Biomedical subjects

Y Engelborghs

Publications and source records attributed to Y Engelborghs.

At least 55 records · Page 3Linked to original sources

PubMed1996

Alterations of rings B and C of colchicine are cumulative in overall binding to tubulin but modify each kinetic step.

The role of the elimination of ring B and/or the modification of ring C of colchicine in tubulin binding kinetics and thermodynamics has been characterized, using four different molecules. These ligands are colchicine (COL); 2-methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-on e (MTC), in which the central ring B has been reduced to one bond; allocolchicine (ALLO), in which ring C has been replaced by a six-membered ring; and 2,3,4-trimethoxy-4'-carbomethoxy-1,1'-biphenyl (TCB), where the same two modifications are made simultaneously. This paper describes the kinetics of association of ALLO with tubulin. The binding is accompanied by a fluorescence increase with slow biphasic kinetics, indicating binding to fast and slow tubulin isotypes. Binding to each of these isotypes occurs in two steps: a fast initial binding followed by a slower isomerization step. The K1 and k2 values for ALLO at 25 degrees C are 14,000 +/- 2,000 and 25,000 +/- 6,000 M-1 (fast and slow isotypes) and 0.055 +/- 0.003 s-1 and 0.013 +/- 0.001 s-1 (fast and slow isotype), respectively. For ALLO the reaction standard enthalpy change of the initial binding is 68 +/- 5 kJ.mol-1 (fast isotype) and 45 +/- 33 kJ.mol-1 (slow isotype) and the activation energy for the second forward step is 58 +/- 14 kJ.mol-1 (fast isotype) and 81 +/- 17 kJ.mol-1 (slow isotype). Displacement kinetics of bound ALLO by podophyllotoxin was monoexponential. The activation energy for the isomerization in the off direction is 107 +/- 7 kJ.mol-1. Comparison of the thermodynamic parameters for all four compounds shows that the modifications of both rings are cumulative with respect to overall binding. For the intermediate state there is a mutual influence of both modifications, suggesting an alteration of the reaction pathway.

Biphenyl Compounds↗

PubMed1996

Different kinetic pathways of the binding of two biphenyl analogues of colchicine to tubulin.

The kinetics of the interaction of tubulin with two biphenyl analogues of colchicine were measured by fluorescence stopped flow. The ligands were 2,3,4-trimethoxy-4'-carbomethoxy-1,1'-biphenyl (TCB) and 2,3,4-trimethoxy-4'-acetyl-1,1'-biphenyl (TKB). The binding of both analogues is accompanied by a fluorescence increase with monophasic kinetics, which indicates that these drugs, unlike colchicine, do not discriminate between the isoforms of tubulin. The observed pseudo-first-order rate constant increases in a nonlinear way with the drug concentration, indicating that the binding of the biphenyl analogues to tubulin occurs, like colchicine, in two steps: a fast reversible equilibrium followed by an isomerization of the initial complex. Kinetic analysis shows that TCB and TKB exhibit differences in their K1 values. At 25 degrees C, these are 114,000 +/- 15,000 M(-1) for TCB and 8,300 +/- 900 M(-1) for TKB. Both molecules show a much higher affinity than colchicine for the initial binding site. Also at 25 degrees C, the k2 value is 0.66 +/- 0.04 s(-1) for TCB and 3.0 +/- 0.2 s(-1) for TKB. From the temperature dependence, a reaction enthalpy change for the initial binding (deltaH(zero)1) of 44 +/- 9 kJ x mol(-1) (TCB) and -40 +/- 14 kJ x mol(-1) (TKB) and an activation energy for the second forward step of 64 +/- 2 kJ x mol(-1) (TCB) and 101 +/- 10 kJ x mol(-1) (TKB) were calculated. The dissociation kinetics were studied by displacement experiments, in which podophyllotoxin was used as a displacing ligand. The rate constant for the second step in the off direction (k(-2)) is 0.25 +/- 0.05 s(-1) for TCB and 0.093 +/- 0.009 s(-1) for TKB at 25 degrees C. The activation energies for the backward isomerization of the complexes were found to be 86 +/- 20 kJ x mol(-1) (TCB) and 79 +/- 5 kJ x mol(-1) (TKB). Combination of these results with the kinetic parameters for association gives a full characterization of the enthalpy pathway for the binding of TCB and TKB. The pathway of TCB binding is shown to differ considerably from that of TKB binding. Since their structural difference is located in ring C', this result points to their use of the ring C' in the first binding step. The competitiveness of the binding of TCB and TKB with those of podophyllotoxin, MTC, and MDL 27048 indicates that the two biphenyls interact as well with the trimethoxyphenyl-specific subsite.

