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J M Andreu

Publications and source records attributed to J M Andreu.

13 recordsLinked to original sources
PubMed1992

Mechanism of binding of the new antimitotic drug MDL 27048 to the colchicine site of tubulin: equilibrium studies.

MDL 27048 [trans-1-(2,5-dimethoxyphenyl)-3-[4-(dimethylamino)phenyl]-2- methyl-2-propen-1-one] fluoresces when bound to tubulin but not in solution. This effect has been investigated and found to be mimicked by viscous solvents. Therefore, MDL 27048 appears to be a fluorescent compound whose intramolecular rotational relaxation varies as a function of microenvironment viscosity. The binding parameters of MDL 27048 to tubulin have been firmly established by fluorescence of the ligand, quenching of the protein fluorescence, and gel equilibrium chromatography. The apparent binding equilibrium constant was (2.75 +/- 0.45) x 10(6)M-1, and the binding site number was 0.81 +/- 0.12 (10 mM sodium phosphate-0.1 mM GTP, pH 7.0, at 25 degrees C). The binding is exothermic. The binding of MDL 27048 overlaps the colchicine and podophyllotoxin binding sites. Binding of MDL 27048 to the colchicine site was also measured by competition with MTC [2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one] , a well-characterized reversibly binding probe of the colchicine site [Andreu et al. (1984) Biochemistry 23, 1742-1752; Bane et al., (1984) J. Biol. Chem. 259, 7391-7398]. In contrast with close analogues of colchicine, MDL 27048 and podophyllotoxin neither affected the far-ultraviolet circular dichroism spectrum of tubulin, within experimental error, nor induced tubulin GTPase activity. Like podophyllotoxin, an excess of MDL 27048 over tubulin induced no abnormal cooperative polymerization of tubulin, which is characteristic of colchicine binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Chemically synthesized 182-235 segment of tau protein and analogue peptides are efficient in vitro microtubule assembly inducers of low apparent sequence specificity.

A 54-amino acid peptide reproducing the first and second repeats and intervening spacer sequence of the tubulin binding motif (residues 182-235) of murine tau protein, and several congeners representing different degrees of sequence scrambling have been prepared by solid phase methods and fully characterized chemically. These double-repeat peptides have been shown to induce microtubule formation at concentrations about one order of magnitude lower than single-repeat controls, under conditions close to the critical concentration needed for tubulin self-assembly. On the other hand, partial loss of microtubule-inducing capacity was observed for peptides with primary structures increasingly disorganized with respect to the canonical peptide. These results call into question the assumption that a high degree of primary structure specificity is involved in the tau-tubulin interaction leading to in vitro microtubule formation.

Amino Acid Sequence↗
PubMed1992

Low resolution structure of microtubules in solution. Synchrotron X-ray scattering and electron microscopy of taxol-induced microtubules assembled from purified tubulin in comparison with glycerol and MAP-induced microtubules.

The structure of microtubules has been characterized to 3 nm resolution employing time-resolved X-ray scattering. This has revealed detailed structural features of microtubules not observed before in solution. The polymerization of highly purified tubulin, induced by the antitumour drug taxol, has been employed as a microtubule model system. This assembly reaction requires Mg2+, is optimal at a 1:1 taxol to tubulin heterodimer molar ratio, proceeds with GTP or GDP and is intrinsically reversible. The X-ray scattering profiles are consistent with identical non-globular alpha and beta-tubulin monomers ordered within the known helical surface lattice of microtubules. Purified tubulin-taxol microtubules have a smaller mean diameter (approx. 22 nm) than those induced by microtubule associated proteins or glycerol (approx. 24 nm), but nearly identical wall substructure to the resolution of the measurements. This is because the majority of the former consist of only 12 protofilaments instead of the typical 13 protofilaments, as confirmed by electron microscopy of thin-sectioned, negatively stained and ice-embedded taxol microtubules. It may be concluded that taxol induces a slight reduction of the lateral contact curvature between tubulin monomers. The main fringe pattern observed in cryo-electron micrographs is consistent with a simple 12 protofilament 3-start skewed lattice model. Cylindrical closure of this lattice can be achieved by tilting the lattice 0.8 degrees with respect to the microtubule axis. The closure implies a discontinuity in the type of lateral contacts between the tubulin monomers (regardless of whether these are of the -alpha-beta- or the -alpha-alpha-/-beta-beta- type), which indicates that lateral contacts and the subunit specificity of taxol binding are, to a large degree, equivalent.

Alkaloids↗
PubMed1991

Physical and spectroscopic methods for the evaluation of the interactions of antimitotic agents with tubulin.

