- Email: [email protected]
A more rapid method for the isolation of the 7S nerve growth factor complex
ANALYTICAL
BIOCHEMISTRY
83, 26-32 (1977)
A More Rapid Method for the Isolation of the 7s Nerve Growth Factor Complex ROBERT W. STACH,BARBARA J. WAGNER,AND BETTIE M. STACH Department
of Biochemistry, 766 Irving
State University of New Avenue, Syracuse, New
York, York
Upstate 13210
Medical
Center,
Received December 9, 1976; accepted June 23, 1977 Nerve growth factor (NGF) is necessary for the growth and development of the sensory and sympathetic nervous systems. The high molecular weight form of NGF, 7s NGF, which is isolated from the male mouse submaxillary glands, is time consuming to isolate. We have designed a different method for the isolation of 7s NGF which is almost nine times more rapid and gives a much higher yield than the old method.
Nerve growth factor (NGF)’ protein is necessary for the growth and development of the sensory and sympathetic nervous system (1,2). The major source of this protein to date is the male mouse submaxillary glands (3). There are two different molecular species which are isolated from this source: (i) the high molecular weight form 7S NGF, which is composed of three different subunits, a, /3, and y, in the ratio of two CYand two y subunits and a /3NGF dimer (4-7); (ii) the low molecular weight pNGF subunit. The PNGF is the only subunit which has NGF activity. In this paper, we discuss a procedure for isolating 7s NGF which is almost nine times as fast and gives a better yield of 7S NGF than the previous procedure (8). METHODS Source of 7s NGF
Male mouse submaxillary glands are extirpated from 67-day-old Swiss Webster mice. The mice are obtained when they are 60 days old and are housed and fed for a week before they are sacrificed. The glands (2026 g) are immediately frozen in liquid nitrogen after extirpation and are stored frozen at -20°C until use. The glands can be stored in this manner for months without any decrease in the yield of 7S NGF. 1 Abbreviations used: NGF, nerve growth factor; 7s NGF, the 7s nerve growth factor complex; PNGF, the p subunit of 7s NGF which is the biologically active subunit; DEAE-cellulose, diethylaminoethyl cellulose; and Tris, tris(hydroxymethyl)aminomethane. 26 Copyright All rights
0 1977 by Academic F’ress. Inc. of reproduction in any form reserved.
ISSN
003-2697
RAPID ISOLATION
Buffers and Column
OF 7s NGF COMPLEX
27
Supplies
The buffers are similar to those used in the original method (8). For the first Sephacryl S-200 chromatography and DEAE-cellulose columns the buffer is 0.05 M Tris-HCl, pH 7.4, at room temperature, containing 0.02% sodium azide. For the second Sephacryl S-200 chromatography, 0.05 M phosphate buffer, pH 6.8, at room temperature, containing 0.02% sodium azide is used. The Tris base was purchased from Schwartz-Mann, Orangeburg, N. Y. The sodium azide was purchased from Fisher Scientific Co., Fair Lawn, N. J. Sephadex G-25 and the Sephacryl S-200 were purchased from Pharmacia Fine Chemicals, Piscataway, N. J. The DEAEcellulose was purchased from Krackeler, Albany, N. Y. and sodium dihydrogen phosphate and disodium hydrogen phosphate were purchased from J. T. Baker, Phillipsburg, N. J. Concentration
Steps
Fractions were concentrated by a modification of the method described by Flodin et al. (9). Concentration steps can be carried out at either 4°C or room temperature. Dry Sephadet G-252 was added to the solution at a rate of 24 g/100 ml with gentle stirring for about 3 min. The thick slurry obtained was then poured into Boerner tubes (A. H. Thomas and Sons, Philadelphia, Pa.) and was centrifuged at 2000 t-pm in an IEC Model CM centrifuge with a 240 head for 10 min. Each tube holds approximately 25 ml of slurry; after centrifugation, there was approximately 10 ml of solution. We have found that Miracloth (Calbiochem, LaJolla, Calif.) is an excellent filter to use in the tubes since the protein recoveries were better using the Miracloth than when using other types of filters. Using this procedure, 350-400 ml of solution can be concentrated to lo- 15 ml in as little as 3 hr. Isolation
of 7s NGF
The following flow chart gives the procedure for the isolation of 7s NGF. Homogenization. Submaxillary glands from 100 mice (20-26 g) were homogenized in 65 ml of cold 0.01 M phosphate buffer, pH 6.8, at room temperature, in a two-speed Waring blender for 90 set at high speed. This solution was centrifuged at 50,000 g for 45 min at 4°C and was filtered through glass wool. The glass wool was washed with a minimum of 0.01 M phosphate buffer, pH 6.8, so that the total volume ‘of the filtrate was not more than 70 ml. Sephacryl S-200 chromatography. The filtrate (65-70 ml) was applied to 2 Sephadex G-25 can be reused after washing and drying according to the instructions given by the manufacturer in Sephadex. Gel Filtration in Theory and Practice, Pharmacia Fine Chemicals, Inc., Piscataway, N. J., pp. 32-33.
