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1.
To probe the substrate specificity of the human metalloproteinase stromelysin (SLN), we determined values of kc/Km for the SLN-catalyzed hydrolysis of substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-MetNH2; SP; kc/Km = 1790 +/- 140 M-1 s-1), 15 analogues of SP, and 17 other peptides. We found a remarkably narrow substrate specificity for SLN: while SP and its analogues could serve as substrates for SLN (hydrolysis occurred exclusively at the Gln6-Phe7 bond), peptides that were not direct analogues could not (kc/Km less than 3 M-1 s-1). From the study of the SLN-catalyzed hydrolysis of SP and its analogues, the following findings emerged: (1) Decreasing the length of SP results in decreases in kc/Km. (2) Conservative amino acid replacements near the scissle bond of SP decrease kc/Km. (3) The SP analogue in which Gly9 is replaced with sarcosine (N-methylglycine) is not hydrolyzed by SLN (kc/Km less than 3 M-1 s-1). (4) Several SP analogues that are not hydrolyzed by SLN are inhibitors of the enzyme. The complexes formed from interaction of SLN with these peptides have dissociation constants that are similar to the Km value for the complex of SLN and SP. Combined, these results suggest that SLN uses the energy that is available from favorable interactions with its substrate to stabilize catalytic transition states but not the Michaelis complex or other stable-state complexes. 相似文献
2.
Integrin-cytoskeletal interactions in migrating fibroblasts are dynamic, asymmetric, and regulated 总被引:22,自引:7,他引:15
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《The Journal of cell biology》1993,123(4):977-991
We have used laser optical trapping and nanometer-level motion analysis to investigate the cytoskeletal associations and surface dynamics of beta 1 integrin, a cell-substrate adhesion molecule, on the dorsal surfaces of migrating fibroblast cells. A single-beam optical gradient trap (laser tweezers) was used to restrain polystyrene beads conjugated with anti-beta 1 integrin mAbs and place them at desired locations on the cell exterior. This technique was used to demonstrate a spatial difference in integrin-cytoskeleton interactions in migrating cells. We found a distinct increase in the stable attachment of beads, and subsequent rearward flow, on the lamellipodia of locomoting cells compared with the retracting portions. Complementary to the enhanced linkage of integrin at the cell lamellipodium, the membrane was more deformable at the rear versus the front of moving cells while nonmotile cells did not exhibit this asymmetry in membrane architecture. Video microscopy and nanometer-precision tracking routines were used to study the surface dynamics of integrin on the lamellipodia of migrating cells by monitoring the displacements of colloidal gold particles coated with anti-beta 1 integrin mAbs. Small gold aggregates were rapidly transported preferentially to the leading edge of the lamellipod where they resumed diffusion restricted along the edge. This fast transport was characterized by brief periods of directed movement ("jumps") having an instantaneous velocity of 37 +/- 15 microns/min (SD), separated by periods of diffusion. In contrast, larger aggregates of gold particles and the large latex beads underwent slow, steady rearward movement (0.85 +/- 0.44 micron/min) (SD) at a rate similar to that reported for other capping events and for migration of these cells. Cell lines containing mutated beta 1 integrins were used to show that the cytoplasmic domain is essential for an asymmetry in attachment of integrin to the underlying cytoskeletal network and is also necessary for rapid, intermittent transport. However, enhanced membrane deformability at the cell rear does not require integrin-cytoskeletal interactions. We also demonstrated that posttranslational modifications of integrin could potentially play a role in these phenomena. These results suggest a scheme for the role of dynamic integrin-mediated adhesive interactions in cell migration. Integrins are transported preferentially to the cell front where they form nascent adhesions. These adhesive structures grow in size and associate with the cytoskeleton that exerts a rearward force on them. Dorsal aggregates more rearward while those on the ventral side remain fixed to the substrate allowing the cell body to move forward. Detachment of the cell rear occurs by at least two modes: (a) weakened integrin- cytoskeleton interactions, potentially mediated by local modifications of linkage proteins, which lead to weakened cell-substratum interactions and (b) ripping of integrins and the highly deformable membrane from the cell body. 相似文献
3.
