8070 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Biochemistry zyxwvu 1985, 24, 8070-8074 Purification of Bovine Glia Maturation Factor and Characterization with Monoclonal Antibody? Ramon Lim,* Joyce zyxwvutsr F. Miller, Danny J. Hicklin, and Andrew A. Andresen Veterans Administration Medical Center and Department zyxwvu of Neurology, Division of Neurochemistry and Neurobiology, University of Iowa, Iowa City, Iowa 52242 Received May 31, I985 ABSTRACT: Glia maturation factor (GMF) is purified 100 000-fold to apparent homogeneity from bovine brains by a procedure consisting of ammonium sulfate precipitation, column chromatography with di- ethylaminoethyl-Sephacel, Sephadex G-75, and hydroxylapatite, and a final step using C4 reverse-phase high-performance liquid chromatography. The product shows a single protein band in sodium dodecyl sulfate-polyacrylamide gel. It has a molecular weight of 14000 and an isoelectric point of pH 5.2. Purified GMF stimulates cultured astroblasts to proliferate and to grow out cell processes with half-maximal activity at 8 ng/mL. A monoclonal antibody raised against partially purified GMF adsorbs the activity of pure G M F and immunologically binds the putative GMF protein band. G l i a maturation factor (GMF),’ first detected in our lab- oratory (Lim et al., 1972, 1973; Lim zyxwvutsr & Mitsunobu, 1974), is an acidic protein endogenous to the adult brain of many species. GMF has been implicated in a number of cellular control mechanisms in the nervous system. It promotes the proliferation and phenotypic expression of astrocytes (Lim, 1980) and Schwann cells (Bosch et al., 1984) in culture and reverses some of the neoplastic growth properties of glial tumor cells (Lim et al., 1981). GMF also stimulates the production of other growth factors or hormones from astrocytes such as interleukin 1 (Fontana et al., 1983) and the prostaglandins (Miller & Lim, 1984). When used in vivo, GMF enhances the morphological recovery of brain tissue following injury to newborn rats (Lim & Miller, 1985). For the most recent review on GMF up to Dec 1983, see article by Lim (1985). Despite attempts to purify this biologically active substance, the chemical nature of GMF remained obscure. We last reported a 10 000-fold partial purification (Lim & Miller, 1984). We now present data on the complete purification of GMF using HPLC as the final step. A monoclonal antibody is obtained that abolishes GMF activity and binds the purified GMF protein. MATERIALS AND METHODS Production of Partially Purified GMF. A 10 000-fold partially purified GMF sample was used as the starting ma- terial for HPLC fractionation. This was prepared from bovine brains by a procedure previously described (Lim & Miller, 1984), which comprises the following steps: ammonium sulfate precipitation, DEAE-Sephacel chromatography, Sephadex G-7 5 chromatography, and hydroxylapatite chromatography (Table I). HPLC Procedure. The HPLC column consisted of a 4.6 mm X 5 cm Vydac C4 reverse-phase column (The Separations Group, Hesperia, CA) having a particle size of 5 pm and a pore size of 300 A. Lyophilized G M F samples were dissolved in the organic solvent (acetonitrile with TFA) immediately before application to the column. The eluted fractions were collected in polypropylene tubes and lyophilized to remove the solvent. Before lyophilization, fractions were stored at -70 OC for up to 2 days. Fractions intended for cell testing were collected in tubes containing BSA to a final concentration of 0.1 mg/mL. These were likewise frozen and lyophilized before use. All other conditions were as described under Results. Bioassay of GMF. GMF was assayed on confluent cultures of astroblasts derived from 17-day-old fetal rat brains. Testing was conducted in F10 medium containing zyx 5% fetal calf serum. G M F activity was based on mitogenicity (increase in [3H]- thymidine incorporation into DNA) and on morphological change (percentage of cells growing out processes), as de- scribed before (Lim & Miller, 1984). SDS-Polyacrylamide Gel Electrophoresis. Lyophilized G M F samples were taken up in a sample buffer consisting of 0.1 M dithiothreitol, 2% SDS, 15% glycerol, 2 mM phenyl- methanesulfonyl fluoride, 2 mM EDTA, 1 mM N-ethyl- maleimide, 1 mM iodoacetic acid, 75 mM Tris-HC1 (pH 6.8), and 0.001% bromophenol blue. After the solution was heated at 100 OC for 5 min, 20 pL was applied to each slot in a gel slab, 70 X 80 X 0.75 mm in dimension, made in a “Mighty Small” apparatus (Hoefer Scientific Instruments). The stacking gel, separation gel, and electrode buffer, all containing 0.1% SDS, were prepared according to Laemmli (1970). Electrophoresis was conducted at a constant current of 20 mA for about 2 h. The gels were fixed and stained with silver nitrate by using the kit purchased from Bio-Rad. Amino Acid Analysis. Protein samples were hydrolyzed for 24 h in 6 N HCl containing 0.2% 2-mercaptoethanol and analyzed with a Beckman 121 MB amino acid analyzer. Cysteine content was determined as cysteic acid after per- ~~ ‘This work was supported by the following to R.L.: VA Merit Review and Career Investigatorship Awards, NSF Grant zyxwvutsr BNS-8308341, NCI Grant CA-3 1796, Diabetes-Endocrinology Research Center Grant AM- 25295, and University of Iowa Cancer Center Grant. This paper is dedicated to Prof. Maurice W. Van Allen, Chairman of Department of Neurology, on the occasion of his retirement. *Author to whom correspondence should be addressed. ___ ~ ___ ’ Abbreviations: BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; GMF, glia maturation factor; HPLC, high-per- formance liquid chromatography; IEF, isoelectric focusing; PAGE, po- lyacrylamide gel electrophoresis; PBS, 10 mM sodium phosphate and 0.15 M NaC1, p H 7.4; SDS, sodium dodecyl sulfate; Tris-HCI, tris(hy- droxymethy1)aminomethane hydrochloride; TBS, 20 mM Tris-HCI and 0.15 M NaC1, pH 7.4; TPBS, 0.05% Tween 20 in PBS; TFA, trifluoro- acetic acid; EDTA, ethylenediaminetetraacetic acid. 0006-2960/85/0424-8070$01.50/0 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 0 1985 American Chemical Society