Microtubule associated proteins of goat brain

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1 J. Biosci., Vol. 4, Number 1, March 1982, pp Printed in India. Microtubule associated proteins of goat brain A. MAJUMDER, S. K. BANERJEE and P. K. SARKAR Department of Cell Biology, Indian Institute of Chemical Biology, Calcutta MS received 22 September 1981; revised 18 January 1982 Abstract. The microtubule associated proteins of goat brain were separated from tubulin on the basis of their thermostability and then fractionated by chromatography on Sepharose 4B column. Analysis of the fractions by SDS-Polyacrylamide gel electrophoresis and assay of their tubulin-assembly-promoting activity indicate that this activity resides primarily in the tauproteins (mol. wt. 55,000-70,000) and a class of even lower molecular weight (25,000-35,000) proteins. Electrophoresis of the microtubule associated protein fractions separated from tubulin by phosphocellulose chromatography are in agreement with the results obtained from fractionation on Sepharose 4B columns. Keywords. Microtubule proteins; goat brain; fractionation. Introduction Microtubule proteins isolated from various sources by cycles of temperaturedependent polymerization and depolymerization (Shelanski et al., 1973) contain in addition to tubulin, several other associated proteins which copolymerize with tubulin. The nature and heterogeneity of these microtubule associated proteins vary in different tissues (Murphy and Borisy, 1975; Fellous et al., 1977; Sloboda e al., 1976; Cleveland et al., 1977, 1979). Previous investigations (Fellous et al., 1977; Sloboda et al., 1976; Cleveland et al., 1977) on the chracterization of these microtubule associated proteins and studies on their in vitro tubulin-assemblypromoting activity indicates that the major activity, resides in two or three high molecular weight (250, ,000) proteins molecular weight and a set of four or five polypeptides of molecular weight 50,000-70,000 referred to as tau-proteins. More recently it has been demonstrated (Herzog and Weber, 1978) that both the high molecular weight proteins and the tau-proteins have independent tubulinassembly-promoting activity but the morphology of the neurotubules formed in these two cases are different. The in vivo association of both tau and the high molecular weight proteins with microtubules in several types of cells has been demonstrated by using immunofluorescent antibodies (Conolloy et al., 1977,1978; Sherline and Schiavone, 1977). However, considerable doubt still remains as to whether all cells types contain the tau and high molecular weight proteins and the physiological role of these accessory proteins that copolymerize with tubulin (Cleveland et al., 1979). In the present communication, we report the isolation of neurotubule proteins from goat brain, their fractionation by two independent procedures and identification of the tubulin-assembly-promoting factors. 61

2 62 Microtubule proteins of goat brain Materials and methods Materials Goat brains, from freshly slaughtered animals, were purchased from the local market. Sodium piperazine-n-n -bis-2-ethane sulphonate, GTP type II, EGTA (ethylene glycol-bis β-amino ethyl ether-n, N -tetra-acetic acid) and phosphocellulose were from Sigma Chemical Co., St. Louis, Missouri, USA. Sephadex G- 25 and Spheres 4B were from Pharmacia, Uppsala, Sweden. Acrylamide,( bis N, N -methylene bisacrylamide) and Temed (Ν,Ν,Ν',Ν'-tetramethylene diamine) were from BioRad, Richmond, California, USA. All other chemicals were locally purchased and were of Analytical grade. Preparation and fractionation of neurotubule proteins from goat brain All Operations except the temperature dependent polymerization of microtubule proteins were done at 4 C. Goat brains totally freed of meninges and blood vessels, were minced into small pieces and blended in a Waring blendor with piperazine ethane sulphonate buffer (0.1 Μ buffer salt, lmm MgCl 2 and 2mM EGTA, ph 6.45) using 1 ml buffer/g of tissue. The material was further homogenized in an all glass Dounce type homogenizer with a B pestle and then centrifuged at g for 15 min. The supernatant was again centrifuged at g for 1 h. From the g supernatants, neurotubule proteins were purified according to the procedure of Shelanski et al. (1973) except that the temperature-dependent polymerization was carried out in the presence of 4 Μ glycerol at 37 C for 1 h and depolymerization at 0 C for 30 min. The final preparation, after two cycles of polymerization and depolymerization was clarified by centrifugation at g for 15 min and used for the isolation of microtubule associated proteins by boiling the purified neurotubule protein (Fellous et al., 1977; Herzog and Weber, 1978). Phosphocellulose chromatography of neurotubule proteins were performed according to the procedure of Weingarten et al. (1975). Miscellaneous procedures SDS-polyacrylamide gel electrophoresis were done using a gradient of 4-15% acrylamide according to the procedure of Laemmli (1970); β-galactosidase, serum albumin, ovalbumin and α-chymotrypsinogen were used as molecular weight markers. Tubulin-assembly-promoting activity of the microtubule assembly proteins were measured by turbidimetric procedure at 350 nm using a thermostatically controlled (37 C) Beckman Model 24 spectrophotometer. The assembly of tubulin was temperature dependent and at a lower temperature, (0-4 C) the polymerized micro-tubules underwent depolymerization. The critical concentration of tubulin required for polymerization was 0.5 mg/ml. The time of equilibration at 37 C was min. Protein was assayed by the procedure of Lowry et al. (1951) using bovine serum albumin as standard. Results In view of the reports (Fellous et al., 1977; Herzog and Weber, 1978) that, most or all of the microtubule associated proteins of rat or porcine brain can be separated from tubulin on the basis of their thermostability, initial isolation of the goat brain

