216

Biochimica et Biophysica Acta, 579 (1979) 216--227

© Elsevier/North-Holland Biomedical Press

BBA 38235 MULTIPLE M O L E C U L A R FORMS OF GLIA M A T U R A T I O N F A C T O R

TAIJI KATO, TIEN-CHENG C.HIU, RAMON LIM *, SHUANG S. TROY and DAVID E. TURRIFF Brain Research Institute and Department of Surgery (Neurosurgery), University of cf~icago, Chicago, IL 60637 (U.S.A.)

(Received December 12th, 1978) Key words: Glia; Maturation factor; Multiple molecular form; (Pig brain)

Summary Glia maturation factor from the pig brain can be detected in t w o molecular forms: the high molecular weight form which is 200 000 dalton in size and the low molecular weight form which is 40 000 dalton in size, as determined by Sephadex gel filtration. The former accounts for 85% of the total biological activity extracted at physiologic pH. The proportion of the low molecular weight form increases following freeze-thawing and ion-exchange chromatography. In addition to the morphological effects, both forms possess mitogenic activity but no esteropeptidase activity. Both forms show similar enzyme susceptibility, being inactivated b y papain, ficin and pronase b u t resistant to subtilisin, thermolysin and trypsin. The high molecular weight form is more resistant to denaturation b y low pH, heating and urea than the low molecular weight form. The high molecular weight factor has an isoelectric point of 4.27 whereas the low molecular weight factor has one of 5.04.

Introduction We previously reported the presence, in the adult brain, of a protein factor which is capable of promoting the morphological and chemical differentiation of glioblasts in culture [1--4]. This factor, designated operationally as the glia maturation factor, stimulates the proliferation of glioblasts while at the same time brings a b o u t dramatic morphological changes, including the extrusion of cell processes which are multipolar, branced and interconnected between cells * To w h o m reprint requests should be addressed. Abbreviations: BAEE, ~-N-benzoyl-L-arginine e t h y l ester; TAME, p-tosyl-L-arginine m e t h y l ester; BTEE, ~-N-benzoyl-L-tyrosine ethyl ester; BAPNA, ~-N-benzoy]-DL-arginlne-p-nitroanilide,

217 [5]. The processes show strong tugging activity whereas the cell bodies undergo rhythmic contractions characteristic of glial cells. The factor stimulates the formation of gliofilaments and other cellular components [6]. The metabolic events that follow the addition of the factor to cultured glioblasts include: the stimulation of DNA, R N A and protein synthesis; the increase in cyclic GMP, cyclic AMP, S-100 protein and glycerophosphate dehydrogenase [7]. In this report, we present evidence for the presence of t w o molecular forms of glia maturation factor: the high molecular weight form and the low molecular weight form. The objective of this work is three-fold: (1) to compare the properties of the t w o molecular forms; (2) to distinguish glia maturation factor from other growth factors on physico-chemical grounds; (3) to obtain sufficient physical and chemical data on the t w o forms of the factor so as to facilitate their complete purification. Materials and Methods

Source of glia maturation factor Pig brains (cerebrum) were used exclusively. Fresh brains were obtained from a local slaughter house within 45 min after killing and transported in ice to the laboratory. As specified in the Results section, the brains were homogenized either fresh or after freezing for at least 48 h. All subsequent operations were carried o u t at 4 ° C. Preparations designated as 'brain extract' were obtained as follows. Pig brains were homogenized in 'Tris-buffered saline' (0.15 M NaC1 containing 0.02 M Tris-HC1, pH 7.4) with a Waring blender, at 23 000 rev./min for 1 min, to make a 25% (w/v) homogenate. The supernatant fraction after spinning the homogenate at 23 000 X g for 16 h in a Spinco No. 15 rotor was the 'brain extract'. Preparations designated as 'ethanol-washed extract' were obtained in the following manner. First, frozen pig brains were used to prepare the brain extract, which was subsequently freeze-thawed three times to encourage the formation of the low molecular weight form. The sample was then dialyzed against several changes of water and lyophilized. The dried powder was washed twice with ice-cold absolute ethanol, each with one ml of ethanol per g wet weight of the original brain tissue. Ethanol was eliminated b y centrifugation at 23 000 X g for 10 min. The activity was subsequently extracted from the pellet with Tris-buffered saline, using 1 ml per 4 g wet weight of the original brain tissue. The aqueous extract after centrifugation was the 'ethanol-washed extract'.