Animals↗

PubMed1996

Kinetics of association and dissociation of two enantiomers, NSC 613863 (R)-(+) and NSC 613862 (S)-(-) (CI 980), to tubulin.

The kinetics of binding of R- and S-enantiomers were studied by the fluorescence stopped-flow technique. For the R-enantiomer, the time course of the increase in fluorescence is best fitted by a sum of two exponentials. In pseudo-first-order conditions, the first observed rate constant showed a linear concentration dependence whereas the second showed a hyperbolic one. The dissociation rate constants were determined independently by displacement experiments with 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC). The two exponential phases were assumed to be due to a two-step binding mechanism: an initial binding followed by a conformational change. This is different from colchicine and MTC binding, where the two phases show a hyperbolic concentration dependence and are attributed to the parallel binding to different isoforms of tubulin [Banerjee, A., & Luduena, R. F. (1992) J. Biol. Chem. 267, 13335-13339]. R-isomer binding did not discriminate between the tubulin isoforms. The temperature dependence of all the rate constants were measured, and the entire thermodynamic reaction path was constructed. For the S-isomer, the direct fluorescence stopped-flow study showed that the signals were largely imputable to the fluorescence of the binding at low-affinity sites [Leynadier, D., Peyrot, V., Sarrazin, M., Briand, C., Andreu, J. M., Rener, G. A., & Temple, C., Jr. (1993) Biochemistry 32, 10674-10682]. Therefore, we exploited the competition between R- and S-isomers to determine the binding kinetics of the S-isomer to the R-site. The observed rate constants for competitive binding showed a linear concentration dependence, thus allowing us to calculate the association rate constant of the S-isomer to the R-site. The kinetics of displacement of the S-isomer by MTC allowed the dissociation rate constant for the S-isomer to be determined. The binding of both enantiomers to tubulin in presence of tropolone methyl ether (analog of the colchicine C ring) was decreased, indicating the involvement of the C subsite.

Animals↗

PubMed1996

Experimental and theoretical study of electrostatic effects on the isoelectric pH and the pKa of the catalytic residue His-102 of the recombinant ribonuclease from Bacillus amyloliquefaciens (barnase).

Barnase, the guanine specific ribonuclease of Bacillus amyloliquefaciens, was subjected to mutations in order to alter the electrostatic properties of the enzyme. Ser-85 was mutated into Glu with the goal to introduce an extra charge in the neighborhood of His-102. A double mutation (Ser-85-Glu and Asp-86-Asn) was introduced with the same purpose but without altering the global charge of the enzyme. A similar set of mutations was made using Asp at position 85. For all mutants the pI was determined using the technique of isoelectric focusing and calculated on the basis of the Tanford-Kirkwood theory. When Glu was used to replace Ser-85, the correlation between the experimental and the calculated values was perfect. However, in the Ser-85-Asp mutant, the experimental pI drop is bigger than the calculated one, and in the double mutant (Ser-85-Asp and Asp-86-Asn) the compensation is not achieved. The effect of the mutations on the pKa of His-102 can be determined from the pH dependence of the kcat/KM for the hydrolysis of dinucleotides, e.g., GpC. The effect can also be calculated using the the method of Honig. In this case the agreement is very good for the Glu-mutants and the single Asp-mutant, but less for the double Asp-mutant. The global stability of the Asp-mutants is, however, the same as the wild type, as shown by stability studies using urea denaturation. Molecular dynamics calculations, however, show that in the double Asp-mutant His-102 (H+) swings out of its pocket to make a hydrogen bridge with Gin-104 which should cause an additional pKa rise. The effect of the Glu-mutations was also tested on all the kinetic parameters for GpC and the cyclic intermediate G > p at pH 6.5, for RNA at pH 8.0, and for poly(A) at pH 6.2. The effect of the mutations is rather limited for the dinucleotide and the cyclic intermediate, but a strong increase of the KM is observed in the case of the single mutant (extra negative charge) with polymeric substrates. These results indicate that the extra negative charge has a strong destabilizing effect on the binding of the polymeric substrates in the ground state and the transition state complex. A comparison with the structure of bound tetranucleotides (Buckle, A.M. and Fersht, A.R., Biochemistry 33:1644-1653, 1994) shows that the extra negative charge points towards the P2 site.

