Biochimica et Biophysica Acta, 405 (1975) 23-26 © Elsevier Scientific Publishing Company, Amsterdam- Printed in The Netherlands BBA 37141 PROPERTIES OF N E R V E G R O W T H F A C T O R F R O M T H E VENOM OF B O T H R O P S ATI~OX
ROBERT E. GLASS* and DEREK V. BANTHORPE Chrktopher lngold Laboratories, Department of Chemistry, University College, London (U.K.) (Received March 19th, 1975)
SUMMARY A glycoprotein fraction isolated in poor yield (approx. 0.04 ~o) from the venom of Bothrops atrox contained nerve growth factor. The material had biological activity, stability, the property of anomalous adsorption onto surfaces, apparent molecular weight and sub-unit structure that were similar to those for nerve growth factor that had been previously purified from the venom of Vipera russelli. Antiserum raised against nerve growth factor from Vipera russelli showed definite cross-Ieactivity with either the material from Bothrops atrox or a nerve growth factor-containing fraction from the venom of Ancistrodon piscivorus piscivorus, while antiserum against r~erve growth factor from mouse had little effect on any of these preparations.
Nerve growth factor is the generic name for a group of proteins that have very similar stimulatory effects both in vivo and in vitro on embryonic and some neonate sensory and sympathetic ganglia [1, 2]. At present, the feasible sources for purification of the factor are mouse submaxillary gland [3-7] and certain snake venoms [8-13]. Although highly purified nerve growth factor from Vipera russelli and mouse (7-S species) evokes closely similar detailed biological responses in the embryonic chick (manuscript in preparation) their chemical [14] and antigenic [15] properties differ widely, and the former material also shows anomalous adsorption properties. We have studied the factor isolated from the readily accessible and relatively cheap venom of Bothrops atrox (Fer de Lance). It has become apparent that the venom contains too low a proportion of nerve growth factor for it to be a feasible source for the isolation of pure material. However, characterisation of our purified fraction has helped to assess the generality of properties of nerve growth factor as well as indicating a possible common subunit structure. Nerve growth factor was purified from the venom of B. atrox through three steps (Table I). The overall yield was only 0.04 ~o. Nerve growth factor is known [9] to be unexpectedly difficult to purify from snake venoms. Heterogeneity may be due to variations or positional isomerism of the carbohydrate residues [16], intraspecies variation, and modification of nerve growth factor by enzymes present in the venom. * To whom correspondence should be addressed at the Department of Molecular Biology, University of Edinburgh, Edinburgh EH9 3JR. U.K.
24 TABLE I PURIFICATION OF NERVE GROWTH FACTOR FROM VENOM OF B. ATROX All operations were carried out at 4 °C. Nerve growth factor was assayed in tissue culture using sensory ganglia of 8-day-old chick embryos [22]. Step 1. B. atrox venom (Sigma Chemical Co. Ltd; 10 g per batch, two batches) was dissolved in 0.025 M Tricine buffer, pH 8.5 (55 ml) and dialyzed overnighl against the same buffer. The insoluble material was removed by centrifugation. After washing the pellet, the combined supernatants (100 ml) were applied to a column (100 x 5 cm) of QAE A-25 Sephadex. Nerve growth factor activity was not absorbed and was eluted with the starting buffer (Fraction I). Step 2. Fraction I (approx. 1.5 g per batch, two batches) in 0.5 M NaC1 (25 ml) was applied to a column (100 × 5 cm) of Sephadex G-100. Nerve growth factor activity (Fraction II) was identified (molecular weight range from 28 000 to 42 000, maximum activity at 35 000). Step 3. Fraction II in 0.075 M Tricine buffer, pH 8.5 (25 ml) was dialyzed overnight against the buffer and applied to a column (75 × 3.4 cm) of SP C-25 Sephadex. Nerve growth factor activity (Fraction III) was eluted with a parabolic salt gradient (1 M NaC1 in starting buffer; 250 ml closed mixing vessel). Step
Fraction
Weight (g)
Recovery of* protein ( ~ )
Activity** level ~g/ml)
1 2 3
Crude venom I, ex QAE A-25 Sephadex II, ex Sephadex G-100 III, ex SP C-25 Sephadex
20.0 2.86 0.54 0.008
66 63 69
10 I 1 0.2 to 0.06***
