asn(289) as playing a critical role in echovirus 1 binding. Asn(289) had not been implicated in previous echovirus 1 binding studies. Taken together, these data reveal the existence of multiple determinants of ligand binding specificities within the alpha(1) and alpha(2) integrin I domains.     

Two synergistic activation mechanisms of alpha2beta1 integrin-mediated collagen binding

Activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate (TPA) induces ligand-independent aggregation of a cell surface collagen receptor, alpha2beta1 integrin. Concomitantly, TPA increases the avidity of alpha2beta1 for collagen and the number of conformationally activated alpha2beta1 integrins. The structural change was shown using a monoclonal antibody 12F1 that recognizes the "open" (active) conformation of the inserted domain in the alpha2 subunit (alpha2I). Amino acid residue Glu-336 in alpha2 subunit is proposed to mediate the interaction between alpha2I domain and beta1 subunit. Glu-336 seems to regulate a switch between open and "closed" conformations, since the mutation alpha2E336A inhibited the TPA-related increase in the number of 12F1 positive integrins. E336A also reduced cell adhesion to collagen. However, E336A did not prevent the TPA-related increase in adhesion to collagen or alpha2beta1 aggregation. Thus, alpha2beta1 integrin avidity is regulated by two synergistic mechanisms, first an alpha2E336-dependent switch to the open alpha2I conformation, and second an alpha2E336-independent mechanism temporally associated with receptor aggregation.     

CD44 cross-linking induces integrin-mediated adhesion and transendothelial migration in breast cancer cell line by up-regulation of LFA-1 (alpha L beta2) and VLA-4 (alpha4beta1)

CD44, a widely expressed cell surface glycoprotein, plays a major role in cell-cell adhesion, cell-substrate interaction, lymphocyte homing, and tumor metastasis. For tumor metastasis to occur through the blood vessel and lymphatic vessel pathway, the tumor cells must first adhere to endothelial cells. Recent studies have shown that high expression of CD44 in certain types of tumors is associated with the hematogenic spread of cancer cells. However, the functional relevance of CD44 to tumor cell metastasis remains unknown. In this study, we investigated the mechanisms of CD44 cross-linking-induced adhesion and transendothelial migration of tumor cells using MDA-MB-435S breast cancer cell line. Breast cancer cells were found to express high levels of CD44. Using flow cytometric analysis and immunofluorescence staining, we demonstrated that cross-linking of CD44 resulted in a marked induction of the expression of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) by exocytosis. These results were also observed with the Hs578T breast cancer cell line. Furthermore, LFA-1- and VLA-4-mediated adhesion and transendothelial cancer cell migration were also studied. Anti-LFA-1 mAb or anti-VLA-4 mAb alone had no effect on adhesion or transendothelial cancer cell migration, but were able to inhibit both of these functions when added together. This shows that CD44 cross-linking induces LFA-1 and VLA-4 expression in MDA-MB-435S cells and increases integrin-mediated adhesion to endothelial cells, resulting in the transendothelial migration of breast cancer cells. These observations provide direct evidence of a new function for CD44 that is involved in the induction of LFA-1 and VLA-4 expression by exocytosis in MDA-MB-435S cells. Because these induced integrins promote tumor cell migration into the target tissue, it may be possible to suppress this by pharmacological means, and thus potentially cause a reduction in invasive capability and metastasis.     

Specific and dual antagonists of alpha(4)beta(1) and alpha(4)beta(7) integrins.

N-(3,5-Dichlorophenylsulfonyl)-(R)-thioprolyl biarylalanine 10a has been identified as a potent and specific antagonist of the alpha(4)beta(1) integrin. Altering the configuration of thioproline from R to S led to a series of dual antagonists of alpha(4)beta(1) and alpha(4)beta(7), and the N-acetyl analogue 8b was found to be the most potent dual antagonist. A binding site model for alpha(4)beta(1) and alpha(4)beta(7) is proposed to explain the structure-activity relationship.     

Adenosine suppresses alpha(4)beta(7) integrin-mediated adhesion of T lymphocytes to colon adenocarcinoma cells