Assay for morphological transformation All the bioassays were conducted on confluent cultures of a homogeneous population of glioblasts, which were obtained from 17-day Sprague-Dawley rat fetuses as follows. The fetuses were aseptically removed from the uteri and chilled at 4 ° C. With the aid of a dissecting microscope, the meningeal layer and surface blood vessels were carefully removed, and the cerebra and cerebella were cut into 1 mm 3 pieces. The cut pieces were washed 15 times with Tyrode solution and stored in a Tyrode-fetal calf serum mixture (1 : 1) at 4°C until all the embryonic brains: were dissected. The dissected tissues were divided into

218

two pools and each pool was treated stepwise as follows: (a) rinsing twice with 5 ml of calcium magnesium-free Tyrode; (b) incubation with 5 ml of calcium magnesium-free Tyrode at 37°C for 15 min; (c) incubation with 5 ml of 0.25% trypsin in GIBCO solution A at 37°C for 30 min. After rinsing three times with a medium consisting of F-10 nutrient containing 20% fetal calf serum, the cells in each pool were dissociated in 2.5 ml of the same medium by trituration with a Pasteur pipet. In the event that the cells failed to disperse with ease,_a 10min incubation with DNAase {0.05 mg/ml Tyrode) usually helped. The two cell pools were mixed and the cells were immediately distributed with a Pasteur pipet into No. 3012 Falcon plastic culture flasks (surface area 25 cm 2) at a ratio of 1 brain per flask. The medium for seeding the cells consisted of 4 ml F-10 and 1 ml fetal calf serum. Penicillin and streptomycin were used at 50 units/ml and 100 pg/ml, respectively, in all media. After incubating the cells at 37°C for two days without disturbance, the medium was changed to 4 ml F-10 supplemented with 10% (v/v) fetal calf serum ('standard medium'). The brain cells became confluent after a week, containing equal numbers o f neuroblasts and glioblasts. The cells in each flask were dislodged with 0.25% trypsin solution (Gibco) by a 30-min incubation, The cells were then collected by low speed centrifugation and subcultured in the 'standard medium' in Lux No. 5218 multiwell plastic trays (Lux Scientific Corp., 1157 Tourmaline Drive, Newbury Park, CA 91320) at a ratio of one Falcon flask per two Lux trays. Each tray contains 8 wells, each of which has a dimension of 3 × 3.5 cm and takes 2 to 2.5 ml medium. The tray cultures were incubated at 37°C in 5% CO2 in air. 20 h later the medium was changed to eliminate the neuroblasts, leaving behind the glioblasts which became confluent in about 4 days. At this time the cells were more than 95% homogeneous with respect to glioblasts and were ready for the morphological assay. The assay was started by replacing the 'standard medium' with 2.5 ml of 'experimental medium' which consisted of 0.5 ml of a test solution containing glia maturation factor and 2 ml of F-10 supplemented with 5% fetal calf serum and antibiotics. The test solutions were passed through 0.22 pm Millipore filters before use. 24 h later the medium was replaced by another 2.5 ml of fresh 'experimental m e d i u m ' in order to restimulate the cells with the factor. After another 16 h the cells were scored for morphological transformation by a person not knowing the identity of the test solution added. 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 per well. All data in this paper are average values from at least two wells. Control wells were exposed to 'experimental m e d i u m ' containing no factor. The scoring of cells after second stimulation rather than the first stimulation as we did before yields more consistent results, even though m a x i m u m response can usually be seen 20 h after a single stimulation.

