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Glia maturation factor in bovine brain: Partial purification and physicochemical characterization
Brain Research, 212 (1981) 393-402 © Elsevier/North-Holland Biomedical Press
393
G L I A M A T U R A T I O N F A C T O R IN BOVINE B R A I N : P A R T I A L PURIFICATION AND PHYSICOCHEMICAL CHARACTERIZATION*
TAIJI KATO, YOKO FUKUI, DAVID E. TURRIFF, SATOE NAKAGAWA, RAMON LIM, BARRY G. W. ARNASON and RYO TANAKA Department of Biochemistry, School of Medicine, Nagoya City University, Mizuho-Ku, Nagoya 467 (Japan) and ( S. N., R.L., B.G.W.A.) Brain Research Institute, Departments of Surgery (Neurosurgery) and Neurology, University of Chicago, Chicago, Ill. 60637 (U.S.A.) and ( D.E.T.) Division of Biomedical Sciences, Rockford School of Medicine, University of Illinois, Rockford, Ill. 61101 (U.S.A.)
(Accepted October 10th, 1980) Key words: glia maturation factor - - maturation - - growth factor - - cell growth - - gila - - bovine
brain
SUMMARY Glia maturation factor ( G M F ) is partially purified from bovine brains by the following procedure: extraction at physiologic pH, dialysis and freeze-drying of the extract, ethanol washing of the dried powder and re-extraction of the ethanol-washed residue with Tris-buffered saline, ion-exchange chromatography with D E A E Sephadex and molecular sieving with Bio-gel P-200. The partially purified protein has an apparent molecular weight of 23,000 and an isoelectric point of 4.75, and retains both morphological transforming and mitogenic activities when tested on glioblasts. Both activities are susceptible to protease digestion and heat inactivation. The procedure results in a 400-fold purification of the morphological activity and a 1400-fold purification of the mitogenic activity. Both activities are detectable when G M F is used in nanogram quantities. The possibility that both functions are expressions of the same factor and the possible role of G M F in the differential or sequential stimulation of cell growth and maturation are discussed.
INTRODUCTION The study of growth or maturation factors in the brain is potentially important for the understanding of the development of the nervous system. In 1972, Lim et al. * A preliminary account of this work was presented at the 1lth Annual Meeting of the American Society for Neurochemis~ry in Houston, Texas, March 2-6, 1980 (Abstr. 92, p. I l 1).
394 first reported the presence of a glia maturation factor (G M F) in the adult brain~L The factor stimulates thc morphological and chemical maturation of glioblasts following an initial increase in D N A synthesis and cell division 7-HJ.t7. G M F isolated from pig brain was previously characterized 5. The present report deals with the study of a similar factor from bovine brain. MATERIALS AND METHODS
Source of bo vine glia maturation fat'tor Fresh bovine brains were obtained from a local slaughter house within 45 min after killing and transported on ice to the laboratory. The brains (500 g) were homogenized in Tris-buffered saline (0.02 M Tris.HCl with 0.15 M NaCI, pH 7.4) with a Waring blender, at 23,000 rpm for 30 sec, to make a 25 I~,;(w/v) homogenate. The supernatant fraction after spinning the homogenate at 23,000 :.: g for 16 h in a Spinco No. 15 rotor was designated as the crude extract. The brain extract was divided into four 500 ml portions in glass bottles and concentrated by the following freeze-thawing procedure. After freezing at --20 ':C for 24 I1, each 500 ml portion was partially thawed by inverting the bottle at room temperature. Two 100 ml portions of liquid werc collected in sequence from each bottle, and the remaining frozen material was discarded. The first 100 ml portions of thawed liquid from the 4 bottles were combined into 400 ml and set aside. The second 100 ml portions were pooled (400 ml total), frozen and partially thawed again to collect 100 ml of liquid. This was then combined with the 400 ml from the first thawing to obtain a total of 500 mi of concentrated material. The concentrated extract was dialyzed against 4 changes of watcr (4 liters each) and then lyophilized to obtain the 'brain powder' which can be stored for long periods of time. The usual yield of the dried powder is 5 g.
Bioassay for gila maturation factor Bioassay was conducted on confluent cultures of a homogeneous population of glioblasts, which were obtained from 17-day Wistar rat fetuses as follows. The cerebra and cerebella were dissected, pooled and dissociated with trypsin in Tyrode solution free of calcium and magnesium for 30 rain at 37 ~'C. The cells were seeded into a tissuc culture flask (Falcon plastic culture flasks, No. 3012, surface area 25 sq. cm) with 20 00 fetal calf serum in F-10 medium. After incubation for 2 days without disturbance, the medium was changed and the culture continued for another 5 days. The brain cells were subcultured in Lux 8-well plastic trays (No. 5218) with 10°,i fctal calf serum in F-10 medium. After 20 h the medium was changed to eliminate the neuroblasts, which were still floating in the medium, leaving behind the glioblasts which became confluent in about 4 days. A more detailed description of the method was presented in a previous paper 5. The morphological assay was startcd by adding 0.5 ml of a test solution containing GM F to 2.0 ml of F-10 containing 5 i!~,fetal call" serum in each culture well. The cells were restimulated with the factor 24 h later. After another 16 h the cells were
395 scored for morphological transformation. A cell possessing at least one process longer than the diameter of the original cell soma was counted as a positive response and the results were expressed as percentage of transformed cells with respect to the total cell population. Assay for mitogenic activity was conducted concurrently with morphological assay by the use of a modification of the method of McLeester and Hall 19-. [Methyl3H]thymidine (0.1/~Ci) was added to each well at the second G M F stimulation. After 16 h of incorporation, the medium was removed and the cells were harvested by treatment with 1.0 ml of Tris-buffered saline containing i.0 mg of trypsin. The cells were pelleted by centrifugation and solubilized with 50 #1 of 0.5 ~ sodium dodecyl sulfate. An aliquot of the cell sample (20/tl) was applied on a paper disc (i.9 cm Whatmann 3MM filter paper, McLeester Research Equipment) which was previously impregnated with 40 #1 of 20 ~ trichloroacetic acid. The paper disc was washed with a cold 5 ~ trichloroacetic acid solution for 10 min and with cold 95 ~ alcohol for 10 min. The radioactivity in the paper disc was counted with a liquid scintillation counter. A more detailed account of this method was previously presented 5.
