Photoaffinity labeling of tubulin subunits with a photoactive analogue of vinblastine

A photoactive, radioactive analogue of vinblastine, N-(p-azido[3,5-3H]benzoyl)-N'-(beta-amino-ethyl)vindesine ([ 3H]NABV), was used to localize the Vinca alkaloid binding site(s) on calf brain tubulin after establishing that its in vitro interactions with tubulin were comparable to those of vinblastine. Microtubule assembly was inhibited by 50% with 2 microM NABV or vinblastine. At higher drug concentrations, NABV and vinblastine both induced tubulin aggregation, and both drugs inhibited tubulin-dependent GTP hydrolysis. Vinblastine and NABV inhibited each other's binding to tubulin, but the binding of neither drug was inhibited by colchicine. Two classes of binding sites for NABV and vinblastine were found on calf brain tubulin. High-affinity sites had apparent KD values of 4.2 and 0.54 microM for NABV and vinblastine, respectively, whereas the low-affinity binding sites showed apparent KD values of 26 and 14 microM for NABV and vinblastine, respectively. Mixtures of tubulin and [3H]NABV were irradiated at 302 nm and analyzed for incorporation of radioactivity into protein. Photolabeling of both the alpha- and beta-subunits of tubulin with increasing concentrations of [3H]NABV exhibited a biphasic pattern characteristic of specific and nonspecific reactions. Nonspecific labeling was determined in the presence of excess vinblastine. Saturable specific covalent incorporation into both subunits of tubulin was observed, with an alpha:beta ratio of 3:2 and maximum saturable incorporation of 0.086 and 0.056 mol of [3H]NABV/mol of alpha-tubulin and beta-tubulin, respectively. Such photolabeling of the tubulin subunits will permit precise localization of Vinca alkaloid binding sites, including identification of the amino acid residues involved, an essential requirement for understanding the interactions of these drugs with tubulin.     

A photoaffinity derivative of colchicine: 6'-(4'-azido-2'-nitrophenylamino)hexanoyldeacetylcolchicine. Photolabeling and location of the colchicine-binding site on the alpha-subunit of tubulin

A photoaffinity analog of colchicine, 6-(4'-azido-2'-nitrophenylamino)hexanoyldeacetylcolchicine, was synthesized by reacting deacetylcolchicine or [3H]deacetylcochicine with N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate. Homogeneity of the photoaffinity analog was established by thin-layer chromatography and high-pressure liquid chromatography. The structure of the photoaffinity analog was determined by 1H and 13C NMR, infrared and ultraviolet-visible spectroscopies, and elemental analysis. Binding of 6-(4'-azido-2'-nitrophenylamino)hexanoyldeacetylcolchicine to bovine renal tubulin was measured by competition with [3H]colchicine. The value of the apparent Ki for the photoaffinity analog was 0.28 microM in the concentration range of 0.8-1.2 microM of the analog. A value of 0.50 microM for the apparent Kd was measured by the direct binding of the tritiated photoaffinity analog to tubulin. The analog is slightly more potent an inhibitor of microtubule formation than colchicine. The photoaffinity analog reacted with renal tubulin upon irradiation with a mercury lamp equipped with a 420-nm cutoff filter. Spectral and radiochemical analyses of the tubulin after photolysis and dialysis have demonstrated a stoichiometric incorporation of the photoaffinity analog in the alpha-subunit of the tubulin. Covalent labeling of tubulin with the photoaffinity analog decreases the extent of [3H]colchicine binding by more than 90%.     

