Characterization of putative cytoskeletal proteins from a trypanosomatid and their comparative binding to microtubules and soluble tubulin.
The corset of microtubules which encloses the cell body of Crithidia fasiculata displays cross-links joining tubules to each other and to plasma membrane. Two proteins, designated COP-33 and COP-61 on the basis of their subunit Mr values, have been considered putative components of this apparatus because of their abundance in isolated cytoskeleton and their ability to cross-link brain microtubules in vitro. The oligomeric structures of the native proteins have now been characterized, and they have been shown to be basic, rather symmetrical, and to require detergent for solubilization. Using monospecific antibodies, enzyme-linked immunoassays of subcellular fractions have shown that each is 5-fold enriched in the microtubule plasma membrane fraction and absent from flagellar and some internal membranes. The binding of each to soluble tubulin and to microtubules has been systematically studied and compared with that of two noncytoskeletal protein ligands (glycolytic enzymes). The observed positive cooperatively was unexpected for binding of these large ligands to microtubules. In each case the maximum number of mol of ligand bound per mol of tubulin (0.5-1.0) was identical whether the latter was dissociated or assembled and so were the dissociation constants (1.3-6.0 x 10(-7) M) for three of the ligand proteins.
Kambadur
R
R
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Lewis
M
M
Chang
S
S
Flavin
M
M
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D003421:Crithidia / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008870:Microtubules / Q000378:metabolism*; D008970:Molecular Weight; D015388:Organelles / Q000378:metabolism*; D017239:Paclitaxel; D011485:Protein Binding; D014404:Tubulin / Q000378:metabolism*
D000470:Alkaloids; D003598:Cytoskeletal Proteins; D046911:Macromolecular Substances; D014404:Tubulin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D017239:Paclitaxel