28
STACH,
WAGNER,
AND
STACH
the bottom of a Sephacryl S-200 column (5 x 100 cm) equilibrated with the Tris-HCl buffer. The column had an upward flow rate of 168 ml/hr. Approximately 20-ml fractions were collected. The fractions were pooled as indicated (Fig. la, S-200-1 fraction). DEAE-cellulose chromatography. The S-200-1 fraction (approximately 350 ml) was applied to the top of a DEAE-cellulose column (5.2 x 2.5 cm; actual column size was 5.2 x 35 cm; the remaining volume after the column was packed was used to load samples and for buffer changes). The column was equilibrated with the Tris HCl buffer and had a flow rate of approximately 1800 mYhr (a Gilson Minipulse four-channel peristaltic pump, Middleton, Wis. with three of the four channels being used to give this fast flow rate). The column was washed with 700 ml of the Tris-HCl buffer containing 0.01 M NaCl. The 7s NGF was eluted with 600 ml of the Tris-HCl buffer containing 0.08 M NaCl. Approximately 20-ml fractions were collected, and those fractions which had absorptions greater than 0.20 were pooled (DEAE fraction). The DEAE fraction (300-400 ml) was concentrated to approximately 15 ml and was applied to a second Sephacryl S-200 column. Sephacryl S-200 chromatography. The DEAE fraction (approximately 15 ml) was applied to the bottom of a Sephacryl S-200 column (2.6 x 100 cm) which was equilibrated with the 0.05 M phosphate buffer. The flow rate was approximately 100 mYhr, and fractions were approximately 4 ml. The fractions were pooled as indicated (Fig. lb, S-200-2 fraction). The purified 7s NGF was concentrated to approximately 10 ml and was stored at 4°C for a short period of time, or was frozen and stored at -20°C for storage longer than 1 or 2 weeks. Bioassay. The NGF activity was determined using 8- to lo-day-old embryonic chick sensory ganglia in Gey’s balanced salts (10) on collagencoated cover slips as previously described (11). Protein determinations. Protein concentrations were determined using the method of Lowry et al. (12) or by using an Efgl”,“, of 15. Either method gave essentially the same results. In most cases, protein concentrations were determined using the above EzEo value. Isoelectric focusing in polyacrylamide gels. Isoelectric focusing in 7.5% polyacrylamide gels (0.5 x 7 cm) was carried out by a modification of the method described by Stach and Shooter (11). Instead of using riboflavin to polymerize the gels, ammonium persulfate was used. The final concentration of ammonium persulfate was approximately 0.8 mglml, and the gels were allowed to polymerize for 2-3 hr at room temperature before being used. Samples were made 4-7 M in urea with 100-150 pug of the protein being analyzed. Gels were stained by the method of Malik and Berrie (13) and were scanned with a Helena Flur-Vis quick-scan densitometer (Helena Instruments, Beaumont, Tex.) using a 595-nm filter.
RAPID ISOLATION
OF 7s NGF COMPLEX
29
I a.
E ; co
cd
Sephacryl
b.
iii a
1.5
s-200-2
SL?ClO.2~ Frocfio” I
3-
I
C.