Synthesis and biological evaluation of aryl azide derivatives of combretastatin A-4 as molecular probes for tubulin

Two new aryl azides, (Z)-1-(3'-azido-4'-methoxyphenyl)-2-(3",4",5"-trimethoxyphenyl)ethene 9 and (Z)-1-(4'-azido-3'-methoxyphenyl)-2-(3",4",5"-trimethoxyphenyl)ethene 5, modeled after the potent antitumor, antimitotic agent combretastatin A-4 (CA-4), have been prepared by chemical synthesis as potentially useful photoaffinity labeling reagents for the colchicine site on beta-tubulin. Aryl azide 9, in which the 3'-hydroxyl group of CA-4 is replaced by an azido moiety, demonstrates excellent in vitro cytotoxicity against human cancer cell lines (NCI 60 cell line panel, average GI50 = 4.07 x 10(-8) M) and potent inhibition of tubulin polymerization (IC50 = 1.4+/-0.1 microM). The 4'-azido analogue 5 has lower activity (NCI 60 cell line panel, average GI50 = 2.28 x 10(-6) M, and IC50 = 5.2+/-0.2 microM for inhibition of tubulin polymerization), suggesting the importance of the 4'-methoxy moiety for interaction with the colchicine binding site on tubulin. These CA-4 aryl azide analogues also inhibit binding of colchicine to tubulin, as does the parent CA-4, and therefore these compounds are excellent candidates for photoaffinity labeling studies.     

Photoaffinity labeling of mitochondrial adenosinetriphosphatase by 2-azidoadenosine 5'-[alpha-32P]diphosphate

2-Azidoadenosine 5'-diphosphate (2-azido-ADP) labeled with 32P in the alpha-position was prepared and used to photolabel the nucleotide binding sites of beef heart mitochondrial F1-ATPase. The native F1 prepared by the procedure of Knowles and Penefsky [Knowles, A. F., & Penefsky, H. S. (1972) J. Biol. Chem. 247, 6617-6623] contained an average of 2.9 mol of tightly bound ADP plus ATP per mole of enzyme. Short-term incubation of F1 with micromolar concentrations of [alpha-32P]-2-azido-ADP in the dark in a Mg2+-supplemented medium resulted in the rapid supplementary binding of 3 mol of label/mol of F1, consistent with the presence of six nucleotide binding sites per F1. The Kd relative to the reversible binding of [alpha-32P]-2-azido-ADP to mitochondrial F1 in the dark was 5 microM in the presence of MgCl2 and 30 microM in the presence of ethylenediaminetetraacetic acid. A linear relationship between the percentage of inactivation of F1 and the extent of covalent photolabeling by [alpha-32P]-2-azido-ADP was observed for percentages of inactivation up to 90%, extrapolating to 2 mol of covalently bound [alpha-32P]-2-azido-ADP/mol of F1. Under these conditions, only the beta subunit was photolabeled. Covalent binding of one photolabel per beta subunit was ascertained by electrophoretic separation of labeled and unlabeled beta subunits based on charge differences and by mapping studies showing one major radioactive peptide segment per photolabeled beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photoaffinity labeling of Torpedo nicotinic receptor with the agonist [3H]DCTA: identification of amino acid residues which contribute to the binding of the ester moiety of acetylcholine

Torpedo marmorata acetylcholine binding sites were photolabeled using 360 nm light, at equilibrium in the desensitized state, with the agonist [3H]DCTA utilizing the CeIV/glutathione procedure described previously (Grutter, et al. (1999) Biochemistry 38, 7476-7484). Photoincorporation of [3H]DCTA was concentration-dependent with a maximum of 7.5% specific labeling on the alpha-subunit and 1.2% on the gamma-subunit. The apparent dissociation constants for labeling of the alpha- and gamma-subunits were 2.2 +/- 1.1 and 3.6 +/- 2.8 microM, respectively. The alpha-chains isolated from receptor-rich membranes photolabeled in the absence or in the presence of carbamylcholine were cleaved with CNBr using an efficient "in gel" procedure. The resulting peptide fragments were purified by HPLC and further submitted to trypsinolysis. The digest was analyzed by HPLC leading to a single radioactive peak which, by microsequencing, revealed two sequences extending from alpha Lys-179 and from alpha His-186, respectively. Radioactive signals could be unambiguously attributed to positions corresponding to residues alpha Tyr-190, alpha Cys-192, alpha Cys-193, and alpha Tyr-198. These four identified [3H]DCTA-labeled residues, which have been also labeled with other affinity and photoaffinity probes including the agonist [3H]nicotine, belong to loop C of the ACh binding site. The chemical structure of [3H]DCTA, together with its well-defined and powerful photochemical reactivity, provides convincing evidence that loop C-labeled residues are primarily involved in the interaction with the ester moiety of acetylcholine.     

