Isolation and Partial Characterization of aand 3-Tubulin from Outer Doublets of Sea-Urchin Sperm and Microtubules of Chick-Embryo Brain ( Strongylocentrotusigel electrophoresis / protein sequencing / evolution )

Two kinds of tubulin (a and ,3) have been described in microtubules from many different systems. In this study a discontinuous acrylamide-gel system containing sodium dodecyl sulfate was used to separate milligram quantities of aand fl-tubulin from microtubules of chick-embryo brain and from outer doublets of sea-urchin sperm. The isolated tubulins were characterized by peptide mapping and automated sequencing of the first 25 NH2-terminal amino acids. Our results show that aand #-tubulin are related but distinctly different proteins and that each one has been highly conserved in the course of evolution. The structural subunit of microtubules is a 100,000-120,000 molecular weight dimer of two 55,000-60,000 molecular weight monomers (1). Recent evidence suggests that there may be at least two kinds of tubulin monomers in microtubules, although it is not clear how they are arranged within the microtubular structure. When microtubules from various sources are solubilized and subjected to electrophoresis on acrylamide gels containing urea or Na dodecyl sulfate, two bands are obtained which contain proteins differing in amino-acid composition and in cyanogen bromide and tryptic peptide maps (2-6). Despite the morphological similarity of microtubules from different sources, it is not known how closely tubulin monomers from different organisms and different types of microtubules resemble each other. Studies using colchicine have shown that microtubules from many different sources bind to this drug with similar affinities, implying the conservation of at least one common site (4, 7-9). Immunological studies have given ambiguous results; some of these studies suggest the presence of a microtubular antigen in a wide variety of organisms (10, 11) and another study suggests that microtubular antigens may vary widely from one organism to another (12). A major obstacle to analyzing the differences between the two forms of tubulin and between tubulins from different sources has hitherto been the difficulty in separating and isolating them in milligram quantities. We report here a method for isolating preparative amounts of each tubulin species on a discontinuous Na dodecyl sulfate gel system, using as sources microtubules from embryonic chick brain and outer doublets of sea-urchin sperm tails. The amino-acid sequences of the two tubulins differ considerably, but relatedness is apparent from their primary structure. Comparison of corresponding tubulin species from each organism indicated that the primary structure of each type of tubulin is strongly conserved in evolution. MATERIAL AND METHODS Preparation of Microtubules. Outer doublets were isolated from sperm flagella of several hundred male sea urchins of the species Strongylocentrotus purpuratus by the method of Stephens (13). Brain microtubule protein was the generous gift of Dr. Leslie Wilson. It was prepared from 16to 18-day-old chick-embryo brains by the method of Bryan and Wilson (4). Protein Was Determined by the method of Lowry et al. (14) as modified by Bailey (15), with bovine-serum albumin as a standard. The intensity of the color that developed was measured on a Klett-Summerson photoelectric colorimeter with a red filter. Carboxymethylation. Protein samples were carboxymethylated by reaction with sodium iodoacetate (16); they were then dialyzed exhaustively in the dark against distilled water and lyophilized. Polyacrylamide Gel Electrophoresis. Continuous urea system. Samples were dissolved in 8 M urea-30 mM Tris HCl (pH 7.8) and run at 200 V for 2 hr on 4.5% polyacrylamide gels, which were 8 M in urea (3). The gels had been previously subjected to electrophoresis at 200 V for 45 min. Discontinuous urea system. This system was based on that of Davis (17) as modified by Yang and Criddle (18) and Shuster (19). The samples were incubated at 1000 for 2-3 min in 1% Na dodecyl sulfate-0.01 M Tris HCl (pH 8.0) and then dialyzed for 6-20 hr at 240 against 10% sucrose, containing 59 mM Tris-phosphate (pH 7.2) before they were layered on the gel. The running buffer was routinely 52 mM Tris-53mM glycine-0.03% Na dodecyl sulfate. The acrylamide to bis ratio in the lower gel was 32:1, and in the upper 4:1. Continuous Na dodecyl sulfate-urea system. The system of Swank and Munkres (20) was used for mapping the products of cyanogen bromide cleavage. Gels from all these systems were stained for 6-12 hr in a solution of 0.03% Coomassie blue in methanol-acetic acid-water (45:9:46) and then rinsed in methanol-acetic acid-water (5:7.5:87.5). Preparative Polyacrylamide-Gel Electrophoresis. The discontinuous Na dodecyl sulfate system was adapted for use with a Buchler model 200 polyprep by adopting suggestions of Chrambach (21) and Rodbard and Chrambach (22). The 3594 * Present address: Department of Pharmacology, Stanford Medical Center, Stanford, Calif. 94305. Isolation and Comparison of Tubulins 3595