Conversion of hydrophilic tubulin into a hydrophobic compound. Evidence for the involvement of membrane proteins.
Brain membranes contain tubulin that can be isolated as a hydrophobic compound by partitioning into Triton X-114. We have previously postulated: (a) that this kind of tubulin is a peripheral membrane protein that arises from microtubules that in vivo interact with membranes and (b) that the hydrophobic behaviour is due to the interaction of tubulin with a membrane component. Here we report the in vitro conversion of hydrophilic into hydrophobic tubulin by incubating microtubule associated proteins (MAPs) free taxol-stabilized microtubules with Triton X-100 solubilized membranes. After incubation, the microtubules were sedimented, depolymerized and subjected to partition into Triton X-114. Part of the tubulin was isolated in the detergent phase and contained, as observed in native membranes, a high proportion of the acetylated isotype. Because of the high proportion of acetylated tubulin the 'in vitro' conversion resembles the 'in vivo' interaction. Electrophoretic analysis of the detergent phase shows, besides tubulin, two major protein bands of 29 and 100 kDa molecular mass. The ability of the solubilized membranes to convert hydrophilic into hydrophobic tubulin is greatly diminished if the solubilized membrane preparation is preincubated in the presence of trypsin or heated at 90 degrees C for 5 min, indicating that the membrane component that confers the hydrophobic behaviour to tubulin is of proteinaceous nature.
Nuñez Fernandez
M
M
Departamento Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Cordoba, Argentina.
Beltramo
D M
DM
Alonso
A C
AC
Barra
H S
HS
D000107:Acetylation; D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D001923:Brain Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D008563:Membrane Lipids; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D008970:Molecular Weight; D017830:Octoxynol; D017239:Paclitaxel; D051381:Rats; D014404:Tubulin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D008563:Membrane Lipids; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D014404:Tubulin; D017830:Octoxynol; D017239:Paclitaxel