Biomedical Research 4 (6) 615-618, 1983
A high molecular weight protein in axoplasm underlying excitable membrane of squid giant axon
SHOICHIRO TSUKITA1, SACHIKO TSUKITA1, TAKAAKI KOBAYASHI2 and GEN MATSUMOTO3 1Department of Anatomy, Faculty of Medicine, University of Tokyo, Bunkyoku, Tokyo 113, 2Department of Biochemistry, Jikei University, School of Medicine, Minatoku, Tokyo i105, and 3Electrotechnical Labora- tory, Tsukuba Science City, Ibaraki 305, Japan
ABSTRACT A high molecular weight protein (M, 260,000), designated 260 K protein, has been shown to play an important role in maintaining the excitability of the axolemma in squid giant axons (5, 6). In this study, we have examined the molecular shape and location of this protein in squid giant axons. In low angle rotary shadowing elec- tron microscopy, the 260 K protein appeared as a straight rod about 100 nm long with a globular head at one end. Taking this observation together with immuno- fluorescence study, we conclude that the 260 K protein is a unique protein located in the axoplasm underlying the excitable membrane of squid giant axons.
One of the major problems we face in the study soluble in 0.6 MKCI, but not in 0.1 MKCI. of membrane excitation is the interaction Taking advantage of this characteristic, five between the ‘excitable membrane and the under- succesive centrifugations separated 260 K pro- lying cytoskeleton. Recently, Matsumoto et al. tein from other cytoskeletal proteins. Since have shown that a high molecular weight protein the squid nerve contains a large amount of (M, 260,000) plays an important role in main- 260 K protein, approximately 0.8 mg of purified taining the excitability of the axolemma in squid 260 K protein was obtained from 100mg (wet giant axons (5, 6). This protein (designated weight) of the nerve. In sodium dodecyl 260 K protein) was first described as a factor for sulfate (SDS)-polyacrylamide gel electrophoresis restoring the membrane excitability of the squid of the purified protein, a single polypeptide with giant axon when it had been destroyed by intra- a molecular weight of 260,000 daltons dominated axonal perfusion of microtubule poison (6, 9). (Fig. 1). Judging from the elution profile of the Most recently, this protein has been partially gel-filtration and the cross-linking study (data purified from squid nerves (7). It became clear not shown), the native 260 K protein seems to that the 260 K protein interacts with micro- exist as a homo-dimer in 0.6 M KC]. tubules to make bundles in vitro, and also that Several high _molecular weight proteins have this protein did not biochemically resemble the been isolated as factors for organizing the three- microtubule-associated proteins (MAPs) ob- dimensional cytoskeletal network inside cells. tained from mammalian brain. T0 further Therefore, we first compared the electrophoretic understand the physiological role of this protein, mobilities of 260 K protein, erythrocyte spectrin, we have attempted to obtain information about brain spectrin (calspectin or fodrin) (1, 2, 4, 10), its molecular shape, antigenicity, and location filamin (14), and myosin in SDS-polyacrylamide in the axoplasm of squid axons. gels (Fig. 1). The electrophoretic banding The 260 K protein was purified from the axon patterns of these proteins clearly differed. bundles of the squid, Doryteuthis bleekeri, Taking this finding together with the amino acid according to the method developed by Kobaya- composition of 260K protein (7), the 260 K shi (see the caption to Fig. 1). The protein is protein seemed to be neither a spectrin-like, a 616 S. TSUKITA et al.
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- . - - - . -...... i§f=%2§§2i=f=f=;2§2f5 2; f§- 2 = -fa; _j Q . __.-;.=,__,_ _ . . _ _ _. . ._ _ _. . "Iiiiitiiiatif-ii:-2=2i€:§@%'?==2£i2¥"_.._'I ”~'5:;2;:=;,:;2;.<;252gi2,,--2;.2;:-2-:=:1:;;;:;:;:;:;2;:;:§;:;:;3,:...252.;2g:;.'.;-:_2'<2g2-:,-1 2.‘ii!$1-'Ffx. . :.-2-.;. -22 .2.;;.2.2. 1:?--I-:'=>;; _§:_:r-fii-.=.=.=._ -:52:-:'5':=':l5:':‘§55 ==~:.==--2 2F=f$I.=;E =-:2.-2- 2;=.=;-22_.'j’2.. . - . 3 . ' ', -_ '- -_ _ j -.'_2.2‘ '.22';'-.‘2--"‘:_§-I __§‘-:_ 2 -' Fig. 1 Electrophoretic banding patterns in 3—12% linear gradient SDS-polyacrylamide gel (3). a, Axoplasm obtained from giant axon by a roller and intra-axonal perfusion with Ca2+ containing solution. b, Purified 260 K protein (0.25 pg). c, Purified 260 K protein (5 pg). d, Erythrocyte spectrin. e, Brain spectrin (calspectin or fodrin). f, Filamin. g, Myo- sin. To purify 260 K protein, squid nerves (fin nerves) were cut in pieces with scissors and incubated in 0.6 M KCl, 10 mM CaCl;, 0.lmM MgCl2, 0.1 mM ATP, 0.1 mM GTP, 1 mM dithiothreitol (DTT), 10 mM K-HEPES buffer (pH 7.2) for 10 min at 4°C. Crude extract was obtained by centrifugation at 37,000 g for 30 min. This extract was diluted with 5 volumes of 1mM MgCl;,, 0.1mM ATP, 0.1 mM GTP, 1mM EGTA, 1mM DTT, and 10 mM K- MES buffer (pH 6.8) (solution A), and centrifuged at 3,000g for 5 min. The pellet was dissolved with 0.6 M KC], 1mM MgCl;, 0.1rnM ATP, 0.1 mM GTP, 1mM EGTA, 1mM DTT, and 10 mM K- MES buffer (pH 6.8) (solution B), and then centrifug- ed at 200,000g for 60 min. The supernatant was diluted with 5 volumes of solution A and centrifuged at 3,000 g for 5 min. The pellet was again dissolved in solution B and the purified 260 K protein (b, c) Fig. 2 Morphology of high molecular weight pro- was recovered