Assay for mitogenic activity DNA synthesis was determined concurrently with morphological assay (see above), the two procedures being carried out on the same glioblast culture. During restimulation of the cells by glia maturation factor, 0.25 gCi of [methyl-

219 3H]thymidine (New England Nuclear; spec. act., 50 Ci/mmol) was added to each well in the Lux plastic tray containing 2.5 ml (total) of culture medium. After 16 h of incorporation and subequent to scoring the cells for morphological response, the medium was removed and the cell layer on the b o t t o m of the well was rinsed twice with Tris-buffered saline. The cells were then detached from the well b y incubating for 30 min (37°C) with 1.0 ml of Tris-buffered saline containing 0.5 mg of trypsin (Sigma Chemical Co. ) (Trypsin treatment at this stage did not affect the incorporation o f thymidine into DNA.) The cells were pelleted in an E p p e n d o r f plastic microtest t u b e (1.5 ml size) by centrifugation at 2000 rev./min for 10 min in the cold. The pellet was solubilized b y the addition of 50 pl of 5% (w/v) sodium dodecyl sulfate followed by sonication for t w o seconds with a Branson sonifier (model W 185D) equipped with a microtip set at 50 W power output. An aliquot (20 pl) of the solubilized cell sample was deposited on a 1.9 cm (diameter) Whatmann No. 3 MM filter paper disc (supplied b y McLeester Research Equip., Inc., 1202 Pontiac Trail, Madison, WI). The paper disc was previously acidified with 40 pl of 20% (w/v) trichloroacetic acid and air dried. The paper disc containing the cell sample was air dried once more and then washed b y immersing the disc in a cold 5% trichloroacetic acid for 10 min. The disc was rinsed twice in cold 95% ethanol, 10 min each time, and was air dried. Thereafter the disc was immersed in a toluene-based scintillation fluid containing 10 g/liter of PPO and 100 mg/liter of POPOP and counted in a liquid scintillation counter. The results reported are counts per min per filter paper disc, and are averaged values from duplicate culture wells. The use of filter paper disc as a support for acid precipitation is a modification of the m e t h o d of McLeester [8].

Column chromatography DEAE Sephadex A-50 p o w d e r (10 g) was suspended in 2 liters of water and the pH brought to above 13 b y adding 100 ml of 4 N NaOH. After 30 min, the suspension was washed in a Buchner funnel with more than 10 liters of water until the pH was close to neutrality. The gel was resuspended in 0.02 M TrisHC1 buffer, pH 7.4, and packed into a column. For regeneration, the material was washed in a beaker with 0.2 N NaOH followed by large amounts of water before repacking. Sephadex G-150 was handled as specified b y the manufacturer. All columns were run at 4 ° C.

Preparation of Sepharose-bound proteases 50 ml of settled Sepharose 4B in water was activated with 100 ml of cyanogen bromide (50 mg/ml water) as the pH was held at 11.0 to 11.3 by the continuous addition of 4 N NaOH for 8 min. After thorough washing with 500 ml of cold 0.1 N NaHCO3 buffer, pH 9.0, in a Buchner funnel [9,10], the solid material was added to a solution containing 10 mg of an enzyme in 20 ml of the bicarbonate buffer. The coupling reaction was allowed to proceed at 0°C for 3 h with constant, gentle, mechanical stirring and then stored at 4°C overnight. On the next day the suspension was packed into a column and washed with several bed volumes of Tris-buffered saline until the absorbance of the effluent at 280 nm was less than 0.04. The a m o u n t of enzyme b o u n d to Sepharose was estimated from the total protein absorbance in the effluent.

220 Usually over 90% of the enzyme protein was coupled. When not in use, the gel was stored at 4°C in Tris-buffered saline containing 0.01% sodium azide. Such gels have been used repeatedly over a period of months without apparent loss of activity.