The interaction of T lymphocytes with tumor cells, a key step in the antitumor immune response, is suppressed by adenosine, a nucleoside produced at increased levels within the hypoxic tumor environment. We have explored the mechanism by which adenosine interferes with the lymphocyte:tumor cell interaction. The adhesion of anti-CD3-stimulated T cells to syngeneic MCA-38 mouse colon adenocarcinoma cells did not involve LFA-1 (alpha(L)beta(2)) or VLA-5 (alpha(5)beta(1)). However, antibodies against either lymphocyte alpha(4) or beta(7) (but not beta(1)) integrin subunits, or against VCAM-1 on the tumor cells, significantly suppressed adhesion, showing that the recognition of MCA-38 cells by T cells is strongly dependent upon the association of alpha(4)beta(7) on the effector cells with VCAM-1 on the tumor targets. This association is modulated by adenosine: The ability of adenosine to suppress T cell adhesion to MCA-38 cells was lost if alpha(4)beta(7) was functionally blocked with anti-alpha(4) antibodies (i) prior to or (ii) during the adhesion assay or if (iii) alpha(+)(4) cells were depleted from the T lymphocyte population. The binding of T cells to fibronectin through alpha(4)beta(1) was not suppressed by adenosine. We conclude that adenosine partially inhibits the interaction of T lymphocytes with tumor cells by blocking the function of integrin alpha(4)beta(7).     

Distinct determinants on collagen support alpha 2 beta 1 integrin-mediated platelet adhesion and platelet activation.

Recent studies have revealed that the sequence of amino acids asp-gly-glu-ala represents an essential determinant of the site within the alpha 1(I)-CB3 fragment of collagen recognized by the alpha 2 beta 1 integrin cell surface collagen receptor (Staatz et al., 1991). Studies employing chemical modifications of collagen amino acid side chains confirm both the essential nature of the acidic side chains of aspartic acid and glutamic acid residues and the nonessentiality of lysine epsilon-amino groups in supporting adhesion mediated by the alpha 2 beta 1 integrin. The approach also indicates the presence of a distinct determinant on collagen separate from the alpha 2 beta 1 recognition site that contains essential lysine side chains and that is necessary for subsequent interactions with the platelet surface that give rise to collagen-induced platelet activation and secretion. The two-step, two-site model for cellular signaling involving both an integrin and a signal-transducing coreceptor suggested by these data may be common to other integrin-mediated processes.     

Circulating integrins: alpha 5 beta 1, alpha 6 beta 4 and Mac-1, but not alpha 3 beta 1, alpha 4 beta 1 or LFA-1.

The alpha 5 beta 1, alpha 6 beta 4 and Mac-1 integrins all participate in the endocytotic cycle. By contrast, alpha 3 beta 1, alpha 4 beta 1 and LFA-1 do so much more slowly, or not at all, in the cell lines examined. This indicates that the alpha-chains appear to determine whether an integrin cycles or not, and that alpha 5 beta 1, alpha 6 beta 4 and Mac-1 can be brought to the leading edge of a moving cell by endocytosis and recycling.     

Distinct recognition of collagen subtypes by alpha(1)beta(1) and alpha(2)beta(1) integrins. Alpha(1)beta(1) mediates cell adhesion to type XIII collagen

Two integrin-type collagen receptors, alpha(1)beta(1) and alpha(2)beta(1), are structurally very similar. However, cells can concomitantly express the both receptors and they might have independent functions. Here, Chinese hamster ovary (CHO) cells, which lack endogenous collagen receptors, were transfected with either alpha(1) or alpha(2) integrin cDNA. Cells were allowed to adhere to various collagen types and their integrin function was tested by observing the progression of cell spreading. The cells expressing alpha(1)beta(1) integrin could spread on collagen types I, III, IV, and V but not on type II, while alpha(2)beta(1) integrin could mediate cell spreading on collagen types I-V. Type XIII is a transmembrane collagen and its interaction with the integrins has not been previously studied. CHO-alpha1beta1 cells could spread on human recombinant type XIII collagen, unlike CHO-alpha2beta1 cells. Integrins alpha(1)beta(1) and alpha(2)beta(1) recognize collagens with the specific alphaI domains. The alpha(1)I and alpha(2)I domains were produced as recombinant proteins, labeled with europium and used in a sensitive solid-phase binding assay based on time-resolved fluorescence. alpha(1)I domain, unlike the alpha(2)I domain, could attach to type XIII collagen. The results indicate, that alpha(1)beta(1) and alpha(2)beta(1) have different ligand binding specificity. Distinct recognition of different collagen subtypes by the alphaI domains can partially explain the differences seen in cell spreading. However, despite the fact that CHO-alpha1beta1 cells could not spread on type II collagen alpha(1)I domain could bind to this collagen type. Thus, the cell spreading on collagens may also be regulated by factors other than the integrins.     

