purification and characterization of glia maturation factor β : a growth regulator for neurons and...

Purification and Characterization of Glia Maturation Factor β: A Growth Regulator forNeurons and GliaAuthor(s): Ramon Lim, Joyce F. Miller and Asgar ZaheerSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 86, No. 10 (May 15, 1989), pp. 3901-3905Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/33984 .

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Proc. Natl. Acad. Sci. USA Vol. 86, pp. 3901-3905, May 1989 Neurobiology

Purification and characterization of glia maturation factor 13: A growth regulator for neurons and glia RAMON LIM*, JOYCE F. MILLER, AND ASGAR ZAHEER

Department of Neurology, Division of Neurochemistry and Neurobiology, University of Iowa College of Medicine and Veterans Administration Medical Center, Iowa City, IA 52242

Communicated by Hewson Swift, February 17, 1989

ABSTRACT A protein has been isolated from bovine brains by using a modification of the procedure used to purify glia maturation factor. The method consists of ammonium sulfate precipitation, chromatography with DEAE-Sephacel, Sephadex G-75, and hydroxylapatite columns, passage through a heparin-Sepharose column, and fmally fractionation by reverse-phase HPLC with a C4 column. The isolated protein reacts strongly with the mouse monoclonal antibody G2-09 and has a molecular weight of =47,000 and an isoelectric point of pH 4.9. The N terminus is blocked, but tryptic digestion releases 28 peptides, 8 of which have been sequenced. The total knQwn residues add up to more than two-thirds of the entire 140-residue protein, estimated from amino acid composition, and show no sequence homology with any known protein. Reversible thermal renaturation greatly enhances its biological activity. The purified protein stimulates differentiation of normal neurons as well as glial cells. It inhibits the proliferation of the N-18 neuroblastoma lihne and the C6 glioma line while promoting their phenotypic expression. We designate this protein glia maturation factor i8.

The trophic function of the nervous system on peripheral organs was detected in an in vivo system as early as 1823 (1). The in vitro counterpart of this experiment was conducted in 1939 (2-4), showing the mitogenic effect of brain homoge- nates on cultured fibroblasts. However, it was not until 1972t that an autoregulatory role of brain-derived growth factors on brain cells was first demonstrated in this laboratory (5, 6). This factor was named glia maturation factor (GMF), based on the bioassay system in which the activity was first observed. Since then this laboratory has engaged in an intensive search for the molecule responsible for this important function. The effort culminated in the isolation of a brain protein with a unique amino acid sequence documented below.

MATERIALS AND METHODS Preliminary Purification of GMF-,f. The published proce-

dure (7, 8) through the Sephadex G-75 step was followed with slight modifications. Briefly, four beef brains [1.0 kg (total wet weight)] were homogenized and centrifuged to obtain the crude extract. The ammonium sulfate precipitate between 45% and 70% saturation was dissolved in 100 ml of water and dialyzed for two 6-hr periods against 10 liters of water. The sample was adjusted to contain 0.02 M Tris HCl (pH 7.45) and applied to a DEAE-Sephacel column (2.5 x 37 cm). After eluting with 1.25 liters of a linear gradient of 0-0.3 M NaCl in the same buffer at 50 ml/hr, the fractions that showed mitogenic and morphologic activities on astrocytes were pooled (500 ml) and concentrated to 50 ml by Amicon PM10 filtration. The sample was applied to a Sephadex G-75

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column (5 x 90 cm) and eluted with 0.15 M NaCl containing the above buffer at 40 ml/hr. The fractions active on astrocytes were pooled (500 ml) and used for the final steps of purification (see Results).