Assay for esteropeptidase activity Esterolytic activity was assayed by the potentiometric method [11] at pH 7.5 and 8.5 at 25°C, using an autotitrator (Radiometer model SBR/SBU/TTT1) with 0.05 M NaOH under flowing N2 gas. The substrates used were a-N-benzoylL-arginine ethyl ester (BAEE), a-N-benzoyl-L-tyrosine ethyl ester (BTEE) and p-tosyl-L-arginine methyl ester (TAME), all at 10 mM concentration. Trypsin was used as a standard enzyme for BAEE and TAME, whereas chymotrypsin and papain were used as standard enzymes for BTEE and BAEE, respectively. All the enzymes and synthetic substrates were products of Sigma Chemical Co. In addition, the assays were repeated by the spectrophotometric method [12] using BAEE (measured at 253 nm) and BTEE (measured at 256 nm), all at 1 mM concentration, at pH 7.5 and 8.5 at 30°C. Amidase activity was measured spectrophotometrically [12] at 410 nm using 1 mM a-N-benzoyl-DLarginine-p-nitroanilide (BAPNA) as substrate and trypsin and papain as the standard enzymes; the assay was conducted at pH 7.5 and at 30°C.

Protein determinations Protein was determined by the method of Lowry et al. [13] except in fractionation procedures where absorbance at 280 nm was used. Results

Demonstration of two molecular forms with Sephadex G-150 When a brain extract prepared from fresh pig brains is fractionated on a Sephadex G-150 column, about 80--90% of the total morphological transforming activity emerges near the void volume, the remainder being eluted in a later peak (Fig. la). However, a relative increase in the second peak can be observed if the brain extract is derived from frozen brains (Fig. lb). When frozen brains are processed up to the step of 'ethanol-washed extract' (see 'Materials and Methods'), a further increase in the second peak occurs (Fig. lc). The first activity peak has an apparent molecular weight of 200 000, and is designated as the high molecular weight glia maturation factor; the second peak has an apparent molecular weight of 40 000, and is designated as the low molecular weight glia maturation factor. Unless otherwise specified, the two molecular forms used for the subsequent studies in this paper were obtained from the Sephadex G-150 peaks upon fractionation of the 'ethanol-washed extract', as in Fig. lc.

Behavior in anion-exchange chromatography When the low molecular form is applied to a DEAE-Sephadex column (Fig. 3a), the major morphological transforming activity emerges as a single peak at 0.2 M NaC1 concentration which is accompanied b y mitogenic activity. When the high molecular form is applied to the same column (Fig. 3b), the morpho-

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Fig. 1. E l u t i o n profiles o f g]ia mattLration factor f r o m Sephadex G-150 column. Samples applied: (a) 10 m l ( 3 0 0 m g p r o t e i n ) o f ' b r a i n e x t r a c t ' o b t a i n e d f r o m 17.5 g ( w e t w e i g h t ) fresh b r a i n ; ( b ) 1 0 m l ( 3 0 0 rag

protein) o f ' b r a i n e x t r a c t ' o b t a i n e d f r o m 1 7 . 5 g f r o z e n h r a i n ; (c) 1 0 m l ( 5 0 m g p r o t e i n ) o f ' e t h a n o l - w a s h e d e x t r a c t ' o b t a i n e d f r o m 1 7 , 5 g f r o z e n b r a i n . T h e c o l u m n , 2.6 × 2 0 0 c m , w a s p r e - e q u i l i b r a t e d a n d e l u t c d w i t h T r i s - b u f f e r e d saline b y u p w a r d f l o w . 3-ml f r a c t i o n s w e r e c o l l e c t e d at a f l o w r a t e o f 14 m l / h . o, p r o t e i n a b s o r b a n c e . Bars, m o r p h o l o g i c a l t r a n s f o r m i n g a c t i v i t y f r o m 0 . 5 m l a l i q u o t s of the f r a c t i o n s .

