DEVELOPMENTAL
BIOLOGY
65, 260-270 (1978)
Synthesis
of Nerve Growth
Factor
ANGELA Department
of Biochemistry,
in Rat Glioma
Cells
M. LONGO
University
of California,
Berkeley,
Received August 8, 1977; accepted in revised form February
California
94720
27, 1978
We have analyzed the incorporation of radioactive amino acids by rat C6 glioma cells into material precipitable with anti-p-nerve growth factor (NGF). We show that amino acids are incorporated into a protein the size of P-NGF which is immunologically related to NGF and which has peptides similar to those of mouse P-NGF. Several lines of evidence obtained in this study support the hypothesis that NGF is produced by the proteolytic cleavage of a higher molecular weight precursor. This evidence includes kinetic studies, demonstration of higher molecular weight material (24,000) immunologically related to NGF, and in vitro processing of the 24,000 MW protein to material of the approximate size of P-NGF. INTRODUCTION
The p form of mammalian nerve growth factor (NGF) is a small protein which selectively enhances growth and differentiation of sympathetic and sensory nerve cells (Levi-Montalcini and Angeletti, 1968). This hormone was originally discovered in extracts of sarcoma tissue (Levi-Montalcini, 1952). It was found later in extracts of mouse submaxillary glands, and this tissue has been the source of most of the material utilized in biochemical studies (e.g., Frazier et al., 1972). Several lines of evidence have indicated that NGF plays an important role in the development and regeneration of the nervous system. The presence and action of NGF in these processes appears independent of the salivary gland (Levi-Montalcini, 1965; Hendry and Iverson, 1973). These observations have led a number of investigators to examine other tissues and cells for the presence of NGF which could potentially be involved in developmental processes. In recent years, material immunologically related to NGF has been detected in rat glioma cells (Long0 and Penhoet, 1974; Longo, 1976) and in tissue culture media conditioned by rat glioma cells (Long0 and Penhoet,
1974),
mouse
L and
3T3
fihro-
blasts (Oger et al., 1974), mouse neuroblas260 OOlZ-X06/78/0652-0260$02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved,
toma cells (Murphy et al., 1975), chick embryo fibroblasts (Young et al., 1975), and human glioma cells (Reynolds and PerezPolo, 1975). Nerve growth factor activity [as detected by the classical dorsal root ganglion assay (Varon and Shooter, 1970)] has been detected in medium conditioned by both rat and human glioma cells (Long0 and Penhoet, 1974; Reynolds and PerezPolo, 1975). Additionally, Varon et al. (1974) have shown that non-neuronal cells of chicken dorsal root ganglia secrete a factor which is required for the development of neurons in vitro and that the activity of this factor is inhibited by antibodies directed against NGF. All of the above mentioned studies imply that NGF is synthesized in the cells or tissues discussed, but thus far no studies of the de nouo synthesis of NGF have been published. Such analyses seem critical to provide a foundation for further studies of the possible developmental role of this hormone. Furthermore, considerable circumstantial evidence has been accumulating which indicates that P-NGF is synthesized in the form of a higher molecular weight precursor. /3-NGF has many physiological and biochemical similarities to insulin (Frazier et al., 1973; Liuzzi et al., 1968) and has extensive amino acid sequence homology to
ANGELA M. LONGO
Glioma Nerve Growth Factor Synthesis
proinsulin (Frazier et al., 1972). Furthermore, P-NGF in the salivary gland exists in a specific multiprotein complex (7 S NGF) with (Yand y subunits (Varon and Shooter, 1970). The y subunit is an arginine esteropeptidase (Greene et al., 1969). Since a number of other polypeptide hormones are generated from larger precursors by the proteolytic action of enzymes with similar specificities (Schachter, 1969; Steiner et al., 1974) and ,&NGF has a carboxyl terminal arginine residue, it has been suggested that P-NGF is produced from a precursor by the arginine esteropeptidase action of y (Server and Shooter, 1976). The major objective of the work presented herein was to demonstrate directly the synthesis of P-NGF in cells. In so doing, we obtained data which support the hypothesis that /3-NGF is derived from a higher molecular weight precursor. MATERIALS
AND
METHODS
Cells. C6 rat glioma cells were a gift of Dr. A. Mamoon, Donner Labs, University of California, Berkeley. The cells were grown in Dulbecco’s modified Eagle medium (DME; Gibco H2) at pH 7.4 supplemented with 10% fetal calf serum (FCS; Irvine Scientific) in an environment with 10% CO1 at 37°C. Stock cultures were maintained in 32-0~ glass prescription bottles. Cells used for labeling experiments were grown in roller bottles containing 200 ml of supplemented medium and a 5% CO2 atmosphere. Cells were pulse labeled with radioactive amino acids by incubation in 50 ml of labeling media (DME minus lysine, leucine, and phenylalanine) containing 5 &i/ml each of [3H]lysine (225 mCi/mmole), [3H]leucine (30 Ci/mmole), and [3H]phenylalanine (7 Ci/mmole) obtained from Schwarz/Mann. Ten percent dialyzed fetal calf serum was used in the pulse-labeling experiments. Cell extracts. To prepare extracts after labeling, cells were washed with phosphatebuffered saline (PBS) and treated for 3-5 min with 0.025% trypsin in PBS. The tryp-
261
sin was inhibited with DME containing 10% FCS. The cells were centrifuged and then washed twice with PBS and resuspended in 1.5 ml of water/roller bottle. The cells were lysed by freezing, thawing, and sonicating for 3-5 min with a microtip on a Branson Model 185 sonicator (setting 4). Cell debris was removed by centrifugation for 20 min at 10,000 rpm in a Sorvall SS-34 rotor.
