Commercial [3H]glutamate contains a contaminant that labels tubulin covalently.
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Animals↗
Biomedical subjects
R H Himes
Publications and source records attributed to R H Himes.
Interaction of a fluorescent paclitaxel analogue with tubulin.
To study the mechanism of binding of the antitumor agent paclitaxel to microtubules and tubulin, we have synthesized a fluorescent analogue of the drug. A dimethylamino group was introduced onto the 3'-N-benzoyl group of paclitaxel. This compound was synthesized from N-debenzoylpaclitaxel and 3-(dimethylamino)benzoyl chloride in 67% yield. N-Debenzoyl-N-[3-(dimethylamino)benzoyl]-paclitaxel has activity similar to paclitaxel in inducing microtubule assembly and binds to tubulin at the paclitaxel-binding site. Under assembly conditions, binding of this paclitaxel analogue to tubulin occurs in a time-dependent manner and is accompanied by a large increase in fluorescence intensity, as well as a large blue shift in the emission maximum. In addition, evidence is presented to show that this compound also binds to tubulin in the dimeric state, but the binding affinity is much lower (Kd = 49 +/- 8 microM at 25 degrees C) than that reported for polymeric tubulin. The fluorescent paclitaxel analogue, with a high quantum yield, will be a useful tool in studying the mechanism of paclitaxel binding to tubulin and the environment of the paclitaxel-binding site on tubulin.
Animals↗
18Oxygen incorporation into inorganic phosphate in the reaction catalyzed by N5,10-methenyltetrahydrofolate synthetase.
The mechanism of the N5,10-methenyltetrahydrofolate synthetase reaction was probed by determining the source of the oxygen atom introduced between the beta- and gamma-phosphates as ATP is converted to ADP and Pi. The reaction was performed using a mixture of [18O]- and [16O]N5-formyltetrahydrofolate in the presence of [16O]H2O and using [16O]N5-formyltetrahydrofolate in the presence of a 1:1 mixture of [18O]H2O and [16O]H2O. 31P NMR spectroscopy was used to examine the products. It was found that 18O from the formyl group was incorporated into Pi, and that 18O was not incorporated from the solvent. The results are consistent with a mechanism involving phosphorylation of the formyl group at the N5-position, followed by displacement of the phosphate by the 10-nitrogen.
Adenosine Diphosphate↗
Polymerization of Antarctic fish tubulins at low temperatures: role of carboxy-terminal domains.
We have proposed previously that the efficient polymerization of tubulins from Antarctic fishes at low, physiological temperatures (-1.8 to +2 degrees C) may result in part from adaptations (e.g., reductions in acidic residues) located in their carboxy-terminal (C-terminal) tails [Detrich & Overton (1986) J. Biol. Chem. 261, 10922-10930]. To test this hypothesis, we have examined the polymerization of Antarctic fish neural tubulins modified at their C termini by proteolysis or by neutralization of carboxyl groups. Addition of subtilisin to low concentrations of Notothenia coriiceps tubulin induced a biphasic assembly reaction: stage I corresponded to the C-terminal cleavage of beta chains to produce alpha beta s dimers, and stage II coincided with the slower, C-terminal cleavage of alpha chains to yield alpha s beta s. Both stage I and stage II polymers consisted of protofilament sheets and microtubules with attached sheets. The critical concentration for assembly of the stage II polymer was at least 10-fold lower than that of untreated tubulin. Neutralization of Glu and Asp carboxyls in Gobionotothen gibberifrons microtubules by the carbodiimide-catalyzed incorporation of glycine ethyl ester (GEE) moieties produced a tubulin, modified largely in its C termini, that assembled more readily than did control tubulin. When 12 GEE groups were incorporated per dimer, the critical concentrations for assembly of modified tubulin at 5-10 degrees C were 2-3-fold smaller than those for the unmodified protein. Comparably modified bovine tubulin (10 GEE/dimer) assembled at 37 degrees C with a critical concentration 2.6-fold lower than that for the unmodified tubulin.(ABSTRACT TRUNCATED AT 250 WORDS)
Adaptation, Physiological↗
The effect of the aromatic rings of taxol on biological activity and solution conformation: synthesis and evaluation of saturated taxol and taxotere analogues.