The adaptation of an analytical procedure for aggrecan based upon gel-permeation chromatography to an FPLC-based protocol has significantly sped up the analysis. The faster assay has permitted determination of the kinetic constants for digestion of human aggrecan by human stromelysin-1. Monomeric aggrecan appeared to be hydrolyzed by stromelysin-1 to multiple forms with lower molecular weight. The disappearance of high-molecular-weight aggrecan was first-order, showing Km much larger than 2 microM and kc/Km = 4000 M-1 s-1 at pH 7.5. The disappearance of high-molecular-weight aggrecan upon hydrolysis by stromelysin-1 at pH 5.5 was also first-order, with kc/Km = 10,700 M-1 s-1. The disappearance of high-molecular-weight aggrecan at pH 7.5 was first-order for digestion by human leukocyte elastase with kc/Km = 230,000 M-1 s-1, by human cathepsin G with kc/Km = 4200 M-1 S-1, and by human plasma plasmin with kc/Km = 2800 M-1 s-1, all with Km much larger than 2 microM. 相似文献
4.
The kinetic parameters of three different molecular forms of urokinase (UK) for the activation of native Glu-plasminogen were compared. The apparent Michaelis constant (Km. app.) of each UK was almost of the same order of magnitude (31-38 microM), but the catalytic constants (kc) were observed to be different: UKh (high molecular weight form, molecular weight 53,000), 2.4 +/- 0.2 s-1; UK+ (low molecular weight form, molecular weight 33,000), 0.83 +/- o.10 s-1, and UKl (trypsin-digested form, molecular weight 36,000), 0.91 +/- 0.18 s-1. The overall second order rate constant, kc/Km calculated for UKh was 7.7 X 10(4) M-1 s-1, higher than for UKl (2.2 X 10(4) M-1 s-1) or UKt (2.4 X 10(4) M-1 s-1), indicating the possibility of a much higher degree of enzymatic specificity and efficiency. 相似文献
5.
An early event of beta(2) integrin activation is the increased diffusion rate of this molecule on the cell surface, thereby providing integrin molecules with a better chance to meet the ligands. The activation of protein kinase C (PKC) stimulates integrin diffusion by releasing the cytoskeletal constraint on integrin molecules. We report here that macrophage-enriched myristoylated alanine-rich C kinase substrate (MacMARCKS), a membrane-associated PKC substrate involved in integrin activation, is required for this PKC-stimulated diffusion of integrin molecules. Using the single-particle tracking technique, we observed that the activation of PKC stimulated an 11-fold increase in the diffusion rate of beta(2) integrins in wild type J774 macrophage cells but not in those expressing mutant MacMARCKS. Further evidence is provided from a MacMARCKS-deficient cell line in which phorbol esters failed to stimulate the diffusion of integrin. Transfection of wild type MacMARCKS into these cells restored the rapid diffusion rate of the beta(2) integrins. The phosphorylation of MacMARCKS is important because transfection of a nonphosphorylatable MacMARCKS mutant or the addition of staurosporine eliminates the rapid diffusion rate of integrin. Furthermore, adding cytochalasin D bypasses the MacMARCKS deficiency and stimulates beta(2) integrin diffusion, suggesting that MacMARCKS's involvement in integrin activation is prior or at the site of cytoskeleton. Therefore, we conclude that MacMARCKS is required for releasing the cytoskeletal constraint on integrin molecules during PKC-mediated integrin activation. 相似文献
6.
7.