Bacillus↗

PubMed1996

Molecular mechanisms of pressure induced conformational changes in BPTI.

We have performed a 800 ps molecular dynamics simulation of bovine pancreatic trypsin inhibitor (BPTI) in water coupled to a pressure bath at 1, 10,000, 15,000, and 20,000 bar. The simulation reproduces quite well the experimental behavior of the protein under high pressure. The protein keeps its globular form, but adopts a different conformation with a very small reduction in volume. Some residues in the hydrophobic core become exposed to water and a large part of the secondary structure of the protein, (60% of the sheet structure and 40% of the helical structure) is denatured between 10 and 15 kbar. This is in good agreement with experimental data (Goossens, K., et al. Eur. J. Biochem, 236:254-262, 1996) that show denaturation of BPTI between 8 and 14 kbar. A further increase of the pressure results in a freezing of the protein as deduced from the large decrease of the mobility of the residues. During the simulation, the normal structure of water changes from an ice Ih-like to an ice VI-like structure, while keeping the liquid state. The driving force of the high pressure induced conformational transition seems be the higher compressibility of the water compared with the protein. This produces a change in the solvent properties and leads to penetration of the solvent into the hydrophobic core.

Aprotinin↗

PubMed1996

Fluorescence study of the conformational properties of recombinant tick anticoagulant peptide (Ornithodorus moubata) using multifrequency phase fluorometry.

Steady-state and multifrequency phase fluorometry were used to characterize the conformational state and conformational dynamics of recombinant tick anticoagulant peptide (Ornithodorus moubata) (TAP). The TAP contains two tryptophan residues at positions 11 and 37. The fluorescence emission varies sigmoidally as a function of pH with a pKa of 6.01 +/- 0.07. This pH dependency suggests that tryptophan fluorescence is quenched by His43 at low pH. This is confirmed by modification of the histidine with diethylpyrocarbonate. At pH 9 the fluorescence decay is well described by a sum of three exponentials (0.52, 1.9 and 5.4 ns), which decrease all three at pH 4 (0.25, 1.61 and 4.4 ns). From the reactivity of the fluorescence lifetimes toward N-bromosuccinimide and from the calculation of the accessibility we can attribute the long lifetime to Trp11, the short one to Trp37 and the middle one to both. The anisotropy decay was resolved into two components of 3.85 ns and 0.27 ns at pH 4 and 4.5 ns and 0.6 ns at pH 9. The long anisotropy decay time corresponds to the rotational correlation time of the protein, the short one to local mobility of the tryptophan residues.

Animals↗

PubMed1995

Phospholipid binding and lecithin-cholesterol acyltransferase activation properties of apolipoprotein A-I mutants.

Recombinant human apolipoprotein A-I (apo A-I) and three deletion mutants: apo A-I(delta Leu44-Leu126), apo A-I(delta Glu139-Leu170), and apo A-I(delta Ala190-Gln243), purified from the periplasmic space of Escherichia coli, were studied. The rate of turbidity decrease following mixing of apo A-I(delta Ala190-Gln243) with dimyristoylphosphatidylcholine (DMPC) vesicles at 23 degrees C was 10-fold lower than that of the other apo A-I proteins, confirming that the carboxy-terminal region of apo A-I plays a role in rapid lipid binding. The Stokes radii of reconstituted high-density lipoproteins (rHDL), containing dipalmitoylphosphatidylcholine and cholesterol, were larger for the three apo A-I mutants [6.3 nm for apo A-I(delta Leu44-Leu126), 6.1 nm for apo A-I(delta Glu139-Leu170), and 6.5 nm for apo A-I(delta Ala190-Gln243)] than for intact apo A-I (5.0 nm). The mutant rHDL all contained 4 apo A-I molecules per particle as compared to 2 for intact apo A-I. Circular dichroism measurements revealed 8 alpha-helices per apo A-I molecule, 5 per apo A-I(delta Leu44-Leu126), 6 per apo A-I(delta Glu139-Leu170), and 4 per apo A-I(delta Ala190-Gln243) molecule as compared to predicted values of 8, 5, 6, and 6 alpha-helices, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

A two-binding-site kinetic model for the ribonuclease-T1-catalysed transesterification of dinucleoside phosphate substrates.

