Biomaterial Database

Probing the ATP binding site of tubulin with thiotriphosphate analogues of ATP. 1998

S Xu, and F Gaskin

Department of Psychiatric Medicine, University of Virginia School of Medicine, Charlottesville 22908, USA.

Tubulin assembly studies with GTP alpha S diastereoisomers have shown that there is stereoselectivity at the alpha-phosphate binding region of tubulin. GTP alpha S(Sp) bound tighter than GTP alpha S(Rp) and promoted nucleation and assembly better than GTP and GTP alpha S(Rp). ATP and dATP have been reported to bind weakly to tubulin and to be less effective than GTP and dGTP in promoting tubulin assembly. This study was done to learn if ATP alpha S(Sp) and dATP alpha S(Sp) are good promoters of tubulin assembly and to compare these ATP thiotriphosphate analogues to the corresponding GTP analogues in tubulin assembly. Studies were also done with ATP alpha S(Rp), GTP, ATP beta S(Sp) and ATP gamma S. At least three cycles of tubulin (25 microM) assembly-disassembly were found with 1 mM ATP alpha S(Sp) and dATP alpha S(Sp) and both nucleotides were incorporated and hydrolyzed in the polymers. Less dATP alpha S(Sp) (25 microM) than ATP alpha S(Sp) (100 microM) promoted assembly to 50% of the maximum value. The critical concentrations (Cc) for assembly with 1 mM nucleotide were low for ATP alpha S(Sp) (3 microM) and dATP alpha S(Sp) (2 microM) and compared favorably with GTP (5 microM), GTP alpha S(Sp) (2 microM) and dGTP alpha S(Sp) (1 microM). Both 1 mM ATP and dATP were poor promoters of tubulin assembly and were not detected in the polymers. The predominant structures induced by 1 mM (ATP alpha S(Sp) and dATP alpha S(Sp) were bundles of sheets and microtubules, which were more stable to the cold and to Ca(II) than microtubules assembled with GTP, ATP or dATP. ATP alpha S(Rp) (1 mM) did not promote assembly suggesting that there is stereoselectivity at the ATP alpha S alpha-phosphate binding region of tubulin as there is with GTP alpha S diastereoisomers. ATP alpha S(Sp) and dATP alpha S(Sp) mimic GTP alpha S(Sp) and dGTP alpha S(Sp) in tubulin assembly since all four nucleotides promote bundles of tubulin in buffer with glycerol, and the deoxy nucleotides have lower Cc, shorter lags and faster rates for tubulin assembly.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D002021	Buffers	A chemical system that functions to control the levels of specific ions in solution. When the level of hydrogen ion in solution is controlled the system is called a pH buffer.	Buffer
D002118	Calcium	A basic element found in nearly all tissues. It is a member of the alkaline earth family of metals with the atomic symbol Ca, atomic number 20, and atomic weight 40. Calcium is the most abundant mineral in the body and combines with phosphorus to form calcium phosphate in the bones and teeth. It is essential for the normal functioning of nerves and muscles and plays a role in blood coagulation (as factor IV) and in many enzymatic processes.	Coagulation Factor IV,Factor IV,Blood Coagulation Factor IV,Calcium-40,Calcium 40,Factor IV, Coagulation
D005990	Glycerol	A trihydroxy sugar alcohol that is an intermediate in carbohydrate and lipid metabolism. It is used as a solvent, emollient, pharmaceutical agent, or sweetening agent.	1,2,3-Propanetriol,Glycerin,1,2,3-Trihydroxypropane,Glycerine
D000255	Adenosine Triphosphate	An adenine nucleotide containing three phosphate groups esterified to the sugar moiety. In addition to its crucial roles in metabolism adenosine triphosphate is a neurotransmitter.	ATP,Adenosine Triphosphate, Calcium Salt,Adenosine Triphosphate, Chromium Salt,Adenosine Triphosphate, Magnesium Salt,Adenosine Triphosphate, Manganese Salt,Adenylpyrophosphate,CaATP,CrATP,Manganese Adenosine Triphosphate,MgATP,MnATP,ATP-MgCl2,Adenosine Triphosphate, Chromium Ammonium Salt,Adenosine Triphosphate, Magnesium Chloride,Atriphos,Chromium Adenosine Triphosphate,Cr(H2O)4 ATP,Magnesium Adenosine Triphosphate,Striadyne,ATP MgCl2
D001665	Binding Sites	The parts of a macromolecule that directly participate in its specific combination with another molecule.	Combining Site,Binding Site,Combining Sites,Site, Binding,Site, Combining,Sites, Binding,Sites, Combining
D013237	Stereoisomerism	The phenomenon whereby compounds whose molecules have the same number and kind of atoms and the same atomic arrangement, but differ in their spatial relationships. (From McGraw-Hill Dictionary of Scientific and Technical Terms, 5th ed)	Molecular Stereochemistry,Stereoisomers,Stereochemistry, Molecular,Stereoisomer
D014404	Tubulin	A microtubule subunit protein found in large quantities in mammalian brain. It has also been isolated from SPERM FLAGELLUM; CILIA; and other sources. Structurally, the protein is a dimer with a molecular weight of approximately 120,000 and a sedimentation coefficient of 5.8S. It binds to COLCHICINE; VINCRISTINE; and VINBLASTINE.	alpha-Tubulin,beta-Tubulin,delta-Tubulin,epsilon-Tubulin,gamma-Tubulin,alpha Tubulin,beta Tubulin,delta Tubulin,epsilon Tubulin,gamma Tubulin
D016244	Guanosine 5'-O-(3-Thiotriphosphate)	Guanosine 5'-(trihydrogen diphosphate), monoanhydride with phosphorothioic acid. A stable GTP analog which enjoys a variety of physiological actions such as stimulation of guanine nucleotide-binding proteins, phosphoinositide hydrolysis, cyclic AMP accumulation, and activation of specific proto-oncogenes.	GTP gamma S,Guanosine 5'-(gamma-S)Triphosphate,gamma-Thio-GTP,GTPgammaS,Guanosine 5'-(3-O-Thio)Triphosphate,gamma S, GTP,gamma Thio GTP