Kinetic intermediates in prothrombin activation. Bovine prethrombin 1 conversion to thrombin by factor X 总被引:1,自引:0,他引:1
Two pathways are possible during the proteolytic formation of alpha-thrombin (alpha-IIa) from prothrombin (II) or prethrombin 1 (P1). One of the pathways, with prethrombin 2 or prethrombin 2 associated with fragment 2 (P2F2) as intermediates, has long been known to exist when activation is catalyzed by Factor Xa (Xa) alone. The second pathway, with meizothrombin or meizothrombin (des fragment 1) (MzIIa(-F1)) as intermediate, has been shown to exist when Factor Va and phospholipids are present with Xa. Until now, MzIIa(-F1) has not been detected in reactions catalyzed by Xa alone. In this study, we demonstrate that P1 activation by Xa alone occurs via both pathways, and we provide rate constants and kinetic equations for calculating the relative contributions of each of the pathways to the formation of alpha-IIa by Xa. Investigation of the initial rates of proteolytic cleavage of P2F2 and P1 by Xa alone indicated first-order dependence on substrate concentration with no evidence of saturation of Xa with either substrate at concentrations as high as 200 microM. Apparent second-order rate constants (kc/Km) of 113 +/- 9 M-1 s-1 for the formation of thrombin from P2F2 and 1,410 +/- 19 M-1 s-1 for the disappearance of P1 were determined at pH 7.5, 25 degrees C, 10 mM CaCl2, 0.15 M ionic strength. A two-step sequential first-order pathway employing these rate constants for thrombin activity production from P1 via P2F2 could not, however, account for the quantity of thrombin that was produced during the early stages of P1 activation. Addition of a parallel first-order reaction to produce thrombin activity from P1 independently of P2F2, tentatively identified as the formation of MzIIa(-F1), yielded progress curves in quantitative agreement with the experimental data. kc/Km for the parallel reaction was estimated to be 98 +/- 10 M-1 s-1. Independent determination of the second-order rate constant for the cleavage of isolated MzIIa (-F1), 15,000 +/- 420 M-1 s-1, indicated that MzIIa(-F1) could meet the kinetic requirements for an intermediate in the parallel activation pathway. The transient formation of MzIIa (-F1), as well as the generation of alpha-IIa, was directly demonstrated during activation of P1 by active site-affinity labeling of the reaction products with a biotin derivative of D-Phe-Pro-Arg chloromethyl ketone and visualization by semiquantitative Western blotting.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
8.
Ali O Guillou H Destaing O Albigès-Rizo C Block MR Fourcade B 《Biophysical journal》2011,(11):2595-2604
Integrins are transmembrane receptors involved in crucial cellular biological functions such as migration, adhesion, and spreading. Upon the modulation of integrin affinity toward their extracellular ligands by cytoplasmic proteins (inside-out signaling) these receptors bind to their ligands and cluster into nascent adhesions. This clustering results in the increase in the mechanical linkage among the cell and substratum, cytoskeleton rearrangements, and further outside-in signaling. Based on experimental observations of the distribution of focal adhesions in cells attached to micropatterned surfaces, we introduce a physical model relying on experimental numerical constants determined in the literature. In this model, allosteric integrin activation works in synergy with the stress build by adhesion and the membrane rigidity to allow the clustering to nascent adhesions independently of actin but dependent on the integrin diffusion onto adhesive surfaces. The initial clustering could provide a template to the mature adhesive structures. Predictions of our model for the organization of focal adhesions are discussed in comparison with experiments using adhesive protein microarrays. 相似文献
9.
Van Wey AS Cookson AL Soboleva TK Roy NC McNabb WC Bridier A Briandet R Shorten PR 《Biotechnology and bioengineering》2012,109(5):1280-1292
The ability for a biofilm to grow and function is critically dependent on the nutrient availability, and this in turn is dependent on the structure of the biofilm. This relationship is therefore an important factor influencing biofilm maturation. Nutrient transport in bacterial biofilms is complex; however, mathematical models that describe the transport of particles within biofilms have made three simplifying assumptions: the effective diffusion coefficient (EDC) is constant, the EDC is that of water, and/or the EDC is isotropic. Using a Monte Carlo simulation, we determined the EDC, both parallel to and perpendicular to the substratum, within 131 real, single species, three-dimensional biofilms that were constructed from confocal laser scanning microscopy images. Our study showed that diffusion within bacterial biofilms was anisotropic and depth dependent. The heterogeneous distribution of bacteria varied between and within species, reducing the rate of diffusion of particles via steric hindrance. In biofilms with low porosity, the EDCs for nutrient transport perpendicular to the substratum were significantly lower than the EDCs for nutrient transport parallel to the substratum. Here, we propose a reaction-diffusion model to describe the nutrient concentration within a bacterial biofilm that accounts for the depth dependence of the EDC. 相似文献
10.