The synthesis and biological evaluation of novel cyclohexyl analogues of taxol and taxotere are detailed. 2-(Cyclohexylcarbonyl)-2-debenzoylbaccatin III (6) was prepared from baccatin III by hydrogenation. Subsequent coupling of 6 with N-t-BOC-3-[(tert-butyldimethylsilyl)oxy]-4-phenyl-2-azetidinone (7), followed by removal of the protecting groups, afforded 2-(cyclohexylcarbonyl)-2-debenzoyltaxotere (9). In a similar synthetic sequence, 3'-cyclohexyl-3'-dephenyltaxol (14) was prepared from N-benzoyl-3-[(tert-butyldimethylsilyl)oxy]-4-cyclohexyl-2-azetidinone (12) and (triethylsilyl)baccatin III. The taxol analogue 15, in which all three taxol phenyl groups are substituted by a cyclohexyl moiety, was synthesized in one step from taxol via hydrogenation. All three analogues (9, 14, and 15) exhibited strong activity in the microtubule assembly assay and cytotoxicity comparable to taxol against B16 melanoma cells. It was also shown that 9, like taxol and taxotere, has an extended side chain in chloroform, but in DMSO/water mixtures preferentially adopts a different conformation in which the 2-(cyclohexylcarbonyl), 3'-phenyl, and 4-acetyl groups cluster. However, this behavior does not appear to occur for 3'-cyclohexyl analogues 14 and 15, in which the side chain conformation remains extended independent of solvent. These results suggest the aromaticity of the 3'-phenyl ring significantly stabilizes the clustered conformation.
Antineoplastic Agents↗
Synthesis of a photoaffinity taxol analogue and its use in labeling tubulin.
A photoaffinity analogue of taxol, N-([3,5-3H]-4-azidobenzoyl)-N-debenzoyltaxol (7), was synthesized and used to photolabel microtubules. Approximately 20% of the noncovalently bound analogue becomes covalently bound upon irradiation at 300 nm. Incorporated label was stable to a 50% ethanol solution and sodium dodecyl sulfate. About 80% of the incorporated label was found in the beta-subunit and 20% in the alpha-subunit. Incorporation did not occur into unpolymerized tubulin, consistent with the fact that taxol binds only to polymerized tubulin, and was decreased by the presence of taxol. Little or no nonspecific labeling occurs. This analogue is currently being used to identify taxol binding site(s) on tubulin.
Affinity Labels↗
The binding of a ciliary microtubule plus-end binding protein complex to microtubules is regulated by ciliary protein kinase and phosphatase activities.
Using a human autoimmune CREST antiserum, we identified a 97-kDa polypeptide at the plus ends of Tetrahymena ciliary microtubules and an antigen associated with mammalian kinetochores (Miller, J.M., Wang, W., Balczon, R., and Dentler, W.L. (1990) J. Cell. Biol. 110, 703-714). The ciliary protein is part of a 1,500-2,000-kDa complex that can be released from ciliary microtubules and from in vitro assembled brain microtubules with ATP and ATP gamma S (Wang, W., Suprenant, K.A., and Dentler, W. L. (1993) J. Biol. Chem. 268, 24796-24807). Here we show that the ATP-dependent release of the 97-kDa protein from microtubules is inhibited, in a concentration-dependent manner, by calf intestine phosphatase and by the protein kinase inhibitor 6-dimethylaminopurine. Sodium orthovanadate, a phosphotyrosine phosphatase inhibitor, stimulated the ATP-dependent release of the 97-kDa protein from microtubules. Therefore, the ciliary fraction contains both protein kinase and phosphatase activities, and selective inhibition of these activities is necessary for the ATP-dependent binding and release of the 97-kDa protein from microtubules. When incubated with [gamma-32P]ATP, a portion of the 97-kDa protein is phosphorylated as are several other polypeptides associated with it. ATP sensitivity requires a low molecular weight heat-stable factor associated with axonemes. These results suggest that the association of the 97-kDa protein with microtubules is regulated by protein phosphorylation by axoneme-associated kinases and phosphatases.
