title: Leishmania tarentolae: purification and characterization of tubulin and its suitability for antileishmanial drug screening.
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.
authors.affiliation: Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Yakovich
authors.forename: Frank L
authors.initials: FL
authors.lastname: Ragone
authors.forename: Juan D
authors.initials: JD
authors.lastname: Alfonzo
authors.forename: Dan L
authors.initials: DL
authors.lastname: Sackett
authors.forename: Karl A
authors.initials: KA
authors.lastname: Werbovetz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002847:Chromatography, Agarose; D004136:Dinitrobenzenes / Q000737:chemistry / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007891:Leishmania / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D008024:Ligands; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013010:Sonication; D013424:Sulfanilamides / Q000737:chemistry / Q000494:pharmacology; D014404:Tubulin / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D050257:Tubulin Modulators / Q000737:chemistry / Q000494:pharmacology
chemical_list: D004136:Dinitrobenzenes; D008024:Ligands; D013424:Sulfanilamides; D014404:Tubulin; D050257:Tubulin Modulators; C012465:oryzalin