Colchicine-binding protein of mammalian brain and its relation to microtubules

Electron microscopic techniques have been used to reveal two classes of subunits of tubulin in ordered arrays. Presumably the two classes correspond to the a and 13 polypeptide chains of tubulin that have been distinguished by chemical criteria. The two types of subunits alternate along individual protofilaments in microtubules, microtubule-precursor sheets, and extended zinc-tubulin sheets. The resolution of the two types of polypeptide chains is achieved by improved negative staining methods which produce micrographs with layer lines at 28 A-' and 84 A-1 in optical or computed transforms, in addition to the layer lines at 21 A-1 and 42 A-1 de- vector points in the same direction for all of the protofilaments of the microtubule. However, for the sheets assembled in the presence of zinc, adjacent protofilaments are staggered by about 21 A and oriented in an antiparallel arrangement with alterate protofilaments related by a 2-fold screw axis. The antiparallel alignment of the protofilaments in the zinc-tubulin sheets accounts for their planarity (no tubular structures are found in the presence of moderate concentrations of zinc), since the intrinsic curvature found with parallel alignment of protofilaments in the absence of zinc would be cancelled by the antiparallel arrangement. Tubulin, the principal component of microtubules, is a molecule with a molecular weight of 110,000 (1). It can be dissociated into a and 1 polypeptide chains which have similar molecular weights (about 55,000) but differ in electrophoretic mobility, amino acid sequence, and extent of phosphorylation (2-4). In terms of the structure of microtubules, morphological units spaced about 40 A along the microtubule axis are observed by electron microscopy and x-ray diffraction (5-9) corresponding in size to about 55,000 molecular weight or the size of a single a or 1 polypeptide chain. In principle, microtubules with 13 strands of protofilaments could be composed of (i) a and ,-chains alternating along protofilaments; (ii) some other regular pattern, such as a2 and 12 homodimers alternating along protofilaments; or (iii) a random arrangement of a and 13 polypeptide chains. Pattern i has been favored on the basis of crosslinking data, which support a heterodimer (a13) structure for tubulin (10), and observations on flagellar microtubules, which give an 80 A-i reflection in transforms suggestive of a pairing of a and 13 chains as would be expected for a heterodimer (7). However, the situation is still ambiguous, since the crosslinking data also reveal appreciable amounts of homodimers (10) and the 80 A reflection could also arise from pairings of polypeptide chains in arrangements other than heterodimers. Direct evidence for pattern i, such as the observation of distinct structures alternating along protofilaments, would settle this issue, and such evidence is presented here.Our evidence for an alternating pattern of a and 13 chains isThe publication costs of this article were defrayed in part by page charge payment. This article must the...

mentioning

confidence: 99%

Differences in α and β polypeptide chains of tubulin resolved by electron microscopy with image reconstruction

Crepeau

McEwen

Edelstein

1978

“…* The colchicine binding activity was measured in parallel in both microtubule-free and corresponding control aliquots within a range of protein concentrations that gave linear colchicine binding when incubated (90 min, 370) with 25 ,uM colchicine (970 Ci/mol) in buffer A containing 1 mM GTP. The colchine bound to tubulin was quantitated by the DEAE-filter method (9,10).…”

mentioning

confidence: 99%

Role of nucleotides in tubulin polymerization: effect of guanylyl 5'-methylenediphosphonate.

Sandoval¹,

MacDonald²,

Jameson³

et al. 1977

Incubation of 48,000 X g rat brain supernatants for 30 min at 37 degrees with 1-2 mM guanylyl 5'-methylenediphosphonate [Gmp(CH2)pp] results in polymerization of 95-98% of the tubulin present. This is considerably more than the 50% polymerization that can be achieved with the natural nucleotide, GTP, under optimal conditions. Gmp(CH2)pp is also much more effective than GTP in inducing polymerization of purified tubulin. Assembly of microtubules with Gmp(CH2)pp occurs at tubulin concentrations one-third of those possible with GTP. Furthermore, with Gmp(CH2)pp, microtubule assembly does not require the high molecular weight basic proteins needed with GTP. Polymerization of tubulin by Gmp(CH2)pp is neither prevented nor reversed by concentrations of calcium (2 mM) that can either prevent microtubule assembly or disrupt already formed microtubules if the nucleotide used is GTP or guanylyl imidodiphosphate. When Ca2+ is added before or after microtubule assembly, electron microscopy of the Gmp(CH2)pp preparations reveals normal microtubules turning into twisted ribbons. Low temperature (4 degrees) can both prevent and disrupt the tubulin assembled Gmp(CH2)pp although disruption proceeds much more slowly when GTP is used.

“…6). 13,16,22,23 Leucine is the only amino acid which differs by more than 10 per cent from at least one of the other four microtubule proteins. Discussion.…”

mentioning

confidence: 99%

Isolation of Microtubule Protein from Cultured Mouse Neuroblastoma Cells

Olmsted¹,

Carlson²,

Klebe³

et al. 1970

273

130

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