3 Majumder et al. 63 microtubule assembly proteins was achieved by boiling purified neurotubule preparations, as described in Methods. The Sepharose-4B elution pattern (figure 1) displayed a definite shoulder between two peaks. Four fractions, as indicated by bars, were collected for analysis by SDS-polyacrylamide gel electrophoresis and for assay of tubulin-assembly-promoting activity. Figure 1. Elution pattern of goat microtubule assembly proteins from Sepharose 4B column. Total boiled factor (4 mg) was applied to the column and the proteins were eluted with piperazine ethane sulphonate buffer. Fractions indicated by bars (I to IV) were pooled separately. The electrophoretic pattern of purified neurotubules show in addition to tubulin, about twenty bands with an extremely faint band in the region of high molecular weight proteins (Figure 2A, lane 2). The residue from the boiling step (lane 3) contains most of these proteins including the band in the region of the high molecular weight proteins. The tau proteins and some other proteins of low molecular weight (25,000 to 35,000) were the only major microtubule assembly proteins recovered (lane 4). The supernatant from the ammonium sulphate precipitation step did not show any distinct band (lane 5). Figure 2b Shows the gel pattern of the Sepharose 4B column fractions as well as the total boiled factor. While no protein band was detectable in the region of the high molecular weight proteins, several faint bands of 100, ,000 molecular weight were present in fractions I and II. These proteins may represent the subunits of the neurofilaments which has recently been shown to copolymerize with tubulin (Runge et al., 1981). The tubulin-assembly-promoting activity of the total boiled factor was assayed in the presence and absence of 4M glycerol using phosphocellulose purified tubulin (phosphocellulose-tubulin). In the presence of glycerol, this tubulin alone underwent some polymerization after a lag of about 4 min (figure 3a). Addition of total boiled factor in increasing amounts promoted both the initial rate and the final level of polymerization with little or no lag. Identical concentrations of tubulin

4 64 Microtubule proteins of goat brain Figure 2. SDS-polyacrylamide gel electrophoretic patterns of goat neurotubule proteins. (A) Lane 1 phosphocellulose tubulin (60 µg); lane 2 neurotubule protein purified by two cycles of polymerization; lane 3 precipitate after heat treatment of neurotubule protein; lane 4 total boiled factor recovered from the supernatant after heat treatment by precipitation with 50% ammonium sulphate; lane 5 supernatant from the ammonium sulphate precipitation step. Protein (60-90 μg) was applied in lanes 2-4. In lane 5, 30 μg supernatant was applied. (b) Lane 1 phosphocellulose-tubulin (75 μg); lane 2,3,4 and 5 represent fractions I to IV (25-45 μg) from the sepharose 4B column respectively; lane 6 total boiled factor (100 μg). Figure 3. Tubulin-assembly-promoting activity of the total boiled factor in piperazine ethane sulphonate buffer in the presence (a) or absence (b) of 4M glycerol. Phosphocellulose tubulin concentration in all cases was 1.2 mg/ml. (a) phosphocellulose tublin alone (O); tubulin: factor ratio, 10:1 ( ); and 5:1 (Δ). (b) Phosphocellulosetubulin alone ( ); tubulin: factor ratio, 10:1 (Δ) and 5:1 ( )