Column chromatography DEAE Sephadex A-50, washed with 0.02 M Tris.HCl buffer containing 0.05 M NaCI, pH 7.4, was packed into a column and equilibrated with the above buffer. Biogel P-200 (100-200 mesh) was packed into a column after complete washing with 0.02 M Tris.HCl buffered saline, pH 7.4. The column was run upward at 4 °C.
lsoelectric focusing lsoelectric focusing was carried out at 4 °C for 16 h in the Pharmacia flat bed apparatus (FBE 3000) at a constant power of 10 W. At the end of the run the gel bed was sectioned into 5 mm-wide zones with the aid of a fractionating grid. The pH was measured after addition of 3 ml water. The suspension was filtered and the residual gel was washed with 2 ml of 0.2 M Tris.HCl, pH 7.4. The absorbance of the combined filtrate and wash was measured at 280 nm after pH adjustment to neutrality with 1 N NaOH.
Assay for esterase activity Esterolytic activity was assayed by the spectrophotometric method t at 253 nm for a-N-benzoyl-t~-arginine ethyl ester (BAEE), at 256 nm for a-N-benzoyl-L-tyrosine ethyl ester (BTEE), and at 256 nm for p-tosyl-L-arginine methyl ester (TAME) as substrates, by the use of a Hitachi UV-spectrophotometer (Model No. 124). Trypsin and chymotrypsin were used as standard enzymes. All enzymes and synthetic substrates were products of Sigma. The assays were conducted at pH 7.5 and at 30 °C.
Preparation of Sepharose-bound porteases All enzymes (papain, ficin, pronase, and trypsin) were coupled to cyanogen bromide-activated Sepharose 4B, which was prepared by the method of Axdn et al. 2 and Porath et al. 13. The coupling reaction was allowed to proceed at 0 °C for 3 h, and then at 4 °C overnight.
396
Protein determination Protein was determined by the method of Lowry ct al. ]] except in fractionation procedures where absorbance at 280 nm was used. RESULTS
Purification procedure Extraction of GMFactivityfrom bovine brain powder (step 1). The dried powder was washed twice with ice-cold absolute ethanol (500 ml). After centrifugation at 10,000 × g for 10 min, the pellet was extracted with 200 ml of Tris-buffered saline by stirring overnight at 4 °C. The aqueous extract after centrifugation at 23,000 /, g for 30 min was the ethanol-washed extract. Ion-exchange column chromatograph)' (step 1I). The ethanol-washed extract (200 ml) was diluted with 2 vols. of 0.02 M Tris.HCl, pH 7.4, to reduce the NaCI concentration to 0.05 M. The sample, concentrated to 100 ml on Amicon PM-10 Diaflo membrane, was applied to a DEAE Sephadex A-50 column (5 :. 45 cm) preequilibrated with 0.02 M Tris.HCl buffer, pH 7.4, containing 0.05 M NaCI. The column was then eluted with a linear gradient of NaCI from 0.05 to 0.7 M in the same buffer (Fig. 1). Both morphological transforming and mitogenic activities appeared from 0.25 to 0.35 M NaCI concentration. The fractions underlined in Fig. 1 were collected and concentrated to 24 ml, and used for the next step. Bio-gel P-200 column chromatography (step Iil). One half of the DEAE-sample (12 ml) derived from 500 g wet weight of bovine brain was applied to a Bio-gel P-200 column which was pre-calibrated with molecular weight standards, including bovine serum albumin, ovalbumin, chymotrypsinogen and ribonuclease. Most of the applied EI.2~-
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50 IOO 150 FRACTION N U M B E R ( I O m l )
200
Fig. I. E l u t i o n profile o f bovine gila m a t u r a t i o n factor f r o m D E A E Sephadex A-50. Ethanol-washed extract (2 g protein), derived f r o m 500 g (wet weight) bovine brain, was applied to a D E A E Sephadex c o l u m n (5 × 45 cm) equilibrated with 0.02 M T r i s - H C I buffer, p H 7.4, containing 0.05 M NaCI. A f t e r washing the sample-charged column with the buffer, the column was eluted with 1800 ml o f the buffer containing a linear gradient o f NaCI f r o m 0.05 to 0.7 M , at a rate o f 60 ml/h. Black bars, m o r p h o l o g i c a l transforming activity; • • , mitogenic activity; . . . . . . . , U V absorbance at 280 nm; .... . theoretical N a C I concentration. The underlined fractions were used f o r the next purification step after pooling and concentration.