logical transforming activity shows up in two peaks, one at 0.2 M NaCl, the other at 0.5 M NaC1, the second being the predominant peak, both containing mitogenic activity. Upon rechromatography on Sephadex G-150, the apparent molecular weight of the peak at 0.2 M NaC1 (whether originated from the low or the high form) is found to be 40 000, corresponding to the low molecular weight factor; whereas that of the peak at 0.5 M NaC1 is 200 000, corresponding to the high molecular weight factor (results not shown).

Isoelectric points Fig. 3 shows the results of isoelectric focusing for the two molecular forms. When the low molecular weight form (Fig. 3a) is focused, a single peak of morphological transforming activity appears in the region of pH 5. When the high molecular weight form (Fig. 3b) is focused, a single peak with the activity appears in the region of pH 4 (although in this instance a shoulder can be observed in the region of pH 5). Both peaks also contain mitogenic activity. The average isoelectric point of the former from seven determinations is 5.04 + 0.17 (S.D.); that of the latter form three determinations is 4.27 + 0.10 (S.D.). Stability toward denaturation The effects of pH, temperature and urea on the two forms of glia maturation factor were compared. The samples were kept at various pH values at 4°C for 16 h. After readjusting the pH back to 7.4 and eliminating any turbidity by centrifugation, the samples were assayed for biological activity. Under these

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Fig. 2. E l u t i o n p r o f i l e s o f t h e . t w o f o r m s o f glia m a t u r a t i o n f a c t o r f r o m D E A E - S e p h a d e x . (a) L o w m o l e c u l a r w e i g h t gila m a t u r a t i o n f a c t o r ( L M W - G M F ) ; (b) high m o l e c u l a r w e i g h t glia m a t u r a t i o n f a c t o r (HMWG M F ) . E t h a n o l - w a s h e d e x t r a c t (2 g p r o t e i n ) , d e r i v e d f r o m 7 0 0 g ( w e t w e i g h t ) f r o z e n pig b r a i n , w a s first ~ a c t i o n a t e d o n a large S e p h a d e x G - 1 5 0 c o l u m n ( 1 0 × 1 0 0 c m ) t o i s o l a t e t h e t w o f o r m s of glla m a t u r a t i o n f a c t o r w h i c h w e r e t h e n s e p a r a t e l y a p p l i e d t o a D E A E - S e p h a d e x c o l u m n (4 × 4 0 c m ) . T h e s a m p l e s a p p l i e d t o t h e c o l u m n w e r e first c o n c e n t r a t e d f r o m 1.5 1 t o 50 m l b y f i l t r a t i o n w i t h A m i c o n PM-10 Diaflo m e m b r a n e a n d d i a l y z e d a g a i n s t 0 . 0 2 M Tris-HCI, p H 7.4. T h e D E A E c o l u m n w a s p r e - e q u i l i b r a t e d with the same buffer. After washing the sample-charged column with the buffer, the c o l u m n was eluted w i t h 8 0 0 m l o f t h e b u f f e r c o n t a i n i n g a linear g r a d i e n t of NaCI f r o m 0 t o 0.7 M, a t a r a t e of 20 m l / h . B l a c k b a r s , m o r p h o l o g i c a l t r a n s f o r m i n g a c t i v i t y ; filled circles, m i t o g e n i c a c t i v i t y ; b o t h f r o m 0 . 5 m l a l i q u o t s o f t h e f r a c t i o n s , o, p r o t e i n a b s o r b a n c e ; . . . . . . , NaCI c o n c e n t r a t i o n .

