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Y Engelborghs

Publications and source records attributed to Y Engelborghs.

At least 73 records · Page 4Linked to original sources
PubMed1992

Fluorescence stopped-flow study of the interaction of tubulin with the antimitotic drug MDL 27048.

The kinetics of the binding of MDL 27048 to tubulin have been studied by fluorescence stopped flow. The binding is accompanied by a fluorescence increase. The time course can be described by a sum of two exponentials, assumed to be due to the presence of two major tubulin isoforms. The observed rate constants depend in a nonlinear way on the concentration of MDL in pseudo-first-order conditions. This concentration dependence can be described by the presence of a fast equilibrium of low affinity, followed by an isomerization of the initial complex. The dissociation kinetics have been studied by displacement experiments, in which MTC was used as a competitive ligand. The reaction enthalpy change for the first binding equilibrium and the activation energies for the forward and reverse steps of the isomerization were determined from the temperature dependence. This was possible for the two tubulin isotype populations. The kinetics of the binding of MDL to tubulin are slowed down in the presence of 3',4',5'-trimethoxyacetophenone, a fast binding analog of the colchicine A-ring, but are not influenced by the binding of tropolone methyl ether, indicating that the binding site of MDL has the A-subsite in common with colchicine, but not the C-subsite.

Animals↗
PubMed1992

Determination of the excited-state lifetimes of the tryptophan residues in barnase, via multifrequency phase fluorometry of tryptophan mutants.

A multifrequency phase fluorometric study is described for wild-type barnase and engineered mutant proteins in which tryptophan residues have been replaced by less fluorescent residues which do not interfere with the determination of the tryptophan emission spectra and lifetimes. The lifetimes of the three tryptophans in the wild-type protein have been resolved. Trp-35 has a single fluorescence lifetime, which varies in the different proteins between 4.3 and 4.8 ns and is pH-independent between pH 5.8 and 8.9. Trp-71 and Trp-94 behave as an energy-transfer couple with both forward and reverse energy transfer. The couple shows two fluorescence lifetimes: 2.42 (+/-0.2) and 0.74 (+/-0.1) ns at pH 8.9, and 0.89 (+/-0.05) and 0.65 (+/-0.05) ns at pH 5.8. In the mutant Trp-94----Phe the lifetime of Trp-71 is 4.73 (+/-0.008) ns at high pH and 4.70 (+/-0.004) ns at low pH. In the mutant Trp-71----Tyr, the lifetime of Trp-94 is 1.57 (+/-0.01) ns at high pH and 0.82 (+/-0.025) ns at low pH. From these lifetimes, one-way energy-transfer efficiencies can be calculated according to Porter [Porter, G.B. (1972) Theor. Chim. Acta 24, 265-270]. At pH 8.9, a 71% efficiency was found for forward transfer (from Trp-71 to Trp-94) and 36% for reverse transfer. At pH 5.8 the transfer efficiency was 86% for forward and 4% for reverse transfer (all +/-2%). These transfer efficiencies correspond fairly well with the ones calculated according to the theory of Förster [Förster, T. (1948) Ann. Phys. (Leipzig) 2, 55-75].(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Structure of a rapidly formed intermediate in ribonuclease T1 folding.

Kinetic intermediates in protein folding are short-lived and therefore difficult to detect and to characterize. In the folding of polypeptide chains with incorrect isomers of Xaa-Pro peptide bonds the final rate-limiting transition to the native state is slow, since it is coupled to prolyl isomerization. Incorrect prolyl isomers thus act as effective traps for folding intermediates and allow their properties to be studied more easily. We employed this strategy to investigate the mechanism of slow folding of ribonuclease T1. In our experiments we use a mutant form of this protein with a single cis peptide bond at proline 39. During refolding, protein chains with an incorrect trans proline 39 can rapidly form extensive secondary structure. The CD signal in the amide region is regained within the dead-time of stopped-flow mixing (15 ms), indicating a fast formation of the single alpha-helix of ribonuclease T1. This step is correlated with partial formation of a hydrophobic core, because the fluorescence emission maximum of tryptophan 59 is shifted from 349 nm to 325 nm within less than a second. After about 20 s of refolding an intermediate is present that shows about 40% enzymatic activity compared to the completely refolded protein. In addition, the solvent accessibility of tryptophan 59 is drastically reduced in this intermediate and comparable to that of the native state as determined by acrylamide quenching of the tryptophan fluorescence. Activity and quenching measurements have long dead-times and therefore we do not know whether enzymatic activity and solvent accessibility also change in the time range of milliseconds. At this stage of folding at least part of the beta-sheet structure is already present, since it hosts the active site of the enzyme. The trans to cis isomerization of the tyrosine 38-proline 39 peptide bond in the intermediate and consequently the formation of native protein is very slow (tau = 6,500 s at pH 5.0 and 10 degrees C). It is accompanied by an additional increase in tryptophan fluorescence, by the development of the fine structure of the tryptophan emission spectrum, and by the regain of the full enzymatic activity. This indicates that the packing of the hydrophobic core, which involves both tryptophan 59 and proline 39, is optimized in this step. Apparently, refolding polypeptide chains with an incorrect prolyl isomer can very rapidly form partially folded intermediates with native-like properties.