The physico-chemical methods for the study of the binding of ligands to tubulin are examined in-depth, emphasizing the assumptions on which they are based and their limitations. The criteria of specificity and linkage to protein self-association are presented. It is shown that, of the direct equilibrium binding techniques, Hummel-Dreyer gel permeation chromatography and rapid ultracentrifugation are applicable only when binding is not linked to protein self-association. Disc filtration is valid only when the reverse unbinding reaction is very low. Binding linked to protein self-association can be measured by batch gel permeation or by dialysis equilibrium. The indirect techniques, such as fluorescence perturbation or difference absorption spectroscopy are discussed in terms of the assumptions on which they are based. They are shown to be used best only after characterization of the binding by direct techniques. Equilibrium binding parameters can also be deduced from careful kinetic experiments. Comparison of calorimetrically measured enthalpies of binding to van't Hoff enthalpies derived from equilibrium measurements indicates that the method of choice is calorimetric, while comparison with van't Hoff analysis can reveal the existence of reaction steps not detected by equilibrium measurements. Use of other indirect approaches, such as titration of an enzymic activity, can also lead to the detection of additional steps. The criteria are set up for the proper data analysis of ligand binding linked to protein self-association and the selection of the proper mode of linkage. It is shown how the thermodynamic contributions of various moieties of a ligand can be established by a rational structural modification of the ligand and the proper analysis of the binding measurements, in which all non-specific entropic contributions are taken into account. It is demonstrated also how a similar analysis of binding data can lead to conclusions about the reaction pathway from a comparison of equilibrium thermodynamic measurements on judiciously modified ligand molecules.

Animals↗
PubMed1990

Tubulin assembly probed with antibodies to synthetic peptides.

Antibodies to synthetic peptides from the alpha and beta-tubulin sequences were employed to study zones of this protein active in microtubule assembly. In purified calf brain tubulin, six short sequences, selected according to their hydrophilicity and conservation, were found to be accessible to their affinity-purified immunoglobulin G (IgG) antibodies, in a competition radioimmunoassay performed under non-assembly native conditions. This indicated that the six sequences are exposed on the surface of the tubulin alpha beta heterodimer. IgG antibodies to the alpha(430-443) and beta(412-431) sequences perturbed substoichiometrically the assembly of purified tubulin, inducing microtubule bundling and the formation of opened up structures. These positions, which are close to the C termini, were accessible to the anti-peptide antibodies in taxol-induced microtubules, Zn2(+)-induced tubulin sheets, Mg2(+)-induced tubulin rings and in PtK2 cell microtubules. This, together with the comparison of the sizes and gross shapes of the antibody probes and microtubules, suggested that these sequences might be located at the protruding parts of the protofilaments. Antibodies to positions alpha(155-168) did not react with microtubules, while the equivalent zone beta(153-165) was accessible. The alpha(214-226) and beta(241-256) sequences were antigenically occluded in the taxol microtubules, Zn2(+)-induced sheets and Mg2(+)-induced ring arrays, as well as in native microtubules from PtK2 cells, though they became reactive by fixation. This result strongly suggested that these two zones are close to tubulin-tubulin contact sites. A working model is proposed in which the positions alpha(214-226) and beta(241-256) are close to the axial contacts between heterodimers, which lead to protofilament formation, while the positions alpha(241-256) and beta(214-226) are suggested to be related to the alpha-beta binding interface within the heterodimer.

Alkaloids↗
PubMed1976

Micrococcus lysodeikticus membrane ATPase. Effect of trypsin on stimulation of a purified form of the enzyme and idenfification of its natural inhibitor.

A soluble purified form of Micrococcus lysodeikticus ATPase (form BAT, from strain B, active, trypsin-stimulated) was stimulated 100% by trypsin and this stimulation was inhibited by preincubation of the protease with phenyl methyl sulphonylfluoride. This form of the enzyme was also stimulated 125-150% by filtration on Sephadex G-200. Analysis by sodium dodecyl sulphate-gel electrophoresis showed that stimulation of this form of M. lysodeikticus ATPase was always accompanied by the disappearance of a subunit of mol. wt. 25000 (epsilon subunit). It suggests that this subunit is the natural inhibitor of M. lysodeikticus ATPase. In the case of ATPase stimulation by trypsin, a partial and limited degradation of the alpha subunit was also observed. The interaction between the epsilon subunit and the rest of the ATPase complex was reversibly affected by pH, suggesting its non-covalent nature.

Adenosine Triphosphatases↗
PubMed1976

Membrane adenosine triphosphatase of Micrococcus lysodeikticus. ISolation of two forms of the enzyme complex and correlation between ezymatic stability, latency and activity.

