GTP analogues interact with the tubulin exchangeable site during assembly and upon binding.
The question of whether nonhydrolyzable nucleotide analogues and other nucleoside triphosphates support tubulin assembly was addressed. Tubulin which contained residual GTP at the exchangeable site polymerized in the absence of added GTP in the presence of DMSO or glycerol. After maximum absorbance was reached, disassembly occurred at a slow rate. When 0.5 mM GMPPCP, GMPPNP, or ATP was included in the assembly reaction, disassembly did not occur, and about 0.1 mol of these nucleotides per mole of tubulin was incorporated into the protein. When 5 mM nucleotide was used or alkaline phosphatase was included in the case of the nonhydrolyzable analogues, a greater amount of assembly occurred and about 0.7-0.8 mol of analogue was incorporated. The products of the assembly reaction were cold-labile microtubules and protofilament ribbons. After cold-depolymerization of the microtubules and ribbons, a second cycle of assembly produced some microtubules, but cold-stable amorphous polymers were the major product. In addition, when GTP at the exchangeable site was first removed by a cycle of assembly, followed by depolymerization, assembly in the presence of GMPPCP, GMPPNP, or ATP produced a mixture of microtubules and cold-stable polymers, both of which contained bound analogue. Incorporation of GMPPCP, GMPPNP, or ATP into polymerized tubulin always occurred at the expense of GDP at the exchangeable site, the content of which decreased correspondingly. Incubation of tubulin with 5 mM GMPPCP, GMPPNP, or ATP under nonassembly conditions also displaced GDP.(ABSTRACT TRUNCATED AT 250 WORDS)
Mejillano
M R
MR
Department of Biochemistry, University of Kansas, Lawrence 66045.
Barton
J S
JS
Nath
J P
JP
Himes
R H
RH
D000469:Alkaline Phosphatase; D000818:Animals; D002417:Cattle; D004121:Dimethyl Sulfoxide; D019204:GTP-Binding Proteins / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D005990:Glycerol; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives*; D006868:Hydrolysis; D007700:Kinetics; D008870:Microtubules / Q000502:physiology* / Q000648:ultrastructure; D014404:Tubulin / Q000378:metabolism* / Q000648:ultrastructure
D014404:Tubulin; D006160:Guanosine Triphosphate; D000469:Alkaline Phosphatase; D019204:GTP-Binding Proteins; D005990:Glycerol; D004121:Dimethyl Sulfoxide