Amino Acid Sequence↗
PubMed1992

Acrylamide quenching of the fluorescence of glyceraldehyde-3-phosphate dehydrogenase: reversible and irreversible effects.

The acrylamide quenching of the tryptophan fluorescence of apo and holo glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was studied. In the case of apo-GAPDH, the steady state fluorescence quenching cannot be described by the classical Stern-Volmer equation: strong cooperative quenching is observed. In the presence of Pi and/or cofactor NAD+, an inaccessible fraction appears. Cooperative quenching is partially suppressed in the presence of Pi and fully absent in the presence of NAD+. The measurements of the fluorescence lifetimes of the holo-enzyme by phasefluorometry allow the resolution of two lifetimes. The long-lived component is quenched by acrylamide, the short-lived component is not. Quenching induces a red shift of the steady state emission peak. The quenching parameters from the lifetime measurements allow the quantitative description of the steady state fluorescence quenching data. In agreement with the observations of Orstan and Gafni (Photochemistry and Photobiology, (1990) 31, 725-731), we find that acrylamide causes a slow, irreversible loss of activity and a reduction of titratable thiol groups when it acts on the apo-enzyme. This inactivation is strongly reduced in the presence of NAD+. We show that this inactivation is also slowed down by the presence of Pi, and that it is accompanied by a loss of the NAD+ binding site. Blocking the thiol groups with 5,5'-dithio-bis-(2-nitrobenzoic acid) does not lead to a protection against the irreversible inactivation by acrylamide, showing that reactions other than thiol modifications are involved in the irreversible effect. A fraction of the inactivation can be reversed by treatment with mercapto-ethanol.

Acrylamide↗
PubMed1992

Allosteric inhibition of human immunodeficiency virus type 1 reverse transcriptase by tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and -thione compounds.

The reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is present in virions and infected cells as an heterodimer (p66/p51). A new class of potent and selective HIV-1 inhibitors, the tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and -thione (TIBO) derivatives, were found to exert their antiviral activity by interacting with monomeric HIV-1 RT (p66) in a way different from that of previously studied RT inhibitors such as azidothymidine 5'-triphosphate. Upon examination of the kinetic properties of the heterodimeric HIV-1 RT and its inhibition by TIBO compounds, a positive cooperativity between the subunits of the enzyme with regard to the 2'-deoxynucleoside 5'-triphosphates and the template/primer was observed. The cooperativity with respect to the template/primer may result from a progressive dimerization in the presence of increasing concentrations of the template/primer, a process referred to as polysteric linkage. Because the cooperativity of p66/p51 was abolished in the presence of TIBO, these compounds behave as allosteric inhibitors.

Benzodiazepines↗
PubMed1991

A fluorescence lifetime study of virginiamycin S using multifrequency phase fluorometry.

Using multifrequency phase fluorometry, fluorescence lifetimes have been assigned to the different protolytic forms of the antibiotic virginiamycin S. These lifetimes are 0.476 +/- 0.005 ns for the uncharged form, 1.28 +/- 0.2 and 7.4 +/- 0.2 ns for the zwitterionic form, 1.19 +/- 0.01 ns for the negatively charged form, and 1.9 +/- 0.1 ns for the double negatively charged form. The assignments are based on lifetime measurements as a function of pH, volume percent ethanol, and excitation wavelength. Excited-state proton transfer is taken into account. It is complete at pH values lower than 1, and no fluorescence of the fully protonated charged form is observed. At pH 8, an excited-state pK* increase is calculated, but proton association is too slow to cause excited-state proton transfer. The addition of divalent cations, at pH 9.4, increases the lifetime of the negatively charged form to a value dependent upon the specific nature of the cation (7.58 +/- 0.06 ns for Mg2+, 6.54 +/- 0.02 ns for Ca2+, and 3.74 +/- 0.05 ns for Ba2+). Monovalent cations do not influence the lifetimes, indicating that their binding to the macrocycle does not influence the fluorescent moiety. The model compound 3-hydroxypicolinamide shows an analogous behavior, but the retrieved lifetime can differ significantly.