Antibody preparation
and analysis. p-
NGF was prepared from mouse submaxillary glands by the method of Mobley et al. (1976). Figure 1 shows a sodium dodecyl sulfate (SDS) gel electrophoretogram of the NGF preparation used as an antigen. Almost all of the Coomassie blue-staining material was localized in a band of the molecular weight of NGF. No heterogeneity was noted on isoelectric focusing gels or upon peptide “fingerprint” analysis. Antibody was prepared in rabbits by the method described by Sarich (1969) using 1.5 mg of protein/injection. As shown in Fig. 2, the antibody was monospecific upon immunoelectrophoresis,
NGF
FIG. 1. An SDS gel electrophoretogram of the NGF preparation used as an antigen. Almost all of the Coomassie blue-staining material was localized in a band of the molecular weight of NGF. This peak is identical with that containing biological activity in the dorsal root ganglion assay.
262
DEVELOPMENTAL BIOLOGY
Mouse NGF antigen
Frc:. 2. Immunoelectrophoresis of anti-NGF IgG against mouse NGF antigen according to techniques described by Arnhum and Wilson (1967). Also observed although not shown here was a monospecific reaction of the antibody against a crude mouse submaxillary gland extract.
which was performed in agar gels on glass slides (1 x 3 in.) according to techniques described by Arnhum and Wilson (1967).
Indirect NGF immunoprecipitation and analysis on SDS-Tris gels. Aliquots of 0.75 ml of antigen-containing solutions (usually cell supernatants) were incubated with 10 ~1 of rabbit anti-NGF IgG, first at 37°C for 30 min and then at 4°C overnight. After addition of 20 ~1 of goat anti-rabbit IgG, further incubation was performed at 37°C for 30 min and at 4°C for at least 4 hr. The resulting immunoprecipitates were spun down at 18,OOOgin a Sorvall SS-34 rotor, washed twice with PBS, and subsequently dissolved in 20 ~1 of 0.1 N NaOH, 40 ~1 of 0.1 M Tris-HCl, pH 6.8, and 20 ~1 of SDS sample buffer containing 20% P-mercaptoethanol @ME), 0.25 M Tris-HCl, pH 6.8, 8% SDS, and 40% glycerol. Before electrophoresis each sample was heated at 98°C for at least 10 min. Electrophoresis was performed on 15% acrylamide-SDS gels according to the method of Laemmli (1970). The proportions of ingredients used were 3.0 ml of 1.5 M Tris-HCl, pH 9.8, 6.0 ml of 30% (W/V) acrylamide-0.8% bis, 0.12 ml of 10% SDS, 4 ~1 of Temed (N,N,N’N-Tetramethylethylenediane) 0.12 ml of 30 mg/ml of ammonium persulfate, and 2.76 ml of water. Gels were poured to a length of 10 cm. A stacking gel containing the following was used: 1.25 ml of 0.5 M Tris, pH 6.8,0.05 ml of 10% SDS, 0.83 ml of 30% acrylamide-0.8% bis, 2 ~1 of Temed, 75 ~1 of water. For analysis the gels were stained in 0.05% Coomassie blue, 10% (v/v) acetic acid, and
VOLUME 65,1978
25% (v/v) isopropanol; destained in 10% acetic acid, and dried. Gels were then cut into 2-mm pieces; swelled in 100 ~1 of water, incubated overnight in 10 ml of a mixture of 143 ml of NCS, 3 kg of toluene, 15.2 g of Omnifluor; and counted on a Packard scintillation counter. In vitro labeling of mouse fi-NGF. Mouse /3-NGF was labeled in vitro according to a method described by Rice and Means (1971) except that [‘4C]acetaldehyde was substituted for [ “C]formaldehyde. The [14C]acetaldehyde was freshly prepared by the acid hydrolysis of [14C]paraldehyde. In general, the procedure gives about 20% of the maximum labeling expected if each amino group were converted to its dialkylamino derivative. Carboxymethylation of NGF. Two milligrams of mouse submaxillary NGF and labeled rat C6 glioma NGF eluted from SDS gels were dissolved in 2 ml of 6 M guanidineHCl, pH 9.3, and reduced using a 50 M excess of dithiothreitol to protein in a nitrogen atmosphere for 4 hr. After adjusting the protein solution to pH 8.0 with 1 N HCl, a neutral solution containing 200 pmole of iodoacetic acid was added. Alkylation was allowed to proceed for 5 min. The reaction was stopped by the addition of a lo-fold molar excess of 2-mercaptoethanol (Angeletti and Bradshaw, 1971). After dialysis against four changes of deionized water for 24 hr, the NGF was recovered by lyophilization. Tryptic digestion. Tryptic digestion of Scarboxymethyl-NGF was carried out using N”-tosylphenylalanine chloromethyl ketone (TPCK)-treated trypsin (Worthington) added, in a ratio of 1:lOO to NGF on a weight basis, three times over a 12-hr period during incubation at 37°C (Angeletti et al., 1973). The pH was adjusted to 8.8 at each 4-hr interval. The small amount of insoluble material remaining after acidification to pH 2 with 6 N HCl was removed by centrifugation. Peptide analysis. The soluble tryptic peptides were fractionated on a 0.9 X 20-cm
