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The colchicine analog 3-chloroacetyl-3-demethylthiocolchicine (3CTC) is a competitive inhibitor of colchicine binding to tubulin, binds to tubulin at 37°C, but not at 0°C, and covalently reacts with ␤-tubulin at 37°C, but not at 0°C, in a reaction inhibited by colchicine site drugs. The approximate intramolecular distance between the oxygen at position C-3 in 3CTC and the chlorine atom of the 3-chloroacetyl group is 3 Å. Using decylagarose chromatography, we purified ␤-tubulin that had reacted with 3-(chloromethyl-[ Tubulin interacts with a wide variety of small ligands, including divalent cations, guanine nucleotides, and antimitotic drugs. Perhaps because of its instability, its tendency to polymerize into pleomorphic structures, and/or its microheterogeneity, tubulin or tubulin-ligand complexes have not been crystallized. A polymeric site for paclitaxel (1), probably on the ␤-subunit (2, 3), has been defined in zinc-induced sheets of antiparallel protofilaments. Efforts to define ligand binding sites have generally involved induction of covalent interactions between tubulin and specific ligands. Usually photoreactive derivatives of the ligand were used (e.g. Refs. 2-5), but "direct photoaffinity" labeling, in which a covalent bond between ligand and tubulin forms under UV irradiation, has also been a valuable technique (6 -8). In most cases the target amino acids in the covalent interaction between protein and ligand were not identified, with localization of the reactive site limited to the tubulin subunit or a peptide fragment.No ligand has attracted more attention than colchicine (9), whose interaction with tubulin permitted the initial isolation of the protein (10). Photoaffinity (4, 5) and direct photoaffinity (7, 11) studies have provided evidence that both the ␣-and ␤-subunits participate in forming the binding site, as has genetic evidence (12-14). In only one of these studies (11) was label localized to specific peptide fragments of tubulin, and the region of colchicine that reacted with the protein was uncertain.We have taken a different approach from concern that use of bulky photoaffinity groups introduces a paradox into the technique. Adequate affinity of such compounds for tubulin implies that the reactive group has been introduced at a position in the ligand of secondary importance for the binding reaction. Thus, covalent reaction(s) may occur at some distance from the binding site itself. While this problem does not arise with the direct photoaffinity method, the precise ligand-peptide reaction has only been established with GTP (6). We decided to explore the use of small, chemically reactive substituents introduced at defined positions in colchicinoids and allocolchicinoids (15, 16). Schmitt and Atlas (17) had used an analog derivatized with the bromoacetyl group in the B ring side chain, but they found extensive nonspecific covalent reactions between their analog and tubulin. We used the isothiocyanate and chloroacetyl groups (16), and introduced them into the side chain, at position C-9 of t...

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Our finding that an analog of paclitaxel (Taxol) modified at position C-2 (2-debenzoyl-2-(m-azidobenzoyl)paclitaxel) was substantially more active than paclitaxel in promoting tubulin assembly [Chaudhary et al. (1994) J. Am. Chem. Soc. 116, 4097-4098] led us to perform an analysis of the modulating effects of microtubule-associated proteins, GTP, and temperature on assembly and polymer stability. The analog always showed superior activity to paclitaxel in inducing polymerization where it fails to occur without drug, probably indicating a greater ability than paclitaxel to "hypernucleate" assembly. In contrast, much smaller differences in effects on polymer stability were observed. The analysis was extended to a large series of derivatives modified at positions C-2, C-7, C-10, and C-3', including docetaxel, a clinically important analog of paclitaxel. While analog stabilization of polymer was frequently observed, neither qualitative nor quantitative analysis of this property reliable predicted whether a compound would have enhanced hypernucleation activity relative to that of paclitaxel. Stabilization was often observed at substoichiometric analog concentrations, while even superstoichiometric concentrations of most compounds failed to induce extensive tubulin polymerization at low temperatures or in the absence of microtubule-associated proteins or GTP. Docetaxel was intermediate in activity between paclitaxel and 2-debenzoyl-2-(m-azidobenzoyl)paclitaxel in promoting assembly reactions. We conclude that the hypernucleation of tubulin assembly and polymer stabilization observed with paclitaxel represent two distinct properties of the drug. Our findings suggest that paclitaxel, docetaxel, and 2-debenzoyl-2-(m-azidobenzoyl)paclitaxel are able to interact with progressively smaller assemblages of tubulin at low temperatures or in the absence of microtubule-associated proteins or GTP.