Adenine↗
Conformation of ATP and ADP bound to N10-formyltetrahydrofolate synthetase determined by TRNOE NMR spectroscopy.
ATP and ADP bind to N10-CHO-H4folate synthetase from Clostridium cylindrosporum at four identical sites. Although both ADP and ATP bind to the enzyme with essentially the same Ka values as the Mg2+.nucleotide complexes, only the Mg2+.nucleotides are kinetically active. Using transferred nuclear Overhauser effect (TRNOE) NMR spectroscopy, we have measured the time-dependent NOE buildup rates of selected protons in ADP and ATP bound to N10-CHO-H4folate synthetase after preirradiating protons H1', H2', H3', and H4'. The results were used to calculate interproton distances. In order to define the conformations of ADP and ATP bound to the enzyme, we used the TRNOE distance constraints in a distance geometry algorithm. The results of the distance geometry calculations suggest that, within experimental error, the conformations of both ADP and ATP (with or without Mg2+) have an average glycosidic torsion angle X (O4'-C1'-N9-C8) of 100 degrees +/- 20 degrees and a sugar pucker angle psi' (C5'-C4'-C3'-O3') of 85 degrees +/- 5 degrees. These values are consistent with a nucleotide structure generated by computer modeling after energy minimization, which has X = 90 degrees +/- 6 degrees and psi' = 81 degrees, indicating a high-anti and C3'-endo conformation.
Adenosine Diphosphate↗
Solvent oxygen is not incorporated into N10-formyltetrahydrofolate in the reaction catalyzed by N10-formyltetrahydrofolate synthetase.
The mechanism of the reaction catalyzed by N10-formyltetrahydrofolate synthetase involves the formation of formyl phosphate as an intermediate which then formylates tetrahydrofolate at the N-10 position. Previous studies demonstrated that the non-enzymic formylation of tetrahydrofolate by formyl phosphate occurs exclusively at the more nucleophilic 5-nitrogen in the reduced pyrazine ring. The experiments described in this report were designed to determine whether N5-formyltetrahydrofolate might be the first product to be formed on the enzyme, followed by formyl transfer to the 10-nitrogen via the cyclic intermediate N5,10-methenyltetrahydrofolate. If this were the case, oxygen from solvent H2O would be incorporated into the formyl group of the N10-derivative. By conducting the reaction in a 1:1 mixture of [16O]H2O and [18O]H2O and using 13C NMR spectroscopy we show that no 18O is incorporated into the product and conclude that the reaction proceeds via a direct formylation of the N-10 position by formyl phosphate.
Catalysis↗
Exchangeable GTP binding site of beta-tubulin. Identification of cysteine 12 as the major site of cross-linking by direct photoaffinity labeling.
After direct photoaffinity cross-linking of [3H]GTP to the beta-subunit of tubulin, followed by tryptic digestion and alkaline phosphatase treatment, we employed cis-diol-specific boronate gel chromatography and reversed-phase high-pressure liquid chromatography to purify a peptide containing most of the covalently bound radioactivity. The sequence of this peptide corresponded to that of residues 3-19 of beta-tubulin. Residue 10 of the peptide, which is Cys-12 in beta-tubulin, could not be identified. The fast atom bombardment mass spectrum of this peptide showed the presence of a predominant species with a molecular mass of 2022 kDa (2021 kDa for the 12C variant), which is 255 Da greater than the molecular mass of the peptide. Fast atom bombardment collision-activated decomposition mass spectrometry analysis produced fragments which are consistent with the beta(3-19) peptide but having a unit of mass of 358 at position 12. Thermolysin digestion of the tryptic peptide restricted the cross-linking site to the 9-amino acid sequence, I(L)QAGQXGNQ. The molecular mass of this peptide was 1174 kDa, which is equal to the mass of the beta(7-15) peptide containing an extra group of mass 255. To explain the molecular masses of the two labeled peptides, which are 26 atomic mass units less than expected, a mechanism of photolabeling is proposed that involves opening of the guanine ring and loss of the C-6 carbonyl function as CO2.