Ribonucleases have been found to have subsites that confer large rate enhancements but do not contribute to substrate binding. In this study, we present a kinetic model that formally explains how subsite binding energy is converted into chemical activation energy. The proposed mechanism takes into account a primary specificity site and a subsite, both of which must be occupied for chemical turnover. An unstable reaction intermediate is formed upon binding of the polymeric substrate monomers at the corresponding subsites. The structure of this reaction intermediate resembles the transition state of the catalysed transphosphorylation reaction. Similar mechanisms may be used by other depolymerizing enzymes including nucleases, glycosidases, and proteases.

Binding Sites↗

PubMed1995

Molecular dynamics simulation of the solution structures of Ha-ras-p21 GDP and GTP complexes: flexibility, possible hinges, and levers of the conformational transition.

Unconstrained molecular dynamics simulations of the GDP and GTP complexes of Ha-ras p21 protein are performed in aqueous environment for 500 ps, using the GROMOS force field. The solvated structures are mutually compared as well as to the X-ray structures [Tong, L. A., de Vos, A. M., Milburn, M. V., & Kim, S. H. (1991) J. Mol. Biol. 217, 503-516; Pai, E. F., Krengel, U., Petsko, G. A., Goody, R. S., Kabsh, W., & Wittinghofer, A. (1990) EMBO J. 9, 2351-2359]. The simulations show areas of flexibility, with deviations from the original structures. The parts that show differences between the two solvated forms are those from residues 12 to 17, 25 to 38, 41 to 51, 57 to 73, 99 to 112, and 120 to 152, coincident with areas of flexibility. Some of these areas also show differences between the X-ray structures and are part of loops on the surface of the protein. Many of the residues in the ends of these loops undergo dihedral transitions during the solvation process. Of all the dihedral transitions observed, 62% occur around the ends of these loops. This suggests that the ends of the areas from 12 to 17, 25 to 38, and 57 to 73 are the hinge points of the conformational transition between the GTP and the GDP forms. The study of the nucleotide interactions in the solution forms shows that residues 29, 30, and 35 establish contacts with the gamma-phosphate and the sugar ring of the GTP and thus these contacts could be proposed as the possible levers of the conformational transition that accompanies GTP hydrolysis.

Amino Acid Sequence↗

PubMed1995

Fluorescence study of the three tryptophan residues of the pore-forming domain of colicin A using multifrequency phase fluorometry.

We have identified the steady-state and time-resolved fluorescence of the three tryptophan residues (Trp-86, Trp-130, and Trp-140) of the pore-forming domain of colicin A using site-directed mutagenesis in order to construct two- and one-tryptophan-containing mutant proteins. Fluorescence lifetimes were measured via multifrequency phase fluorometry. The fluorescence of the pore-forming domain of colicin A is dominated by Trp-140 which contributes almost 53% to the fluorescence intensity. Mutation of Trp-140 results in a decrease in fluorescence quantum yield and average lifetime. Colicin A wild-type and all mutant proteins display multiple lifetimes which belong to three different lifetime classes: 0.38-0.57 ns for tau 1, 1.6-1.87 ns for tau 2, and 3.6-4.41 ns for tau 3 at pH 5. At pH 7, the three classes are 0.64-0.89 ns for tau 1, 2.01-2.19 ns for tau 2, and 4.23-4.94 ns for tau 3. This pH effect influences all the lifetimes and must be attributed to a general conformational change. In wild-type colicin A, tau 3 originates mainly from Trp-140 while Trp-86 and Trp-130 both provide a major contribution to tau 2. The pH dependence of the fluorescence intensity gives rise to a pKa of 5.2. The different lifetime components of two of the three single-tryptophan-containing mutants show different quenching properties toward acrylamide, indicating that each lifetime is coupled to a different microenvironment. The linear combination of the lifetimes of the single tryptophans into pairs simulates very well the behavior of the two-tryptophan-containing mutants except for one, the mutant containing Trp-86 and Trp-130. The lifetimes of the wild-type protein can only be obtained by the linear combination of the lifetimes from the mutant containing the tryptophan pair Trp-86/Trp-130 and the mutant containing Trp-140. Mutual energy transfer between Trp-86 and Trp-130 is assumed to be the explanation of this deviation since the mutant proteins display no structural or dynamic aberrances. The calculated energy transfer efficiency amounts to 65% for energy transfer from Trp-86 to Trp-130 and 21% for the reverse transfer and is in agreement with our measurements.

