A rapid method for quantitative determination of microtubule protein using DEAE-cellulose filters (original) (raw)

1972, Analytical Biochemistry

The purpose of this report is to describe a rapid quantitative method for determining the amount of microtubule protein in cell extracts. The method is based upon two properties: (a) the specific affinity of microtubule protein subunits for thr plant, alkaloid, colchicine, and (b) the strong absorption of microtubule protein to DEAE ion exchangers at neutral pH and moderate ionic strength. A version of the present, method was described in studies on the mechanism of action of colchicine (1) and subsequent modifications have been used with some success (2,3) ; however, neither a detailed account of the DEAE filter assay nor a consideration of the relevant parameters which affect the reliability of the assay has (hitherto) been published. This report will describe the current version of the method as applied to microtubule protein derived from porcine brain tissue. Colchicine is best, known for its specific inhibition of cell division through its disruptive action on the mitotic spindle (4,5). The drug apparently exerts its antimitotic action by depolymerizing spindle microt,ubules (6), and there is increasing evidence which supports the hypothesis that the other biological actions of colchicine are also caused by a disruptive action on microtubules (7). EarIier studies showed that colchicine was reversibly bound to a set of cellular sites in the human cell line, strain KB, sea urchin eggs, brain tissue, and neuroblastoma cells (8-10). By using affinity for colchicine as an assay, a procedure was developed which allowed the isolation of a highly purified form of colchitine-binding protein from homogenates of whole mammalian brain (2). On the basis of its molecular weight, sedimentation coefficient, amino acid composition, and number of guanosine triphosphate binding sites, the colchicine-binding protein was found to be very similar to the protein obtained from microtubules of sea urchin sperm tails and Tetrahymena cilia (U-13). Recently, it was shown that the colchicine-binding protein 373