ANGELA M. LONGO
Glioma Nerve Growth Factor Synthesis
column of Aminex A-5 equilibrated with 0.05 N pyridine-acetate (pH 2.5) and eluted with a double linear gradient of pyridine-acetate buffers at 50°C (Bradshaw et al., 1969). The column flowed at a rate of 30 ml/l-u and 4.5-n-J fractions were collected. A 1.5~ml aliquot of each fraction was evaporated in a glass scintillation vial and redissolved in 0.5 ml of water and 7.5 ml of toluene-omnifluor-TritonX-100 (16:1:8) mixture. The remaining 3.0 ml in each fraction was used for protein ninhydrin determination. Determination of peptide concentration
using ninhydrin reagent with alkaline hydrolysis of peptides. Three milliliters of each fraction was dried in a vacuum oven. Then 0.5 ml of 0.5 M NaOH was added and oven dried overnight. This was neutralized with 0.5 ml of 0.5 M acetic acid. Next, the ninhydrin reagent was freshly prepared: 2.0 g of ninhydrin and 0.3 g of hydrindantin were dissolved in 75 ml of methyl Cellosolve (peroxide free); then 25 ml of 4 M sodium acetate buffer (pH 5.5) was added. Of this solution, 0.5 ml was added to each tube which was then covered with aluminum foil and placed in an uncovered boiling water bath for 15 min. After cooling, 1 ml of 50% ethanol was added to each tube with Vortex mixing. The tubes were spun down in an International centrifuge at 1000 rpm for 10 min to remove precipitable material and then carefully decanted into a cuvette and read at 570 nm. RESULTS
Synthesis of NGF by C6 Rat Glioma Cells in Tissue Culture The most direct demonstration of the de novo synthesis of a protein is the incorporation of radioactive amino acids into a purified preparation of the protein. To obtain such a demonstration for NGF we labeled C6 cells in tissue culture with radioactive amino acids, continuously for 23 hr. We then made cell extracts, precipitated NGF with a specific antiserum, and analyzed the immunoprecipitates by SDS gel electrophoresis on 15% polyacrylamide gels
263
as described under Materials and Methods. As seen in Fig. 3A, the procedure employed resulted in the precipitation and resolution of several proteins, one of which comigrated with a 14C-labeled mouse NGF standard. The presence of other peaks of higher molecular weight was confusing at first, since our antibody preparation was monospecific by immunoelectrophoretic analysis. However, a series of control experiments cleared up much of the confusion and allowed tentative identification of the other peaks. An 18,000g supernatant of glioma cell extract was incubated under the same conditions as were used in the indirect immunoprecipitation assay, with the omission of antiNGF serum. A precipitate resulted from this incubation. Analysis of this material, shown in Fig. 3B, demonstrated the presence of self-aggregating proteins. It is likely that the first peak is tubulin. The tendency of this protein to form aggregates at 37°C is well documented (Wiche et al., 1972; Wiche and Cole, 1976), and a sample of purified neuroblastoma tubulin (gift of Dr. G. Wiche) comigrated with this peak. The second high molecular weight peak seen in self-aggregating controls (Wiche et al., 1972) is presumed to be actin, which was seen copurifying with microtubules in some but not all C6 clones (Wiche and Cole, 1976). Because of the high concentration of the heavy and light chains of the antibody, the exact positions of the high molecular weight peaks were variable. Evidence to be presented below suggests that the third peak of approximately 24,000 MW seen in the immunoprecipitate is a precursor to 2.5 S NGF. An additional control consisting of normal rabbit IgG with goat anti-rabbit IgG, incubated under the same conditions as used in the indirect immunoprecipitation assay of Fig. 3A, was observed to be similar to the control shown in Fig. 3B.