5 Majumder et al. 65 and the boiled factor were tested for polymerization in the absence of glycerol (Figure 3b); here, phosphocellulose tubulin alone was totally inactive and the activity of the boiled factor was about half of that observed in the presence of glycerol. In view of the marked effect of glycerol on the assembly of phosphocellulose-tubulin, all subsequent assays were performed m the presence of 4 Μ glycerol. Figure 4 shows the relative tubulin-assembly-promoting activity of the total boiled factor, fraction II (peak I) and fraction IV (peak II) from the Sepharose column. These two fractions were assayed since the electrophoretic pattern of fraction II was very similar to that of fraction I and that of fraction III was almost identical to that of fraction IV (see Figure 2B). The results (figure 4) show that Figure 4. Tubulin-assembly-promoting activity of total boiled factor and the fractions eluted from the Sepharose 4B column in 4 Μ glycerol -piperazine ethane sulphonate buffer. (Δ), phosphocellulose-tubulin (1.45 mg/ml)alone; ( ), plus 0,1 mg of total boiled factor; (ο),plus 0.1 mg fraction II from sepharose 4B column, ( ) plus 0.1 mg of fraction IV from sepharose 4B column. both fraction II which is enriched with tau proteins, and fraction IV which is enriched with the low molecular weight proteins are individually more active than the unfractionated factor and that fraction IV is relatively more active than fraction II, The decreased activity of the unfractionated factor is likely to be due to the presence of inactive 100, ,000 molecular weight proteins which are seen primarily in fraction I, reduced in fraction II, and are totally absent in fraction IV (see figure 2B). To examine if the dalton proteins in the goat microtubule assembly proteins are artifacts of boiling or proteolytic degradation products of high molecular weight proteins, we have also fractionated the goat microtubule assembly proteins by chromatography on phosphocellulose columns (Weingarten

6 66 Microtubule proteins of goat brain et al., 1978) using buffers containing 0.01 Μ phenylmethyl sulphonyl fluoride. Following the elution of all the tubulin from the columns, the microtubule assembly proteins were eluted in five steps using 0.1,0.15,0.2,0.25 and 0.8 Μ NaCl. These fractions were concentrated by precipitation with 50% ammonium sulphate, dialysed against SDS-containing sample buffer (Laemmli, 1970) and subjected to electrophoresis in polyacrylamide gels. The electrophoretic pattern of these fractions (figure 5) displayed considerable similarity to those of the boiled factors, primarily showing two major classes of bands the tau proteins and the low molecular weight proteins. In 0.15 Μ and 0.8 Μ NaCl (Lanes 2 and 5), a few proteins of molecular weight 100, ,000 appeared, but in none of the fractions the typical high molecular weight proteins were detectable. These later proteins however were consistently present in microtubule assembly proteins from embryonic or adult chick brain, fractionated by phosphocellulose chromatography under identical conditions. Figure 5. SDS-polyacrylamide gel electrophoretic pattern of the microtubule assembly protein fractions eluted from the phosphocellulose column by increasing concentrations of sodium chloride. Lanes 1-5 represent fractions eluted with 0. 1, 0. 15, 0.2, 0.25 and 0.8M sodium chloride respectively, (50-70 µg proteins applied in each slot). Lane 6-phosphocellulose-tubulin (20 µg). Discussion In this communication, we show that microtubule assembly proteins from goat brain are characteristically different from those isolated from other sources in at least two aspects. The major difference between goat and other vertebrate brain microtubule assembly proteins is with respect to the high molecular weight proteins-which are