* Due to the nature of the assay it is not possible to express the results in terms of recovery of biological activity. ** Concentration required to give the standard response [22] in vitro. *** Glass or polystyrene pipettes used in the multiple-pipette method of assay, respectively. T h e purified active fraction c o m p r i s e d two m a i n c o m p o n e n t s as i n d i c a t e d by p o l y a c r y l a m i d e gel electrophoresis at p H 9.5 [17]; these c o m p o n e n t s were glycoproteins, as i n d i c a t e d by the p e r i o d a t e - S c h i f f ' s reagent [18] a n d c o n t a i n e d a p p r o x . 12 (w/w) c a r b o h y d r a t e (gas-liquid c h r o m a t o g r a p h y o f trimethylsilyl derivatives [19]) m a d e u p o f N - a c e t y l g l u c o s a m i n e (6 ~ ) , glucose (3.4 ~ ) , m a n n o s e (2.5 ~o) a n d galactose (0.1 ~ ) . H i g h l y purified nerve g r o w t h f a c t o r f r o m V. russelli is also r e p o r t e d [9] to be a g l y c o p r o t e i n . T h e isoelectric p o i n t s (pI 8.0 a n d 9.0) o f these c a r b o h y d r a t e staining b a n d s on p o l y a c r y l a m i d e gel electrofocussing [9] i n d i c a t e d basic species similar to those o f nerve g r o w t h factor f r o m V. russelli a n d m o u s e (10.5 a n d 9.3, respectively) b u t different to t h a t o f nerve g r o w t h f a c t o r i s o l a t e d f r o m Naja naja (6.8) (cf. ref. 14). The m o l e c u l a r weight o f nerve g r o w t h factor-active m a t e r i a l f r o m B. atrox was a p p r o x . 35 000 as m e a s u r e d on a c a l i b r a t e d S e p h a d e x c o l u m n . T h o u g h glycop r o t e i n s usually b e h a v e a n o m a l o u s l y in gel filtration [20], a m o l e c u l a r weight o f 37 000 for V. russelli nerve g r o w t h f a c t o r was o b t a i n e d f r o m either gel filtration o r u l t r a c e n t r i f u g a t i o n studies [9]. The p o s s i b l e occurrence o f s u b - u n i t structure was exp l o r e d using p o l y a c r y l a m i d e gel electrophoresis in s o d i u m d o d e c y l s u l p h a t e [21]. The active f r a c t i o n f r o m B. atrox c o n t a i n e d a m a j o r species o f m o l e c u l a r weight o f a p p r o x . 19 000. In c o m p a r i s o n , m o u s e 7-S nerve g r o w t h f a c t o r t h a t is k n o w n [7] to c o n t a i n sub-units o f a p p r o x . 13 000 gave e l e c t r o p h o r e t i c b a n d s c o r r e s p o n d i n g to species o f m o l e c u l a r weight o f a p p r o x . 12 000, 16 000 and 26 000 u n d e r the same conditions. H i g h l y purified nerve g r o w t h factor f r o m V. russelli t h a t h a d been considered to
25 be a single species o f a b o u t 37 000 gave major bands o f approx. 18 000 and 42 000. These results certainly suggest that nerve growth factor f r o m snake venom, like that f r o m mouse, contains sub-units (cf. ref. 13). The purified fraction evoked the standard response in tissue culture at 2.10 - 7 6.10 -8 g/ml, depending on whether glass or plastic pipettes were used in the m o s t reliable m e t h o d [22] of assay. This activity, in its lowest range, is similar to that reported for nerve growth factor f r o m v e n o m o f Ancistrodon piscivorus piscivorus [10, 11] but is 5-20-fold less than that o f highly purified material from mouse or V. russelli [22]. The dose response characteristics were similar to those of nerve growth factor f r o m the latter source [22] in that use o f the single-pipette method o f assay gave spurious results p r o b a b l y due to a n o m a l o u s adsorption effects. Such effects were not found for mouse nerve growth factor. The stability o f the biological activity o f our fraction closely resembled that o f nerve growth factor f r o m V. russelli [9] and whereas the activity o f the fraction was unaltered by incubation with crude v e n o m f r o m B. atrox, similar treatment destroyed most nerve growth factor activity in the 7-S material from mouse. The biological activity o f the active fractions f r o m venoms of both B. atrox and A. piscivorus were either unaffected or were blocked by only low dilutions o f the antiserum to nerve growth factor from mouse, whereas the antiserum raised against nerve growth factor f r o m V. russelli destroyed the activity at high dilutions (cross-reactivity o f 15-60 ~o) (Table II). This lack o f cross-over between antigens and antisera f r o m different species seems, therefore, to be a general phenomenon, (see also ref. 15).