Profile of Tryptic Digestion of In Vivo Labeled C6 Rat Glioma NGF and Mouse ,8 NGF In order to confirm that the peak of ra-
264
DEVELOPMENTAL
BIOLOGY
VOLUME 65, 1978
IO GEL SLICE NUMBER FIG. 3. (A) Profile of 15% SDS-Tris gel analysis of an indirect immunoprecipitate obtained from soluble C6 glioma cell extract and anti-NGF as described under Materials and Methods. Cells were continuously labeled for 23 hr with 25% of the normal level of lysine, leucine, and phenylalanine in DME and 5 ,aCi/ml each of ]‘H]lysine, r’H]leucine, and [“Hlphenylalanine in the presence of 5% CO:! atmosphere. Gels were stained, dried, cut, and counted in NCS-toluene-Omnifluor. H refers to the position of the heavy chain (50,000 MW) of the antibodies, and L, to the position of the light chain (25,000 MW). (B) Control: Profile of 15% SDS-Tris gel profile of self-aggregating proteins obtained from 18,OOOgcentrifugation of glioma cell supernatant incubated under the same conditions as were used in the indirect immunoprecipitation assay with the omission of antiNGF serum.
dioactive material which migrated similarly to /?-NGF on polyacrylamide gels represented the rat glioma equivalent to mouse P-NGF, the tryptic peptides of the glioma protein were compared to those of mouse P-NGF by chromatography on Aminex A-5 columns as shown in Fig. 4. The peptides of the mouse /?-NGF were detected by assay for ninhydrin-positive material and the peptides from the putative glioma NGF were labeled with [“Hlphenylalanine, [“HIleucine, and [“Hllysine. The pattern we obtained for mouse NGF is similar to that obtained by other investigators (Angeletti et al., 1973). The pattern obtained for the
rat C6 NGF is very similar with a number of coincident or overlapping peaks. Since the rat C6 NGF was labeled nonuniformly, it would not be expected to have similar proportions of peaks as compared to mouse NGF. Also, since rat NGF may be slightly larger than mouse NGF, we might expect one or two extra peaks, one of which appears around fraction 396. Immunological data we have reported previously show that the rat glioma NGF amino acid sequence appears to differ from that of mouse submaxillary /3-NGF by about lo%, so that the peaks that differ by a few fractions can be explained as manifestations of the small
ANGELA
M.
LONGO
C--J
k-1
(JON
Glioma Nerve Growth Factor Synthesis
Wd3 H,
3SflOWFJ) NIUIAHNAN
-‘%
265
266
DEVELOPMENTAL BIOLOGY
sequence differences 1974).
(Long0 and Penhoet,
Immunological Reactivity of Proteins Isolated from SDS Gel Electrophotograms Radioactive peaks corresponding to pNGF and the next larger peak were cut from an SDS gel and were extracted by shaking in a warm room (37°C) overnight as indicated in Table 1. As designated therein, both the 14,000 MW peak (P-NGF) and the 24,000 MW peak are immunologically related to mouse P-NGF.