:0 FRACTION
NUMBER
FIG. 1 Sephacryl and Sephadex chromatography steps in the isolation of 7S NGF. (a) Sephacryl S-200 chromatography of gland homogenate (see Methods). Dashed lines indicate fractions which were pooled to give the S-200-1 fraction. (b) Sephacryl S-200 chromatography of DEAE fraction, obtained as described under Methods. Dashed lines indicate fractions which were pooled to give the purified 7S NGF. (c) Sephadex G-150 chromatography of DEAE fraction, obtained as described in Ref. (8). Dashed lines indicate fractions which were pooled to give purified 7S NGF.
RESULTS
This new method for isolating 7s NGF is approximately nine times faster, in total hours, than the previous method (Table 1). The first chromatographic step in this new method uses Sephacryl S-200 instead of Sephadex G-100 and is three times faster than the old (Table l), but there is little or no difference in the elution profile, as can be seen (Fig. la). The dashed lines indicate where the sample is pooled to give the S-200-1 fraction. Isoelectric focusing in polyacrylamide gels showed only these fractions to contain any appreciable amount of the 7s NGF complex. Chromatography of this S-200-1 fraction on a DEAE-cellulose column gives the same elution profile as does the previous method (data not shown), even though the flow rate is approximately four times faster (Table 1).
30
STACH,
WAGNER,
AND
TABLE THE
TIME
I T TAKES
1
7S NGF
T O ISOLATE
COMPARED
T O THE
Method NW Original
b
0.025 0.025
Centrifugation I I
Lyophiliz&ion 0 16
BY THE
ORIGINAL
Duration
Homogenization
STACH
s-200- I or G-100~
NEW
METHOD
AS
METHOD
of each step (hr) DEAEcehlose
5 I5
1 4
Concentration 3 48
s-20&2 cl1 G-150’ 3 23
CONWl-
tration
Total
1.5 24
14.5 131
a In the original method (8) Sephadex G-100 and G-150 were used ’ &pared as described by Vuon er a[. (8).
Another point of departure from the original method is the second Sephacryl S-200 chromatographic step. In the original method, Sephadex G-150 chromatography was used as a final purification step (8). Usually the elution profile for this column showed two peaks (Fig. lc); sometimes, a shoulder as the start of a third peak could also be seen (arrow) and the column would take almost 24 hr to develop (Table 1). However, with Sephacry1 S-200 the time it takes to develop the column is cut by almost a factor of eight, and the separation seems to be much better (Fig. lb). The sample from the second Sephacryl S-200 column is pooled as indicated (Fig. lb) and is concentrated to give the purified 7s NGF. The concentration steps are the places where the most change has occurred. These steps can be carried out at either 4°C or room temperature. When concentration is carried out at 4°C the protein recovery is slightly greater than when the concentration is carried out at room temperature (85-95+% recovery as compared to 80-90% recovery, respectively). However, how the concentration steps are carried out makes no difference in the amount of purified 7s NGF obtained, whether at 4°C or room temperature. Figure 2, a and b, compares the 7s NGF isolated by this new procedure to that isolated by the old procedure as analyzed on isoelectric focusing gels. It can be seen that there is very little, if any, difference in the two 7s NGFs with respect to isoelectric focusing gels. This is also true with respect to the biological activity of the 7s NGF isolated by this new method as compared to 7s NGF isolated by the old method; both have the same biological activity. An interesting side effect of this new method was observed. It was noticed that the quantity of protein from the DEAE-cellulose chromatography was 1.5-2 times as much as when the old method was used. This increase in the amount of protein was still present after concentration and also after chromatography on the second Sephacryl S-200 column. We have been able to obtain as much as 90 mg of purified 7s NGF from approximately 26 g of submaxillary glands, as compared to approximately 45 mg
RAPID ISOLATION
31
OF 7s NGF COMPLEX
MIGRATION
( cm)
w
FIG. 2. Analysis of purified 7s NGF in isoelectric focusing polyacrylamide gels. Gels were run for 1 hr at 100 and 200 V until the cytochrome c standard ran offthe end of the gel (approximately 2.5 hr); migration is from left to right. Gels were stained and scanned as described under Methods. (a) Purified 7s NGF as isolated by this new method. (b) Purified 7s NGF as isolated by the old method(l).