Cations↗
PubMed1991

Interaction between virginiamycin S and ribosomes is partly provided by a salt bridge with a Mg2+ ion.

Type B streptogramins, such as virginiamycin S (VS), are cyclic hexadepsipeptides, inhibiting protein synthesis in prokaryotes. L-Thr connects a 3-hydroxypicolinyl residue (3-OH-Pic) to the peptide lactone ring. The fluorescence intensity of 3-OH-Pic is strongly increased by chelation to alkaline earth cations or binding to ribosomes. Similar behavior of the ribosome-VS complex and the VS-Mg chelate provides strong evidence for the presence of a VS-Mg chelate within the ribosomal binding site. Different models involving the ribosome binding of either members of the VS-Mg2+ chelate or both have been tested by fluorescence lifetime measurements, equilibrium titrations, and stopped-flow spectrofluorometry. Our data strongly suggest that (a) the interaction between VS and the ribosome is partly provided by a salt bridge between suitable acceptor atoms of the ribosome and the 3-OH-Pic residue, (b) Mg2+ can be exchanged by Mn2+ without dissociation of the ribosome-VS complex, (c) Mg2+ coordinates to the negative form of the 3-OH-Pic residue, probably via an interaction with the phenolate oxygen and the amide carboxyl group, and (d) the picolinyl residue is essential for the biological activity, as indicated by the lack of activity when the latter is replaced by a serine derivative.

Binding Sites↗
PubMed1990

Kinetics of conformational changes in Nereis sarcoplasmic calcium-binding protein upon binding of divalent ions.

The sarcoplasmic calcium-binding protein (SCP) of the sandworm Nereis possesses three Ca2(+)-Mg2+ sites but no Ca2(+)-specific site. Binding of Mg2+, but not of Ca2+, displays a marked positive cooperativity. The apparent cooperativity of Ca2+ binding in the presence of Mg2+ results from the allostery in Mg2+ dissociation. Binding of the first Ca2+ or Mg2+ induces all the conformational change, monitored by Trp fluorescence. In displacement reactions the conformational changes occur in the step SCP.Mg3----SCP.Ca1Mg2. Stopped-flow experiments indicate that Trp fluorescence changes upon Ca2(+)-binding are instantaneous whereas Mg2(+)-binding involves a fast pre-equilibrium (Keq = 28 M-1), followed by two slow consecutive conformational changes with k1 = 13.5 s-1 and k2 = 0.21 s-1. The fluorescence change after dissociation of Ca2+ from SCP is monophasic with k = 0.02 s-1; that after Mg2+ dissociation is biphasic with k1 = 0.8 s-1 and k2 = 0.1 s-1. Trp life time measurements also indicate that Ca2(+)- and Mg2(+)-induced conformational changes are completely different. Displacement of bound Ca2+ by Mg2+ can be described by two consecutive reactions in which the first (without fluorescence change) corresponds to the dissociation of the last Ca2+ (k1 = 2.4 s-1) and the second (k2 = 0.45 s-1) to the final conformational change observed upon direct Mg2+ binding. Displacement of bound Mg2+ by Ca2+ follows the kinetic scheme of simple competition; the conformational rate constant approaches asymptotically (up to the limit of 129 s-1) the dissociation rate of Mg2+ as the concentration of Ca2+ increases. In summary, after fast dissociation of Ca2+ or Mg2+, Nereis SCP slowly converts to the metal-free configuration, but in Ca2(+)-Mg2+ exchange reactions, the conformational changes are nearly as fast as the cation dissociation reactions.

Animals↗
PubMed1990

Analysis of the binding of glyceraldehyde-3-phosphate dehydrogenase to microtubules, the mechanism of bundle formation and the linkage effect.