Kinetic Evidence for a Precursor to p NGF In a series of experiments TABLE
designed to
1
DIHECT IMMIJNOLOGICAI. REPIIRCIPITATION OF [.‘H]NGF AND ,‘H PHEC~JI
BIOLOGY
65, 260-270 (1978)
Synthesis
of Nerve Growth
Factor
ANGELA Department
of Biochemistry,
in Rat Glioma
Cells
M. LONGO
University
of California,
Berkeley,
Received August 8, 1977; accepted in revised form February
California
94720
27, 1978
We have analyzed the incorporation of radioactive amino acids by rat C6 glioma cells into material precipitable with anti-p-nerve growth factor (NGF). We show that amino acids are incorporated into a protein the size of P-NGF which is immunologically related to NGF and which has peptides similar to those of mouse P-NGF. Several lines of evidence obtained in this study support the hypothesis that NGF is produced by the proteolytic cleavage of a higher molecular weight precursor. This evidence includes kinetic studies, demonstration of higher molecular weight material (24,000) immunologically related to NGF, and in vitro processing of the 24,000 MW protein to material of the approximate size of P-NGF. INTRODUCTION
The p form of mammalian nerve growth factor (NGF) is a small protein which selectively enhances growth and differentiation of sympathetic and sensory nerve cells (Levi-Montalcini and Angeletti, 1968). This hormone was originally discovered in extracts of sarcoma tissue (Levi-Montalcini, 1952). It was found later in extracts of mouse submaxillary glands, and this tissue has been the source of most of the material utilized in biochemical studies (e.g., Frazier et al., 1972). Several lines of evidence have indicated that NGF plays an important role in the development and regeneration of the nervous system. The presence and action of NGF in these processes appears independent of the salivary gland (Levi-Montalcini, 1965; Hendry and Iverson, 1973). These observations have led a number of investigators to examine other tissues and cells for the presence of NGF which could potentially be involved in developmental processes. In recent years, material immunologically related to NGF has been detected in rat glioma cells (Long0 and Penhoet, 1974; Longo, 1976) and in tissue culture media conditioned by rat glioma cells (Long0 and Penhoet,
1974),
mouse
L and
3T3
fihro-
blasts (Oger et al., 1974), mouse neuroblas260 OOlZ-X06/78/0652-0260$02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved,
toma cells (Murphy et al., 1975), chick embryo fibroblasts (Young et al., 1975), and human glioma cells (Reynolds and PerezPolo, 1975). Nerve growth factor activity [as detected by the classical dorsal root ganglion assay (Varon and Shooter, 1970)] has been detected in medium conditioned by both rat and human glioma cells (Long0 and Penhoet, 1974; Reynolds and PerezPolo, 1975). Additionally, Varon et al. (1974) have shown that non-neuronal cells of chicken dorsal root ganglia secrete a factor which is required for the development of neurons in vitro and that the activity of this factor is inhibited by antibodies directed against NGF. All of the above mentioned studies imply that NGF is synthesized in the cells or tissues discussed, but thus far no studies of the de nouo synthesis of NGF have been published. Such analyses seem critical to provide a foundation for further studies of the possible developmental role of this hormone. Furthermore, considerable circumstantial evidence has been accumulating which indicates that P-NGF is synthesized in the form of a higher molecular weight precursor. /3-NGF has many physiological and biochemical similarities to insulin (Frazier et al., 1973; Liuzzi et al., 1968) and has extensive amino acid sequence homology to
ANGELA M. LONGO
Glioma Nerve Growth Factor Synthesis
proinsulin (Frazier et al., 1972). Furthermore, P-NGF in the salivary gland exists in a specific multiprotein complex (7 S NGF) with (Yand y subunits (Varon and Shooter, 1970). The y subunit is an arginine esteropeptidase (Greene et al., 1969). Since a number of other polypeptide hormones are generated from larger precursors by the proteolytic action of enzymes with similar specificities (Schachter, 1969; Steiner et al., 1974) and ,&NGF has a carboxyl terminal arginine residue, it has been suggested that P-NGF is produced from a precursor by the arginine esteropeptidase action of y (Server and Shooter, 1976). The major objective of the work presented herein was to demonstrate directly the synthesis of P-NGF in cells. In so doing, we obtained data which support the hypothesis that /3-NGF is derived from a higher molecular weight precursor. MATERIALS