in supernatant after centrifugation at teins in rotary shadowed preparations (12). a—g, 200,000 g for 60 min. All procedures were carried 260 K protein from squid nerve. h, i, Spectrin from out at 4°C. Erythrocyte spectrin (13) (d), brain human erythrocytes. j, k, Calspectin from rat brain. spectrin (1) (e), filamin (14) (f), and myosin (8) (g) 1, m, Filamin from chicken gizzard. n, o, Myosin were purified from human erythrocytes, rat brain, from rabbit skeletal muscle. The isolation proce- chicken gizzard, and rabbit skeletal muscle, respec- dures are described in Fig. 1. a, ><60,000; b—o, tively. Note that the electrophoretic banding pat- >< 140,000 terns of these proteins clearly differ. Arrows indi- cate molecular weights of 260,000, 200,000, and 43,000 from the top. and brain spectrin dimers, two polypeptide chains were loosely intertwined and tightly joined at both ends to form a strand about filamin-like, nor a myosin-like protein. This 100 nm long (Fig. 2, h-k), and filamin dimers finding was further confirmed by comparison of about 80 nm long appeared to be joined only at the molecular shapes using low angle rotary one end (Fig. 2, l and m). Myosin molecules shadowing technique (12) (Fig. 2). As pre- about 150 nm long showed a characteristic Y- viously reported (1, 2, 11), in both erythrocyte shaped structure with two heads apart (Fig. 2, A UNIQUE PROTEIN ASSOCLATED WITH AXOLEMMA 617 n and o). In contrast to these proteins, the 260 K protein appeared as a straight rod about 100n1n long with a globular head at one end (Fig. 2, a-d). Occasionally, this molecule formed a ring structure by head-to-tail associa- tion within one molecule (Fig. 2, f and g). At higher magnification, some molecules were seen to be composed of two strands arranged parallel with each other (Fig. 2e). In order to find the location of 260 K protein in squid giant axons, antiserum to the 260K protein was produced in rabbits. First, using the Ouchterlony double-diffusion method, we Fig. 3 Ouchterlony double-diffusion plate showing tested the ability of the axoplasm obtained from reactions between antiserum against 260K protein (a) and purified 260 K protein (b), axoplasm obtained a giant axon, 260 K protein, spectrin, calspectin, from giant axon (c), spectrin from human erythro- filamin, and myosin to react with the antiserum cytes (d), filamin from chicken gizzard (e), myosin (Fig. 3). The axoplasm and the purified 260 K from rabbit skeletal muscle (f), and calspectin from protein reacted with this antiserum with pre- rat brain (g). The isolation procedures are described cipitin lines of antigenic identity, but the other in the caption to Fig. 1. proteins showed no cross-reactivity. The loca- tion of the 260 K protein was studied by indirect immunofluorescence microscopy (Fig. 4). homogeneously stained. It would be interesting When frozen transverse sections of squid nerves to know if the axoplasm in giant axons is were used, intense fiuo.rescence appeared at the differentiated into two regions, central and periphery of the axoplasm of the squid giant peripheral, and if the peripheral region contain- axon, indicating that 260 K protein was located ing a large amount of 260 K protein is specialized in the axoplasm underlying the excitable mem- to maintain the excitability of the axolemma. brane (axolemma). Weaker staining was also The results obtained in this study lead us to detected in the central region of the axoplasm conclude that the 260 K protein is a unique in giant axons. On the other hand, in small protein located in the axoplasm underlying the axons around the giant axon, the axoplasm was excitable membrane in squid giant axons. At I Fig. 4 Immunofluorescence microscopic localization of 260 K protein in a transverse frozen section of squid nerve containing giant axon. a, Phase-contrast microscopy. b, Indirect immunofluorescence microscopy. >< 140. The rabbit anti-260 K protein antibody was labelled by incubating the sections with FITC-conjugated goat anti-rabbit IgG. *, Axoplasm of giant axon. Arrowheads, small axons 618 S. TSUKITA et al. present, there is very little information on how microtubules necessary for generation of sodium the 260 K protein regulates the membrane current in squid giant axons. I. J. Membrane excitability, but we believe that further studies on Biol. (in press) the molecular architecture of the axoplasm MATSUMOTO G., Konxvxsm T. and SAKAI H. (1979) Restoration of the excitability of squid underlying the axolemma in squid giant axons giant axon by tubulin-tyrosine ligase and micro- will provide a clear picture of the molecular tubule proteins. J. Biochem. 86, 1155-1158 mechanism of the membrane excitation. Muuor-"usm H., MINAMI Y., MATSUMOTO G. and SAKAI H. (1983) Bundling of microtubles in vitro We wish to thank Professor H. Ishikawa, Depart- by a high molecular weight protein prepared ment of Anatomy, Faculty of Medicine, Gunma from the squid axon. J. Biochem. 93, 639-650 University, for his valuable discussions. This study RICHARDS E. G., CHUNG C.-S., MENZEL D. B. was supported in part by a research grant from the and OLcoTT H. S. (1967) Chromatography of Mitsubishi Foundation. myosin on diethylaminoethyl-sephadex A-50. Biochemistry 6, 528-540 Receivedfor publication 4 November 1983 SAKAI H. and MATSUMOTO G. (1978) Tubulin and other proteins. from squid giant axon. J. 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