Effect of guanine nucleotides on the hydrophobic interaction of tubulin.
The influence of guanine nucleotides on the binding of tubulin to hydrophobic components is investigated. Tubulin binds to a hydrophobic phenyl-Sepharose gel in a reversible, nucleotide-dependent way. Assembly-competent tubulin is released with ion-free water as eluent. It contains one guanosine triphosphate per dimer. More denatured tubulin needs a mixture of ethanol-water to elute. Consequently, hydrophobic interaction chromatography over phenyl-Sepharose represents an easy method for preparing polymerizable tubulin free of nucleotides at the exchangeable sites. While, in the absence of guanine nucleotide, the binding of tubulin to phenyl-Sepharose is rapid and immediately reversible on nucleotide addition, the binding of the nucleotide-dependent hydrophobic sites of tubulin to 1,8-ANS is slow, and its dissociation on nucleotide addition is poor. No differences are observed between the shielding of hydrophobic sites in the presence of GTP or GDP. Neither inorganic phosphate nor A1F4- is found to directly influence guanine nucleotides in their ability to shield hydrophobic sites.
Hanssens
I
I
Interdisciplinary Research Centre, Katholieke Universiteit Leuven, Campus Kortrijk, Belgium.
Baert
J
J
Van Cauwelaert
F
F
D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D005453:Fluorescence; D006150:Guanine Nucleotides / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006868:Hydrolysis; D012685:Sepharose / Q000031:analogs & derivatives; D013552:Swine; D014404:Tubulin / Q000502:physiology* / Q000648:ultrastructure; D014867:Water
D006150:Guanine Nucleotides; D014404:Tubulin; D014867:Water; C020927:phenyl-sepharose; D006160:Guanosine Triphosphate; D012685:Sepharose