The activation dependent adhesion of macrophages to laminin involves cytoskeletal anchoring and phosphorylation of the alpha 6 beta 1 integrin 总被引:36,自引:16,他引:20
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Macrophages require activation with either PMA (Mercurio, A. M., and L. M. Shaw. 1988. J. Cell Biol. 107:1873-1880) or interferon-gamma (Shaw, L. M., and A. M. Mercurio. 1989. J. Exp. Med. 169:303-308) to adhere to a laminin substratum. In the present study, we identified an integrin laminin receptor on macrophages and characterized cellular changes that occur in response to PMA activation that facilitate laminin adhesion. A monoclonal antibody (GoH3) that recognizes the integrin alpha 6 subunit (Sonnenberg, A., H. Janssen, F. Hogervorst, J. Calafat, and J. Hilgers. 1987. J. Biol. Chem. 262:10376-10383) specifically inhibited adhesion to laminin-coated surfaces. This antibody precipitated an alpha 6 beta 1 heterodimer (Mr 130/110 kD) from 125I surface-labeled macrophages. The amount of radiolabeled receptor on the cell surface did not increase after PMA activation. Thus, the induction of laminin adhesion cannot be attributed to de novo or increased surface expression of alpha 6 beta 1. By initially removing the Triton X-100-soluble fraction of macrophages and then disrupting the remaining cytoskeletal framework, we observed that 75% of the alpha 6 beta 1 heterodimer on the cell surface is anchored to the cytoskeleton in macrophages that had adhered to a laminin substratum in response to PMA. Significant cytoskeletal anchoring of this receptor was not observed in macrophages that had adhered to fibronectin or tissue culture plastic, nor was it seen in nonadherent cells. PMA also induced phosphorylation of the cytoplasmic domain of the alpha 6 subunit, but not the beta 1 subunit. Phosphorylated alpha 6 was localized to the cytoskeletal fraction only in macrophages plated on a laminin substratum. In summary, our results support a mechanism for the regulation of macrophage adhesion to laminin that involves specific and dynamic matrix integrin-cytoskeletal interactions that may be facilitated by integrin phosphorylation. 相似文献
11.
Acetyl phosphate is hydrolyzed by the calcium ATPase of leaky sarcoplasmic reticulum vesicles from rabbit skeletal muscle with Km = 6.5 mM and kcat = 7.9 s-1 in the presence of 100 microM calcium (180 mM K+, 5 mM MgSO4, pH 7.0, 25 degrees C). In the absence of calcium, hydrolysis is 6% of the calcium-dependent rate at low and 24% at saturating concentrations of acetyl phosphate. Values of K0.5 for calcium are 3.5 and 2.2 microM (n = 1.6) in the presence of 1 and 50 mM acetyl phosphate, respectively; inhibition by calcium follows K0.5 = 1.6 mM (n approximately 1.1) with 50 mM acetyl phosphate and K0.5 = 0.5 mM (n approximately 1.3) with 1.5 mM ATP. The calcium-dependent rate of phosphoenzyme formation from acetyl phosphate is consistent with Km = 43 mM and kf = 32 s-1 at saturation; decomposition of the phosphoenzyme occurs with kt = 16 s-1. The maximum fraction of phosphoenzyme formed in the steady state at saturating acetyl phosphate concentrations is 43-46%. These results are consistent with kc congruent to 30 s-1 for binding of Ca2+ to E at saturating [Ca2+], to give cE.Ca2, in the absence of activation by ATP. Phosphoenzyme formed from ATP and from acetyl phosphate shows the same biphasic reaction with ADP, rate constants for decomposition that are the same within experimental error, and similar or identical activation of decomposition by ATP. It is concluded that the reaction pathways for acetyl phosphate and ATP in the presence of Ca2+ are the same, with the exception of calcium binding and phosphorylation; an alternative, faster route that avoids the kc step is available in the presence of ATP. The existence of three different regions of dependence on ATP concentration for steady state turnover is confirmed; activation of hydrolysis at high ATP concentrations involves an ATP-induced increase in kt. 相似文献
12.