The binding of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to microtubules was analysed by the determination of the concentration of the free enzyme in equilibrium with the complex. At low ionic strength (0.03 M) the binding data are best described by a sum of high (Kd = 0.28 microM) and low affinity (Kd = 7.14 microM) sites, showing positive cooperativity. Addition of 1 mM adenosine 5'-[beta,gamma-imido]triphosphate (p[NH]ppA) or increasing the ionic strength to 0.1 M reduces the binding constant of the high-affinity sites considerably. Adding microtubule-associated proteins (at I = 0.1 M) does not appreciably influence the affinities. Total stoichiometries vary over 2.1-1.2 tubulin dimers involved in a binding site for GAPDH. Bundling is reduced concomitantly with the reduction of the affinities and the increase of the stoichiometry to close to 1 mol GAPDH/mol tubulin dimer. The critical concentration of tubulin is practically not influenced by the binding of the enzyme. This behaviour is discussed in terms of the concept of linked functions. p[NH]ppA dissociates the bundles very rapidly. Analytical sedimentation studies showed that the dissociation of the bundles by p[NH]ppA is not due to the dissociation of the tetrameric enzyme. Bundling slows down association and dissociation of microtubules. The rate of bundle formation, after addition of GAPDH to preformed microtubules, is not dependent on the GAPDH concentration.

Adenosine Triphosphate↗
PubMed1990

Kinetics of the spontaneous organization of microtubules in solution.

Optically anisotropic zones occur spontaneously in solutions of microtubules. These tactoids, in which microtubules are arranged in parallel arrays, can be visualized by their birefringence. With microtubules assembled in the presence of associated proteins (MAPs), birefringence appears immediately after nucleation of polymerization, even at relatively low protein concentrations. It is not dependent on whether the assembly is initiated by temperature jump or by isothermal addition of GTP. With pure tubulin, assembled in buffers containing 25% glycerol or 4% dimethylsulfoxide and/or taxol, birefringence appears within a few hours, but it can be speeded up by gentle agitation. With tubulin assembled in the presence of MAPs, spontaneous orientation occurs simultaneously with polymerization. This may be due to the existence of more pronounced repulsive forces between microtubules when they are covered with MAPs. A simple calculation of the covolume, suggests that tactoid formation is expected for microtubules of lengths of 5 to 10 microns at protein concentrations in the range 1 to 3 mg/ml (as observed), and that repulsive forces will promote tactoid formation at even lower protein concentrations.

Alkaloids↗
PubMed1990

Evidence that folliculo-stellate cells do not impede the permeability of intercellular spaces to molecular diffusion in three-dimensional aggregate cell cultures of rat anterior pituitary.

The permeability of intercellular spaces within the anterior pituitary (AP) and the influence of folliculo-stellate (FS) cells on compartmentalization within this tissue, has become a matter of debate. In reaggregated pituitary cell cultures as well as in the AP in situ the intercellular gaps and follicle-like structures remain accessible to molecular diffusion, whereas in some studies FS cells were reported to form tight epithelia that impede macromolecular transport through the spaces between the epithelial cells. In the present study the permeability of AP cell reaggregates was examined using fluorescent BSA as a tracer. Using confocal scanning laser microscopy a direct visualization of the permeation process was achieved. Quantitative estimation of the effective diffusion coefficient (Deff) for fluorescein-BSA within the aggregates was obtained using the fluorescence photobleaching recovery technique. Deff was 1.33 +/- 0.31 x 10(-7) cm2/sec (mean +/- SD) in aggregates from 14-day-old female rats and 2.45 +/- 0.55 x 10(-7) cm2/sec in aggregates from adult female rats. These values are about three times lower than in free solution. Calculation of the time-dependent concentration distribution inside the aggregate for a Deff = 2 x 10(-7) cm2/sec revealed that the concentration of the fluorescent tracer in the center of the aggregate reaches 90% of the concentration outside the aggregate after 0.5 min for aggregates with a radius of 50 microns and 6 min for aggregates with a radius of 150 microns. Aggregates enriched in FS cells, in which we previously showed a sustained inhibition of secretory responses to stimulatory and inhibitory agents as compared to total population aggregates, showed a diffusion coefficient (Deff = 1.85 +/- 0.77 x 10(-7) cm2/sec) which was not significantly different from that in the total population aggregates. The present study shows that AP cell aggregates are fully permeable to diffusing molecules within minutes and that in a three-dimensional tissue configuration FS cells, which were reported to tonically inhibit AP hormone release in response to various secretagogues, do not impede molecular diffusion to an extent which would account for sustained inhibition of hormone release.

Animals↗
PubMed1989

Secondary structure analysis of tubulin and microtubules with Raman spectroscopy.

Raman spectroscopy is used to study the secondary structure of tubulin in the assembled and the dissociated states from the analysis of the amide-I band. Essentially two states are recognized: the GTP- and the GDP-bound state, differing in alpha-helix and antiparallel beta-sheet content. Microtubules give a spectrum which is very similar to the GDP-bound state. MAPs and temperature have minor effects, while increasing the pH up to 8 causes a reduction in alpha-helix content and a increase in antiparallel beta-sheet. The binding of demecolcine also induces structural changes which are similar to the GDP-bound state.