from the same weight of glands using the old method of isolation. However, usual yields are in the range of 70-80 mg of the purified 7s NGF. DISCUSSION
We have demonstrated that it is possible to isolate 7s NGF much more rapidly than had been done previously. The time it takes to isolate 7s NGF has been cut by almost a factor of nine, and the yield has been increased by almost a factor of two. This faster isolation method has no deleterious effect on the composition or the biological activity of the 7s NGF or on the biological activity of its P-subunit when it is isolated from the 7s NGF complex. ACKNOWLEDMENTS This work was supported by USPHS Grant No. NS 12325 and General Research Support Grant No. RR05402. We wish to thank Hester A. McFadden for excellent assistance by doing the bioassay. We also wish to thank the graduate students who took the Biochemical Methods course and verified the results presented here.
32
STACH, WAGNER,
AND STACH
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Levi-Montalcini, R., and Angeletti, P. U. (1968) Phys. Rev. 48, 534-569. Herrup, K., Stickgold, R., and Shooter, E. M. (1974)Ann. N. Y. Acad. Sci. 228,381-392. Cohen, S. (1960) Proc. Nat. Acad. Sci. USA 46, 302-311. Stach, R. W., and Shooter, E. M., manuscript in preparation. Stach, R. W., Server, A. C., Pignatti, P. F., Piltch, A., and Shooter, E. M. (1976) Biochemistry 15, 1455-1461. Stach, R. W., and Shooter, E. M., manuscript in preparations. Varon, S., Nomura, .I., and Shooter, E. M. (1968) Biochemistry 7, 1296-1303. Varon, S., Nomura, J., and Shooter, E. M. (1967) Biochemistry 6, 2202-2209. Flodin, P., Gelotte, B., and Porath, J. (1960) Nature (London) 188, 493-494. Gey, G. O., and Gey, M. K. (1936) Amer. J. Cancer 27, 45-76. Stach, R. W., and Shooter, E. M. (1974) .I. Biol. Chem. 249, 6668-6674. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951)J. Biol. Chem. 193, 265-275. Malik, N., and Berrie, A. (1972) Anal. Biochem. 49, 173-176.
BIOCHEMISTRY
83, 26-32 (1977)
A More Rapid Method for the Isolation of the 7s Nerve Growth Factor Complex ROBERT W. STACH,BARBARA J. WAGNER,AND BETTIE M. STACH Department
of Biochemistry, 766 Irving
State University of New Avenue, Syracuse, New
York, York
Upstate 13210
Medical
Center,
Received December 9, 1976; accepted June 23, 1977 Nerve growth factor (NGF) is necessary for the growth and development of the sensory and sympathetic nervous systems. The high molecular weight form of NGF, 7s NGF, which is isolated from the male mouse submaxillary glands, is time consuming to isolate. We have designed a different method for the isolation of 7s NGF which is almost nine times more rapid and gives a much higher yield than the old method.
Nerve growth factor (NGF)’ protein is necessary for the growth and development of the sensory and sympathetic nervous system (1,2). The major source of this protein to date is the male mouse submaxillary glands (3). There are two different molecular species which are isolated from this source: (i) the high molecular weight form 7S NGF, which is composed of three different subunits, a, /3, and y, in the ratio of two CYand two y subunits and a /3NGF dimer (4-7); (ii) the low molecular weight pNGF subunit. The PNGF is the only subunit which has NGF activity. In this paper, we discuss a procedure for isolating 7s NGF which is almost nine times as fast and gives a better yield of 7S NGF than the previous procedure (8). METHODS Source of 7s NGF
Male mouse submaxillary glands are extirpated from 67-day-old Swiss Webster mice. The mice are obtained when they are 60 days old and are housed and fed for a week before they are sacrificed. The glands (2026 g) are immediately frozen in liquid nitrogen after extirpation and are stored frozen at -20°C until use. The glands can be stored in this manner for months without any decrease in the yield of 7S NGF. 1 Abbreviations used: NGF, nerve growth factor; 7s NGF, the 7s nerve growth factor complex; PNGF, the p subunit of 7s NGF which is the biologically active subunit; DEAE-cellulose, diethylaminoethyl cellulose; and Tris, tris(hydroxymethyl)aminomethane. 26 Copyright All rights
0 1977 by Academic F’ress. Inc. of reproduction in any form reserved.