AND
METHODS
Cells. C6 rat glioma cells were a gift of Dr. A. Mamoon, Donner Labs, University of California, Berkeley. The cells were grown in Dulbecco’s modified Eagle medium (DME; Gibco H2) at pH 7.4 supplemented with 10% fetal calf serum (FCS; Irvine Scientific) in an environment with 10% CO1 at 37°C. Stock cultures were maintained in 32-0~ glass prescription bottles. Cells used for labeling experiments were grown in roller bottles containing 200 ml of supplemented medium and a 5% CO2 atmosphere. Cells were pulse labeled with radioactive amino acids by incubation in 50 ml of labeling media (DME minus lysine, leucine, and phenylalanine) containing 5 &i/ml each of [3H]lysine (225 mCi/mmole), [3H]leucine (30 Ci/mmole), and [3H]phenylalanine (7 Ci/mmole) obtained from Schwarz/Mann. Ten percent dialyzed fetal calf serum was used in the pulse-labeling experiments. Cell extracts. To prepare extracts after labeling, cells were washed with phosphatebuffered saline (PBS) and treated for 3-5 min with 0.025% trypsin in PBS. The tryp-
261
sin was inhibited with DME containing 10% FCS. The cells were centrifuged and then washed twice with PBS and resuspended in 1.5 ml of water/roller bottle. The cells were lysed by freezing, thawing, and sonicating for 3-5 min with a microtip on a Branson Model 185 sonicator (setting 4). Cell debris was removed by centrifugation for 20 min at 10,000 rpm in a Sorvall SS-34 rotor.
Antibody preparation
and analysis. p-
NGF was prepared from mouse submaxillary glands by the method of Mobley et al. (1976). Figure 1 shows a sodium dodecyl sulfate (SDS) gel electrophoretogram of the NGF preparation used as an antigen. Almost all of the Coomassie blue-staining material was localized in a band of the molecular weight of NGF. No heterogeneity was noted on isoelectric focusing gels or upon peptide “fingerprint” analysis. Antibody was prepared in rabbits by the method described by Sarich (1969) using 1.5 mg of protein/injection. As shown in Fig. 2, the antibody was monospecific upon immunoelectrophoresis,
NGF
FIG. 1. An SDS gel electrophoretogram of the NGF preparation used as an antigen. Almost all of the Coomassie blue-staining material was localized in a band of the molecular weight of NGF. This peak is identical with that containing biological activity in the dorsal root ganglion assay.
262
DEVELOPMENTAL BIOLOGY
Mouse NGF antigen
Frc:. 2. Immunoelectrophoresis of anti-NGF IgG against mouse NGF antigen according to techniques described by Arnhum and Wilson (1967). Also observed although not shown here was a monospecific reaction of the antibody against a crude mouse submaxillary gland extract.
which was performed in agar gels on glass slides (1 x 3 in.) according to techniques described by Arnhum and Wilson (1967).
Indirect NGF immunoprecipitation and analysis on SDS-Tris gels. Aliquots of 0.75 ml of antigen-containing solutions (usually cell supernatants) were incubated with 10 ~1 of rabbit anti-NGF IgG, first at 37°C for 30 min and then at 4°C overnight. After addition of 20 ~1 of goat anti-rabbit IgG, further incubation was performed at 37°C for 30 min and at 4°C for at least 4 hr. The resulting immunoprecipitates were spun down at 18,OOOgin a Sorvall SS-34 rotor, washed twice with PBS, and subsequently dissolved in 20 ~1 of 0.1 N NaOH, 40 ~1 of 0.1 M Tris-HCl, pH 6.8, and 20 ~1 of SDS sample buffer containing 20% P-mercaptoethanol @ME), 0.25 M Tris-HCl, pH 6.8, 8% SDS, and 40% glycerol. Before electrophoresis each sample was heated at 98°C for at least 10 min. Electrophoresis was performed on 15% acrylamide-SDS gels according to the method of Laemmli (1970). The proportions of ingredients used were 3.0 ml of 1.5 M Tris-HCl, pH 9.8, 6.0 ml of 30% (W/V) acrylamide-0.8% bis, 0.12 ml of 10% SDS, 4 ~1 of Temed (N,N,N’N-Tetramethylethylenediane) 0.12 ml of 30 mg/ml of ammonium persulfate, and 2.76 ml of water. Gels were poured to a length of 10 cm. A stacking gel containing the following was used: 1.25 ml of 0.5 M Tris, pH 6.8,0.05 ml of 10% SDS, 0.83 ml of 30% acrylamide-0.8% bis, 2 ~1 of Temed, 75 ~1 of water. For analysis the gels were stained in 0.05% Coomassie blue, 10% (v/v) acetic acid, and