Identification of the cysteine residue of beta-tubulin alkylated by the antimitotic agent 2,4-dichlorobenzyl thiocyanate, facilitated by separation of the protein subunits of tubulin by hydrophobic column chromatography

The mechanism of action of the antimitotic drug 2,4-dichlorobenzyl thiocyanate (DCBT) has been examined in detail. Shown in previous studies to inhibit tubulin polymerization [Abraham, I., Dion, R. L., Duanmu, C., Gottesman, M. M., & Hamel, E. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6839-6843] and to form a covalent bond preferentially with beta-tubulin [Bai, R., Duanmu, C., & Hamel, E. (1989) Biochim. Biophys. Acta 994, 12-20], DCBT has now been documented to interact at low concentrations with a high degree of specificity at cysteine residue 239 of beta-tubulin. These low DCBT concentrations also result in the partial inhibition of tubulin polymerization. Such findings strongly indicate that cysteine-239 of beta-tubulin is essential for microtubule assembly. Although alpha-tubulin is alkylated almost as well as beta-tubulin when the drug:tubulin ratio = 5:1 (Bai et al., 1989), beta-tubulin is alkylated about 25 times as extensively as alpha-tubulin, almost exclusively at Cys-239, when the drug:tubulin ratio = 1:5. In addition, we find that low concentrations of DCBT do not affect the binding of colchicine to tubulin but that colchicine and related compounds do reduce the alkylation of tubulin by DCBT. This suggests that Cys-239 of beta-tubulin is not involved in the binding of colchicine to tubulin but that this amino acid residue is at least partially masked by the drug when it is bound to the protein. We also describe a column chromatography procedure (hydrophobic chromatography on decylagarose) useful for the preparative resolution of unalkylated, although denatured, alpha- and beta-tubulin.     

Two photoaffinity analogues of the tripeptide, hemiasterlin, exclusively label alpha-tubulin

A synthetic analogue of the tripeptide hemiasterlin, designated HTI-286, depolymerizes microtubules, is a poor substrate for P-glycoprotein, and inhibits the growth of paclitaxel-resistant tumors in xenograft models. Two radiolabeled photoaffinity analogues of HTI-286, designated 4-benzoyl-N,beta,beta-trimethyl-l-phenylalanyl-N(1)-[(1S,2E)-3-carboxy-1-isopropylbut-2-enyl]-N(1),3-dimethyl-l-valinamide (probe 1) and N,beta,beta-trimethyl-l-phenylalanyl-4-benzoyl-N-[(1S,2E)-3-carboxy-1-isopropyl-2-butenyl]-N,beta,beta-trimethyl-l-phenylalaninamide (probe 2), were made to help identify HTI-286 binding sites in tubulin. HTI-286, probe 1, and probe 2 had similar affinities for purified tubulin [apparent K(D(app)) = 0.2-1.1 microM], inhibited polymerization of purified tubulin approximately 80%, and were potent inhibitors of cell growth (IC(50) = 1.0-22 nM). Both radiolabeled probes labeled exclusively alpha-tubulin. Labeling by [(3)H]probe 1 was inhibited by probe 1, HTI-286, vinblastine, or dolastatin 10 (another peptide antimitotic agent that depolymerizes microtubules) but was either unaffected or enhanced (at certain temperatures) by colchicine or paclitaxel. [(3)H]Probe 1 also labeled exclusively tubulin in cytosolic extracts of whole cells. The major, if not exclusive, contact site for probe 1 was mapped to residues 314-339 of alpha-tubulin and corresponds to the sheet 8 and helix 10 region. This region is known to (1) have longitudinal interactions with beta-tubulin across the interdimer interface, (2) have lateral interactions with adjacent protofilaments, and (3) contact the N-terminal region of stathmin, a protein that induces depolymerization of tubulin. Binding of probe 1 to this region may alter the conformation of tubulin outside the labeling domain, since enzymatic removal of the C-terminus of only alpha-tubulin by subtilisin after, but not before, photolabeling is blocked by probe 1. These results suggest that hemiasterlin is in close contact with alpha-tubulin and may span the interdimer interface so that it contacts the vinblastine- and dolastatin 10-binding sites believed to be in beta-tubulin. In addition, we speculate that antimitotic peptides mimic the interaction of stathmin with tubulin.     