ISSN
003-2697
RAPID ISOLATION
Buffers and Column
OF 7s NGF COMPLEX
27
Supplies
The buffers are similar to those used in the original method (8). For the first Sephacryl S-200 chromatography and DEAE-cellulose columns the buffer is 0.05 M Tris-HCl, pH 7.4, at room temperature, containing 0.02% sodium azide. For the second Sephacryl S-200 chromatography, 0.05 M phosphate buffer, pH 6.8, at room temperature, containing 0.02% sodium azide is used. The Tris base was purchased from Schwartz-Mann, Orangeburg, N. Y. The sodium azide was purchased from Fisher Scientific Co., Fair Lawn, N. J. Sephadex G-25 and the Sephacryl S-200 were purchased from Pharmacia Fine Chemicals, Piscataway, N. J. The DEAEcellulose was purchased from Krackeler, Albany, N. Y. and sodium dihydrogen phosphate and disodium hydrogen phosphate were purchased from J. T. Baker, Phillipsburg, N. J. Concentration
Steps
Fractions were concentrated by a modification of the method described by Flodin et al. (9). Concentration steps can be carried out at either 4°C or room temperature. Dry Sephadet G-252 was added to the solution at a rate of 24 g/100 ml with gentle stirring for about 3 min. The thick slurry obtained was then poured into Boerner tubes (A. H. Thomas and Sons, Philadelphia, Pa.) and was centrifuged at 2000 t-pm in an IEC Model CM centrifuge with a 240 head for 10 min. Each tube holds approximately 25 ml of slurry; after centrifugation, there was approximately 10 ml of solution. We have found that Miracloth (Calbiochem, LaJolla, Calif.) is an excellent filter to use in the tubes since the protein recoveries were better using the Miracloth than when using other types of filters. Using this procedure, 350-400 ml of solution can be concentrated to lo- 15 ml in as little as 3 hr. Isolation
of 7s NGF
The following flow chart gives the procedure for the isolation of 7s NGF. Homogenization. Submaxillary glands from 100 mice (20-26 g) were homogenized in 65 ml of cold 0.01 M phosphate buffer, pH 6.8, at room temperature, in a two-speed Waring blender for 90 set at high speed. This solution was centrifuged at 50,000 g for 45 min at 4°C and was filtered through glass wool. The glass wool was washed with a minimum of 0.01 M phosphate buffer, pH 6.8, so that the total volume ‘of the filtrate was not more than 70 ml. Sephacryl S-200 chromatography. The filtrate (65-70 ml) was applied to 2 Sephadex G-25 can be reused after washing and drying according to the instructions given by the manufacturer in Sephadex. Gel Filtration in Theory and Practice, Pharmacia Fine Chemicals, Inc., Piscataway, N. J., pp. 32-33.