TABLE II IMMUNOLOGICAL RELATIONSHIP BETWEEN NERVE GROWTH FACTOR FROM VARIOUS SOURCES Antiserum was diluted with rabbit serum and the resulting solutions assayed using explanated sensory ganglia in a medium consisting of medium No. 199 (Wellcome Reagents Ltd), antiserum solution, and nerve growth factor solution (1 : 1 : 1, v/v). One biological unit of activity, as defined as that concentration of nerve growth factor required to produce a standard response of 2.5 on an arbitrary, semi-quantitative scale of 0 to 5 [22], was used in all cases. Antisera were gifts from Dr D. C. Edwards (Burroughs-Wellcome labs, Beckingham, Kent). A purified fraction containing nerve growth factor activity was isolated [11] from the venom of A. piscivorus (Sigma Chemical Co. Ltd) by A. R. Berry (U.C.L.). Antigen nerve growth factor (NGF)
Reciprocal dilution of antibody titre Antiserum to mouse 7-S nerve growth factor
Antiserum to V. russelli nerve growth factor
Mouse 7-S
NGF V. russelli NGF B. atrox NGF A. piscivorus NGF
1000
2
1
600
No effect
60-120
1
200-500
26 The a n o m a l o u s a b s o r p t i o n properties of snake v e n o m nerve growth factor have been attributed to the presence of the c a r b o h y d r a t e moiety [22]. This moiety, which m a y have a protective function, might also be the cause of the lack of antigenic cross-reactivity. However, the presence of the carbohydrate moiety a p p a r e n t l y has no effect o n the biological action of nerve growth factor; the response in tissue culture of whole sensory ganglia to nerve growth factor from V. russelli, B. atrox or mouse were indistinguishable when the same n u m b e r of biological units of each material was tested. O u r present results indicate that a l t h o u g h nerve growth factor from differing sources shows very similar biological effects a n d p r o b a b l e similarities in s u b - u n i t structure, there are p r o f o u n d i m m u n o l o g i c a l differences. These differences may largely reflect the absence or variation to the c a r b o h y d r a t e moiety. ACKNOWLEDGEMENT
We thank the Science Research Council for a grant to R.E.G. (1969-1972). REFERENCES 1 Levi-Montalcini, R. and Angeletti, P. U. (1968) Physiol. Rev. 48, 534-569 2 Zaimis, E. and Knight, J. (1972) Nerve Growth Factor and its Antiserum, p. 273, Athlone Press, London 3 Varon, S., Nomura, J. and Shooter, E. M. (1968) Biochemistry 7, 1296-1303 4 Smith, A. P., Varon, S. and Shooter, E. M. (1968) Biochemistry 7, 3259-3268 5 Levi-Montalcini, R., Tentori, L., Vivaldi, G., Angeletti, P. U. and Morini-Betiolo, G. B. (1965) Gazz. Chim. Ital. 95, 333-337 6 Bocchini, V. and Angeletti, P. U. (1969) Prec. Natl. Acad. Sci. U.S. 64, 787-794 7 Angeletti, R. H. and Bradshaw, R. A. (1971) Prec. Natl. Acad. Sci. U.S. 68, 2417-2420 8 Angeletti, R. H. (1970) Prec. Natl. Acad. Sci. U.S. 65, 668-674 9 Pearce, F. L., Banks, B. E. C., Banthorpe, D. V., Berry, A. R., Davies, H. ft. S. and Vernon, C. A. (1972) Eur. J. Biochem. 29, 417--425 10 Cohen, S. (1959) J. Biol. Chem. 234, 1129-1137 11 Banks, B. E. C., Banthorpe, D. V., Berry, A. R., Davies, H. ft. S., Doonan, S., Lament, D. M., Shipolini, R. and Vernon, C. A. (1968) Biochem. J. 108, 157-158 12 Angeletti, P. U., Calissaro, P., Chen, J. S. and Levi-Montalcini, R. (1967) Biochim. Biophys. Acta 147, 180-182 13 Angeletti, R. H. (1968) J. Chromatogr. 