Factors influencing the polymerization of outer fibre microtubule protein

Abstract

Microtubule proteins have recently been prepared and characterized from a number of diverse sources. Renaud, Rowe & Gibbons (1966, 1968) have obtained a protein from acetone powders of Tetrahymena pyriformis cilia and from isolated outer fibre doublets of this same species. The protein has an actin-like amino acid composition, a minimum subunit weight of 55000, and 7·5 free sulphydryl groups per mole of monomer. Stephens (1968a) has fractionated sea-urchin flagella, following the procedure of Gibbons (1965), to obtain the isolated outer fibres and has found the protein to have properties virtually identical to those of its ciliary counterpart. Both the flagellar and the ciliary outer-fibre proteins contain 1 mole of bound guanine nucleotide per mole of protein subunit (Stephens, Renaud & Gibbons, 1967).

Selective Extraction of Isolated Mitotic Apparatus

“…Mixtures of equal parts of the two proteins gave a band identical in appearance and mobility to the bands of either protein alone . The estimated molecular weight, averaged from all determinations, was 52,000 for both proteins, in reasonable agreement with values of 56,000-66,000 previously reported for outer doublet microtubule protein from sea urchin sperm (5,17,19) .…”

“…The constituent proteins of microtubules from several cell types have recently been identified on the basis of their binding of colchicine (1)(2)(3)(4)(5)(6)(7)(8)(9)(10), their precipitation by vinblastine (7)(8)(9)(11)(12)(13), and, in cilia and flagella, by selective solubilization procedures monitored by electron microscopy (3,5,(14)(15)(16)(17)(18)(19) . These proteins show a striking resemblance in physicochemical properties (3-6, 9, 14-17) which serves to confirm the interpretation that they are in fact microtubule proteins .…”

Serological Similarity of Flagellar and Mitotic Microtubules

“…Prolonged dialysis against low ionicstrength buffer containing ethylenediaminetetraacetate (EDTA) solubilized central fibers, dynein, and matrix proteins (10,41) . Usually dialysis was preceded or followed by a detergent extraction to remove the flagellar membranes, using either 1 % Triton X-100 (41), 0.05% Sarkosyl (40), or 0 .5% digitonin (10,46) . Membrane protein could also be extracted by storing the preparations, after dialysis, at -20 °C in 50% glycerol (40) .…”

“…Usually dialysis was preceded or followed by a detergent extraction to remove the flagellar membranes, using either 1 % Triton X-100 (41), 0.05% Sarkosyl (40), or 0 .5% digitonin (10,46) . Membrane protein could also be extracted by storing the preparations, after dialysis, at -20 °C in 50% glycerol (40) . Outer fibers were recovered from glycerol by dilution (at least 1 :4 in Tris-DTT) and centrifugation.…”

“…An acetone powder was prepared from sperm flagella (33,40), and extracted to obtain the portion (35-40%) soluble in Tris-DTT .…”