397
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,
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7'0 FRACTION
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I10
130
NUMBER ( 6 m l )
Fig. 2. Elution profile of bovine GMF from Bio-gel P-200 column. The column (2.6 x 150 cm) was eluted by reverse flow at 12 ml/h. For explanation and symbols, see legend to Fig. 1.
brain protein came out at the high molecular weight region (larger than 40,000 Daltons), but morphological transforming activity showed up at an apparent molecular weight of 23,000 ± 3000 which was accompanied by mitogenic activity (Fig. 2.) The purification procedure and increase in specific activity are summarized in Table I. Dose-response curves Fig. 3 shows the d o s e - r e s p o n s e relationship o f G M F at various stages o f purification. A t the high c o n c e n t r a t i o n range, the a p p a r e n t mitogenic activity showed a p a r a d o x i c a l inhibition, a l t h o u g h this was o v e r c o m e u p o n further increase in G M F concentration. N o such p a r a d o x i c a l effect was seen with the m o r p h o l o g i c a l t r a n s f o r m ing activity. The lowest detectable protein c o n c e n t r a t i o n for mitogenic activity was 100 ng/ml whereas that for m o r p h o l o g i c a l activity was 300 ng/ml. Heat lability G M F s a m p l e s from D E A E Sephadex were incubated at p H 7.4 at 70 °C for various lengths o f time and i m m e d i a t e l y chilled in ice water. T h e r e a c t i o n mixture was centrifuged in the cold and the clear s u p e r n a t a n t used for bioassays (Fig. 4). A t 20 min TABLE 1 Purification of GMF from bovine brain The data presented are calculated on the basis of 500 g (wet weight) bovine brain as the starting material. For both the mitogenic and morphological activities, one unit is defined as that exhibited by 1 mg protein in the crude extract. Protein recovered (mE)
Crude extract DEAESephadex Bio-gel P-200
9312.0 50.4 1.5
Mitogenic activity
Morphological activity
Activity recovered (units)
Specific activity (units/mg)
Activity recovered (units)
Specific activity (units~rag)
9312 2974 2108
1 59 1405
9312 1361 600
1 27 400
398
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PROTEIN ( m g / W E L L ) Fig. 3. D o s e - r e s p o n s e curves of glia m a t u r a t i o n factor. A : mitogenic activity. B: morphological transf o r m i n g activity. All experiments were conducted on the s a m e batch o f glioblasts in s e c o n d a r y culture. !3 . . . . . IZI, crude bovine brain extract; 0 - - - 0 , G M F at step 11 ( D E A E Sephadex); ---C~, GMF at step 1II (Bio-gel 1:'-200).
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INCUBATION TIME A T 7 0 " (rain)
Fig. 4. Heat lability o f gila m a t u r a t i o n factor. G M F (0.8 mg/ml) were incubated at pH 7.4 and 70 ~C for various lengths of time. Finally the samples incubated for 60 rain were boiled for 10 rain. 0.5 ml aliquots of s u p e r n a t a n t were taken for cell tests. _1, the incorporation o f [3H]thymidine into unheated controls. All other s y m b o l s are'explained in legend to Fig. I.
399 TABLE II
Effect of proteases on GMF GMF (2.3 mg) was mixed with 1.5 mg of an enzyme bound to Sepharose in 3 ml of Tris-buffered saline (pH 7.4) and incubated at 37 °C with continuous shaking. After incubation, the immobilized enzyme was removed by membrane filtration and the filtrate assayed for morphological and mitogenic activities. Dithiothreitol (DTT), 1 raM, was included in the incubation mixture when papain or ficin was used. Values for papain and ficin treatment are expressed as percentage of control in the presence of DT/'.
Enzyme treatment
After 1 h incubation: Papain Trypsin After 16 h incubation: Papain Trypsin Pronase Ficin
Morphological activity (% remaining)
Mitogenic activity (% remaining)
10 80
48.5 92.1
0 0 0 10
31.2 28.2 27.7 55.7
most of the mitogenic and morphological t r a n s f o r m i n g activities were lost. Boiling completely inactivated the two activities.
Protease susceptibility Table II shows the relative susceptibility of bovine G M F to the proteases. G M F was fairly resistant to trypsin b u t sensitive to p a p a i n after 1 h of incubation. A l t h o u g h the morphological t r a n s f o r m i n g activity entirely disappeared after 16 h of i n c u b a t i o n , some mitogenic activity still remained.
Esterase activity T o distinguish G M F from other growth factors such as epidermal growth /-
B
6
~-/ 8~ x
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4 o~ ~ 2 ~ -'J40'~o ° m
3 I
0
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I0 20 FRACTION NUMBER
1
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/0~
30
Fig. 5. Isoelectric focusing of bovine GMF. Isoelectric focusing was conducted on a flat bed of 5 (w/v) Sephadex G-75 (superfine) in the presence of 10~ (w/v) sucrose and 1.2~ (v/v) Pharmalyte (pH range 4-6.5). The sample was obtained from a Bio-gel P-200 column as described in legend to Fig. 2. . . . . , pH. All other symbols are explained in legend to Fig. 1.
400 factor 3,14-~ and nerve growth factor4, ts which have esteropeptidase activity in one of the subunits, esterase activity was measured using BAEE, BTEE, and TAME as substrates. No esterase activity was detected in any of the G M F sample.