Two new forms of the plasma membrane ATP-ase of Micrococcus lysodeikticus NCTC 2665 were isolated from a sub-strain of the microorganism by polyacrylamide gel electrophoresis. One of them had a mol.wt of 368,000 and a very low specific activity (0.80 mumol.min-1.mg protein-1) that could not be stimulated by trypsin. This form has been called B1 (strain B, inactive). If the elctrophoresis was carried out in the presence of reducing agents (i.e., dithiothreitol) and the pH of the effluent maintained at a value of 8.5 another form of the enzyme was obtained. This had a mol.wt of 385,000 and a specific activity of 2.5-5.0 mumol.min-1.mg protein-1 that could be stimulated by trypsin to 5-10 mumol.min-1.mg protein-1. This preparation of the ATPase has been called from BA (strain B, enzyme active). The subunit composition of both forms has been studied by sodium dodecyl sulphate and urea gel electrophoresis and compared to that of the enzyme previously purified from the original strain (form A). The three forms of the enzyme had similar beta and delta subunits, with mol.wt of about 50,000 and 30,000 dalton, respectively. They also had in common the component(s) of relative mobility 1.0, whose status as true subunit(s) of the enzyme remains yet to be established. However, subunit alpha, that had a mol.wt of about a 52,500 in form A (ANDREU et al. Eur. J. Biochem. (1973) 37, 505-515), had a mol.wt similar to beta in form B1 and about 60,000 in form BA. Furthermore BA usually showed two types of this subunit (alpha' and alpha") and an additional peptide chain E) with a mol.wt of about 25,000 dalton. This latter subunit seemed to account for the stimulation by trypsin of form BA. Forms BA could be converted to B1 by storage and freezing and thawing. Conventional protease activity could not be detected in any of the purified ATPase forms and addition of protease inhibitors to form BA failed to prevent its conversion to form B1. The low activity form (B1) was more stable than the active forms of the enzyme and also differeed in its circular dichroism. These results show that M. lysodeikticus ATPase can be isolated in several forms. Although these variations may be artifacts caused by the purification procedures, they provide model systems for understanding the structural and functional relationships of the enzyme and for drawing some speculations about its function in vivo.

Adenosine Triphosphatases↗
PubMed1975

Conformational and molecular responses to pH variation of the purified membrane adenosine triphosphatase of Micrococcus lysodeikticus.

A preparation of ATPase from the membranes of Micrococcus lysodeikticus, solubilized and more than 95% pure, showed two main bands in analytical polyacrylamide gel electrophoresis. They did not correspond to isoenzymes because one band could be converted into the other by exposure to a mildly alkaline pH value. The conversion was paralleled by changes in molecular weight, circular dichroism and catalytic properties. Denaturation by pH at 25 degrees C was followed by means of circular dichroism, ultracentrifugation and polyacrylamide gel electrophoresis. A large conformational transition took place in the acid range with midpoints at about pH = 3.6 (I = 10(-4) M), 4.3 (I = 0.03 M) and 5.3 (I = 0.1 M). The transition was irreversible. Strong aggregation of the protein occurred in this range of pH. The final product was largely random coil, but even at pH 1.5 dissociation into individual subunits was not complete. However, partial dissociation took place at pH 5 (I = 0.028 M). At this pH value the enzyme was inactive, but 20-30% of the activity could be recovered when the pH was returned to 7.5. In the alkaline region the midpoint of the transition occurred near pH = 11 (I = 0.028 M). The pK of most of the tyrosine residues of the protein was about 10.9. The unfolding was irreversible and the protein was soon converted into peptide species with molecular weights lower than those determined for the subunits by gel electrophoresis in the presence of sodium dodecyl sulphate. Conventional proteolysis did not account for the transformation.

Adenosine Triphosphatases↗
PubMed1975

Micrococcus lysodeikticus ATPase. Purification by preparative gel electrophoresis and subunit structure studied by urea and sodium dodecylsulfate gel electrophoresis.

Micrococcus lysodeikticus ATPase was purified by preparative gel electrophoresis after its "shodk wash" release from the membrane. The method afforded the highest yield of pure protein in the minimum time as compared with former purification procedures. The pure protein had a specific activity of 7 mumol Pi-min- minus 1-mg- minus 1 with incubation times not longer than 3 min, 345 000 mol. wt and was not stimulated by trypsin. By gel electrophoresis at alkaline pH (8.5) in 8 M urea or in sokium dodecylsulfate, the ATPase revealed a complex pattern with two major subunits (alpha and beta) and two minor ones (gamma and delta). The non-identity between the major subunits was demonstrated.

Adenosine Triphosphatases↗