Association of fodrin with brain microtubules.
We have compared the polypeptide composition of microtubules isolated from bovine brain by the conventional in vitro reassembly method with those obtained by direct isolation of brain microtubules into a stabilizing buffer. The stabilizing buffer included 6.7 M glycerol to limit the rate of subunit exchange between assembled and unassembled states. The microtubule-associated proteins normally found by in vitro reassembly are also found in the stabilized preparation, but in smaller proportions. Fodrin, a brain membrane-associated protein believed to be homologous to spectrin, was found to be the most abundant component after tubulin in the stabilized microtubules. The ratio of tubulin to fodrin, 16:1 by mass, was almost constant at each stage of the preparation. Some actin was initially present in the stabilized microtubules, but was gradually lost during purification. When stabilized microtubules were diluted into cold aqueous buffer, they depolymerized and the recovered microtubule protein could then be purified by in vitro reassembly. The composition after this treatment resembled that of microtubules prepared initially by reassembly in vitro. The missing fodrin was found to be removed in the preliminary centrifugation and was unavailable for incorporation into growing microtubules during the in vitro assembly step. This suggests that the standard in vitro reassembly procedure for purification of microtubules may distort the composition of microtubule-associated proteins.
D000818:Animals; D001923:Brain Chemistry; D002021:Buffers; D002118:Calcium; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002458:Cell Fractionation / Q000379:methods; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism; D046911:Macromolecular Substances; D008840:Microfilament Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D013049:Spectrin / Q000302:isolation & purification
D002021:Buffers; D002118:Calcium; D002352:Carrier Proteins; D046911:Macromolecular Substances; D008840:Microfilament Proteins; D008869:Microtubule-Associated Proteins; D013049:Spectrin