Amides↗
PubMed1987

Kinetics of binding of macrolides, lincosamides, and synergimycins to ribosomes.

The synergistic effect of type A (virginiamycin M (VM)) and type B (virginiamycin S (VS)) synergimycins and their antagonistic effect against erythromycin (a 14-membered macrolide) for binding to the large ribosomal subunit (50 S) have been related. This investigation has now been extended to 16-membered macrolides (leucomycin A3 and spiramycin) and to lincosamides (lincomycin). A dissociation of VS-ribosome complexes was induced as well by 16-membered macrolides as by lincosamides. The observed dissociation rate constant of VS-ribosome complexes was identified with the kappa-vs in the case of 16-membered macrolides, but linearly related to lincomycin concentration, suggesting a direct binding of the latter antibiotic to VS-ribosome complexes and the triggering of a conformational change of particles entailing VS release. Two different mechanisms were also involved in the VM-promoted reassociation to ribosomes of VS previously displaced by either macrolides or lincosamides. By binding to lincosamide-ribosome complexes, VM induced a conformational change of ribosomes resulting in higher affinity for VS and lower affinity for lincosamides. On the contrary, an incompatibility for a simultaneous binding of VM and 16-membered macrolides to ribosomes was observed. These results have been interpreted by postulating specific (nonoverlapping) and aspecific (overlapping) antibiotic binding sites at the peptidyltransferase domain. All the kinetic constants of five antibiotic families (type A and B synergimycins, 14- and 16-membered macrolides, and lincosamides) and a topological model of peptidyltransferase are presently available.

Erythromycin↗
PubMed1987

A fluorescence stopped flow study of the competition and displacement kinetics of podophyllotoxin and the colchicine analog 2-methoxy-5-(2',3',4'-trimethoxyphenyl) tropone on tubulin.

The colchicine analog 2-methoxy-5-(2',3',4'-trimethoxyphenol) tropone (AC) was used as a fluorescent probe to study the binding kinetics of podophyllotoxin at high concentrations. The observed pseudo-first order rate constant showed a linear concentration dependence up to 1 mM. The bimolecular rate constant (195 M-1 s-1 at 15 degrees C) and the activation energy (57 kJ/mol) correspond perfectly with those previously determined in the submicromolar range (Cortese, F., Bhattacharyya, B., and Wolf, J. (1977) J. Biol. Chem. 252, 1134-1140). Displacement kinetics of bound AC by podophyllotoxin, allow the determination of the dissociation rate constants for AC. By studying the temperature dependence, and combining with the binding rate constants previously determined (Engelborghs, Y., and Fitzgerald, T.J. (1986) Ann. N.Y. Acad. Sci. 466, 709-717) a full characterization of the kinetic pathway is possible. This is shown to differ considerably from the pathway of colchicine binding.

Animals↗
PubMed1987

The immune response towards beta-adrenergic ligands and their receptors--VII. Equilibrium and kinetic binding studies of l-alprenolol to a monoclonal anti-alprenolol antibody.

Binding of the catecholamine beta-adrenergic antagonist, l-alprenolol, by the IgGl anti-alprenolol monoclonal antibody 37A4 was examined using the radioligand 3H-dihydroalprenolol as an extrinsic signal and the increase in antibody fluorescence upon l-alprenolol binding as intrinsic signal. Equilibrium binding studies based on both signals indicated that the binding process was exothermic with a positive entropy change. The difference in the affinity constants obtained by radioligand binding studies and by fluorescence analysis could be ascribed to the higher affinity of the hydrogenated tritiated l-dihydroalprenolol compared to the unsaturated l-alprenolol. The association rate constants determined by both signals were 10(4)-10(5)/M/sec and showed a high activation enthalpy (8-10 kcal/mol), thus excluding a diffusion controlled reaction. At low temp (7 degrees C), the fluorescence stopped-flow studies showed non-linear pseudo first order kinetics, indicating the existence of a fast pre-equilibrium of low affinity, followed by a conformational change leading to the tight binding of the ligand. The dissociation rate constants determined using both signals were very similar. Thus, the differences in affinity between the hydrogenated and non-saturated l-alprenolol could be ascribed to the association rate constants. Affinity constants and thermodynamic parameters calculated from the kinetic data were in close agreement with those determined by equilibrium binding. The mechanisms of ligand binding are discussed in terms of the interactions of idiotypes and anti-idiotypes in the anti-catecholamine immune response.