Affinity Labels↗
Sequence and expression of the gene for N10-formyltetrahydrofolate synthetase from Clostridium cylindrosporum.
Sau3 A and Hind III restriction fragments of Clostridium cylindrosporum genomic DNA were used to isolate clones containing 80% of the N10-H4folate synthetase gene in a 5' fragment and the remaining 20% of the gene in the 3' fragment. These fragments were joined at a common SnaB I restriction site and expressed in Escherichia coli at a level equivalent to what is normally found in C. cylindrosporum. Sequence comparisons show a large degree of homology with genes from two other clostridial species, including a thermophile. Certain conserved sequences found in the three clostridial proteins and in the N10-H4folate synthetase portion of eukaryotic C1-H4folate synthases may represent consensus sequences for nucleotide and H4folate binding.
Amino Acid Sequence↗
A comparison of microtubule assembly in brain extracts from young and old rats.
Microtubule assembly was examined in the high-speed supernatant from homogenates of young (2-4 months old) and old (more than 24 months old) rat brains and significant age-related differences in microtubule assembly were found in the absence of exogenous GTP. In extracts from young brains, the increase in absorbance at 350 nm, which reflects the assembly reaction, was characterized by three phases (lag, elongation, and steady state) superimposed on a slow continuous increase due to non-specific aggregation. However, assembly in extracts from old brains, was very sluggish, in some cases barely more rapid than the non-specific aggregation reaction. Two to three times as much protein was assembled into cold-labile microtubules in extracts from young brains than from old brains. When 1 mM GTP was included in the assembly solutions the patterns of assembly in extracts from young and old brains became similar, with about the same amount of protein assembled into cold-labile microtubules. The assembly of tubulin purified from rat brains showed no differences between young and old. Results showed that extracts from old brains contained a higher GTPase activity than extracts from young brains. The sluggish assembly in extracts from old brains could be due to a deficiency in GTP or an inefficient regeneration of GTP.
Age Factors↗
Conformational properties of the beta(400-436) and beta(400-445) C-terminal peptides of porcine brain tubulin.
Two peptides from the C-terminal region of the major beta-tubulin isotype (400-436 and 400-445) that include the critical areas for interaction with MAP2 and tau were examined to determine their conformations in aqueous solution. Despite a high theoretical potential for alpha-helix formation, CD spectroscopy showed that these peptides consisted primarily of random coil with some reverse turn. This was unaffected by the presence of counterions to the negatively charged side chains (Ca2+, Mg2+), but did change when the side-chain charges were neutralized by lowering the pH; under these conditions, the alpha-helix content of the longer peptide rose to 25% and the C-terminal truncated peptide to 15%. The peptides also adopt alpha-helical structure in the presence of trifluoroethanol, the truncated peptide again attaining a lower maximum percentage. The beta(400-445) peptide was also studied by 1-D and 2-D NMR techniques. The results indicate that at pH 5.6 or 7 in an aqueous solution the peptide is extremely flexible and lacks regular secondary structure, consistent with the CD results. Both peptides inhibited microtubule-associated protein-stimulated tubulin assembly, with the longer peptide being about 4 times as inhibitory as the smaller peptide. Neither was inhibitory in the absence of microtubule-associated proteins, indicating that interaction with this species was necessary for inhibition. The greater activity of the longer peptide could be due to the extra negative charges in this peptide and/or the greater tendency of this peptide to form an alpha-helical structure under the appropriate conditions.