Bioassay and Cell Testing. Bioassay of GMF activity during purification was performed on cultured astrocytes as described (7). Intact dorsal root ganglia (DRGs) were obtained from 12-day chicken embryos and cultured on a collagen surface in Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g of glucose per liter, 1% glutamine, and 10% (vol/vol) heat-inactivated fetal calf serum. Dissociated DRG neurons were obtained from 20-day rat fetuses and cultured on a poly(D-lysine)-coated surface (12-well plastic plates) in the above nutrient with supplements. Mouse neuroblastoma line clone N-18 (from M. Nirenberg, National Institutes of Health) was seeded in 24-well plastic plates and tested in RPMI 1640 medium containing 10% (vol/vol) heat-inactivated fetal calf serum. Rat glioma line clone C6 (American Type Culture Collection) was seeded in 8-well plastic plates and tested in F12/DMEM (1:1) containing 5% (vol/vol) fetal calf serum.

Production of Antibodies and Immunoassay. The mouse monoclonal antibody G2-09 (an IgG2b) against bovine GMF was produced as described (8). Rabbit polyclonal antibodies were obtained by immunizing rabbits with 200 ,ug of GMF-pB in 0.5 ml of complete Freund's adjuvant by injections into eight hind toe pads. Three weeks later, each rabbit was given a booster injection with 600 ,tg of GMF-f3 in 2 ml of incom- plete Freund's adjuvant by intradermal injections at four sites on the back. Antisera were collected after another week. The serum designated 88-02 was used in the current experiment. ELISA were camed out by using a peroxidase-labeled sec- ond antibody and 2,2'-azinobis(3-ethylbenzothiazoline- 6-sulfonic acid), diammonium salt, (ABTS) as substrate (9); results were read at 415 nm from 96-well microtiter plates by using a Bio-Tek microplate reader.

Amino Acid Composition. Protein samples were hydro- lyzed at 110?C in 6 M HCl under argon for 24, 48, and 72 hr and derivatized with phenylisothiocyanate. The phenylthio- carbamyl amino acids were separated with HPLC, using a Waters Pico-Tag amino acid analyzer.

Peptide Mapping and Sequence Determination. GMF-pB was extensively reduced and alkylated (10) and subsequently digested with trypsin using an enzyme/substrate ratio of 1:100 (wt/wt). The resulting tryptic peptides were separated on a C8 reverse-phase HPLC column (2.1 mm x 10 cm, Applied Biosystems RP-300) at a flow rate of 200 ,ul/min, using a linear gradient of 2-80% (vol/vol) acetonitrile con-

Abbreviations: DRG, dorsal root ganglion; FGF, fibroblast growth factor; GMF, glia maturation factor; F3CCOOH, trifluoroacetic acid. *To whom correspondence and reprint requests should be addressed at: Department of Neurology, University of Iowa College of Med- icine, Iowa City, IA 52242.

tLim, R., Li, W. K. P. & Mitsunobu, K. (1972) Abstracts of the SecondAnnual Meeting of the SocietyforNeuroscience, Oct. 8-11, 1972, Houston, TX, p. 181.

3901



3902 Neurobiology: Lim et al. Proc. Natl. Acad. Sci. USA 86 (1989)

.12 : 0.6

E .08 0.4 0.8

0 E B

coG 1.Hdoyaaieclm hoaorpy nytepo

U - ~~~~~~~~~~~~~0 b.04 0.2 0.4 4ia

taining 0 to o -~~~~~~~~~~~~~

20 40 60

FRACTION NUMBER (Smi)

FIG. 1. Hydroxylapatite column chromatography. Only the profile of the phosphate gradient elution is shown. Fractions of 5 ml were collected. Protein concentration was measured at 280 nm. ELISA determination of GMF-io immunoreactivity with monoclonal antibody G2-09 is expressed as absorbance at 415 nm. Vertical bars represent thymidine incorporation when tested on astrocytes. Dou- ble-headed arrow indicates fractions pooled.

tanming 0.1% trifluoroacetic acid (F3CCOOH) over 45 min. The amino acid sequence of the peptides were determined with an Applied Biosystems 477 gas-phase microsequencer with on-line analysis of phenyithiohydantoin amino acid derivatives.