Isoelectric focusing Fig. 5 shows the results of preparative isoelectric focusing, using the narrow pH range Pharmalyte (ampholyte) from pH 4 to 6.5. Both mitogenic and morphological transforming activities focused at pH 4.75 l: 0.30. DISCUSSION We previously demonstrated that G M F exists in the fresh pig brain mainly in a large molecular weight form (200,000 Daltons) and that attempts at purification frequently results in the appearance of molecules of smaller sizes, notably one of 40,000 Daltons 5. On this basis we postulated that G M F exists in situ as a large mother molecule and that from this a family of smaller molecules of similar function can be generated as a result of either dissociation or cleavage of covalent bonds. In the present paper we demonstrated that G M F activity is found in the fresh bovine brain in association with a 23,000 molecular weight component. That this molecule is smaller that what was found in the pig brain may simply imply that native bovine G M F is more readily dissociable or degradable than native porcine GMF. Other than the difference in molecular size, G M F from the two sources show very similar physicochemical and biological properties. Thus, both are acidic proteins and both possess morphological and mitogenic activities on glioblasts which are susceptible to heat and protease inactivation. Both are devoid of esterase activity. Bovine G M F appears to be more suitable for purification than porcine G M F because the former is readily obtainable in a uniform, low molecular weight species which, upon molecular sieving, separates easily from the bulk of brain proteins which are larger in molecular size. Besides, the isolation of a smaller protein is expected to bc more amenable to chemical characterization and sequencing than a larger one. The biological activity of G M F was originally defined in terms of its ability to promote the morphological differentiation of glioblasts 9. Subsequently, we observed that a mitogenic activity always accompanies the morphological function and suggested that G M F could be both a mitogenic and maturation factor ~, as is nerve growth factor, in the present work, although both activities were co-purified, the mitogenic function was obtained at a higher state of purity than the morphological transforming function. This finding, however, does not contradict our suggestion, since the two functions could reside in two different active sites in the same or different protein subunits, and that one active site (in this case that of morphological activity) may be less stable than the other. Alternatively, it is possible that the morphological function requires cofactors which are partially eliminated during the fractionation procedures. The exact answers to these questions must await the complete purification of GMF. Before the problem is resolved it is best to assay the two activities together
401 because one method complements the other. Morphological assay seems to work better at high G M F concentrations while mitogenic assay appears to be more sensitive as the purity of the factor increases. It is noteworthy that, in terms of mitogenicity, the specific activity of G M F is comparable to that of other growth factors such as fibroblast growth factor. It is also important to point out that at the high concentration range a paradoxical inhibition is observed with mitogenic activity. Assuming that both activities are effects of the same factor, then the differential expression of mitogenic and morphological functions at different G M F concentrations may have real biological significance in the sequential or reciprocal control of growth and differentiation of glial ceils. The fact that, at low G M F concentrations, mitogenic changes can occur without subsequent morphological differentiation is evidence that cell division per se is not a sufficient cause for cell maturation. In other words, the morphological transformation brought about by G M F is not an obligatory event following and secondary to cell division. We previously purified G M F from the pig brain to a 400-fold purity 9. However, a major criticism of that procedure is that trypsin digestion was employed in the principal step and this may introduce uncertainties due to possible structural alterations. In the current work no trypsin was used. One additional advantage with the present work is that GM F is more active in bovine brain than in pig brain, so that even at the same degree of purification bovine G M F has a higher specific activity than pig GMF. The current work underlines the similarity in biological functions between pig and bovine brain GMF, and offers the latter as an altetnative source for the purification of the factor. ACKNOWLEDGEMENTS This work was supported by a Grant-in-Aid for Scientific Research (Ministry of Education) and the Naito Research Grant for 1978 from Japan; and by N I H Grant CA-27031 and NSF Grant BNS 79-00352 from the U.S.A.
REFERENCES 1 Arnon, R., Papain. In G. E. Perlman and L. Lorand (Eds.), Methods in Enzymology, Vol. 19, Academic Press, New York, 1970, pp. 226-244. 2 Ax6n,R., Porath, J. and Ernback, S., Chemicalcoupling of peptides and proteins to polysaccharides by means of cyanogen halides, Nature (Lond.), 214 (1967) 1302-1304. 3 Cohen, S., Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal, J. biol. Chem., 237 (1962) 1555-1562. 4 Greene, L. A., Shooter, E. M. and Varon, S., Enzymatic activities of mouse nerve growth factor and its subunits, Proc. nat. Acad. Sci. (Wash.), 60 (1968) 1383-1388. 5 Kato, T., Chiu, T.-C., Lim, R., Troy, S. S. and Turriff, D. E., Multiple molecular forms of glia maturation factor, Biochim. biophys. Acata (Amst.), 579 (1979) 216-227. 6 Lim, R., Li, W. K. P. and Mitsunobu, K., Morphological transformation of dissociated embryonic brain cells in the presence of brain extracts, Neurosci. Abstr., 2 (1972) 181.
402 7 Lira, R., Mitsunobu, K. and Li, W. K. P., Maturation-stimulating effect of brain extract and dibutyryl cyclic AMP on dissociated embryonic brain cells in culture, Exp. Cell Res., 79 (1973) 243 246. 8 Lim, R. and Mitsunobu, K., Brain cells in culture: morphological transformation by a protein, Science, 185 (1974) 63-66. 9 Lim, R. and Mitsunobu, K., Partial purification of a morphological transforming factor from pig brain, Biochim. biophys. Acta ( Amst.), 400 (1975) 200-207. 10 Lim, R., Turriff, D. E., Troy, S. S., Moore, B. W. and Eng, L. F., Glia maturation factor: effect on chemical differentiation of glioblasts in culture, Science, 195 (1977) 195 - 196. I 1 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. bioL Chem., 193 (1951) 265-275. 12 McLeester, R. C. and Hall, T. C., Simplification of amino acid incorporation and other assays using filter paper techniques, Analyt. Biochem., 79 (1977) 627--630. 13 Porath, J., Ax6n, R. and Ernback, S.~ Chemical coupling of proteins to agarose, Nature (Lond.), 215 (1967) 1491-1492. 14 Taylor, J. M., Cohen, S. and Mitchell, W. M., Epidermal growth factor: high and low molecular weight forms, Proc. nat. Acad. Sci. (Wash.), 67 (1970) 164 171. 15 Taylor, J. M., Mitchell, W. M. and Cohen, S., Epidermal growth factor. Physical and chemical properties, J. biol. Chem., 247 (1972) 5928-5934. 16 Taylor, J. M., Mitchell, W. M. and Cohen, S., Characterization of the binding protein for epidermal growth factor, J. biol. Chem., 249 (1974) 2188-2194. 17 Turriff, D. E. and Lim, R., Glia maturation factor increases cyclic GMP in glioblasts, Brain Research, 166 (1979) 436-441. 18 Varon, S., Nomura, J. and Shooter, E. M., The isolation of the mouse nerve growth factor protein in a high molecular weight form, Biochemistrv, 6 (1967) 2202- 2209.