28
STACH,
WAGNER,
AND
STACH
the bottom of a Sephacryl S-200 column (5 x 100 cm) equilibrated with the Tris-HCl buffer. The column had an upward flow rate of 168 ml/hr. Approximately 20-ml fractions were collected. The fractions were pooled as indicated (Fig. la, S-200-1 fraction). DEAE-cellulose chromatography. The S-200-1 fraction (approximately 350 ml) was applied to the top of a DEAE-cellulose column (5.2 x 2.5 cm; actual column size was 5.2 x 35 cm; the remaining volume after the column was packed was used to load samples and for buffer changes). The column was equilibrated with the Tris HCl buffer and had a flow rate of approximately 1800 mYhr (a Gilson Minipulse four-channel peristaltic pump, Middleton, Wis. with three of the four channels being used to give this fast flow rate). The column was washed with 700 ml of the Tris-HCl buffer containing 0.01 M NaCl. The 7s NGF was eluted with 600 ml of the Tris-HCl buffer containing 0.08 M NaCl. Approximately 20-ml fractions were collected, and those fractions which had absorptions greater than 0.20 were pooled (DEAE fraction). The DEAE fraction (300-400 ml) was concentrated to approximately 15 ml and was applied to a second Sephacryl S-200 column. Sephacryl S-200 chromatography. The DEAE fraction (approximately 15 ml) was applied to the bottom of a Sephacryl S-200 column (2.6 x 100 cm) which was equilibrated with the 0.05 M phosphate buffer. The flow rate was approximately 100 mYhr, and fractions were approximately 4 ml. The fractions were pooled as indicated (Fig. lb, S-200-2 fraction). The purified 7s NGF was concentrated to approximately 10 ml and was stored at 4°C for a short period of time, or was frozen and stored at -20°C for storage longer than 1 or 2 weeks. Bioassay. The NGF activity was determined using 8- to lo-day-old embryonic chick sensory ganglia in Gey’s balanced salts (10) on collagencoated cover slips as previously described (11). Protein determinations. Protein concentrations were determined using the method of Lowry et al. (12) or by using an Efgl”,“, of 15. Either method gave essentially the same results. In most cases, protein concentrations were determined using the above EzEo value. Isoelectric focusing in polyacrylamide gels. Isoelectric focusing in 7.5% polyacrylamide gels (0.5 x 7 cm) was carried out by a modification of the method described by Stach and Shooter (11). Instead of using riboflavin to polymerize the gels, ammonium persulfate was used. The final concentration of ammonium persulfate was approximately 0.8 mglml, and the gels were allowed to polymerize for 2-3 hr at room temperature before being used. Samples were made 4-7 M in urea with 100-150 pug of the protein being analyzed. Gels were stained by the method of Malik and Berrie (13) and were scanned with a Helena Flur-Vis quick-scan densitometer (Helena Instruments, Beaumont, Tex.) using a 595-nm filter.
RAPID ISOLATION
OF 7s NGF COMPLEX
29
I a.
E ; co
cd
Sephacryl
b.
iii a
1.5
s-200-2
SL?ClO.2~ Frocfio” I
3-
I
C.
:0 FRACTION
NUMBER
FIG. 1 Sephacryl and Sephadex chromatography steps in the isolation of 7S NGF. (a) Sephacryl S-200 chromatography of gland homogenate (see Methods). Dashed lines indicate fractions which were pooled to give the S-200-1 fraction. (b) Sephacryl S-200 chromatography of DEAE fraction, obtained as described under Methods. Dashed lines indicate fractions which were pooled to give the purified 7S NGF. (c) Sephadex G-150 chromatography of DEAE fraction, obtained as described in Ref. (8). Dashed lines indicate fractions which were pooled to give purified 7S NGF.
RESULTS
This new method for isolating 7s NGF is approximately nine times faster, in total hours, than the previous method (Table 1). The first chromatographic step in this new method uses Sephacryl S-200 instead of Sephadex G-100 and is three times faster than the old (Table l), but there is little or no difference in the elution profile, as can be seen (Fig. la). The dashed lines indicate where the sample is pooled to give the S-200-1 fraction. Isoelectric focusing in polyacrylamide gels showed only these fractions to contain any appreciable amount of the 7s NGF complex. Chromatography of this S-200-1 fraction on a DEAE-cellulose column gives the same elution profile as does the previous method (data not shown), even though the flow rate is approximately four times faster (Table 1).
30
STACH,
WAGNER,
AND
TABLE THE
TIME
I T TAKES
1
7S NGF
T O ISOLATE
COMPARED
T O THE
Method NW Original
b
0.025 0.025
Centrifugation I I
Lyophiliz&ion 0 16
BY THE
ORIGINAL
Duration
Homogenization
STACH
s-200- I or G-100~
NEW
METHOD
AS
METHOD
of each step (hr) DEAEcehlose
5 I5
1 4
Concentration 3 48
s-20&2 cl1 G-150’ 3 23
CONWl-
tration
Total
1.5 24
14.5 131
a In the original method (8) Sephadex G-100 and G-150 were used ’ &pared as described by Vuon er a[. (8).