Angiotensin converting enzyme (ACE) gene I\D polymorphism has been associated with high altitude (HA) disorders as well as physical performance. We, however, envisage that the polymorphism may be associated with adaptation to the hypobaric hypoxia of altitude, thus facilitating physical performance. For this purpose, three unrelated adult male groups, namely (1) the Ladakhis (HLs), who reside at and above a height of 3600 m, (2) lowlanders, who migrated to Ladakh (MLLs), and (3) resident lowlanders (LLs), have been investigated. The HLs had significantly ( p 0n001) greater numbers of the II homozygotes and the ID heterozygotes than the DD homozygotes, the genotype distribution being 0n46, 0n43 and 0n11 for II, ID and DD genotypes respectively. The MLLs comprised 60 % II homozygotes, which was higher ( p 0n001) than the HLs (46 %). In the LLs, the heterozygotes were greater ( p 0n001) in number than the II and DD homozygotes. The I allele frequency was 0n72 in the MLLs, 0n67 in the HLs and 0n55 in the LLs. Polymorphism study suggested that the II genotype could be associated with altitude adaptation, which might influence physical efficiency. People living at sea level experience various levels of physical discomfort at high altitude (HA) due to hypobaric hypoxia and cold. The effects of hypoxia on cardio-pulmonary function (Sutton et al. 1988 ; Mirrakhimov & Winslow, 1996) are most evident during hard physical work when the already taxed oxygen transport system needs to meet increased metabolic demands. This results in somewhat reduced levels of performance of individuals when faced with tasks demanding high levels of physical and mental performance. However, long-term in-

The pattern of angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism in the Indian population is poorly known. In order to determine the status of the polymorphism, young unrelated male army recruits were screened. The population had cultural and linguistic differences and lived in an environment that varied significantly from one region to another. Analysis of the genotype, showed higher frequency of the insertion allele in four of the five groups i.e. I allele frequency was significantly higher (P < 0.05) in Dogras, Assamese and Kumaonese. The deletion allele frequency was comparatively higher in the fifth group that belonged to Punjab. A correlation was observed between the genotype and enzyme activity. Involvement of a single D allele in the genotype enhanced the activity up to 37.56 3.13%. The results suggested ethnic heterogeneity with a significant gene cline with higher insertion allele frequency. Such population-based data on various polymorphisms can ultimately be exploited in pharmacogenomics.

Microtubule-associated-protein-dependent assembly of tubulin with GDP in the exchangeable site (tubulin-GDP) can occur with minimal free Mg2' (<3 pM). This reaction is totally inhibited by EDTA and by GTP concentrations over 2 mM and stimulated by MgC1,. Quantitative aspects of this stimulation are affected by both the Mg" and GTP concentrations, but no relationship exists between reaction rates and relative amounts of different magnesium and GTP species. GTP binding to tubulin-GDP, while maximally stimulated 2 -3-fold by exogenous MgCl,, was inhibited less than SO% by EDTA, and the amount of GTP bound increased as its concentration rose to levels that inhibited polymerization. Studies on the binding of Mg2+ to tubulin-GDP in the presence and absence of GTP showed that the increase in the amount of tubulin-associated Mg" was substoichiometric to the amount of GTP bound (maximum stoichiometry of additional Mg2+ to GTP bound, 0.7). Upon polymerization the increased Mg2+ content of tubulin was reduced, indicating its loss during GTP hydrolysis. Mg2+ thus plays a critical role in assembly distinct from its enhancement of GTP binding to the exchangeable site. If magnesium is present in trace amounts, this role must either be catalytic during polymerization or limited to nucleation.Exogenous Mg" plays a variable role in tubulin polymerization. Reaction conditions in which exogenous cation is essential include one system with high concentrations of tubulin in Pipes [l] and another in which dimethyl sulfoxide + Pipes induces polymerization [2, 31. Generally, however, substantial, albeit suboptimal, assembly occurs without exogenous Mg", whether it is induced with MAPs + Mes or Pipes, with glutamate, or with glycerol + Mes [4-61.There are at least four complications to understanding the role of Mg2' in tubulin polymerization. First, a broad range of GTP concentrations can be used to support assembly, from near-stoichiometric with tubulin to millimolar. Second, GTPMg binds to tubulin in the exchangeable nucleotide site more readily than does unliganded GTP, while GDP binding is little affected by Mg2+ [5, Al, Sn, and Cd (unpublished observations). These weaker interactions may be mediated through the sulfhydryl groups of tubulin 1151 and/or the highly acidic carboxyl termini of a-andor P-tubulin [7, 161. Correspondence to E. Hamel, Building 37, Room 5C25, National Institutes of Health, Bethesda, Maryland 20892, USA Fax: +301 496 5839. Abbreviations. MAPs, microtubule-associated proteins ; tubulin-GDP, a tubulin preparation with GDP in the exchangeable site and GTP in the nonexchangeable site; SE, standard error.We have described effects of Be on tubulin-nucleotide interactions and on microtubule-associated protein (MAP)-dependent microtubule assembly [6, 171. Relative to Mg, Be had a reduced ability to stimulate binding of GTP to unpolymerized tubulin, but near-stoichiometric amounts of guanine nucleotide derived from [ 8-I4C]GTP were recovered in microtubules formed in the presence of either Be or Mg. With Mg, at le...

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