Specific Vinca alkaloid-binding polypeptides identified in calf brain by photoaffinity labeling

A radioactive, photoactive Vinca alkaloid, N-(p-azido-[3,5-3H]-benzoyl)-N'-beta-aminoethylvindesine [( 3H]NABV) with pharmacological and biological activities similar to vinblastine was synthesized and used to identify specific Vinca alkaloid macromolecular interactions in calf brain homogenate by photoaffinity labeling. The most prominent photolabeled species were 54.3- and 21.5-kDa polypeptides. The Vinca alkaloid-binding specificity of these polypeptides was confirmed by competitive blocking of specific photolabeling by vinblastine but not by colchicine or daunorubicin. The 54.3- and 21.5-kDa polypeptides exhibited specific half-maximum saturable photolabeling at 2.1 and 0.95 X 10(-7) M [3H]NABV, respectively. Relative vinblastine and NABV association constants (Ka vinblastine/Ka NABV) for the 54.3- and 21.5-kDa polypeptides were estimated to be 0.86 and 1.4, respectively. The 54.3-kDa component was found in both high speed (100,000 X g; 1 h) pellet and supernatant fractions, whereas the 21.5-kDa component was located primarily in the high speed pellet. Photolabeling of both components was maximal after 12-min UV light exposure, linear up to 120 micrograms of homogenate protein and only slightly affected by the nitrene scavenger p-aminobenzoic acid. The 54.3-kDa polypeptides of [3H]NABV-photolabeled calf brain high speed supernatant and detergent-solubilized high speed pellet fractions were identified as tubulin subunits by immunoprecipitation with monoclonal antibodies to alpha- or beta-tubulin subunits. Although the identity and function of the 21.5-kDa polypeptide is not known, this polypeptide may have a role in membrane-related effects of the Vinca alkaloids. These results demonstrate that [3H]NABV is an attractive tool for identifying and characterizing specific high affinity vinblastine cellular polypeptide acceptors which may initiate or mediate known and unknown mechanisms of Vinca alkaloid action.     

A photoactive phosphonamide derivative of GTP for the identification of the GTP-binding domain in beta-tubulin

A GTP photoaffinity probe (125I-APTG) was developed that incorporated an [125I]-N-(4-azidophenyl)-2-amino-3-(4-hydroxy-3-iodophenyl)propionamide group at the gamma-position of GTP through a phosphonamide linkage. A combination of saturation and GTP protection studies (90% protection at 25 microM GTP with an apparent Kd of 5 microM) validated the use of this new probe as a satisfactory GTP mimic. This probe offered the advantage of possessing an 125I radiolabel external to the GTP moiety, in contrast to the previously reported [gamma 32P]-8-N3GTP that possessed an internal 32P radiolabel. This novel feature accommodated the purification of photolabeled peptides using a combination of ion-exclusion, gel filtration, and HPLC techniques. [125I]APTG was used to identify a peptide (beta:65-79) in the exchangeable GTP-binding domain of the beta-subunit of tubulin.     