RESULTS

Final Purification of GMF-a. The partially purified GMF sample (500 ml) from the Sephadex 6-75 step was applied to a hydroxylapatite (Bio-Gel HT) column having a gel volume of 50 ml (5 cm diameter x 2.5 cm height). After washing the charged column with 1 column volume of 0.15 M NaCl in 0.02 M Tris'HCI (pH 7.45), the bulk of the protein was eliminated with 150 ml of 0.05 M potassium phosphate (pH 7.45). This was followed by a 390-ml gradient of 0.05 M-0.3 M potassium phosphate (pH 7.45) at a flow rate of 40 ml/hr (Fig. 1). Fractions containing mitogenic (and morphologic) activity on astrocytes were pooled (120 ml).

At this stage the sample was divided into three portions. A 40-ml aliquot was passed through a 5-ml heparin-Sepharose column to eliminate the contaminating fibroblast growth factors (FGFs). Flow rate was set at 20 m/lhr. The heparin column was regenerated with 20 ml of 2 M NaCl in 0.01 M TrisHCi (pH 7.0) and, before use, equilibrated with 20 ml of 0.1 M potassium phosphate (pH 7.45).

The heparin-treated sample (40 ml) was filtered through a Millipore Millex GV (0.22-gim pore size) and adjusted to contain 0.1% F3CCOOH. The sample was further divided

A B 0.8 80s

E p .

i - ~~~~~~~~~~~~~~~~~E ;0.4 - - 40S 0

* --- - S~~~~~~~~~~~~~~~~0 4 l

0 0 E

4 8 12 16 4 8a 2 1

FRACTION NUMBER (1.5mI)

FIG. lr%l 2. H PLC - fractionation_ of GM-1 by- actoitil grdin

Table 1. Purification steps and protein recovery

Protein Step recovered, mg

Crude extract 13,900 (NH4)2SO4 fraction 2,900 DEAE-Sephacel 627 Sephadex G-75 49 Hydroxylapatite 2.3 Heparin-Sepharose 1.3 HPLC 0.12

Values are based on 1 kg of bovine brain. It is impractical to estimate purification fold because of the presence of other growth factors in the cruder preparations that overlap the biological activities of GMF and because of a tendency for GMF-,3 to denature during the last two steps (see text). Protein was determined by the bicinchoninic acid method (11).

into four portions. A 10-ml aliquot was loaded on a Vydac C4 reverse-phase HPLC column (4.6 mm x 25 cm) (particle size, 5 ,um; pore size, 300 A) at a rate of 1.5 ml/min. Material from the charged HPLC column was eluted at the same speed using the following program: 0% acetonitrile for 4 min, 0-30% (vol/vol) acetonitrile gradient for 10 min, and finally 30-45% (vol/vol) acetonitrile gradient for 15 min, all in the presence of 0.1% TFA. The major peak that emerged at 40% (vol/vol) acetonitrile and reacted posi ively with the monoclonal antibody G2-09 was designated GMF-f3 (Fig. 2A).

The GMF-f3 peaks from four HPLC runs were pooled and purified once more through the same HPLC column. To do this the pool was diluted 1:1 with water containing TFA to achieve a final concentration of 20% (vol/vol) acetonitrile and 0.1% TFA. The sample was loaded to the column and material was eluted as before. The GMF-,B peak obtained at this time, representing the yield from one-third of the starting material, was the final product (Fig. 2B). The overall purification scheme is outlined in Table 1.

During the purification of GMF-f3, a minor peak preceding GMF-f3 in the HPLC profile (Fig. 2A) that also reacted positively with G2-09 was designated GMF-a. It has a