Real-time analysis of the assembly of ligand, receptor, and G protein by quantitative fluorescence flow cytometry. 总被引:4,自引:0,他引:4
We describe a general approach for the quantitative analysis of the interaction among fluorescent peptide ligands (L), receptors (R), and G proteins (G) using fluorescence flow cytometry. The scheme depends upon the use of commercially available fluorescent microbeads as standards to calibrate the concentration of fluorescent peptides in solution and the receptor number on cells in suspension. We have characterized a family of fluoresceinated formyl peptides and analyzed both steady-state and dynamic aspects of ligand formyl peptide-receptor interactions in digitonin-permeabilized human neutrophils. Detailed receptor-binding studies were performed with the pentapeptide N-formyl-Met-Leu-Phe-Phe-Lys-fluorescein. Equilibrium studies showed that GTP [S] caused a loss of binding affinity of approximately two orders of magnitude, from approximately 0.04 nM (LRG) to approximately 3 nM (LR), respectively. Kinetic studies revealed that this change in affinity was principally due to an increase in the dissociation rate constants from approximately 1 x 10(-3) s-1 (LRG) to approximately 1 x 10(-1) s-1 (LR). In contrast, the association rate constants in the presence and absence of guanine nucleotide (approximately 3 x 10(7) s-1 M-1) were statistically indistinguishable and close to the diffusion limit. In the presence of guanine nucleotide (LR), the kinetic data were adequately fit by a single-step reversible-binding model. In the absence of guanine nucleotides, not all receptors have rapid access to G to form the LRG ternary complex. Mathematically, those R that have rapid access to G are either precoupled to R or the association of G with R is fast compared to the association of L with R. The physiological consequences of coupling heterogeneity are discussed. 相似文献
13.
Comparative behavior of membrane protein-antibody complexes on motile fibroblasts: implications for a mechanism of capping 总被引:4,自引:3,他引:1
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《The Journal of cell biology》1990,111(6):2499-2512
A characteristic feature of fibroblast locomotory activity is the rearward transport across the leading lamella of various materials used to mark the cell surface. The two processes most frequently invoked as explanations for this transport phenomenon, called capping, are (a) retrograde membrane flow arising from directed membrane insertion and (b) rearward cortical cytoskeletal flow arising from cytoskeletal assembly and contraction. The retrograde lipid flow hypothesis, the most current form of the membrane flow scheme, makes explicit predictions about the movement of membrane proteins subjected to the postulated rearward lipid flow. Several of these predictions were tested by comparing the behavior of four membrane proteins, Pgp-1, Thy- 1, H-2, and influenza HA0, identified by fluorescent antibodies. With the exception of Pgp-1, these proteins were uniformly distributed under nonaggregated conditions but were capped when aggregated into patches. In contrast, Pgp-1 was capped in similar time frames in both nonaggregated and aggregated states where the lateral diffusion coefficients were very different. Furthermore, the capping behavior of two tagged membrane proteins was markedly different yet both had similar diffusion coefficients. The results from these tests disprove the bulk membrane flow hypothesis and are at odds with explicit predictions of the retrograde lipid flow hypothesis for the mechanism of capping. This work, therefore, supports the alternative cytoskeletal- based mechanism for driving capping. Requirements for coupling cytoskeletal movement to membrane components are discussed. 相似文献
14.