Characterization of the Taxol binding site on the microtubule. Identification of Arg(282) in beta-tubulin as the site of photoincorporation of a 7-benzophenone analogue of Taxol

Photoaffinity labeling methods have allowed a definition of the sites of interaction between Taxol and its cellular target, the microtubule, specifically beta-tubulin. Our previous studies have indicated that [(3)H]3'-(p-azidobenzamido)Taxol photolabels the N-terminal 31 amino acids of beta-tubulin (Rao, S., Krauss, N. E., Heerding, J. M., Swindell, C. S., Ringel, I., Orr, G. A., and Horwitz, S. B. (1994) J. Biol. Chem. 269, 3132-3134) and [(3)H]2-(m-azidobenzoyl)Taxol photolabels a peptide containing amino acid residues 217-233 of beta-tubulin (Rao, S., Orr, G. A., Chaudhary, A. G., Kingston, D. G. I., and Horwitz, S. B. (1995) J. Biol. Chem. 270, 20235-20238). The site of photoincorporation of a third photoaffinity analogue of Taxol, [(3)H]7-(benzoyldihydrocinnamoyl) Taxol, has been determined. This analogue stabilizes microtubules polymerized in the presence of GTP, but in contrast to Taxol, does not by itself enhance the polymerization of tubulin to its polymer form. CNBr digestion of [(3)H]7-(benzoyldihydrocinnamoyl)Taxol-labeled tubulin, with further arginine-specific cleavage by clostripain resulted in the isolation of a peptide containing amino acid residues 277-293. Amino acid sequence analysis indicated that the photoaffinity analogue cross-links to Arg(282) in beta-tubulin. Advances made by electron crystallography in understanding the structure of the tubulin dimer have allowed us to visualize the three sites of photoincorporation by molecular modeling. There is good agreement between the binding site of Taxol in beta-tubulin as determined by photoaffinity labeling and electron crystallography.     

Mapping of nucleotide-depleted mitochondrial F1-ATPase with 2-azido-[alpha-32P]adenosine diphosphate. Evidence for two nucleotide binding sites in the beta subunit

Photolabeling of nucleotide binding sites in nucleotide-depleted mitochondrial F1 has been explored with 2-azido [alpha-32P]adenosine diphosphate (2-N3[alpha-32P] ADP). Control experiments carried out in the absence of photoirradiation in a Mg2+-supplemented medium indicated the presence of one high affinity binding site and five lower affinity binding sites per F1. Similar titration curves were obtained with [3H]ADP and the photoprobe 3'-arylazido-[3H]butyryl ADP [( 3H]NAP4-ADP). Photolabeling of nucleotide-depleted F1 with 2-N3[alpha-32P]ADP resulted in ATPase inactivation, half inactivation corresponding to 0.6-0.7 mol of photoprobe covalently bound per mol F1. Only the beta subunit was photolabeled, even under conditions of high loading with 2-N3[alpha-32P]ADP. The identification of the sequences labeled with the photoprobe was achieved by chemical cleavage with cyanogen bromide and enzymatic cleavage by trypsin. Under conditions of low loading with 2-N3[alpha-32P]ADP, resulting in photolabeling of only one vacant site in F1, covalently bound radioactivity was located in a peptide fragment of the beta subunit spanning Pro-320-Met-358 identical to the fragment photolabeled in native F1 (Garin, J., Boulay, F., Issartel, J.-P., Lunardi, J., and Vignais, P. V. (1986) Biochemistry 25, 4431-4437). With a heavier load of photoprobe, leading to nearly 4 mol of photoprobe covalently bound per mol F1, an additional region of the beta subunit was specifically labeled, corresponding to a sequence extending from Gly-72 to Arg-83. The isolated beta subunit also displayed two binding sites for 2-N3-[alpha-32P]ADP. When F1 was first photolabeled with a low concentration of NAP4-ADP, leading to the covalent binding of 1.5 mol of NAP4-ADP/mol F1, with the bound NAP4-ADP distributed equally between the alpha and beta subunits, a subsequent photoirradiation in the presence of 2-N3[alpha-32P]ADP resulted in covalent binding of the 2-N3[alpha-32P]ADP to both alpha and beta subunits. It is concluded that each beta subunit in mitochondrial F1 contains two nucleotide binding regions, one of which belongs to the beta subunit per se, and the other to a subsite shared with a subsite located on a juxtaposed alpha subunit. Depending on the experimental conditions, the subsite located on the alpha subunit is either accessible or masked. Unmasking of the subsite in the three alpha subunits of mitochondrial F1 appears to proceed by a concerted mechanism.     