Alprenolol↗
PubMed1984

Analysis of fluorescence quenching of ribosome-bound virginiamycin S.

The two virginiamycin components VM and VS interact synergistically with bacterial ribosomes in vitro and in vivo. Ribosome affinity for virginiamycin S increases about 10-fold upon incubation with virginiamycin M. This effect has been previously traced by spectrofluorimetric measurement based on the enhancement of virginiamycin S fluorescence upon its binding to the 50 S ribosomal subunit. In the present work the action of two virginiamycin S fluorescence quenchers, acrylamide and iodide, has been explored to gather information about the accessibility of ribosome-bound virginiamycin S and the variation of the accessibility level in the presence of virginiamycin M. Both acrylamide (non-ionized quencher) and iodide (ionized quencher) proved powerful quenchers of free virginiamycin S solutions. Since a comparable effect was obtained on 3- hydroxypicolinamide , the latter was indicated as the part of the molecule involved in the fluorescence effect. Fluorescence quenching by either agent was of the dynamic, i.e. collisional, type. Such an inference was based on the fact that these quenchers merely modified the emission spectrum (not the absorption spectrum), the bimolecular rate constant for the quenching process decreased linearly with the viscosity of the medium (static-type quenching is viscosity-independent), and that linear Stern-Volmer plots were obtained. The quenching ability of both agents underwent a sharp decrease in the presence of ribosomes; however, the Stern-Volmer equation was followed only in the case of acrylamide, whereas Lehrer 's relationship had to be applied in the case of iodide. When ribosomes were incubated with virginiamycin M, the fluorescence quenching ability of acrylamide and iodide was significantly reduced. Conclusions are as follows: a) the 3- hydroxypicolinyl residue of virginiamycin S is buried within an open well on the ribosome surface and is likely to be involved in the interaction with the binding site; b) the accessibility to the well is partly controlled by electrostatic forces; c) interaction of ribosomes with virginiamycin M entails a conformational change whereby the access to the well is reduced. These findings provide a molecular explanation for the previously observed increase of the association constant of virginiamycin S to ribosomes incubated with virginiamycin M which was found to be due to the decrease of the dissociation rate constant (the association rate constant remains practically the same).

Anti-Bacterial Agents↗
PubMed1983

Fluorescence stopped flow analysis of the interaction of virginiamycin components and erythromycin with bacterial ribosomes.

The kinetics of the interaction between the 50 S subunits (R) of bacterial ribosomes and the antibiotics virginiamycin S (VS), virginiamycin M (VM), and erythromycin have been studied by stopped flow fluorimetric analysis, based on the enhancement of VS fluorescence upon its binding to the 50 S ribosomal subunit. Virginiamycin components M and S exhibit a synergistic effect in vivo, which is characterized in vitro by a 5- to 10-fold increase of the affinity of ribosomes for VS, and by the loss of the ability of erythromycin to displace VS subsequent to the conformational change (from R to R*) produced by transient contact of ribosomes with VM. Our kinetic studies show that the VM-induced increase of the ribosomal affinity for VS (K*VS = 25 X 10(6) M-1 instead of KVS = 5.5 X 10(6) M-1) is due to a decrease of the dissociation rate constant (k*-VS = 0.008 s-1 instead of 0.04 s-1). The association rate constant remains practically the same (k+VS approximately k*+VS = 2.8 X 10(5) M-1 s-1), irrespective of the presence of VM. VS and erythromycin bind competitively to ribosomes. This effect has been exploited to determine the dissociation rate constant of VS directly by displacement experiments from VS . 50 S complexes, and the association rate constant of erythromycin (k+Ery = 3.2 X 10(5) M-1 S-1) on the basis of competition experiments for binding of free erythromycin and VS to ribosomes. By making use of the change in competition behavior of erythromycin and VS, after interaction of ribosomes with VM, the conformational change induced by VM has been explored. Within the experimentally available concentration region, the catalytic effect of VM has been shown to be coupled to its binding kinetics, and the association rate constant of VM has been determined (k+VM = 1.4 X 10(4) M-1 S-1). Evidence is presented for a low affinity binding of erythromycin (K*Ery approximately 3.3 X 10(4) M-1) to ribosomes altered by contact with VM. A model involving a sequence of 5 reactions has been proposed to explain the replacement of ribosome-bound erythromycin by VS upon contact of 50 S subunits with VM.

Erythromycin↗