37, 62-69 14 Pearce, F. L., Banks, B. E. C., Banthorpe, D. V., Berry, A. R., Davies, H. ft. S. and Vernon, C. A. (1972) Nerve Growth Factor and its Antiserum, pp. 3-18, Athlone Press, London 15 Banks, B. E. C., Banthorpe, D. V., Charlwood, K. A., Pearce, F. L., Vernon, C. A. and Edwards, D. C. (1973) Nature 246, 503-504 16 Spire, R. G. (1970) Annu. Rev. Biochem. 39, 599-638 17 Hjerten, S., Jerstedt, S. and Tiselius, A. (1965) Anal. Biochem. 11, 219-232 18 Clarke, J. T. (1964) Ann. N.Y. Acad. Sci. 121, 428-433 19 Bolton, C. H., Clamp, J. R. and Hough, L. (1965) Biochem. J. 96, 5c 20 Andrews, P. (1965) Biochem. J. 96, 595-606 21 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406--4412 22 Pearce, F. L., Banthorpe, D. V., Cook, J. M. and Vernon, C. A. (1973) Eur. J. Biochem. 32. 569575
ROBERT E. GLASS* and DEREK V. BANTHORPE Chrktopher lngold Laboratories, Department of Chemistry, University College, London (U.K.) (Received March 19th, 1975)
SUMMARY A glycoprotein fraction isolated in poor yield (approx. 0.04 ~o) from the venom of Bothrops atrox contained nerve growth factor. The material had biological activity, stability, the property of anomalous adsorption onto surfaces, apparent molecular weight and sub-unit structure that were similar to those for nerve growth factor that had been previously purified from the venom of Vipera russelli. Antiserum raised against nerve growth factor from Vipera russelli showed definite cross-Ieactivity with either the material from Bothrops atrox or a nerve growth factor-containing fraction from the venom of Ancistrodon piscivorus piscivorus, while antiserum against r~erve growth factor from mouse had little effect on any of these preparations.
Nerve growth factor is the generic name for a group of proteins that have very similar stimulatory effects both in vivo and in vitro on embryonic and some neonate sensory and sympathetic ganglia [1, 2]. At present, the feasible sources for purification of the factor are mouse submaxillary gland [3-7] and certain snake venoms [8-13]. Although highly purified nerve growth factor from Vipera russelli and mouse (7-S species) evokes closely similar detailed biological responses in the embryonic chick (manuscript in preparation) their chemical [14] and antigenic [15] properties differ widely, and the former material also shows anomalous adsorption properties. We have studied the factor isolated from the readily accessible and relatively cheap venom of Bothrops atrox (Fer de Lance). It has become apparent that the venom contains too low a proportion of nerve growth factor for it to be a feasible source for the isolation of pure material. However, characterisation of our purified fraction has helped to assess the generality of properties of nerve growth factor as well as indicating a possible common subunit structure. Nerve growth factor was purified from the venom of B. atrox through three steps (Table I). The overall yield was only 0.04 ~o. Nerve growth factor is known [9] to be unexpectedly difficult to purify from snake venoms. Heterogeneity may be due to variations or positional isomerism of the carbohydrate residues [16], intraspecies variation, and modification of nerve growth factor by enzymes present in the venom. * To whom correspondence should be addressed at the Department of Molecular Biology, University of Edinburgh, Edinburgh EH9 3JR. U.K.