Characterization of photoaffinity labeling of benzodiazepine binding sites

The specific photoaffinity labeling of membrane-bound and detergent-solubilized benzodiazepine binding sites has been investigated using UV irradiated [3H] flunitrazepam as a photochemical probe. The time course and the regional and pharmacological specificity of the photolabeling reaction has been determined for "brain-specific" benzodiazepine binding sites; "peripheral-type" binding sites treated in an identical manner were not specifically labeled. Comparison of the number of sites labeled and blocked by [3H]flunitrazepam photolabeling of detergent-solubilized preparations indicated that about one site was blocked and unavailable for reversible binding for each site photolabeled. In contrast, when membrane-bound sites were photolabeled, about four sites were inactivated for each site photolabeled. Examination of photolabeled binding sites from various brain regions including cortex, striatum, and hippocampus using sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave only a single labeled band of apparent Mr = 48,000.     

Kinetics of colchicine binding to purified beta-tubulin isotypes from bovine brain.

Tubulin, the constituent protein of microtubules, is an alpha beta heterodimer; both alpha and beta exist in several isotypic forms whose functional significance is not precisely known. The antimitotic alkaloid colchicine binds to mammalian brain tubulin in a biphasic manner under pseudo-first-order conditions in the presence of a large excess of colchicine (Garland, D. L. (1978) Biochemistry 17, 4266-4272). We have studied the kinetics of colchicine binding to purified beta-tubulin isotypes and find that each of the purified beta-tubulin isotypes binds colchicine in a monophasic manner. The apparent on-rate constants for the binding of colchicine to alpha beta II-, alpha beta III-, and alpha beta IV-tubulin dimers are respectively 132 +/- 5, 30 +/- 2, and 236 +/- 7 M-1 s-1. When the isotypes are mixed, the kinetics become biphasic. Scatchard analysis revealed that the isotypes differ significantly in their affinity constants (Ka) for binding colchicine. The affinity constants are 0.24 x 10(6), 0.12 x 10(6), and 3.31 x 10(6) M-1, respectively, for alpha beta II-, alpha beta III-, and alpha beta IV-tubulin dimers. Our results are in agreement with the hypothesis that the beta-subunit of tubulin plays a major role in the interaction of colchicine with tubulin. Our binding data raise the possibility that the tubulin isotypes might play important regulatory roles by interacting differently with other non-tubulin proteins in vivo, which in turn, may regulate microtubule-based functions in living cells.     

Photoaffinity labeling of the recBCD enzyme of Escherichia coli with 8-azidoadenosine 5'-triphosphate

The recB and recD subunits of the recBCD enzyme (exonuclease V) from Escherichia coli were covalently photolabeled with the ATP photoaffinity analogue [alpha-32P]8-azido-ATP. The labeling was specific for ATP binding sites by the following criteria. Saturation occurs at high 8-azido-ATP concentrations with dissociation constants of 30 and 120 microM for the recD and recB subunits, respectively; ATP strongly inhibits the photolabeling; 8-azido-ATP is hydrolyzed by the recBCD enzyme and supports its double-stranded DNA exonuclease activity; and the label is largely confined to two peptides obtained by tryptic digestion of the photolabeled holoenzyme; one is derived from the recB subunit and the other from the recD subunit.     

Tubulin sequence region beta 155-174 is involved in binding exchangeable guanosine triphosphate

Assembly-competent microtubule protein was directly photoaffinity labeled with [alpha-32P]guanosine triphosphate by UV irradiation. The labeled tubulin was digested with trypsin. The radioactive fragments were isolated and sequenced, revealing beta-tubulin residues 155-174 to be the major labeled region. An antibody to a synthetic peptide comprising residues beta 154-165 inhibits GTP incorporation and tubulin polymerization.     

