Interaction of tubulin with guanosine 5'-O-(1-thiotriphosphate) diastereoisomers: specificity of the alpha-phosphate binding region.
The exchangeable nucleotide-binding site of tubulin has been studied using diastereoisomers A (Sp) and B (Rp) of guanosine 5'-O-(1-thiotriphosphate) (GTP alpha S) in which the phosphorus atom to which sulfur is attached is chiral. GTP alpha S(A) (10 microM) nucleated assembly of purified tubulin (20 microM) into microtubules in buffer containing 0.1 M 2-(N-morpholino)ethanesulfonic acid with 3 mM Mg2+ and 1 mM EGTA, pH 6.6 at 37 degrees C. With 0.2 mM GTP alpha S(A), the critical concentration (Cc; minimum protein concentration required for assembly) was 8 microM tubulin. Neither 0.2 mM GTP nor GTP alpha S(B) promoted microtubule assembly in buffer with 0.5-6.75 mM Mg2+ and 20-70 microM tubulin. The Cc values for GTP alpha S-(A)-induced assembly of tubulin in buffer with 30% glycerol and of microtubule protein (tubulin and microtubule-associated proteins) in buffer were lower than for GTP. GTP alpha S(A)-induced microtubules were more stable to the cold and to Ca2+. GTP alpha S(A) and GTP but not GTP alpha S(B) bound tightly to tubulin at 4 degrees C. Although GTP alpha S(B) did not nucleate assembly, it did bind to tubulin since it was incorporated into the growing microtubule. Both isomers were hydrolyzed in the microtubules. These studies show that GTP alpha S(A) promotes tubulin assembly better than GTP and GTP alpha S(B) and that there is stereoselectivity at the alpha-phosphate binding region of tubulin. The stereoselectivity may be due to different MgGTP alpha S(A) and -(B) interactions with tubulin.
Xu
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S
Department of Psychiatric Medicine, University of Virginia School of Medicine, Charlottesville 22908.
Gaskin
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F
D000818:Animals; D001921:Brain; D002021:Buffers; D005990:Glycerol / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000031:analogs & derivatives* / Q000378:metabolism; D006868:Hydrolysis; D008274:Magnesium / Q000494:pharmacology; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism; D010755:Organophosphates / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D013237:Stereoisomerism; D013552:Swine; D014404:Tubulin / Q000378:metabolism*
D002021:Buffers; D008869:Microtubule-Associated Proteins; D010755:Organophosphates; D014404:Tubulin; C034882:guanosine 5'-O-(1-thiotriphosphate); D016244:Guanosine 5'-O-(3-Thiotriphosphate); D008274:Magnesium; D005990:Glycerol