24 TABLE I PURIFICATION OF NERVE GROWTH FACTOR FROM VENOM OF B. ATROX All operations were carried out at 4 °C. Nerve growth factor was assayed in tissue culture using sensory ganglia of 8-day-old chick embryos [22]. Step 1. B. atrox venom (Sigma Chemical Co. Ltd; 10 g per batch, two batches) was dissolved in 0.025 M Tricine buffer, pH 8.5 (55 ml) and dialyzed overnighl against the same buffer. The insoluble material was removed by centrifugation. After washing the pellet, the combined supernatants (100 ml) were applied to a column (100 x 5 cm) of QAE A-25 Sephadex. Nerve growth factor activity was not absorbed and was eluted with the starting buffer (Fraction I). Step 2. Fraction I (approx. 1.5 g per batch, two batches) in 0.5 M NaC1 (25 ml) was applied to a column (100 × 5 cm) of Sephadex G-100. Nerve growth factor activity (Fraction II) was identified (molecular weight range from 28 000 to 42 000, maximum activity at 35 000). Step 3. Fraction II in 0.075 M Tricine buffer, pH 8.5 (25 ml) was dialyzed overnight against the buffer and applied to a column (75 × 3.4 cm) of SP C-25 Sephadex. Nerve growth factor activity (Fraction III) was eluted with a parabolic salt gradient (1 M NaC1 in starting buffer; 250 ml closed mixing vessel). Step
Fraction
Weight (g)
Recovery of* protein ( ~ )
Activity** level ~g/ml)
1 2 3
Crude venom I, ex QAE A-25 Sephadex II, ex Sephadex G-100 III, ex SP C-25 Sephadex
20.0 2.86 0.54 0.008
66 63 69
10 I 1 0.2 to 0.06***
* Due to the nature of the assay it is not possible to express the results in terms of recovery of biological activity. ** Concentration required to give the standard response [22] in vitro. *** Glass or polystyrene pipettes used in the multiple-pipette method of assay, respectively. T h e purified active fraction c o m p r i s e d two m a i n c o m p o n e n t s as i n d i c a t e d by p o l y a c r y l a m i d e gel electrophoresis at p H 9.5 [17]; these c o m p o n e n t s were glycoproteins, as i n d i c a t e d by the p e r i o d a t e - S c h i f f ' s reagent [18] a n d c o n t a i n e d a p p r o x . 12 (w/w) c a r b o h y d r a t e (gas-liquid c h r o m a t o g r a p h y o f trimethylsilyl derivatives [19]) m a d e u p o f N - a c e t y l g l u c o s a m i n e (6 ~ ) , glucose (3.4 ~ ) , m a n n o s e (2.5 ~o) a n d galactose (0.1 ~ ) . H i g h l y purified nerve g r o w t h f a c t o r f r o m V. russelli is also r e p o r t e d [9] to be a g l y c o p r o t e i n . T h e isoelectric p o i n t s (pI 8.0 a n d 9.0) o f these c a r b o h y d r a t e staining b a n d s on p o l y a c r y l a m i d e gel electrofocussing [9] i n d i c a t e d basic species similar to those o f nerve g r o w t h factor f r o m V. russelli a n d m o u s e (10.5 a n d 9.3, respectively) b u t different to t h a t o f nerve g r o w t h f a c t o r i s o l a t e d f r o m Naja naja (6.8) (cf. ref. 14). The m o l e c u l a r weight o f nerve g r o w t h factor-active m a t e r i a l f r o m B. atrox was a p p r o x . 35 000 as m e a s u r e d on a c a l i b r a t e d S e p h a d e x c o l u m n . T h o u g h glycop r o t e i n s usually b e h a v e a n o m a l o u s l y in gel filtration [20], a m o l e c u l a r weight o f 37 000 for V. russelli nerve g r o w t h f a c t o r was o b t a i n e d f r o m either gel filtration o r u l t r a c e n t r i f u g a t i o n studies [9]. The p o s s i b l e occurrence o f s u b - u n i t structure was exp l o r e d using p o l y a c r y l a m i d e gel electrophoresis in s o d i u m d o d e c y l s u l p h a t e [21]. The active f r a c t i o n f r o m B. atrox c o n t a i n e d a m a j o r species o f m o l e c u l a r weight o f a p p r o x . 19 000. In c o m p a r i s o n , m o u s e 7-S nerve g r o w t h f a c t o r t h a t is k n o w n [7] to c o n t a i n sub-units o f a p p r o x . 13 000 gave e l e c t r o p h o r e t i c b a n d s c o r r e s p o n d i n g to species o f m o l e c u l a r weight o f a p p r o x . 12 000, 16 000 and 26 000 u n d e r the same conditions. H i g h l y purified nerve g r o w t h factor f r o m V. russelli t h a t h a d been considered to
25 be a single species o f a b o u t 37 000 gave major bands o f approx. 18 000 and 42 000. These results certainly suggest that nerve growth factor f r o m snake venom, like that f r o m mouse, contains sub-units (cf. ref. 13). The purified fraction evoked the standard response in tissue culture at 2.10 - 7 6.10 -8 g/ml, depending on whether glass or plastic pipettes were used in the m o s t reliable m e t h o d [22] of assay. This activity, in its lowest range, is similar to that reported for nerve growth factor f r o m v e n o m o f Ancistrodon piscivorus piscivorus [10, 11] but is 5-20-fold less than that o f highly purified material from mouse or V. russelli [22]. The dose response characteristics were similar to those of nerve growth factor f r o m the latter source [22] in that use o f the single-pipette method o f assay gave spurious results p r o b a b l y due to a n o m a l o u s adsorption effects. Such effects were not found for mouse nerve growth factor. The stability o f the biological activity o f our fraction closely resembled that o f nerve growth factor f r o m V. russelli [9] and whereas the activity o f the fraction was unaltered by incubation with crude v e n o m f r o m B. atrox, similar treatment destroyed most nerve growth factor activity in the 7-S material from mouse. The biological activity o f the active fractions f r o m venoms of both B. atrox and A. piscivorus were either unaffected or were blocked by only low dilutions o f the antiserum to nerve growth factor from mouse, whereas the antiserum raised against nerve growth factor f r o m V. russelli destroyed the activity at high dilutions (cross-reactivity o f 15-60 ~o) (Table II). This lack o f cross-over between antigens and antisera f r o m different species seems, therefore, to be a general phenomenon, (see also ref. 15).