Amino Acid Sequence↗
Synthesis of biologically active taxol analogues with modified phenylisoserine side chains.
Taxol (1) is a highly potent antitumor agent, exerting its mechanism of action by promoting the assembly of stable microtubules in cells. We are reporting on the first synthesis and biological evaluation of taxol derivatives with substituted phenyl rings at the C-13 N-benzoyl-(2'R,3'S)-3'-phenylisoserine side chain of taxol (1). Two taxol derivatives were synthesized, one possessing a N-(p-chlorobenzoyl)-(2'R,3'S)-3'-phenylisoserine side chain (2) and the other one a N-benzoyl-(2'R,3'S)-3'-(p-chlorophenyl)isoserine side chain (3). The synthesis of the novel phenylisoserine side chains was achieved through the asymmetric synthesis of 3-hydroxy-4-aryl-2-azetidinone derivatives via the ester enolate-imine cyclocondensation reaction. The 2-azetidinones 14 and 15 were acylated with p-chlorobenzoyl chloride and benzoyl chloride, respectively, to form the N-acyl beta-lactams 16 and 17. Subsequent coupling of 16 and 17 to 7-(triethylsilyl)baccatin III (6) in the presence of pyridine and DMAP afforded, after removal of the protecting groups, the desired taxol analogues 2 and 3 in excellent yields. The newly synthesized derivatives 2 and 3 were tested in the tubulin assembly assay and also evaluated for their cytotoxicity against B16 melanoma cells. It was found that the taxol derivatives 2 and 3 had activity comparable to taxol (1).
Animals↗
The conversion of tubulin carboxyl groups to amides has a stabilizing effect on microtubules.
In a recent study, we demonstrated that the conversion of carboxyl residues in the C-termini of tubulin to neutral amides with glycine ethyl ester enhanced the ability of the protein to assemble into microtubules and decreased its interaction with microtubule-associated proteins (MAPs). In this work, we investigated the effects of carboxyl modification on the dynamic behavior of microtubules at polymer mass steady state. After steady state, microtubules assembled from unmodified tubulin were sheared, and the mean polymer lengths decreased to 5 microns and then increased to 29 microns within 130 min. In contrast, lengths of sheared microtubules polymerized from tubulin containing 23 modified carboxyl groups increased by only 2-fold. Stabilization of polymer lengths was also observed directly by video-enhanced light microscopy of microtubules grown off of axonemes. Rapid shortening was seen in microtubules composed of unmodified but not modified tubulin. Further evidence for the less dynamic behavior of microtubules as a result of carboxyl modification was obtained from kinetic studies of the elongation phase during assembly which showed a 3-fold lower off-rate constant, k-, for modified microtubules. Another effect of the modification was a 12-fold reduction in the steady-state rate constant for GTP hydrolysis (165 s-1 for unmodified and 14 s-1 for modified). These results suggest that reduction of the negative charges in the C-termini by modification of the acidic residues stabilizes microtubules against depolymerization. MAPs may stabilize microtubules in an analogous manner.
Amides↗
Cellular uptake and tubulin binding properties of four Vinca alkaloids.
The in vitro effects of four Vinca alkaloids, vinblastine (VLB), vincristine (VCR), vindesine (VDS) and vinepidine (VPD), on B16 melanoma proliferation, binding to bovine brain tubulin and B16 melanoma cell extracts, and uptake by the B16 cells were compared. The relative binding affinities to bovine brain tubulin were VPD greater than VCR congruent to VDS greater than VLB with the Ka for VPD being about 4-fold higher than that for VLB. On the other hand, the relative effects on B16 cell proliferation were exactly the opposite. Differences were found in the degree of concentration of the four alkaloids by the cells: 100-fold for VLB, 50-fold for VCR and VDS, and 20-fold for VPD. At the extracellular concentrations of drugs which inhibit proliferation by 50%, the intracellular concentration would still be far less than the tubulin concentration. Thus, it is likely that all of the Vinca alkaloids would be bound to tubulin and difference in uptake rather than Ka values is the major factor in determining the relative effectiveness of the drugs. L cells showed 50% the sensitivity of B16 melanoma cells toward VLB and 30% the sensitivity toward VPD. The L cells also concentrated these drugs to a lesser extent than did the B16 cells.
