Isolation of Microtubule Protein from Cultured Mouse Neuroblastoma Cells

Abstract

Abstract. The addition of vinblastine to high-speed supernatants derived from homogenates of cultured mouse neuroblastoma cells results in the formation of a precipitate which has been characterized as microtubule protein by the following criteria: colchicine-binding activity, molecular weight, amino acid composition, and electrophoretic mobility. The method therefore permits the rapid isolation of microtubule protein from crude supernatants of neuroblastoma cells.Recent work has shown that the application of vinblastine or vincristine to various cell types results in the breakdown of microtubules into filaments and the formation of large cytoplasmic crystals which appear to be composed of microtubule protein and bound vinblastine.'-7 Since vinblastine caused this crystallization of microtubule protein in vivo, it seemed reasonable to determine if this drug would also cause the aggregation of microtubule protein in vitro.7'8 In this report, we describe studies in which the treatment of high-speed supernatants of microtubule-rich neuroblastoma cells with vinblastine causes the precipitation of colchicine-binding protein. In addition to a high specific activity of colchicine binding, the precipitate has been characterized as microtubule protein by its molecular weight, amino acid composition, and electrophoretic mobility similar to that of microtubule protein isolated from Chlamydomonas flagella.Materials and Methods. Cell culture: A murine neuroblastoma tumor line was adapted to in vitro culture in November 1967, and was maintained in either suspension or monolayer culture.9Electron microscopy: Electron microscopy of the cultured cells was carried out using the fixation, flat-embedding, and staining procedures described by Brinkley et al.1OCell fractionation: Cells from suspension cultures were harvested by low-speed centrifugation at room temperature and were washed once with ST (0.24 M sucrose, 0.01 M Tris, pH 7.0); all succeeding operations were performed at 40C. Cells, 3 X 108, were homogenized in 3.0 ml ST or ST with 20 mM MgCI2 (SMT), centrifuged at 2500 X g for 10 min, the pellet discarded, and the supernatant centrifuged at 35,000 X g for 30 min. The resulting supernatant was centrifuged at 150,000 X g for 90 min, and the 150,000 X g supernatant used for the isolation of microtubule protein.Precipitation of microtubule protein with vinblastine: Vinblastine11 was added to the 150,000 X g supernatant to a final concentration of 2 X 10-1 M. If cell homogenization was carried out in ST, MgCl2 was added to a final concentration of 2.5 mM following the addition of a vinblastine. The solution became turbid immediately but 129

Isolation of tubulin from pig platelets

“…In our studies with platelets we have found that the more conventional procedures for tubulin isolation based upon ammonium sulphate fractionation and DEAE-ion exchange chromatography [ IO,1 3] were inapplicable and gave poor yields of tubulin with large amounts of actin. Similarly, the use of the Vinca alkaloid, Vinblastine sulphate to precipitate tubulin from the platelet soluble phase, (a technique sometimes used to isolate tubulin from other sources [29] ) resulted in very impure preparations in which actin again predominated.…”

Phosphorylation of Vinblastine‐Isolated Microtubules from Chick‐Embryonic Muscles

“…5) that nearly 50 % of the 32P radioactivity is eluted at a volume corresponding to a molecular weight of approximately 120 000, coinciding with the colchicine binding. The remaining 32P radioactivity emerged with the void volume, indicating that it is a high-molecular-weight aggregate of the kind which is known to form with vinblastine [24]. Protein kinase activity (measured with casein as substrate) was found to emerge with a molecular weight of 54000.…”

Colchicine-Binding Protein and the Secretion of Thyroid Hormone

“…Moreover, specific removal of the central pair of microtubules from sea urchin sperm tails progressively removes the colchicine-binding activity from the sperm tails and is accompanied by a corresponding increase of such activity in the extract (26) More direct evidence derives from experiments of Kirkpatrick et al (22) who showed that the major protein obtained from isolated brain microtubules characterized on the basis of ultrastructure behaves electrophoretically like the colchicine-binding protein isolated directly from brain and has the same molecular weight Furthermore, antibodies made to preparations from Tetrahymena cilia of the type that yield colchicinebinding protein appear to combine with intact bundles of microtubules (27). Using another approach, it has been shown that vinblastine-induced paracrystalline precipitates of microtubules (28)(29)(30) show in vivo radioautographic localization of eolchicine-"H (31) The isolated paracrystals also have colchicine-binding activity in vitro and have an amino acid composition similar to that reported for colchicine-bindmg protein and other presumptive microtubular subunits (32).…”