conditions the low molecular weight form starts to lose its morphological effect at pH 4 whereas the high molecular weight form starts to lose its activity at pH 2. On the alkaline side, both begin to be inactivated at pH 12. For temperature effect we incubated the samples at pH 7.4 at 80°C for various lengths of time, and tested them after clearing up the turbidity by centrifugation. We found that there is a progressive decrease in the morphological effect of the low molecular weight form starting from 10 min on. At 60 min only 10% of the original activity remains. For the high molecular weight form a slower rate of inactivation is noticed starting from 20 min, resulting in a 50% loss at 60 min. If boiled for 10 min, both molecular forms are completely inactivated. For urea denaturation, the samples were exposed to the agent at pH 7.4 and at 4°C for 16 h. The recovery of activity was assessed after elimination of urea by dilution and reconcentration with Diaflo PM-10 membrane. Our results show that the low molecular weight form loses 50% of its morphological effect at 4 M urea

223

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Fig. 3. I s o e l e c t r i c f o c u s i n g o f t h e t w o f o r m s o f glLa m a t u r a t i o n f a c t o r . (a) L o w m o l e c u l a r w e i g h t gila m a t u r a t i o n f a c t o r ( L M W - G M F ) ; (b) high m o l e c u l a r w e i g h t gila m a t u r a t i o n f a c t o r ( H M W - G M F ) . T h e t w o m o l e c u l a r f o r m s w e r e s e p a r a t e l y f o c u s e d o n a fiat b e d of 5% S e p h a d e x G-75 ( s u p e r f i n e ) in t h e p r e s e n c e o f 10% s u c r o s e a n d 1.2% A m p h o l l n e ( p H r a n g e 3 . 5 t o 1 0 ) i n a n L K B M u l t i p h o r m o d e l 2 1 1 7 . T h e s a m p l e s u s e d w e r e o b t a i n e d f r o m a S e p h a d e x 6 - 1 5 0 c o l u m n as d e s c r i b e d in l e g e n d t o Fig. 2 a n d c o n c e n t r a t e d f r o m 3 0 0 m l t o 1 0 m l w i t h P M - 1 0 Diaflo m e m b r a n e . T h e s a m p l e s w e r e e v e n l y a p p l i e d t o t h e gel b e d a n d f o c u s e d a t 4 ° C f o r 16 h at a c o n s t a n t p o w e r o f 10 W. T h e gel b e d w a s t h e n p a r t i t i o n e d i n t o 5 - r a m strips w i t h a f r a c t i o n a t i n g grid. E a c h f r a c t i o n w a s d i l u t e d w i t h 3 m l w a t e r a n d t h e p H m e a s u r e d . T h e gel susp e n s i o n w a s f i l t e r e d a n d t h e r e s i d u a l gel w a s h e d w i t h 5 m l o f 0.2 M Tris-HC1, p H 7.4, T h e f i l t r a t e a n d w a s h w e r e c o m b i n e d a n d a d j u s t e d to n e u t r a l i t y w i t h 1 N N a O H or HCI b e f o r e use f o r p r o t e i n d e t e r m i n a t i o n a n d cell t e s t . T h e p r e s e n c e o f A m p h o l i n e in t h e t e s t s a m p l e s did n o t a f f e c t t h e cells. - . . . . . , p H . All o t h e r s y m b o l s are e x p l a i n e d in l e g e n d t o Fig. 2.

and 75% at 8 M concentration; the corresponding losses for the high molecular weight form are 0% and 25%. For all these studies the mitogenic activity roughly parallels the morphological activity. Susceptibility to proteolytic e n z y m e s

Table I shows that the low molecular weight form, in general, is more susceptible to proteolytic degradation than the high molecular weight form. However, they both show the same relative susceptibility with respect to the enzymes, both being most sensitive to papain and ficin, fairly sensitive to pronase, and rather resistant to subtilisin, thermolysin and trypsin. Papain and ficin are thioproteases. Although both are nonspecific, there seems to be a preference for acidic residues. Both subtilisin and trypsin are serine enzymes, and the selectivity of trypsin for lysine and arginine is well known. Thus, the relative susceptibility to the proteases may be partially explained b y the fact