Animals↗
Synthesis and evaluation of some water-soluble prodrugs and derivatives of taxol with antitumor activity.
The synthesis and evaluation of some 2'- and 7-amino acid derivatives of taxol (1) are reported. Reaction of taxol with N-protected amino acids gave 2'-N-protected amino acid esters of taxol. However, deprotection of the amino group and subsequent isolation of products were complex and only successful when formic acid was used to deprotect a t-BOC protecting group. Esterification of taxol using N,N-dialkylated amino acids gave 2'-amino acid esters of taxol, 2'-(N,N-dimethylglycyl)taxol (4) and 2'-[3-(N,N-diethylamino)propionyl]taxol as its methanesulfonic acid salt (5b), in good yield. The 7-derivatives, 7-(N,N-dimethylglycyl)taxol (9) and 7-L-alanyltaxol (12), were prepared by two alternate methods. In the first approach, the 2'-hydroxyl group was protected using the [(2,2,2-trichloro-ethyl)oxy]carbonyl, or troc, protecting group followed by the esterification of the 7-hydroxyl and subsequent deprotection of the amino and troc groups. In the second approach, taxol was allowed to react with more than 2 molar equiv of the N-protected amino acids or N,N-dialkylated amino acids to give 2',7-diamino acid esters of taxol. For the protected amino acids, the deprotection of the amino group followed by removal of the 2'-substituent gave the 7-amino acid esters of taxol. The methanesulfonic acid salts of both 2'- and 7-amino acid esters showed improved solubility ranging from 2 to greater than 10 mg/mL. The 7-derivatives were effective in promoting microtubule assembly in vitro while 2'-derivatives showed little in vitro activity. The derivatives 2'-(N,N-dimethylglycyl)taxol (4) and 2'-[3-(N,N-diethylamino)propionyl]taxol (5) inhibited proliferation of B16 melanoma cells to an extent similar to that of taxol, while the other derivatives were about 50% as cytotoxic. In a mammary tumor screen, 2'-[3-(N,N-diethylamino)propionyl]taxol showed the greatest antitumor activity compared to the other analogues. The lower activities of the 7-derivatives in inhibiting tumor growth and melanoma cell proliferation (although they were almost as active as taxol in inducing microtubule assembly in vitro) may be due to differences in drug uptake by the cells. The similar cytotoxic and antitumor activities of the 2'-analogues and taxol can be explained by their conversion to taxol or an active taxol metabolite. Therefore, the 2'-analogues appear to behave as prodrugs and have the potential to be developed as chemotherapeutic agents.
Alkaloids↗
The interaction of [13C]-enriched colchicine with tubulin as determined by NMR spectroscopy.
[13C]Colchicine, labeled at the tropolone ring methoxy carbon, was used to study interactions with tubulin containing either Mg2+ or Mn2+ at the high affinity divalent cation binding site. Similar experiments were carried out in the presence of excess free divalent cation. The results show that: (1) when Mn2+ occupies the N-site, the 13C signal of the colchicine methoxy carbon of protein-bound colchicine is not broadened, indicating that in protein-bound colchicine the tropolone methoxy group is not close to the N-site cation; (2) when excess Mn2+ is present in solution this 13C signal is severely broadened, indicating that a low affinity divalent cation site or the exchangeable site (E-site) divalent cation is situated near the colchicine binding site; and (3) in the absence of paramagnetic ions a downfield chemical shift is observed for the tropolone methoxy carbon of colchicine upon binding to tubulin, suggesting that colchicine binds near an aromatic group(s) on tubulin.
Animals↗