a b c d 1 2 3 4

0 ! I 11 11 1 A~I C,

0. 3 C.5 o.7 0.10 .e' 4.6 I

.0

0~~~~~~~~~~~~~~~ CD4.4 -A 0

0.3A 0.5 0.7 B

1 Relative Mobility Retention Time (min)

FIG. 3. Determination of molecular mass. (A) SDS/PAGE under reducing conditions in 15% polyacrylamide gel (12). Bands: a, ovalbumin (44 kDa); b, a-chymotrypsinogen (25.7 kDa); c, 13- lactoglobulin (18.4 kDa); d, lysozyme (14.3 kDa). Arrow pointing upward indicates protein band of GMF-13 (200 ng). Arrow pointing downward indicates position of GMF-,3 in the semilog plot. (B) Size-exclusion HPLC on a Bio-Sil TSK 125 column (Bio-Rad, 7.5 mm x 30 cm), using 0.1 M Na2SO4/0.02 M NaH2PO4, pH 6.8, at 1 ml/min. Arrows: 1, thyroglobulin (670 kDa); 2, y-globulin (158 kDa); 3, ovalbumin (44 kDa); 4, myoglobin (17 kDa). Elution profile shows the position of GMF-f3S, which coincides with that of myoglobin. With both methods the molecular weight of GMF-j3 is estimated to be 17,250.



Neurobiology: Lim et al. Proc. Natl. Acad. Sci. USA 86 (1989) 3903

Table 2. Amino acid composition of GMF-3

Amino Residue(s) per Amino Residue(s) per acid molecule acid molecule

Asx 14 Tyr 5 Glx 24 Val 10 Ser 8 Ile 7 Gly 5 Leu 12 His 2 Phe 8 Arg 8 Lys 12 Thr 6 Cys* 3 Ala 6 Met* 3 Pro 6 Trpt 1

The total number of residues is 140 and the calculated molecular weight is 16,375. *Determined after performic acid oxidation. tData from sequence analysis.

molecular weight of =16,000 and an isoelectric point of pH 4.9. Further characterization has not been performed.

Chemical Characterization of GMF-,B. GMF-pB behaves as a single band on SDS/PAGE under reducing conditions, indicating a single polypeptide chain with an apparent molecular weight of =17,000 (Fig. 3). The isoelectric point as determined on an LKB Ampholine PAG plate is pH 4.9. The amino acid composition (Table 2) shows a predominance of acidic amino acids and contains one tryptophan, three me- thionines, and three cysteines (half cystines), with an estimated length of 140 residues. The N terminus is blocked. Tryptic digestion releases 28 peptides (Fig. 4), 8 of which have been sequenced (Fig. 5). The total number of known residues adds up to more than two-thirds of the entire protein. A homology search through the Protein Identification Re- source database (November 1988; release no. 17) showed no match with any of the known proteins, including other growth factors isolated from the nervous system.

The secondary structure of GMF-,B was measured with far-ultraviolet circular dichroism (Fig. 6). The profile obtained at various temperatures indicates reversible thermal denaturation. The renatured GMF-,3, when obtained after gradually heating to 80?C and cooling back to 20?C (both at a rate of 2?C/min), exhibits a higher helical content than the original protein. This process greatly increases the biological activity. For this reason the biological characterization of GMF-pB in this report was conducted with the heat-treated, renatured protein.

Immunological Characterization. When GMF-pB was com- pared with acidic FGF for immunologic cross-reactivity using ELISA, acidic FGF has 0.1% the reactivity of GMF-,3, if

1.0 - 1 I i

0.0 6 28

0.6l15 .ll 220 nm

0A 20 a nm

< 0.4 17-

17 23~~~~~~~~1 0.2 -10 12 14 k

4 A 1~~~ 1 J,25

6 0 1 1 8 1 20 22

Absorbance~~ ~ pek aelbee fo t 8

10 T12= ETNNMIIMK T13= NKLVQTAELT K T14= VFEIR T15= FIVYSYK T17= LGFFH T18= NTEDLTEEWL R 20 T24= LVVLDEELEG ISPDELKDEL PER T28= VSYPLCFIFS SPVGCKPEQQ MMYAGSK

FIG. 5. Amino acid sequence of eight of the tryptic peptides of GMF-P (see Fig. 4). The single-letter code for the amino acids was used.

tested with monoclonal antibody G2-09, and has 2% of the reactivity of GMF-pB, if tested with polyclonal antibody 88-02 (Fig. 7). The following growth factors, when tested with antiserum 88-02, all exhibited less than 1% the reactivity of GMF-,B: basic FGF, interleukin 1, tumor necrosis factor, nerve growth factor, insulin, insulin-like growth factor II, epidermal growth factor, and S-100 protein.