VOLUME 65,1978
25% (v/v) isopropanol; destained in 10% acetic acid, and dried. Gels were then cut into 2-mm pieces; swelled in 100 ~1 of water, incubated overnight in 10 ml of a mixture of 143 ml of NCS, 3 kg of toluene, 15.2 g of Omnifluor; and counted on a Packard scintillation counter. In vitro labeling of mouse fi-NGF. Mouse /3-NGF was labeled in vitro according to a method described by Rice and Means (1971) except that [‘4C]acetaldehyde was substituted for [ “C]formaldehyde. The [14C]acetaldehyde was freshly prepared by the acid hydrolysis of [14C]paraldehyde. In general, the procedure gives about 20% of the maximum labeling expected if each amino group were converted to its dialkylamino derivative. Carboxymethylation of NGF. Two milligrams of mouse submaxillary NGF and labeled rat C6 glioma NGF eluted from SDS gels were dissolved in 2 ml of 6 M guanidineHCl, pH 9.3, and reduced using a 50 M excess of dithiothreitol to protein in a nitrogen atmosphere for 4 hr. After adjusting the protein solution to pH 8.0 with 1 N HCl, a neutral solution containing 200 pmole of iodoacetic acid was added. Alkylation was allowed to proceed for 5 min. The reaction was stopped by the addition of a lo-fold molar excess of 2-mercaptoethanol (Angeletti and Bradshaw, 1971). After dialysis against four changes of deionized water for 24 hr, the NGF was recovered by lyophilization. Tryptic digestion. Tryptic digestion of Scarboxymethyl-NGF was carried out using N”-tosylphenylalanine chloromethyl ketone (TPCK)-treated trypsin (Worthington) added, in a ratio of 1:lOO to NGF on a weight basis, three times over a 12-hr period during incubation at 37°C (Angeletti et al., 1973). The pH was adjusted to 8.8 at each 4-hr interval. The small amount of insoluble material remaining after acidification to pH 2 with 6 N HCl was removed by centrifugation. Peptide analysis. The soluble tryptic peptides were fractionated on a 0.9 X 20-cm
ANGELA M. LONGO
Glioma Nerve Growth Factor Synthesis
column of Aminex A-5 equilibrated with 0.05 N pyridine-acetate (pH 2.5) and eluted with a double linear gradient of pyridine-acetate buffers at 50°C (Bradshaw et al., 1969). The column flowed at a rate of 30 ml/l-u and 4.5-n-J fractions were collected. A 1.5~ml aliquot of each fraction was evaporated in a glass scintillation vial and redissolved in 0.5 ml of water and 7.5 ml of toluene-omnifluor-TritonX-100 (16:1:8) mixture. The remaining 3.0 ml in each fraction was used for protein ninhydrin determination. Determination of peptide concentration
using ninhydrin reagent with alkaline hydrolysis of peptides. Three milliliters of each fraction was dried in a vacuum oven. Then 0.5 ml of 0.5 M NaOH was added and oven dried overnight. This was neutralized with 0.5 ml of 0.5 M acetic acid. Next, the ninhydrin reagent was freshly prepared: 2.0 g of ninhydrin and 0.3 g of hydrindantin were dissolved in 75 ml of methyl Cellosolve (peroxide free); then 25 ml of 4 M sodium acetate buffer (pH 5.5) was added. Of this solution, 0.5 ml was added to each tube which was then covered with aluminum foil and placed in an uncovered boiling water bath for 15 min. After cooling, 1 ml of 50% ethanol was added to each tube with Vortex mixing. The tubes were spun down in an International centrifuge at 1000 rpm for 10 min to remove precipitable material and then carefully decanted into a cuvette and read at 570 nm. RESULTS
Synthesis of NGF by C6 Rat Glioma Cells in Tissue Culture The most direct demonstration of the de novo synthesis of a protein is the incorporation of radioactive amino acids into a purified preparation of the protein. To obtain such a demonstration for NGF we labeled C6 cells in tissue culture with radioactive amino acids, continuously for 23 hr. We then made cell extracts, precipitated NGF with a specific antiserum, and analyzed the immunoprecipitates by SDS gel electrophoresis on 15% polyacrylamide gels
263