Another point of departure from the original method is the second Sephacryl S-200 chromatographic step. In the original method, Sephadex G-150 chromatography was used as a final purification step (8). Usually the elution profile for this column showed two peaks (Fig. lc); sometimes, a shoulder as the start of a third peak could also be seen (arrow) and the column would take almost 24 hr to develop (Table 1). However, with Sephacry1 S-200 the time it takes to develop the column is cut by almost a factor of eight, and the separation seems to be much better (Fig. lb). The sample from the second Sephacryl S-200 column is pooled as indicated (Fig. lb) and is concentrated to give the purified 7s NGF. The concentration steps are the places where the most change has occurred. These steps can be carried out at either 4°C or room temperature. When concentration is carried out at 4°C the protein recovery is slightly greater than when the concentration is carried out at room temperature (85-95+% recovery as compared to 80-90% recovery, respectively). However, how the concentration steps are carried out makes no difference in the amount of purified 7s NGF obtained, whether at 4°C or room temperature. Figure 2, a and b, compares the 7s NGF isolated by this new procedure to that isolated by the old procedure as analyzed on isoelectric focusing gels. It can be seen that there is very little, if any, difference in the two 7s NGFs with respect to isoelectric focusing gels. This is also true with respect to the biological activity of the 7s NGF isolated by this new method as compared to 7s NGF isolated by the old method; both have the same biological activity. An interesting side effect of this new method was observed. It was noticed that the quantity of protein from the DEAE-cellulose chromatography was 1.5-2 times as much as when the old method was used. This increase in the amount of protein was still present after concentration and also after chromatography on the second Sephacryl S-200 column. We have been able to obtain as much as 90 mg of purified 7s NGF from approximately 26 g of submaxillary glands, as compared to approximately 45 mg
RAPID ISOLATION
31
OF 7s NGF COMPLEX
MIGRATION
( cm)
w
FIG. 2. Analysis of purified 7s NGF in isoelectric focusing polyacrylamide gels. Gels were run for 1 hr at 100 and 200 V until the cytochrome c standard ran offthe end of the gel (approximately 2.5 hr); migration is from left to right. Gels were stained and scanned as described under Methods. (a) Purified 7s NGF as isolated by this new method. (b) Purified 7s NGF as isolated by the old method(l).
from the same weight of glands using the old method of isolation. However, usual yields are in the range of 70-80 mg of the purified 7s NGF. DISCUSSION
We have demonstrated that it is possible to isolate 7s NGF much more rapidly than had been done previously. The time it takes to isolate 7s NGF has been cut by almost a factor of nine, and the yield has been increased by almost a factor of two. This faster isolation method has no deleterious effect on the composition or the biological activity of the 7s NGF or on the biological activity of its P-subunit when it is isolated from the 7s NGF complex. ACKNOWLEDMENTS This work was supported by USPHS Grant No. NS 12325 and General Research Support Grant No. RR05402. We wish to thank Hester A. McFadden for excellent assistance by doing the bioassay. We also wish to thank the graduate students who took the Biochemical Methods course and verified the results presented here.
32
STACH, WAGNER,
AND STACH
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Levi-Montalcini, R., and Angeletti, P. U. (1968) Phys. Rev. 48, 534-569. Herrup, K., Stickgold, R., and Shooter, E. M. (1974)Ann. N. Y. Acad. Sci. 228,381-392. Cohen, S. (1960) Proc. Nat. Acad. Sci. USA 46, 302-311. Stach, R. W., and Shooter, E. M., manuscript in preparation. Stach, R. W., Server, A. C., Pignatti, P. F., Piltch, A., and Shooter, E. M. (1976) Biochemistry 15, 1455-1461. Stach, R. W., and Shooter, E. M., manuscript in preparations. Varon, S., Nomura, .I., and Shooter, E. M. (1968) Biochemistry 7, 1296-1303. Varon, S., Nomura, J., and Shooter, E. M. (1967) Biochemistry 6, 2202-2209. Flodin, P., Gelotte, B., and Porath, J. (1960) Nature (London) 188, 493-494. Gey, G. O., and Gey, M. K. (1936) Amer. J. Cancer 27, 45-76. Stach, R. W., and Shooter, E. M. (1974) .I. Biol. Chem. 249, 6668-6674. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951)J. Biol. Chem. 193, 265-275. Malik, N., and Berrie, A. (1972) Anal. Biochem. 49, 173-176.