Biological Characterization. The biological effect of GMF-.3 was tested on normal and neoplastic cells derived from the nervous system. On normal astrocytes, GMF-pB promotes proliferation and morphological differentiation as described (8). In addition, GMF-p stimulates neuritic outgrowth from intact DRGs in culture (Fig. 8). On dissociated DRGs where fibroblasts and glial cells have been eliminated by means of cytosine arabinoside, GMF-pB promotes neuronal survival and stimulates the outgrowth of neurites (Fig. 9). When tested on the neuroblastoma line N-18 and the glioma line C6, GMF-P exhibits a strong antimitotic effect while enhancing their phenotypic expression (Fig. 10). The N-18 cells respond to GMF-P by outgrowth of neuritic processes, whereas the C6 cells respond by transformation from a polygonal to a spindle-shaped (fusiform) pattern, both consistent with morphological maturation. Fig. 11 depicts the dose-response relationship of GMF-f3 with respect to the two tumor lines. The half-maximal dose is estimated to be -8 ng/ml for N-18 cells and 40 ng/ml for C6 cells.

DISCUSSION

The current work is a continuation of an earlier effort in this laboratory to isolate and identify GMF. Although we purified GMF to a single protein band on an SDS/polyacrylamide gel (8), subsequent attempts to sequence it proved unsuccessful, suggesting microheterogeneity. As an improvement to the earlier procedure, we now incorporated three additional

0

0 -4000 6800 C~2 C

-12000 ~ ~ 20

200 220 240

Nanometers

FIG. 6. Circular dichroism spectra of GMF-f3 in 50 mM sodium phosphate (pH 7.0) at various temperatures. At 20?C the native protein shows major ellipticity bands around 210 nm and 220 nm, which represent a major contribution of the secondary structural content. These bands disappear as the temperature is raised to 60?C and 80?C, consistent with protein denaturation. However, gradually heating and recooling the protein (80aCron 200C) results in renaturation, leading to a more active conformation th ththe original protein.



3904 Neurobiology: Lim et al. Proc. Natl. Acad. Sci. USA 86 (1989)

1.0 LA (monoclonal) ,

0.6

0.0

E 0.4 -

0.2- ;i X ~~~~aFGF i o.@ B ,

00. (polyolonal) 9 o.6 Wddni

0.4 -

0.2 -

O >~~~~aO 0.2 1 10 100 600

Amount of Antigen (ng/well)

FIG. 7. Immunologic comparison by an ELISA between GMF-,B (solid circles) and acidic FGF (open circles). (A) Mouse monoclonal antibody G2-09 against GMF-,/. (B) Rabbit polyclonal antiserum 88-02 against GMF-,8.

features in the scheme. (i) We enhanced protein resolution by using a gradient elution for the hydroxylapatite column and by increasing the length of the HPLC column from 5 cm to 25 cm. (ii) We included a heparin-Sepharose step to exclude the FGFs (13) from the final product; these heparin-binding proteins were a source of confusion in the study of GMF. (iii) We utilized the monoclonal antibody G2-09 to monitor the final steps of purification. We have demonstrated (17) that monoclonal antibody G2-09 recognizes a brain-specific protein localized in astrocytes and Schwann cells (14-16) and that the protein is expressed in Schwann cells only after nerve injury, implying a role in axonal regeneration. The combina- tion of these improvements allowed us to isolate the brain protein GMF-,f.

A number of growth factors have been isolated from the nervous system, the most notable examples of which are the

f

FIG. 8. Effect of GMF-13 on an intact DRG. (A) GMF-,B was added at 50 ng/ml. (B) Control without GMF 13i Micrographs were taken after 48 hr of testing. (Phase-contrast montage. Bar =1 mm.)