224

TABLE I E F F E C T OF P R O T E A S E S O N G L I A M A T U R A T I O N F A C T O R T h e l o w m o l e c u l a r w e i g h t ( 2 . 0 m g ) or t h e high m o l e c u l a r w e i g h t f o r m ( 1 . 5 rag) was m i x e d w i t h 3 m g of a n e n z y m e b o u n d t o S e p h a r o s e , in T r i s - b u f f e r e d saline ( p H 7.4) in a t o t a l v o l u m e o f 5 m l , a n d i n c u b a t e d at 37°C w i t h c o n t i n u o u s stirring. A f t e r i n c u b a t i o n , t h e i m m o b i l i z e d e n z y m e was r e m o v e d b y c e n t r i f u g a t i o n , and the s u p e r n a t a n t f r a c t i o n a s s a y e d for m o r p h o l o g i c a l a n d m i t o g e n i c activities. T h e r e m a i n i n g activities are p r e s e n t e d as p e r c e n t a g e o f c o n t r o l s w i t h o u t e n z y m e . C y s t e i n e , 5 m M , was i n c l u d e d in the i n c u b a t i o n m i x t u r e w h e n p a p a l n or fiein was u s e d ; c y s t e i n e itself did n o t a f f e c t the cell tests. Enzyme treatment

Low molecular weight form

High m o l e c u l a r w e i g h t f o r m

Morphological activity (% r e m a i n i n g )

Mitogenic activity (% r e m a i n i n g )

Morphological activity (% r e m a i n i n g )

Mitogenic activity (% r e m a i n i n g )

After 1 h incubation Papain Ficin Pronase Sub tilisin Thermolysin Trypsin

5 10 55 70 70 75

20 45 55 70 70 95

10 20 75 75 75 95

20 15 50 65 100 70

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0 25

30 45

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that the two forms of glia maturation factor are acidic proteins, containing more acidic than basic residues. On the other hand, trypsin treatment does not enhance the activity of either.

A bsence of esteropeptidase activity The two molecular forms were each assayed for esteropeptidase activity. Neither esterase nor amidase was detectable in either form after six separate determinations. Based on the sensitivity of the machines used, the specific activity of the enzymes in glia maturation factor, if any, is less than 1/50 000 that of the standard enzymes. The overall results of the current study are summarized in Table II.

T A B L E II C O M P A R I S O N OF TWO F O R M S O F G L I A M A T U R A T I O N F A C T O R

Molecular weight Isoelectric point p H stability H e a t stability Stability in urea S u s c e p t i b i l i t y to p r o t e a s e s Esteropeptidase activity