Colicins↗

PubMed1995

Immobilizing and imaging microtubules by atomic force microscopy.

Microtubules isolated from pig brains have been immobilized on an inorganic substrate for use in AFM studies. The method employs 4-aminobutyldimethylmethoxysilane and glutaraldehyde to activate a silicon wafer for binding the biopolymer. The covalent bond ensures the positional stability of the tubules on the substrate, and allows reproducible scanning probe experiments. Microtubules have been imaged both by atomic force and scanning tunneling microscopy, yielding results very similar to electron microscopy. The average apparent height of the tubules is smaller than observed with transmission electron microscopy (25 nm) and is smaller in buffer solution (10 nm) than in air (15 nm). The biopolymer surface is softer under buffer than in air. The highest resolution was obtained with the tapping mode where surface features as small as 10 nm in X and Y have been resolved. Gold-coated tubules bound on silicon have been successfully imaged by STM, while images of uncertain origin were generated for tubules deposited on graphite. It is shown that artefacts imaged on a blank graphite surface can easily be confounded with collapsed tubules.

Animals↗

PubMed1995

Interaction between human alpha1-acid glycoprotein (orosomucoid) and 2-p-toluidinylnaphthalene-6-sulfonate.

The interaction between human alpha1-acid glycoprotein (orosomucoid) and the fluorescent probe, 2-p-toluidinylnaphthalene-6-sulfonate (TNS) has been studied. An association constant of 16.7 (+/- 3) x 10(3) M-1 was obtained for the complex at 20 degrees C with a stoichiometry of 1:1. From the effect of temperature on the binding process, the standard enthalpy change for the binding is calculated to be delta H0 = -18 +/- 3 kJ mol-1 and the standard entropy change delta S0 = 19 +/- 12 J K-1 mol-1. The tryptophan fluorescence of the protein can be described by a sum of three exponentials. Upon TNS binding, the average fluorescence lifetime of the protein in the complex changes much less than the fluorescence intensity. The bound TNS is therefore a very efficient acceptor for the protein fluorescence. The TNS bound to orosomucoid present two fluorescence lifetimes 11 and 4.3 ns. The possible origins of the two lifetimes are discussed.

Fluorescent Dyes↗

PubMed1994

The role of Glu-60 in the specificity of the recombinant ribonuclease from Bacillus amyloliquefaciens (barnase) towards dinucleotides, poly(A) and RNA.

A computer model of the complex between G2'p5'G and barnase, the recombinant ribonuclease of Bacillus amyloliquefaciens, was constructed, based on the known structure of the complex RNAase T1.G2'p5'G. This model suggests that the conserved residue Glu-60 plays an important role in the specificity of barnase for guanosine. A barnase mutant was therefore made in which Glu-60 was replaced by Gln. This mutation increases the Km for the dinucleotides GpC and GpA, by a factor of 10, but does not change the kcat. For ApA, the kcat/Km decreases by a similar factor, but the individual parameters could not be determined. The mutation, however, has no influence on the kcat and the Km of barnase action towards RNA and poly(A). This demonstrates that the interactions between the substrate and the residue at position 60 must be different in the case of ApA and poly(A). For RNA, this conclusion is also likely, but not absolutely certain, because barnase/RNA might be a Briggs-Haldane type enzyme/substrate pair. Therefore, if the effect of the mutation were limited to an increase of the dissociation rate constant of the substrate (k-1), this would not be evident in Km or kcat/Km. In view of the clear cut situation with poly(A), the pH profile for and the effect of salt concentration on the kinetic parameters of the mutant barnase were studied for this substrate. The influence of salt on the Km can be interpreted via the linked function concept and shows a cooperative dissociation of 7-10 counterions upon poly(A) binding. The binding of the substrate is strongly reduced at high pH, and the pKa involved decreases strongly at high salt concentrations. Poly(A) and RNA show a pH dependency of their absorbance spectrum, indicating a pH-dependent change of base stacking, which may influence the catalytic parameters.