-~~~~~~. 3

FIG. 9. Effect of GMF-,3 on neurons dissociated from DRGs. Ganglion cells were dissociated with trypsin and seeded at 6000 cells per well. The cells were grown in the presence of cytosine arabinoside (10 AM) to eliminate nonneuronal cells. (A) Control without GMF-,B. (B) GMF-,3 at 50 ng/ml. Micrographs were taken after 48 hr of exposure to GMF-13. Note larger total cell number per field and larger percentage of neurite-bearing cells in the presence of GMF-,B. (Phase-contrast. Bar = 40 Am.)

acidic and basic FGFs (13). The two proteins show 55% homology and both are 30% homologous with interleukin 1. The FGFs exhibit a broad range of target-cell specificity, including endothelial cells, fibroblasts, astrocytes, neurons, and prostatic cells. In fact, their actions are so diverse that many growth factor activities separately detected in brain extracts turned out to be acidic FGF or a slight variant of it. Therefore, the FGF family represents a large proportion of growth-regulating activities in the brain. Since cruder preparations of GMF showed functions overlapping those of FGF, a fact partly explainable in retrospect by cross- contamination, it became a pressing question as to whether GMF was also another FGF in disguise. Obviously, a clearcut answer could only be obtained by comparing the

e i a~~~~~~~~~~e

FIG. 10. Effect of GMF-,8 on tumor cell lines. (A and A') N-18 neuroblastoma. (B and B') C6 glioma. (A and B) Controls without GMF-13. (A' and B') GMF-,3 added at 250 ng/ml. Cells were seeded at 2 x 105 cells per well and allowed to attach for 4 hr before testing. Micrographs were taken after 60 hr of testing. (Phase-contrast. Bars = 40 ,Lm.)



Neurobiology: Lim et al. Proc. Natl. Acad. Sci. USA 86 (1989) 3905

40-

40

20 -~~~~~~~ 120< 2

V % S~~~~~

0 ~ ~ ~ ~~~~2 o~~~~~~~~~~s

20 - lE l l 2

0 100 200

GMF-P (ng/ml)

FIG. 11. Dose-response curves of GMF-,8. (A) N-18 neuroblastoma line. (B) C6 glioma line. Cells were seeded at 2 x 105 cells per well and allowed to attach for, 4 hr. The cells were then exposed to GMF-8 for 6(0 hr and scored. For N-18, cells with one or more neurites longer than one cell diameter were scored positive. For C6, cell wit sidesaewrscored positive. Four hundred cells from representative areas of each well were scored and the results are expressed as percent total population. Cell number 'per well was' determined with a Coulter counter after trypsin'treatment. All values are averages of at least four wells with a range of S% or less.

amino acid sequence of the two proteins. In the current work this has been accomplished. A search for homology using the known sequ'ence from GMF-,B tryptic peptides demonstrated that at least more'than two-thirds of the aminio acid sequence of GMF-/3 is distinct from that of the FGFs. Furthermore, based on information from partial sequence, we have syn- thesized oligonucleotide probes and have screened and cloned the cDNA for GMF-,B. The deduced complete sequence of GMF-,B again shows no homology with the FGFs and i'n fact with any'known protein entered in the Protein Identificatio'n Resource data base (Kaplan, R., Jaye, M. & R.L., unpublished results).

The lack of horhology is corroborated by the absence of immunologi. cross-reactivity between GMF-,8 and the other pwltleins tested, using the polyclonal anwtibody developed against GMF-,o. Polyclonal antibodies are a better measure of cross-reactivity than monoclonal antibodies because they are directed toward multiple epitopes dirstributed across the entire length of the proteeln antigen.