Fibronectin fragmentation promotes alpha4beta1 integrin-mediated contraction of a fibrin-fibronectin provisional matrix

In injured tissues, the fibrin-fibronectin (FN) provisional matrix provides a framework for cell adhesion, migration, and repair. Effective repair and remodeling require a proper balance between extracellular matrix (ECM) deposition, contraction, and turnover. We utilized a three-dimensional (3D) fibrin-FN provisional matrix model to determine the contributions of the FN-binding integrin receptors alpha5beta1 and alpha4beta1 to matrix contraction. CHOalpha5 cells expressing alpha5beta1, a receptor for FN's RGD cell-binding domain, were highly contractile, and cells were well spread on a 3D fibrin-FN matrix. In contrast, CHOalpha4 cells expressing the alpha4beta1 receptor for FN's alternatively spliced V region attached less efficiently to FN and were deficient in fibrin-FN matrix contraction. Surprisingly, cell adhesion and matrix contraction by CHOalpha4 cells were dramatically enhanced, to levels equivalent to CHOalpha5 cells, when proteolyzed FN was used in place of intact FN in the fibrin-FN matrix. Similar enhancement was observed when ligand binding by alpha4beta1 integrins was activated by treatment with Mn(++), but not by stimulation of actin organization with LPA. Therefore, alpha4beta1-dependent cell responses to the provisional matrix are modulated by cleavage of matrix components.     

An angiogenic laminin site and its antagonist bind through the alpha(v)beta3 and alpha5beta1 integrins.

Angiogenesis is important for wound healing, tumor growth, and metastasis. Endothelial cells differentiate into capillary-like structures on a laminin-1-rich matrix (Matrigel). We previously identified 20 angiogenic sites on laminin-1 (alpha1beta1gamma1) by screening 559 overlapping synthetic peptides. C16, the most potent gamma1 chain peptide, blocked laminin-1-mediated adhesion and was the only gamma1 chain peptide to block attachment to both collagen I and fibronectin. This suggested that C16 was acting via a receptor common to these substrates. We demonstrated that C16 is angiogenic in vivo. Affinity chromatography identified the integrins alpha5beta1 and alpha(v)beta3 as surface receptors. Blocking antibodies confirmed the role of these receptors in C16 adhesion. C16 does not contain an RGD sequence and, as expected, an RGD-containing peptide did not block C16 adhesion nor did C16 act via MAP kinase phosphorylation. Furthermore, we identified a C16 scrambled sequence, C16S, which antagonizes the angiogenic activity of bFGF and of C16 by binding to the same receptors. Because the laminin gamma1 chain is ubiquitous in most tissues, C16 is likely an important functional site. Since the biological activity of C16 is blocked by a scrambled peptide, C16S may serve as an anti-angiogenic therapeutic agent.     

Caveolin-1 regulates glioblastoma aggressiveness through the control of alpha(5)beta(1) integrin expression and modulates glioblastoma responsiveness to SJ749, an alpha(5)beta(1) integrin antagonist

Caveolin-1 plays a checkpoint function in the regulation of processes often altered in cancer. Although increased expression of caveolin-1 seems to be the norm in the glioma family of malignancies, populations of caveolin-1 positive and negative cells coexist among glioblastoma specimens. As no data are available to date on the contribution of such cells to the phenotype of glioblastoma, we manipulated caveolin-1 in the glioblastoma cell line U87MG. We showed that caveolin-1 plays a critical role in the aggressiveness of glioblastoma. We identified integrins as the main set of genes affected by caveolin-1. We reported here that the phenotypic changes observed after caveolin-1 modulation were mediated by alpha(5)beta(1) integrins. As a consequence of the regulation of alpha(5)beta(1) levels by caveolin-1, the sensitivity of cells to the specific alpha(5)beta(1) integrin antagonist, SJ749, was affected. Mediator of caveolin-1 effects, alpha(5)beta(1) integrin, is also a marker for glioma aggressiveness and an efficient target for the treatment of glioma especially the ones exerting the highest aggressive phenotype.     

Kinetic studies on partially liganded species of carboxyhemoglobin: (alpha 1CO-beta 1CO)alpha 2 beta 2 or (alpha 2CO beta 2CO)alpha 1 beta 1

Kinetics of CO combination with and dissociation from isomer III, (alpha 1CO beta 1CO)alpha 2 beta 2 or alpha 1 beta 1 (alpha 2CO beta 2CO), and Hb Rothschild have been studied using the double mixing and microperoxidase methods. Isomer III was prepared in a manner so that it was the only reactive species in the reaction mixture. The biphasic reaction time course in both the "on" and "off" reactions of isomer III and the CO combination reaction of Hb Rothschild are attributed to slow relaxation between the fast and slow CO-reacting species in the two proteins: isomer III: l'f = 6 x 10(6) M-1 s-1, l'dimer = 1.7 x 10(6) M-1 s-1, l's = 2.2 x 10(5) M-1 s-1, lf = 0.15 s-1, ls = 0.01 s-1; Hb Rothschild: l'f = 2.8 x 10(6) M-1 s-1; l's = 2.7 x 10(5) M-1 s-1.