as described under Materials and Methods. As seen in Fig. 3A, the procedure employed resulted in the precipitation and resolution of several proteins, one of which comigrated with a 14C-labeled mouse NGF standard. The presence of other peaks of higher molecular weight was confusing at first, since our antibody preparation was monospecific by immunoelectrophoretic analysis. However, a series of control experiments cleared up much of the confusion and allowed tentative identification of the other peaks. An 18,000g supernatant of glioma cell extract was incubated under the same conditions as were used in the indirect immunoprecipitation assay, with the omission of antiNGF serum. A precipitate resulted from this incubation. Analysis of this material, shown in Fig. 3B, demonstrated the presence of self-aggregating proteins. It is likely that the first peak is tubulin. The tendency of this protein to form aggregates at 37°C is well documented (Wiche et al., 1972; Wiche and Cole, 1976), and a sample of purified neuroblastoma tubulin (gift of Dr. G. Wiche) comigrated with this peak. The second high molecular weight peak seen in self-aggregating controls (Wiche et al., 1972) is presumed to be actin, which was seen copurifying with microtubules in some but not all C6 clones (Wiche and Cole, 1976). Because of the high concentration of the heavy and light chains of the antibody, the exact positions of the high molecular weight peaks were variable. Evidence to be presented below suggests that the third peak of approximately 24,000 MW seen in the immunoprecipitate is a precursor to 2.5 S NGF. An additional control consisting of normal rabbit IgG with goat anti-rabbit IgG, incubated under the same conditions as used in the indirect immunoprecipitation assay of Fig. 3A, was observed to be similar to the control shown in Fig. 3B.
Profile of Tryptic Digestion of In Vivo Labeled C6 Rat Glioma NGF and Mouse ,8 NGF In order to confirm that the peak of ra-
264
DEVELOPMENTAL
BIOLOGY
VOLUME 65, 1978
IO GEL SLICE NUMBER FIG. 3. (A) Profile of 15% SDS-Tris gel analysis of an indirect immunoprecipitate obtained from soluble C6 glioma cell extract and anti-NGF as described under Materials and Methods. Cells were continuously labeled for 23 hr with 25% of the normal level of lysine, leucine, and phenylalanine in DME and 5 ,aCi/ml each of ]‘H]lysine, r’H]leucine, and [“Hlphenylalanine in the presence of 5% CO:! atmosphere. Gels were stained, dried, cut, and counted in NCS-toluene-Omnifluor. H refers to the position of the heavy chain (50,000 MW) of the antibodies, and L, to the position of the light chain (25,000 MW). (B) Control: Profile of 15% SDS-Tris gel profile of self-aggregating proteins obtained from 18,OOOgcentrifugation of glioma cell supernatant incubated under the same conditions as were used in the indirect immunoprecipitation assay with the omission of antiNGF serum.
dioactive material which migrated similarly to /?-NGF on polyacrylamide gels represented the rat glioma equivalent to mouse P-NGF, the tryptic peptides of the glioma protein were compared to those of mouse P-NGF by chromatography on Aminex A-5 columns as shown in Fig. 4. The peptides of the mouse /?-NGF were detected by assay for ninhydrin-positive material and the peptides from the putative glioma NGF were labeled with [“Hlphenylalanine, [“HIleucine, and [“Hllysine. The pattern we obtained for mouse NGF is similar to that obtained by other investigators (Angeletti et al., 1973). The pattern obtained for the
rat C6 NGF is very similar with a number of coincident or overlapping peaks. Since the rat C6 NGF was labeled nonuniformly, it would not be expected to have similar proportions of peaks as compared to mouse NGF. Also, since rat NGF may be slightly larger than mouse NGF, we might expect one or two extra peaks, one of which appears around fraction 396. Immunological data we have reported previously show that the rat glioma NGF amino acid sequence appears to differ from that of mouse submaxillary /3-NGF by about lo%, so that the peaks that differ by a few fractions can be explained as manifestations of the small
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DEVELOPMENTAL BIOLOGY
sequence differences 1974).
(Long0 and Penhoet,
Immunological Reactivity of Proteins Isolated from SDS Gel Electrophotograms Radioactive peaks corresponding to pNGF and the next larger peak were cut from an SDS gel and were extracted by shaking in a warm room (37°C) overnight as indicated in Table 1. As designated therein, both the 14,000 MW peak (P-NGF) and the 24,000 MW peak are immunologically related to mouse P-NGF.
Kinetic Evidence for a Precursor to p NGF In a series of experiments TABLE
designed to
1
DIHECT IMMIJNOLOGICAI. REPIIRCIPITATION OF [.‘H]NGF AND ,‘H PHEC~JI