393
G L I A M A T U R A T I O N F A C T O R IN BOVINE B R A I N : P A R T I A L PURIFICATION AND PHYSICOCHEMICAL CHARACTERIZATION*
TAIJI KATO, YOKO FUKUI, DAVID E. TURRIFF, SATOE NAKAGAWA, RAMON LIM, BARRY G. W. ARNASON and RYO TANAKA Department of Biochemistry, School of Medicine, Nagoya City University, Mizuho-Ku, Nagoya 467 (Japan) and ( S. N., R.L., B.G.W.A.) Brain Research Institute, Departments of Surgery (Neurosurgery) and Neurology, University of Chicago, Chicago, Ill. 60637 (U.S.A.) and ( D.E.T.) Division of Biomedical Sciences, Rockford School of Medicine, University of Illinois, Rockford, Ill. 61101 (U.S.A.)
(Accepted October 10th, 1980) Key words: glia maturation factor - - maturation - - growth factor - - cell growth - - gila - - bovine
brain
SUMMARY Glia maturation factor ( G M F ) is partially purified from bovine brains by the following procedure: extraction at physiologic pH, dialysis and freeze-drying of the extract, ethanol washing of the dried powder and re-extraction of the ethanol-washed residue with Tris-buffered saline, ion-exchange chromatography with D E A E Sephadex and molecular sieving with Bio-gel P-200. The partially purified protein has an apparent molecular weight of 23,000 and an isoelectric point of 4.75, and retains both morphological transforming and mitogenic activities when tested on glioblasts. Both activities are susceptible to protease digestion and heat inactivation. The procedure results in a 400-fold purification of the morphological activity and a 1400-fold purification of the mitogenic activity. Both activities are detectable when G M F is used in nanogram quantities. The possibility that both functions are expressions of the same factor and the possible role of G M F in the differential or sequential stimulation of cell growth and maturation are discussed.
INTRODUCTION The study of growth or maturation factors in the brain is potentially important for the understanding of the development of the nervous system. In 1972, Lim et al. * A preliminary account of this work was presented at the 1lth Annual Meeting of the American Society for Neurochemis~ry in Houston, Texas, March 2-6, 1980 (Abstr. 92, p. I l 1).
394 first reported the presence of a glia maturation factor (G M F) in the adult brain~L The factor stimulates thc morphological and chemical maturation of glioblasts following an initial increase in D N A synthesis and cell division 7-HJ.t7. G M F isolated from pig brain was previously characterized 5. The present report deals with the study of a similar factor from bovine brain. MATERIALS AND METHODS
Source of bo vine glia maturation fat'tor Fresh bovine brains were obtained from a local slaughter house within 45 min after killing and transported on ice to the laboratory. The brains (500 g) were homogenized in Tris-buffered saline (0.02 M Tris.HCl with 0.15 M NaCI, pH 7.4) with a Waring blender, at 23,000 rpm for 30 sec, to make a 25 I~,;(w/v) homogenate. The supernatant fraction after spinning the homogenate at 23,000 :.: g for 16 h in a Spinco No. 15 rotor was designated as the crude extract. The brain extract was divided into four 500 ml portions in glass bottles and concentrated by the following freeze-thawing procedure. After freezing at --20 ':C for 24 I1, each 500 ml portion was partially thawed by inverting the bottle at room temperature. Two 100 ml portions of liquid werc collected in sequence from each bottle, and the remaining frozen material was discarded. The first 100 ml portions of thawed liquid from the 4 bottles were combined into 400 ml and set aside. The second 100 ml portions were pooled (400 ml total), frozen and partially thawed again to collect 100 ml of liquid. This was then combined with the 400 ml from the first thawing to obtain a total of 500 mi of concentrated material. The concentrated extract was dialyzed against 4 changes of watcr (4 liters each) and then lyophilized to obtain the 'brain powder' which can be stored for long periods of time. The usual yield of the dried powder is 5 g.
Bioassay for gila maturation factor Bioassay was conducted on confluent cultures of a homogeneous population of glioblasts, which were obtained from 17-day Wistar rat fetuses as follows. The cerebra and cerebella were dissected, pooled and dissociated with trypsin in Tyrode solution free of calcium and magnesium for 30 rain at 37 ~'C. The cells were seeded into a tissuc culture flask (Falcon plastic culture flasks, No. 3012, surface area 25 sq. cm) with 20 00 fetal calf serum in F-10 medium. After incubation for 2 days without disturbance, the medium was changed and the culture continued for another 5 days. The brain cells were subcultured in Lux 8-well plastic trays (No. 5218) with 10°,i fctal calf serum in F-10 medium. After 20 h the medium was changed to eliminate the neuroblasts, which were still floating in the medium, leaving behind the glioblasts which became confluent in about 4 days. A more detailed description of the method was presented in a previous paper 5. The morphological assay was startcd by adding 0.5 ml of a test solution containing GM F to 2.0 ml of F-10 containing 5 i!~,fetal call" serum in each culture well. The cells were restimulated with the factor 24 h later. After another 16 h the cells were
395 scored for morphological transformation. A cell possessing at least one process longer than the diameter of the original cell soma was counted as a positive response and the results were expressed as percentage of transformed cells with respect to the total cell population. Assay for mitogenic activity was conducted concurrently with morphological assay by the use of a modification of the method of McLeester and Hall 19-. [Methyl3H]thymidine (0.1/~Ci) was added to each well at the second G M F stimulation. After 16 h of incorporation, the medium was removed and the cells were harvested by treatment with 1.0 ml of Tris-buffered saline containing i.0 mg of trypsin. The cells were pelleted by centrifugation and solubilized with 50 #1 of 0.5 ~ sodium dodecyl sulfate. An aliquot of the cell sample (20/tl) was applied on a paper disc (i.9 cm Whatmann 3MM filter paper, McLeester Research Equipment) which was previously impregnated with 40 #1 of 20 ~ trichloroacetic acid. The paper disc was washed with a cold 5 ~ trichloroacetic acid solution for 10 min and with cold 95 ~ alcohol for 10 min. The radioactivity in the paper disc was counted with a liquid scintillation counter. A more detailed account of this method was previously presented 5.