Studies of a plasma membrane steroid receptor in Xenopus oocytes using the synthetic progestin RU 486

A steroid binding protein (Mr = 110,000) has previously been identified in the plasma membrane of Xenopus laevis oocytes by photoaffinity labeling with [3H]R5020. In order to further characterize this steroid receptor, the photoaffinity labeled receptor protein was solubilized with 0.1% Brij 35. The solubilized labeled receptor yielded an approximate mol. wt of 102,000 +/- 2,000 by sucrose density gradient centrifugation, suggesting that the solubilized receptor exists as a monomer. RU 486, a synthetic progestin antagonist for mammalian cytosolic receptor systems, inhibited up to 70% of [3H] R5020 photoaffinity binding to the 110,000-Dalton receptor with an IC50 of 5 microM and induced germinal vesicle breakdown (GVBD) with an EC50 of 9.0 +/- 0.6 microM. GVBD induced by RU 486 was slower than with progesterone, and RU 486 was less powerful than progesterone. Micromolar concentrations of RU 486 also potentiated GVBD induced by sub-optimal concentrations of progesterone or R5020. Furthermore, RU 486 inhibited oocyte plasma membrane adenylate cyclase with an apparent IC50 of 7.5 +/- 2.5 microM. The close correlation of the EC50 value for RU 486 induction of GVBD with the IC50 values for inhibition of [3H]R5020 photoaffinity labeling of the 110,000-Dalton receptor and inhibition of adenylate cyclase activity further supports the physiological significance of the oocyte plasma membrane steroid receptor.     

Mechanism of vascular relaxation by thaligrisine: functional and binding assays

In the present study we examine the mechanism by which thaligrisine, a bisbenzyltetrahydroisoquinoline alkaloid, inhibits the contractile response of vascular smooth muscle. The work includes functional studies on rat isolated aorta and tail artery precontracted with noradrenaline or KCl. In other experiments rat aorta was precontracted by caffeine in the presence or absence of extracellular Ca2+. In order to assess whether thaligrisine interacts directly with calcium channel binding sites or with alpha-adrenoceptors we examined the effect of the alkaloid on [3H]-(+)-cis diltiazem, [3H]-nitrendipine and [3H]-prazosin binding to cerebral cortical membranes. The functional studies showed that the alkaloid inhibited in a concentration-dependent manner the contractile response induced by depolarization in rat aorta (IC50 = 8.9+/-2.9 microM, n=5) and in tail artery (IC50 = 3.04+/-0.3 microM, n=6) or noradrenaline induced contraction in rat aorta (IC50 = 23.0+/-0.39 microM, n=9) and in tail artery (IC50 = 3.8+/-0.9 microM, n=7). In rat aorta, thaligrisine concentration-dependently inhibited noradrenaline-induced contraction in Ca2+-free solution (IC50 = 13.3 microM, n=18). The alkaloid also relaxed the spontaneous contractile response elicited by extracellular calcium after depletion of noradrenaline-sensitive intracellular stores (IC50 = 7.7 microM, n=4). The radioligand receptor-binding study showed that thaligrisine has higher affinity for [3H]-prazosin than for [3H]-(+)-cis-diltiazem binding sites, with Ki values of 0.048+/-0.007 microM and 1.5+/-1.1 microM respectively. [3H]-nitrendipine binding was not affected by thaligrisine. The present work provides evidence that thaligrisine shows higher affinity for [3H]-prazosin binding site than [3H]-(+)-cis-diltiazem binding sites, in contrast with tetrandrine and isotetrandrine that present similar affinity for both receptors. In functional studies thaligrisine, acted as an alpha1-adrenoceptor antagonist and as a Ca2+ channel blocker, relaxing noradrenaline or KCl-induced contractions in vascular smooth muscle. This compound specifically inhibits the refilling of internal Ca2+-stores sensitive to noradrenaline, by blocking Ca2+-entry through voltage-dependent Ca2+-channels.     