'One important finding in the current work is that GMF-,8 undergoes reversible alterations in cenformation during the purification procedure; full biological activity can best be demonstraed wih y Cuter-het c teater trypsrenattion. If this point weres ofetleas one could have missed the biologica l activity entirely and thereby failed to recognize the purified protein as a growth factor. Asthouh GMF was originally detected as' a growth factor for glial cells, we have, now documented that the purified GMF-ty is Et rowth regulator for neurons as well. For a growth factor that has biological action on glial cells it is always debatable as to whether the observed neuronal effrom ia sequ dec actionw or an indirect effec through glial cells. Two pieces of evidence favor a direct action. (i) In dissociated normal neurons from

DRGs, where glial and fibroblast elements had been mostly eliminated through the use of an antimitotic agent, GMF-/3 enhanced neuronal survival and neurite outgrowth (Fig. 9). (ii) In the cloned neuroblastoma cell line N-18 where no glial cells existed, the neurite-promoting effect of GMF-,B still showed up (Fig. 10). In any event, the direct and indirect effects of GMF-P on neurons are not necessarily mutually exclusive.

In this paper, we also documented the antineoplastic effect of GMF-p on both neuronal and glial tumors. In both cases GMF-pB arrests the proliferation and promotes the phenotypic expression of the cells-i.e., neurite outgrowth in neuroblastoma cells (Fig. 1OA) and spindle-shaped morphology in glioma cells (Fig. lOB). It should be pointed out that the antimitotic action of GMF-/3 on C6 cells is contrary to our earlier observation of a mitogenic effect of crude GMF on the same cell line (18). This discrepancy is probably due to contamination of the crude preparations by other growth factors, such as FGF. It is possible that some other biological differences between GMF-p and crude GMF may exist, but this is beyond the scope of the present paper and will be the subject of future investigation. Inasmuch as pure GMF-,B is now available, a thorough reevaluation of GMF function is in order, and it is only after this that the complete range of GMF activity, in comparison with other growth factors, will be unfolded.

We thank the following for technical assistance: B. A. Baggens- toss, M. A. Midthun, B. Fink, W. Zhong, and Y. Liu. Amino acid composition was carried out by A. Bergold at the University of Iowa Protein Structure Facility. Protein sequencing was performed by W. Lane at the Harvard Microchemistry Facility. This work was sup- ported by the following grants to R.L.: Veteran's Administration Merit Review Award, Grant BNS-8607283 from the National Science Foundation, and Grant DK-25295 from the Diabetes-Endocrinology Research Center.

1. Todd, J. T. (1823) J. Sci. Lit. Arts 16, 84-96. 2. Trowell, 0. A. & Willmer, E. N. (1939) J. Exp. Biol. 16, 60-70. 3. Hoffman, R. S., Tenenbaum, E. & Doljanski, L. (1940) Growth

4, 207-221. 4. Hoffman, R. S. (1940) Growth 4, 361-376. 5. Lim, R., Mitsunobu, K. & Li, W. K. P. (1973) Exp. Cell Res.

79, 243-246. 6. Lim, R. & Mitsunobu, K. (1974) Science 185, 63-66. 7. Lim, R. & Miller, J. F. (1984) J. Cell. Physiol. 119, 255-259. 8. Lim, R., Miller, J. F., Hicklin, D. J. & Andresen, A. A. (1985)

Biochemistry 24, 8070-8074. 9. Groome, N. P. (1980) J. Neurochem. 35, 1409-1417.

10. Kasper, C. B. (1975) in Molecular Biology, Biochemistry and Biophysics, ed. Needleman, S. B. (Springer, New York), Vol. 8, pp. 114-161.

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12. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 13. Thomas, K. A. (1987) FASEB J. 1, 434-440. 14. L'im, R., Hicklin, D. J., Ryken, T. C. & Miller, J. F. (1987)

Dev. Brain Res. 33, 49-57. 15. Lim, R., Hicklin, D. J., Miller, J. F., Williams, T. H. &

Crabtree, J. B. (1987) Dev. Brain Res. 33, 93-100. 16. Lim, R., Hicklin, D. J., Ryken, T. C., Miller, J. F. & Bosch,

E. P. (1988) Dev. Brain Res. 40, 277-284. 17. Lim, R., Zhong, W., Bosch, E. P. & Miller, J. F. (1988) Trans.

Am. Soc. Neurochem. 19, 83. 18. Lim, R., Hicklin, D. J., Ryken, T. C., Han, X. M., Liu, K. N.,

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