Bacillus↗

PubMed1994

Interaction of desacetamidocolchicine, a fast binding analogue of colchicine with isotypically pure tubulin dimers alpha beta II, alpha beta III, and alpha beta IV.

Desacetamidocolchicine (DAAC) is a colchicine analogue that lacks the acetamido side chain in the ring-B of colchicine. Unlike colchicine, it binds to tubulin very quickly, and yet it has powerful antimitotic properties. It has been demonstrated that the beta-tubulin isoforms differ significantly in their interactions with colchicine. In an effort to understand the role of the ring-B of colchicine, we have studied the interaction of DAAC with purified beta-tubulin isoforms. The association was studied fluorometrically using a stopped-flow instrument under pseudo-first-order conditions in the presence of a large excess of drug. The observed pseudo-first-order rate constants increased in a nonlinear way with the drug concentration, indicating that the binding of DAAC to tubulin isoforms occur in two steps as is true for the binding of colchicine to tubulin (Garland, D.L. (1978) Biochemistry 17, 4266-4272), [formula: see text] where the first step is a fast reversible binding and the second step is a slow conformational change leading to the formation of the stable complex (T.DAAC)*. Kinetic analysis shows that the tubulin isoforms exhibit very little differences in their K1 values, which are 5794 +/- 670, 7109 +/- 1800, and 8993 +/- 1780 M-1 for alpha beta II, alpha beta III, and alpha beta IV, respectively. The k2 values for alpha beta II, alpha beta III, and alpha beta IV are 0.67 +/- 0.05, 0.05 +/- 0.006, and 0.59 +/- 0.07 s-1, respectively. The apparent on-rate constants (k(on,app) = K1k2) for alpha beta II, alpha beta III, and alpha beta IV are 3907 +/- 530, 376 +/- 80, and 5305 +/- 1200 M-1 s-1, respectively. The off-rate constants as measured by the loss of fluorescence of drug-tubulin complexes in the presence of a large excess of podophyllotoxin are 6.3 x 10(-4), 5.2 x 10(-4), and 5.7 x 10(-4) s-1, respectively, for alpha beta II, alpha beta III, and alpha beta IV. The affinity constants as determined by Scatchard analyses are 2.5 x 10(6), 1.5 x 10(6), and 4 x 10(6) M-1 for alpha beta II, alpha beta III, and alpha beta IV, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

Cloning, expression and purification of a sarcoplasmic calcium-binding protein from the sandworm Nereis diversicolor via a fusion product with chloramphenicol acetyltransferase.

A gene coding for the Nereis sarcoplasmic calcium-binding protein (NSCP) was synthesized and expressed in Escherichia coli. The sequence of the gene was derived from the protein sequence by reverse translation. It possesses a number of unique, regularly spaced, restriction endonuclease cleavage sites to facilitate future site-directed mutagenesis. For the cloning strategy the gene sequence was divided into four parts. Three parts were cloned by ligation of hybridized oligomers and one part by inverse PCR. The protein was expressed as a fusion protein with the bacterial chloramphenicol acetyl-transferase (CAT), which could be easily purified by affinity chromatography. At the junction of the CAT and NSCP moieties a recognition site for the proteolytic enzyme factor Xa was built in. However, the distance between the moieties appeared to be crucial to warrant cleavage. A kinetic analysis showed that NSCP prepared from the sandworm and the one expressed by E. coli behaved in the same way. This system provides a basis for site-specific mutagenesis studies, in order to elucidate the molecular mechanism of cation binding and concomitant conformational changes.

Amino Acid Sequence↗

PubMed1994

A fluorescence study of tryptophan-histidine interactions in the peptide anantin and in solution.