Column chromatography DEAE Sephadex A-50, washed with 0.02 M Tris.HCl buffer containing 0.05 M NaCI, pH 7.4, was packed into a column and equilibrated with the above buffer. Biogel P-200 (100-200 mesh) was packed into a column after complete washing with 0.02 M Tris.HCl buffered saline, pH 7.4. The column was run upward at 4 °C.
lsoelectric focusing lsoelectric focusing was carried out at 4 °C for 16 h in the Pharmacia flat bed apparatus (FBE 3000) at a constant power of 10 W. At the end of the run the gel bed was sectioned into 5 mm-wide zones with the aid of a fractionating grid. The pH was measured after addition of 3 ml water. The suspension was filtered and the residual gel was washed with 2 ml of 0.2 M Tris.HCl, pH 7.4. The absorbance of the combined filtrate and wash was measured at 280 nm after pH adjustment to neutrality with 1 N NaOH.
Assay for esterase activity Esterolytic activity was assayed by the spectrophotometric method t at 253 nm for a-N-benzoyl-t~-arginine ethyl ester (BAEE), at 256 nm for a-N-benzoyl-L-tyrosine ethyl ester (BTEE), and at 256 nm for p-tosyl-L-arginine methyl ester (TAME) as substrates, by the use of a Hitachi UV-spectrophotometer (Model No. 124). Trypsin and chymotrypsin were used as standard enzymes. All enzymes and synthetic substrates were products of Sigma. The assays were conducted at pH 7.5 and at 30 °C.
Preparation of Sepharose-bound porteases All enzymes (papain, ficin, pronase, and trypsin) were coupled to cyanogen bromide-activated Sepharose 4B, which was prepared by the method of Axdn et al. 2 and Porath et al. 13. The coupling reaction was allowed to proceed at 0 °C for 3 h, and then at 4 °C overnight.
396
Protein determination Protein was determined by the method of Lowry ct al. ]] except in fractionation procedures where absorbance at 280 nm was used. RESULTS
Purification procedure Extraction of GMFactivityfrom bovine brain powder (step 1). The dried powder was washed twice with ice-cold absolute ethanol (500 ml). After centrifugation at 10,000 × g for 10 min, the pellet was extracted with 200 ml of Tris-buffered saline by stirring overnight at 4 °C. The aqueous extract after centrifugation at 23,000 /, g for 30 min was the ethanol-washed extract. Ion-exchange column chromatograph)' (step 1I). The ethanol-washed extract (200 ml) was diluted with 2 vols. of 0.02 M Tris.HCl, pH 7.4, to reduce the NaCI concentration to 0.05 M. The sample, concentrated to 100 ml on Amicon PM-10 Diaflo membrane, was applied to a DEAE Sephadex A-50 column (5 :. 45 cm) preequilibrated with 0.02 M Tris.HCl buffer, pH 7.4, containing 0.05 M NaCI. The column was then eluted with a linear gradient of NaCI from 0.05 to 0.7 M in the same buffer (Fig. 1). Both morphological transforming and mitogenic activities appeared from 0.25 to 0.35 M NaCI concentration. The fractions underlined in Fig. 1 were collected and concentrated to 24 ml, and used for the next step. Bio-gel P-200 column chromatography (step Iil). One half of the DEAE-sample (12 ml) derived from 500 g wet weight of bovine brain was applied to a Bio-gel P-200 column which was pre-calibrated with molecular weight standards, including bovine serum albumin, ovalbumin, chymotrypsinogen and ribonuclease. Most of the applied EI.2~-
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.- ""
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u
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50 IOO 150 FRACTION N U M B E R ( I O m l )
200
Fig. I. E l u t i o n profile o f bovine gila m a t u r a t i o n factor f r o m D E A E Sephadex A-50. Ethanol-washed extract (2 g protein), derived f r o m 500 g (wet weight) bovine brain, was applied to a D E A E Sephadex c o l u m n (5 × 45 cm) equilibrated with 0.02 M T r i s - H C I buffer, p H 7.4, containing 0.05 M NaCI. A f t e r washing the sample-charged column with the buffer, the column was eluted with 1800 ml o f the buffer containing a linear gradient o f NaCI f r o m 0.05 to 0.7 M , at a rate o f 60 ml/h. Black bars, m o r p h o l o g i c a l transforming activity; • • , mitogenic activity; . . . . . . . , U V absorbance at 280 nm; .... . theoretical N a C I concentration. The underlined fractions were used f o r the next purification step after pooling and concentration.
397
E_ . 2 0 o co od t_.15
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o
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rn
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-
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IZ?
