Leishmania tarentolae: purification and characterization of tubulin and its suitability for antileishmanial drug screening |
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Authors: | Yakovich Adam J Ragone Frank L Alfonzo Juan D Sackett Dan L Werbovetz Karl A |
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Institution: | Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA. |
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Abstract: | Previously, tubulin has been purified from Leishmania amazonensis and used to identify novel molecules with selective antimitotic activity. However, L. amazonensis is pathogenic and requires a relatively expensive medium for large-scale cultivation. Herein, the purification and characterization of tubulin from the non-pathogenic Leishmania tarentolae is reported, together with the sequence of alpha- and beta-tubulin from this organism. This protein was purified by sonication, diethylaminoethyl-Sepharose chromatography, and one assembly disassembly cycle in 1% overall recovery based on total cellular protein. Leishmania tarentolae tubulin was indistinguishable from the corresponding L. amazonensis protein in terms of binding affinity for dinitroaniline sulfanilamides and sensitivity to assembly inhibition by these compounds. The amino acid sequences derived from the L. tarentolae alpha- and beta-tubulin genes were 99.6 and 99.4% identical to the corresponding amino acid sequences from the Leishmania major Friedlin strain. These results indicate that tubulin from L. tarentolae is suitable for use in drug screening. |
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Keywords: | Leishmania Tubulin Dinitroaniline Drug discovery DEAE diethylaminoethyl DMSO dimethyl sulfoxide EGTA ethyleneglycol-bis(β-aminoethyl ether) GB-II-5 N1-phenyl-3 5-dinitro-N4 N4-di-n-propylsulfanilamide GB-II-150 N1-phenyl-3 5-dinitro-N4 N4-di-n-butylsulfanilamide PBS phosphate-buffered saline PME buffer consisting of 0 1 M Pipes (pH 6 9) 1 mM MgCl2 and 1 mM EGTA |
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