Anantin is a heptadecapeptide in which the C-terminal peptide chain pierces the covalently cyclized peptide ring formed by an amide link between the alpha-NH2 end group and the beta-carboxyl group of Asp(8). It contains a tryptophan and a histidine at positions 5 and 12, respectively. Des-Phe(17)-anantin lacks the C-terminal phenylalanine. Fluorescence emission intensity as a function of pH follows the ionization of a single residue. The pKa amounts to 7.23 +/- 0.03 for anantin and is attributed to His(12). At pH 9 the quantum yield is 0.12 +/- 0.01 for anantin, whereas at pH 4.5 the quantum yield decreases more than two-fold (0.05 +/- 0.01). Practically identical parameters are observed for des-Phe(17)-anantin. This pH dependency reveals intramolecular quenching of the excited indole ring of Trp(5) by the imidazole of His(12), which results in a marked decrease of the tryptophan fluorescence at low pH. In a multifrequency phase fluorometric study the fluorescence lifetimes for both peptides at pH 4.5 and pH 9 are determined. At both, pH fluorescence decay is well described by a sum of two exponentials. For anantin at pH 4.5 the lifetimes are 0.72 +/- 0.07 ns and 1.67 +/- 0.07 ns. At pH 9 the lifetimes are 1.11 +/- 0.12 ns and 2.55 +/- 0.03 ns. In methanol we find two lifetimes for anantin: 0.68 +/- 0.01 ns and 2.57 +/- 0.01 ns. The lifetimes are found to be slightly dependent upon emission wavelength. For des-Phe(17)-anantin practically the same values are observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

On the mechanism of the activation of human plasminogen by recombinant staphylokinase.

The mechanism of activation of human plasminogen by recombinant staphylokinase (STAR) was studied using the active site titrant p-nitrophenyl-p'-guanidinobenzoate (NPGB). NPGB prevented active site exposure in equimolar mixtures of plasminogen and STAR but reacted stoichiometrically with mixtures preincubated in the absence of titrant. Active site generation occurred progressively, with a marked initial lag phase followed by an exponential growth phase, and was associated with the conversion of single-chain plasminogen to two-chain plasmin. Incubation of mixtures of plasminogen and STAR with catalytic amounts (< 0.2% molar ratio) of preformed plasmin.STAR complex or of urokinase shortened the lag hase, whereas catalytic amounts (5% molar ratio) of the plasmin inhibitor alpha 2-antiplasmin delayed active site generation. The following kinetic model for the activation of plasminogen (P) by STAR (S) fits the experimental data, [formula: see text] and is described by [formula: see text] or [formula: see text] In this model, plasminogen and STAR produce an inactive complex (P.S), in which active plasmin.STAR (p.S) is generated in a rate limiting step, which is accelerated by plasminogen activators and delayed by plasmin inhibitors. At room temperature in a 0.1 M Veronal buffer, pH 8.3, containing 0.1 M arginine, the data are adequately fitted by the integrated equation with k1 = 4.0 x 10(-7) s-1 and k2 = 1.3 x 10(-2) microM-1 s-1. The k1 value could be explained by contamination of the plasminogen preparation with 3 ppm plasmin, converted by S to p.S. It is concluded that STAR activates plasminogen via a mechanism which differs in several essential aspects from that of streptokinase.

Amino Acid Sequence↗

PubMed1993

Evidence for an alternative pathway for colchicine binding to tubulin, based on the binding kinetics of the constituent rings.

The kinetics of tropolone methyl ether binding to tubulin were measured by following the loss of colchicine binding capacity upon preincubation of tubulin with tropolone methyl ether. At 25 degrees C a bimolecular association rate constant of 2.7 (+/- 0.2) M-1 min-1 was determined, and from the temperature dependence an activation energy of 37 (+/- 8) kJ.mol-1 was calculated. By displacement experiments a dissociation rate constant of 2.9 (+/- 0.6) x 10(-2) min-1 was determined at 25 degrees C. The effect of 3',4',5'-trimethoxyacetophenone (TMA) is 2-fold. TMA reduces the apparent association rate constant of colchicine, indicating that it equilibrates very rapidly and reversibly with the colchicine binding site. From this reduction the binding constant for TMA can be obtained. At 25 degrees C a value of 112 (+/- 13) M-1 is estimated. The binding of TMA is practically thermoneutral. Preincubation of tubulin with TMA over 30 min not only reduces the subsequent binding rate constant of colchicine but also the amplitude. This indicates that TMA also binds slowly in a second mode or site. Stopped-flow kinetic studies reveal that fast TMA binding competes for the initial binding of colchicine. From these results it is concluded that colchicine binds initially with its trimethoxybenzene ring and in a subsequent step with the tropolone ring.

Acetophenones↗