Cell adhesion to extracellular matrix regulates the life cycle of integrins. 总被引:3,自引:0,他引:3
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S L Dalton E Scharf R Briesewitz E E Marcantonio R K Assoian 《Molecular biology of the cell》1995,6(12):1781-1791
The expression of alpha 5 beta 1 integrin on the surface of fibroblasts requires adhesion to substratum. We have examined the basis for this adhesion-dependent surface expression by comparing the life cycle of integrins in parallel cultures of adherent and nonadherent cells. Results of biosynthetic labeling experiments in NRK fibroblasts showed that the synthesis and biosynthetic processing of the beta 1 integrin subunit proceed in the absence of cell attachment; however, when examining the behavior of preexisting cell surface integrins, we observed that the alpha beta 1 integrins are internalized and degraded when adhesion to substratum is blocked. A kinetic analysis of integrin internalization in cycloheximide-treated NRK cells showed that each of the fibroblast integrins we examined (in both the beta 1 and beta 3 families) are lost from the cell surface after detachment from substratum. Thus, the default integrin life cycle in fibroblasts involves continuous synthesis, processing, transport to the cell surface, and internalization/degradation. Interestingly, studies with NIH-3T3 cells expressing alpha 1 beta 1 integrin showed that the loss of cell-surface alpha 5 beta 1 integrin is blocked by adhesion of cells to dishes coated with type IV collagen (a ligand for alpha 1 beta 1 integrin) as well as fibronectin. Similarly, adhesion of these cells to dishes coated with type IV collagen stabilizes the surface expression of alpha 5 beta 1 as well as alpha 1 beta 1 integrin. We propose that the adhesion of fibroblasts to extracellular matrix protein alters the integrin life cycle and permits retention of these proteins at the cell surface where they can play important roles in transmitting adhesion-dependent signals. 相似文献
15.
Urokinase-catalysed plasminogen activation. Effects of ligands binding to the AH-site of plasminogen
U Christensen 《Biochimica et biophysica acta》1988,957(2):258-265
The kinetics of activation of Lys-plasminogen (Lys-77-Asn-790) and miniplasminogen (Val-442-Asn-790) catalysed by low-molecular-weight urokinase (LMW-urokinase) was investigated in the presence and absence of ligands that bind to the AH-site of the plasminogens. 6-Aminohexanoic acid and alpha-N-acetyl-L-lysine methyl ester (AcLysMe) were used. Saturation of the AH-sites of the plasminogens result in similar, but rather small positive effects on the kinetics of activation of the two plasminogens. Michaelis constants decrease approx. 2-fold and second-order rate constants (kc/Km)Pg increase approx. 1.2-fold. Michaelis constants (KPg values) were obtained using a new approach; the values were determined from the competing effects of the plasminogens on urokinase-catalysed hydrolysis of a synthetic substrate. In the pH range 7.4-8.0, only minor alterations of the values of the kinetic parameters are observed. At 25 degrees C, values of (kc/Km)Pg are approx. 3-fold less than the value at 37 degrees C, whereas KPg is not changed. We conclude that kc/Km values are approx. 10(5) M-1.s-1 and that KPg values are approx. 40 microM of urokinase-catalysed conversions of Lys- and miniplasminogen to their respective plasmins. 相似文献
16.
E A Permiakov A V Ostrovski? L P Kalinichenko G Iu De?kus 《Molekuliarnaia biologiia》1987,21(4):1017-1022
Dissociation kinetics of parvalbumin complexes with calcium and magnesium ions were studied by means of stopped-flow method employing intrinsic protein fluorescence registration. In the temperature range from 10 to 30 degrees C the kinetic curves of Ca2+ and Mg2+ dissociation are best fitted with a sum of two exponential terms, each term is ascribed to a dissociation process in one of two bindings sites of parvalbumin. Dissociation rate constants in this temperature range increase from 0.03 to 0.8 s-1 and from 0.18 to 5 s-1 for Ca2+, and from 0.9 to 4.5 s-1 and from 4 to 33 s-1 for Mg2+. Parvalbumin equilibrium binding constants of Ca2+ and Mg2+ were also measured in the same temperature range. It makes possible to estimate the rate constants of association of Ca2+ and Mg2+. In the case of Ca2+ the rate of association approaches the diffusion controlled limit. 相似文献
17.