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)     

Colchicine binding in the free-living nematode Caenorhabditis elegans

The [3H]colchicine-binding activity of a crude supernatant of the free-living nematode Caenorhabditis elegans was resolved into a non-saturable component and a tubulin-specific component after partial purification of tubulin by polylysine affinity chromatography. The two fractions displayed opposing thermal dependencies of [3H]colchicine binding, with non-saturable binding increasing, and tubulin binding decreasing, at 4 degrees C. Binding of [3H]colchicine to C.elegans tubulin at 37 degrees C is a pseudo-first-order rate process with a long equilibration time. The affinity of C. elegans tubulin for [3H]colchicine is relatively low (Ka = 1.7 x 10(5) M(-1)) and is characteristic of the colchicine binding affinities observed for tubulins derived from parasitic nematodes. [3H]Colchicine binding to C. elegans tubulin was inhibited by unlabelled colchicine, podophyllotoxin and mebendazole, and was enhanced by vinblastine. The inhibition of [3H]colchicine binding by mebendazole was 10-fold greater for C. elegans tubulin than for ovine brain tubulin. The inhibition of [3H]colchicine binding to C. elegans tubulin by mebendazole is consistent with the recognised anthelmintic action of the benzimidazole carbamates. These data indicate that C. elegans is a useful model for examining the interactions between microtubule inhibitors and the colchicine binding site of nematode tubulin.     

Roles of colchicine rings B and C in the binding process to tubulin

The interactions of tubulin with colchicine analogues in which the tropolone methyl ether ring had been transformed into a p-carbomethoxybenzene have been characterized. The analogues were allocolchicine (ALLO) and 2,3,4-trimethoxy-4'-carbomethoxy-1,1'-biphenyl (TCB), the first being transformed colchicine and the second transformed colchicine with ring B eliminated. The binding of both analogues has been shown to be specific for the colchicine binding site on tubulin by competition with colchicine and podophyllotoxin. Both analogues bind reversibly to tubulin with the generation of ligand fluorescence. The binding of ALLO is slow, the fluorescence reaching a steady state in the same time span as colchicine; that of TCB is rapid. The displacement of ALLO by podophyllotoxin proceeds with a half-life of ca. 40 min. Binding isotherms generated from gel filtration and fluorescence measurements have shown that both analogues bind to tubulin with a stoichiometry of 1 mol of analogue/mol of alpha-beta tubulin. The equilibrium binding constants at 25 degrees C have been found to be (9.2 +/- 2.5) x 10(5) M-1 for ALLO and (1.0 +/- 0.2) X 10(5) M-1 for TCB. Binding of both analogues was accompanied by quenching of protein fluorescence, perturbation of the far-ultraviolet circular dichroism of tubulin, and induction of the tubulin GTPase activity, similarly to colchicine binding. Both inhibited microtubule assembly in vitro, ALLO substoichiometrically, and both induced the abnormal cooperative polymerization of tubulin, which is characteristic of the tubulin-colchicine complex. Analysis in terms of the simple bifunctional ligand binding mechanism developed for colchicine [Andreu, J.M., & Timasheff, S.N. (1982) Biochemistry 21, 534-543] and comparison with the binding of the colchicine two-ring analogue, 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one [Andreu, J. M., Gorbunoff, M. J., Lee, J. C., & Timasheff, S. N. (1984) Biochemistry 23, 1742-1752], have shown that transformation of the tropolone methyl ether part of colchicine into p-carbomethoxybenzene weakens the standard free energy of binding to tubulin by 1.4 +/- 0.1 kcal/mol, while elimination of ring B weakens it by 1.0 +/- 0.1 kcal/mol. The roles of rings C and B of colchicine in the thermodynamic and kinetic mechanisms of binding to tubulin were analyzed in terms of these findings.