The SH-SS exchange reaction between the Ellman's reagent and protein containing SH groups as a method for determining conformational states: tubulin.
Reactivity of tubulin SH groups, estimated by the slope to the curve in the SH-SS exchange reaction with 5-5' dithiobis (2-nitrobenzoic acid), was compared with that of bovine serum albumin (BSA) and studied in presence of various ligands. A small part of tubulin SH groups (12%) showed a higher reactivity, while the remaining portion had a smaller reactivity than that of BSA. The SH group reactivity of tubulin but not the total amount (12.8 mu/mole) diminished when assayed with colchicine and MgCl2 (0.1 and 0.2 mM, respectively); 0.2 mM CaCl2 increased the reactivity at the maximum level. On the basis of the biological role of tubulin SH groups and of the opposite effects exerted by Ca++ and Mg++ on the protein, the results presented here seem to indicate a correlation between conformational states of tubulin and its biological functions.
Di Simplicio
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Dipartimento di Biologia Ambientale, Università di Siena.
Tiezzi
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Moscatelli
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Bianco
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MT
Cresti
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D000818:Animals; D002122:Calcium Chloride / Q000494:pharmacology; D002417:Cattle; D003078:Colchicine / Q000494:pharmacology; D004220:Disulfides / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D009579:Nitrobenzoates; D011487:Protein Conformation; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism*
D004220:Disulfides; D009579:Nitrobenzoates; D013438:Sulfhydryl Compounds; D014404:Tubulin; D015636:Magnesium Chloride; D012710:Serum Albumin, Bovine; D006160:Guanosine Triphosphate; D004228:Dithionitrobenzoic Acid; D008274:Magnesium; D002122:Calcium Chloride; D003078:Colchicine