Low molecular weight form

High molecular weight form

40 000 5.04 p H 5--11 Less stable Less stable More s u s c e p t i b l e None

200 000 4.27 pH 3--11 More stable More stable Less s u s c e p t i b l e None

225 Discussion In this article we demonstrated the presence of two molecular forms of glia maturation factor from pig brain, one with a molecular weight of a b o u t 200 000 and an isoelectric point of a b o u t pH 4, the other with a molecular weight of a b o u t 40 000 and an isoelectric point of about pH 5. The former is the predominant species at physiologic pH and apparently is the thermodynamically more stable form, as evidenced by its resistance to various denaturing procedures. The data also indicate the conversion of the high molecular weight form to the low form, although the reverse process has not been demonstrated. In considering the relationship between the t w o forms, three possibilities exist. First, the high molecular form could be the native molecule or molecular complex, and the low molecular form could be an enzymatic degradation p r o d u c t during isolation. Secondly, the formation of the low molecular form could be the result of dissociation where the reverse process, for one reason or another, has a slow equilibrium. This type of apparent irreversibility has been reported before [14]. Thirdly, the low molecular weight form could be the native molecule and the high molecular form could be an artifact of isolation produced by the aggregation of the former. However, the fact that in a fresh homogenate almost all the activity resides in the high .molecular form seems to speak against this. It is not u n c o m m o n for enzymes or other biologically active proteins to be detected in multiple forms and it is not always easy to decide which of these forms is the biologically native molecule. One example is the recent observation [15--17] that the protein nerve growth factor can be detected in at least six different forms in the homogenate of mouse submandibular gland, and that a newly detected species, rather than the well-known '7 S' nerve growth factor, m a y indeed be the naturally occurring protein in the salivary gland. It is interesting to note that the morphological effect of glia maturation factor is always accompanied by a mitogenic activity, suggesting that it qualifies as one of the growth factors. However, it is not clear as to whether the two biological functions are expressions of the same polypeptide chain. A number of growth factors have been identified b y others. Both nerve growth factor [18--20] and epidermal growth factor [21--24] are complex protein molecules possessing a trypsin-like esteropeptidase activity. Although glia maturation factor is also a complex protein, it is devoid of esteropeptidase activity either in the large form or in the small molecular form. Besides, the active subunit of nerve growth factor responsible for neuritic outgrowth is a basic polypeptide [19] whereas both forms of glia maturation factor are acidic proteins. The mitogenic activity of epidermal growth factor differs from that of glia maturation factor in being completely heat-resistant, even with boiling [25]. Epidermal growth factor stimulates cell proliferation in fibroblasts [26-28] and glial cells [29] as well as in epidermal cells. On the other hand, nerve growth factor has no observable biological effect on glial cells [ 5 ]. Fibroblast growth factor [30--32] is a p o t e n t mitogenic factor which acts n o t only on fibroblasts b u t also on other cell types including giia [29]. It has no esteropeptidase activity. It is a small basic protein (13 000 daltons) and has n o t been reported to exist in any larger form. The factor is very sensitive to trypsin and heat inactivation.

226

The 'platelet factor' is a group of heterogeneous polypeptides released by platelets during the coagulation process [29,33,34]. It has a mitogenic effect on glia. However, it differs from glia maturation factor in being a serum factor, and in being trypsin-labile and heat-stable. Somatomedin B is a group of polypeptides found in human plasma which can stimulate glial proliferation [29, 35]. They all differ from glia maturation factor in having a molecular size of only 5000 daltons. Furthermore, we have failed to induce glial differentiation b y using sera or plasma from different sources and at different concentrations [36]. Human osteosarcoma cells secret a factor which can stimulate glial proliferation [29]. The factor is trypsin-sensitive and heat-stable. It has recently been reported [37] that sympathetic neurons from chick embryos contain a mitogenic factor which can act on homologous non-neuronal cells, presumably glia. The chemical nature of this factor is n o t known. Thus, glial cells are responsive to a number of growth and/or maturation factors, many of which appear to differ from glia maturation factor in physicochemical properties. Whether all of these are biologically significant in vivo and how the various control mechanisms interact remain to be clarified. On the other hand, their c o m m o n mitogenic effect on glial cells may suggest the possibility that some of these factors may be interrelated in a manner not currently apparent to us.

Acknowledgements This work was supported by U.S. Public Health Service grants No. NS-09228, NS-14316, NS-07376 and CA-19266. Drs. Kato, Chiu, Troy and Turriff (NIH postdoctoral fellowship No. NS-05017) were Research Associates in Dr. Lim's laboratory. We thank Drs. John H. Law and Ferenc J. K6zdy for the use of their autotitrator. The technical assistance of Mr. Morgan Jenkins and Mr. William Carron is gratefully acknowledged. References 1 2 3 4 5 6 7 6 9 10 11 12 13 14 15 16

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Multiple molecular forms of glia maturation factor.

216 Biochimica et Biophysica Acta, 579 (1979) 216--227 © Elsevier/North-Holland Biomedical Press BBA 38235 MULTIPLE M O L E C U L A R FORMS OF GLIA...
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