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,
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0 50
7'0 FRACTION
90
I10
130
NUMBER ( 6 m l )
Fig. 2. Elution profile of bovine GMF from Bio-gel P-200 column. The column (2.6 x 150 cm) was eluted by reverse flow at 12 ml/h. For explanation and symbols, see legend to Fig. 1.
brain protein came out at the high molecular weight region (larger than 40,000 Daltons), but morphological transforming activity showed up at an apparent molecular weight of 23,000 ± 3000 which was accompanied by mitogenic activity (Fig. 2.) The purification procedure and increase in specific activity are summarized in Table I. Dose-response curves Fig. 3 shows the d o s e - r e s p o n s e relationship o f G M F at various stages o f purification. A t the high c o n c e n t r a t i o n range, the a p p a r e n t mitogenic activity showed a p a r a d o x i c a l inhibition, a l t h o u g h this was o v e r c o m e u p o n further increase in G M F concentration. N o such p a r a d o x i c a l effect was seen with the m o r p h o l o g i c a l t r a n s f o r m ing activity. The lowest detectable protein c o n c e n t r a t i o n for mitogenic activity was 100 ng/ml whereas that for m o r p h o l o g i c a l activity was 300 ng/ml. Heat lability G M F s a m p l e s from D E A E Sephadex were incubated at p H 7.4 at 70 °C for various lengths o f time and i m m e d i a t e l y chilled in ice water. T h e r e a c t i o n mixture was centrifuged in the cold and the clear s u p e r n a t a n t used for bioassays (Fig. 4). A t 20 min TABLE 1 Purification of GMF from bovine brain The data presented are calculated on the basis of 500 g (wet weight) bovine brain as the starting material. For both the mitogenic and morphological activities, one unit is defined as that exhibited by 1 mg protein in the crude extract. Protein recovered (mE)
Crude extract DEAESephadex Bio-gel P-200
9312.0 50.4 1.5
Mitogenic activity
Morphological activity
Activity recovered (units)
Specific activity (units/mg)
Activity recovered (units)
Specific activity (units~rag)
9312 2974 2108
1 59 1405
9312 1361 600
1 27 400
398
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PROTEIN ( m g / W E L L ) Fig. 3. D o s e - r e s p o n s e curves of glia m a t u r a t i o n factor. A : mitogenic activity. B: morphological transf o r m i n g activity. All experiments were conducted on the s a m e batch o f glioblasts in s e c o n d a r y culture. !3 . . . . . IZI, crude bovine brain extract; 0 - - - 0 , G M F at step 11 ( D E A E Sephadex); ---C~, GMF at step 1II (Bio-gel 1:'-200).
~=4f-
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40
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60
INCUBATION TIME A T 7 0 " (rain)
Fig. 4. Heat lability o f gila m a t u r a t i o n factor. G M F (0.8 mg/ml) were incubated at pH 7.4 and 70 ~C for various lengths of time. Finally the samples incubated for 60 rain were boiled for 10 rain. 0.5 ml aliquots of s u p e r n a t a n t were taken for cell tests. _1, the incorporation o f [3H]thymidine into unheated controls. All other s y m b o l s are'explained in legend to Fig. I.
399 TABLE II
Effect of proteases on GMF GMF (2.3 mg) was mixed with 1.5 mg of an enzyme bound to Sepharose in 3 ml of Tris-buffered saline (pH 7.4) and incubated at 37 °C with continuous shaking. After incubation, the immobilized enzyme was removed by membrane filtration and the filtrate assayed for morphological and mitogenic activities. Dithiothreitol (DTT), 1 raM, was included in the incubation mixture when papain or ficin was used. Values for papain and ficin treatment are expressed as percentage of control in the presence of DT/'.
Enzyme treatment
After 1 h incubation: Papain Trypsin After 16 h incubation: Papain Trypsin Pronase Ficin
Morphological activity (% remaining)
Mitogenic activity (% remaining)
10 80
48.5 92.1
0 0 0 10
31.2 28.2 27.7 55.7
most of the mitogenic and morphological t r a n s f o r m i n g activities were lost. Boiling completely inactivated the two activities.
Protease susceptibility Table II shows the relative susceptibility of bovine G M F to the proteases. G M F was fairly resistant to trypsin b u t sensitive to p a p a i n after 1 h of incubation. A l t h o u g h the morphological t r a n s f o r m i n g activity entirely disappeared after 16 h of i n c u b a t i o n , some mitogenic activity still remained.
Esterase activity T o distinguish G M F from other growth factors such as epidermal growth /-
B
6
~-/ 8~ x
~
4 o~ ~ 2 ~ -'J40'~o ° m
3 I
0
I
I
I
I0 20 FRACTION NUMBER
1
I
0
/0~
30
Fig. 5. Isoelectric focusing of bovine GMF. Isoelectric focusing was conducted on a flat bed of 5 (w/v) Sephadex G-75 (superfine) in the presence of 10~ (w/v) sucrose and 1.2~ (v/v) Pharmalyte (pH range 4-6.5). The sample was obtained from a Bio-gel P-200 column as described in legend to Fig. 2. . . . . , pH. All other symbols are explained in legend to Fig. 1.
400 factor 3,14-~ and nerve growth factor4, ts which have esteropeptidase activity in one of the subunits, esterase activity was measured using BAEE, BTEE, and TAME as substrates. No esterase activity was detected in any of the G M F sample.