7 Majumder et al. 67 either absent or present in quantities too small to detect in the goat brain. The in vitro tubulin-assembly-activity of both the tau proteins and the high molecular weight proteins have been well characterized in the case of neurotubules from calf (Sloboda et al., 1976), porcine (Murphy and Borisy, 1975; Cleveland et al., 1977), rat (Fellous et al., 1977) and chick (Cleveland et al., 1979) brain, and their association in vivo has also been well established (Conolly et al., 1977, 1978, Sherline and Schiavone, 1977). High molecular weight proteins have been reported to be absent in the neuroblastoma (Nagle et al., 1977) and other nonneural cell lines (Weatherbee et al., 1978) and present in minute amounts in lamb brain (Maccionia and Seeds, 1978). Fractionation of porcine brain microtubule assembly proteins by phosphocellulose chromatography showed (Herzog and Weber, 1978) that the high molecular weight proteins, free of tau, emerge from the column in buffers containing Μ sodium chloride and the tau polypeptides appear at higher salt concentrations. In contrast, phosphocellulose chromatography of goat brain microtubule assembly proteins showed tau as the major protein in both 0.1 Μ and 0.15 Μ salt concentrations, no distinct band for high molecular weight protein was detected in these fractions. These findings and the polymerization activity data of the fractionated boiled factors (figure 4) indicate that the high molecular weight proteins, if present, represent a negligible fraction of the total goat microtubule assembly proteins and are unlikely to have any important physiological role. The second characteristic feature in goat microtubule assembly proteins is the presence of the dalton proteins in addition to the more commonly present tau proteins. These proteins are not degradation products or results of heat denaturation of the neurotubule proteins since they appeared in microtubule assembly proteins isolated by two different procedures and in buffers containing the protease inhibitor phenyl methyl sulphonyl fluoride. Comparison of the tubulin-assembly-promoting activity of these proteins with the tau proteins indicated that both classes of proteins are active (figure 4); however, quantitation of their relative activity require complete separation of these two classes of proteins which is yet to be achieved. References Cleveland D. W., Hwo, S. Y. and Kirshner, M. W. (1977), J. Mol Biol., 116, 207. Cleveland, D. W., Spiegelman, B. M. and Kirshner, Μ. W. (1979), J. Biol Chem., 254, Conolly, J. Α., Kalanis, V. J., Cleveland, D. W. and Kirshner, M. W. (1977), Proc. Natl Acad. Sci. U.S.A., 74, Conolly, J. Α., Kalanis, V. J., Cleveland, D. W. and Kirshner, M. W. (1978), J. Cell Biol., 76, 781. Fellous, Α., Francon, J., Lenon, A. M. and Nunez, J. (1977) Eur. J. Biochem., 78, 167. Herzog, W. and Webber, K. (1978), Eur. J. Biochem., 92, 1. Laemmli, U. K. (1970), Nature (London), 227, 680. Lowry, Ο. Η., Rosebrough, Ν. J., Farr, Α. L. and Randall, R. J. (1951), J. Biol Chem., 193, 265. Maccioni, R. B. and Seeds, N. W. (1978), Arch. Biochem. Biophys., 185, 262. Murphy, D. B. and Borisy, G. G. (1975), Proc. Natl. Acad. Sci. U.S.A., 72, Nagle, Β. W., Doenges, Κ. Η. and Bryan, J. (1977), Cell, 12, 573. Runge, Μ. S., Schlaepfer, W. W. and Williams, R. C. Jr. (1981), Biochemistry, 20,170.

8 68 Microtubule proteins of goat brain Shelanaki, M. L., Gaskin, F. and Cantor, C. R. (1973), Proc. Natl. Acad. Sci. U.S.A., 70, 765. Sherline, P. and Schiavone, K. (1977) Science (Wash. D.C.), 198, Sloboda, R. D., Dentler, W. L. and Rosenbaum,.J. L. (1976), Biochemistry, 15, Weatherbee, J. Α., Luftig, and Weihing, R. R. (1978), J. Cell Biol., 78, 47. Weingarten, Μ. D., Lockwood, Α. Η., How, S. Υ. and Kirshner, Μ. W. (1975). Proc. Natl. Acad. Sci. U.S.A., 72,1858.