Rates and equilibria for a photoisomerizable antagonist at the acetylcholine receptor of Electrophorus electroplaques 总被引:1,自引:0,他引:1
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M E Krouse H A Lester N H Wassermann B F Erlanger 《The Journal of general physiology》1985,86(2):235-256
Voltage-jump and light-flash experiments have been performed on isolated Electrophorus electroplaques exposed simultaneously to nicotinic agonists and to the photoisomerizable compound 2,2'-bis-[alpha-(trimethylammonium)methyl]-azobenzene (2BQ). Dose-response curves are shifted to the right in a nearly parallel fashion by 2BQ, which suggests competitive antagonism; dose-ratio analyses show apparent dissociation constants of 0.3 and 1 microM for the cis and trans isomers, respectively. Flash-induced trans----cis concentration jumps produce the expected decrease in agonist-induced conductance; the time constant is several tens of milliseconds. From the concentration dependence of these rates, we conclude that the association and dissociation rate constants for the cis-2BQ-receptor binding are approximately 10(8) M-1 s-1 and 60 s-1 at 20 degrees C; the Q10 is 3. Flash-induced cis----trans photoisomerizations produce molecular rearrangements of the ligand-receptor complex, but the resulting relaxations probably reflect the kinetics of buffered diffusion rather than of the interaction between trans-2BQ and the receptor. Antagonists seem to bind about an order of magnitude more slowly than agonists at nicotinic receptors. 相似文献
18.
We have previously shown that the glycosylphosphatidyl-inositol (GPI)-linked urokinase-type plasminogen activator receptor (uPAR) reversibly associates with the integrins complement receptor type 3 (CR3; alphaMbeta2) and CR4 (alphaxbeta2) during leukocyte motility. These receptor-to-receptor interactions could potentially be accounted for by diffusion-controlled reactions or by directed transport phenomena. To address these alternatives, we have used computer simulation techniques. Our results show that a diffusion-controlled interaction between uPAR and CR4 during accumulation at lamellipodia is not physically reasonable. This suggests that a directed transport mechanism participates in establishing uPAR-integrin association. 相似文献
19.
Modified trypsin kallikrein inhibitor (I*), with the reactive-site peptide bond Lys-15--Ala-16 split, reacts with alpha-chymotrypsin (E) via an intermediate X to the stable tetrahedral complex C:E + I in equilibrium X leads to C. Formation X constitutes a fast pre-equilibrium (equilibrium constant Kx = 7 X 10(-5) M, association rate constant kx = 4 X 10(3)M-1s-1) to the slow reaction X leads to C (rate constant kc = 2 X 10(-3) s-1), all values at pH 7.5. No intermediate X is observed when alpha-chymotrypsin reacts with I*-OMe in which the carboxyl group of Lys-15 is esterified by methanol. This observation as well as the different pH dependence of the overall association rate constants in the case of I* and I*-OMe indicate tha formation of X precedes formation of the acyl enzyme in the catalytic pathway. The data are compared to the similar results obtained with beta-trypsin and I* or I*-OMe. 相似文献
20.
Specific integrin alpha and beta chain phosphorylations regulate LFA-1 activation through affinity-dependent and -independent mechanisms 总被引:1,自引:0,他引:1
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Integrins are adhesion receptors that are crucial to the functions of multicellular organisms. Integrin-mediated adhesion is a complex process that involves both affinity regulation and cytoskeletal coupling, but the molecular mechanisms behind this process have remained incompletely understood. In this study, we report that the phosphorylation of each cytoplasmic domain of the leukocyte function-associated antigen-1 integrin mediates different modes of integrin activation. alpha Chain phosphorylation on Ser1140 is needed for conformational changes in the integrin after chemokine- or integrin ligand-induced activation or after activation induced by active Rap1 (Rap1V12). In contrast, the beta chain Thr758 phosphorylation mediates selective binding to 14-3-3 proteins in response to inside-out activation through the T cell receptor, resulting in cytoskeletal rearrangements. Thus, site-specific phosphorylation of the integrin cytoplasmic domains is important for the dynamic regulation of these complex receptors in cells. 相似文献