Isoelectric focusing Fig. 5 shows the results of preparative isoelectric focusing, using the narrow pH range Pharmalyte (ampholyte) from pH 4 to 6.5. Both mitogenic and morphological transforming activities focused at pH 4.75 l: 0.30. DISCUSSION We previously demonstrated that G M F exists in the fresh pig brain mainly in a large molecular weight form (200,000 Daltons) and that attempts at purification frequently results in the appearance of molecules of smaller sizes, notably one of 40,000 Daltons 5. On this basis we postulated that G M F exists in situ as a large mother molecule and that from this a family of smaller molecules of similar function can be generated as a result of either dissociation or cleavage of covalent bonds. In the present paper we demonstrated that G M F activity is found in the fresh bovine brain in association with a 23,000 molecular weight component. That this molecule is smaller that what was found in the pig brain may simply imply that native bovine G M F is more readily dissociable or degradable than native porcine GMF. Other than the difference in molecular size, G M F from the two sources show very similar physicochemical and biological properties. Thus, both are acidic proteins and both possess morphological and mitogenic activities on glioblasts which are susceptible to heat and protease inactivation. Both are devoid of esterase activity. Bovine G M F appears to be more suitable for purification than porcine G M F because the former is readily obtainable in a uniform, low molecular weight species which, upon molecular sieving, separates easily from the bulk of brain proteins which are larger in molecular size. Besides, the isolation of a smaller protein is expected to bc more amenable to chemical characterization and sequencing than a larger one. The biological activity of G M F was originally defined in terms of its ability to promote the morphological differentiation of glioblasts 9. Subsequently, we observed that a mitogenic activity always accompanies the morphological function and suggested that G M F could be both a mitogenic and maturation factor ~, as is nerve growth factor, in the present work, although both activities were co-purified, the mitogenic function was obtained at a higher state of purity than the morphological transforming function. This finding, however, does not contradict our suggestion, since the two functions could reside in two different active sites in the same or different protein subunits, and that one active site (in this case that of morphological activity) may be less stable than the other. Alternatively, it is possible that the morphological function requires cofactors which are partially eliminated during the fractionation procedures. The exact answers to these questions must await the complete purification of GMF. Before the problem is resolved it is best to assay the two activities together
401 because one method complements the other. Morphological assay seems to work better at high G M F concentrations while mitogenic assay appears to be more sensitive as the purity of the factor increases. It is noteworthy that, in terms of mitogenicity, the specific activity of G M F is comparable to that of other growth factors such as fibroblast growth factor. It is also important to point out that at the high concentration range a paradoxical inhibition is observed with mitogenic activity. Assuming that both activities are effects of the same factor, then the differential expression of mitogenic and morphological functions at different G M F concentrations may have real biological significance in the sequential or reciprocal control of growth and differentiation of glial ceils. The fact that, at low G M F concentrations, mitogenic changes can occur without subsequent morphological differentiation is evidence that cell division per se is not a sufficient cause for cell maturation. In other words, the morphological transformation brought about by G M F is not an obligatory event following and secondary to cell division. We previously purified G M F from the pig brain to a 400-fold purity 9. However, a major criticism of that procedure is that trypsin digestion was employed in the principal step and this may introduce uncertainties due to possible structural alterations. In the current work no trypsin was used. One additional advantage with the present work is that GM F is more active in bovine brain than in pig brain, so that even at the same degree of purification bovine G M F has a higher specific activity than pig GMF. The current work underlines the similarity in biological functions between pig and bovine brain GMF, and offers the latter as an altetnative source for the purification of the factor. ACKNOWLEDGEMENTS This work was supported by a Grant-in-Aid for Scientific Research (Ministry of Education) and the Naito Research Grant for 1978 from Japan; and by N I H Grant CA-27031 and NSF Grant BNS 79-00352 from the U.S.A.
REFERENCES 1 Arnon, R., Papain. In G. E. Perlman and L. Lorand (Eds.), Methods in Enzymology, Vol. 19, Academic Press, New York, 1970, pp. 226-244. 2 Ax6n,R., Porath, J. and Ernback, S., Chemicalcoupling of peptides and proteins to polysaccharides by means of cyanogen halides, Nature (Lond.), 214 (1967) 1302-1304. 3 Cohen, S., Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal, J. biol. Chem., 237 (1962) 1555-1562. 4 Greene, L. A., Shooter, E. M. and Varon, S., Enzymatic activities of mouse nerve growth factor and its subunits, Proc. nat. Acad. Sci. (Wash.), 60 (1968) 1383-1388. 5 Kato, T., Chiu, T.-C., Lim, R., Troy, S. S. and Turriff, D. E., Multiple molecular forms of glia maturation factor, Biochim. biophys. Acata (Amst.), 579 (1979) 216-227. 6 Lim, R., Li, W. K. P. and Mitsunobu, K., Morphological transformation of dissociated embryonic brain cells in the presence of brain extracts, Neurosci. Abstr., 2 (1972) 181.
402 7 Lira, R., Mitsunobu, K. and Li, W. K. P., Maturation-stimulating effect of brain extract and dibutyryl cyclic AMP on dissociated embryonic brain cells in culture, Exp. Cell Res., 79 (1973) 243 246. 8 Lim, R. and Mitsunobu, K., Brain cells in culture: morphological transformation by a protein, Science, 185 (1974) 63-66. 9 Lim, R. and Mitsunobu, K., Partial purification of a morphological transforming factor from pig brain, Biochim. biophys. Acta ( Amst.), 400 (1975) 200-207. 10 Lim, R., Turriff, D. E., Troy, S. S., Moore, B. W. and Eng, L. F., Glia maturation factor: effect on chemical differentiation of glioblasts in culture, Science, 195 (1977) 195 - 196. I 1 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. bioL Chem., 193 (1951) 265-275. 12 McLeester, R. C. and Hall, T. C., Simplification of amino acid incorporation and other assays using filter paper techniques, Analyt. Biochem., 79 (1977) 627--630. 13 Porath, J., Ax6n, R. and Ernback, S.~ Chemical coupling of proteins to agarose, Nature (Lond.), 215 (1967) 1491-1492. 14 Taylor, J. M., Cohen, S. and Mitchell, W. M., Epidermal growth factor: high and low molecular weight forms, Proc. nat. Acad. Sci. (Wash.), 67 (1970) 164 171. 15 Taylor, J. M., Mitchell, W. M. and Cohen, S., Epidermal growth factor. Physical and chemical properties, J. biol. Chem., 247 (1972) 5928-5934. 16 Taylor, J. M., Mitchell, W. M. and Cohen, S., Characterization of the binding protein for epidermal growth factor, J. biol. Chem., 249 (1974) 2188-2194. 17 Turriff, D. E. and Lim, R., Glia maturation factor increases cyclic GMP in glioblasts, Brain Research, 166 (1979) 436-441. 18 Varon, S., Nomura, J. and Shooter, E. M., The isolation of the mouse nerve growth factor protein in a high molecular weight form, Biochemistrv, 6 (1967) 2202- 2209.