TABLE II IMMUNOLOGICAL RELATIONSHIP BETWEEN NERVE GROWTH FACTOR FROM VARIOUS SOURCES Antiserum was diluted with rabbit serum and the resulting solutions assayed using explanated sensory ganglia in a medium consisting of medium No. 199 (Wellcome Reagents Ltd), antiserum solution, and nerve growth factor solution (1 : 1 : 1, v/v). One biological unit of activity, as defined as that concentration of nerve growth factor required to produce a standard response of 2.5 on an arbitrary, semi-quantitative scale of 0 to 5 [22], was used in all cases. Antisera were gifts from Dr D. C. Edwards (Burroughs-Wellcome labs, Beckingham, Kent). A purified fraction containing nerve growth factor activity was isolated [11] from the venom of A. piscivorus (Sigma Chemical Co. Ltd) by A. R. Berry (U.C.L.). Antigen nerve growth factor (NGF)
Reciprocal dilution of antibody titre Antiserum to mouse 7-S nerve growth factor
Antiserum to V. russelli nerve growth factor
Mouse 7-S
NGF V. russelli NGF B. atrox NGF A. piscivorus NGF
1000
2
1
600
No effect
60-120
1
200-500
26 The a n o m a l o u s a b s o r p t i o n properties of snake v e n o m nerve growth factor have been attributed to the presence of the c a r b o h y d r a t e moiety [22]. This moiety, which m a y have a protective function, might also be the cause of the lack of antigenic cross-reactivity. However, the presence of the carbohydrate moiety a p p a r e n t l y has no effect o n the biological action of nerve growth factor; the response in tissue culture of whole sensory ganglia to nerve growth factor from V. russelli, B. atrox or mouse were indistinguishable when the same n u m b e r of biological units of each material was tested. O u r present results indicate that a l t h o u g h nerve growth factor from differing sources shows very similar biological effects a n d p r o b a b l e similarities in s u b - u n i t structure, there are p r o f o u n d i m m u n o l o g i c a l differences. These differences may largely reflect the absence or variation to the c a r b o h y d r a t e moiety. ACKNOWLEDGEMENT
We thank the Science Research Council for a grant to R.E.G. (1969-1972). REFERENCES 1 Levi-Montalcini, R. and Angeletti, P. U. (1968) Physiol. Rev. 48, 534-569 2 Zaimis, E. and Knight, J. (1972) Nerve Growth Factor and its Antiserum, p. 273, Athlone Press, London 3 Varon, S., Nomura, J. and Shooter, E. M. (1968) Biochemistry 7, 1296-1303 4 Smith, A. P., Varon, S. and Shooter, E. M. (1968) Biochemistry 7, 3259-3268 5 Levi-Montalcini, R., Tentori, L., Vivaldi, G., Angeletti, P. U. and Morini-Betiolo, G. B. (1965) Gazz. Chim. Ital. 95, 333-337 6 Bocchini, V. and Angeletti, P. U. (1969) Prec. Natl. Acad. Sci. U.S. 64, 787-794 7 Angeletti, R. H. and Bradshaw, R. A. (1971) Prec. Natl. Acad. Sci. U.S. 68, 2417-2420 8 Angeletti, R. H. (1970) Prec. Natl. Acad. Sci. U.S. 65, 668-674 9 Pearce, F. L., Banks, B. E. C., Banthorpe, D. V., Berry, A. R., Davies, H. ft. S. and Vernon, C. A. (1972) Eur. J. Biochem. 29, 417--425 10 Cohen, S. (1959) J. Biol. Chem. 234, 1129-1137 11 Banks, B. E. C., Banthorpe, D. V., Berry, A. R., Davies, H. ft. S., Doonan, S., Lament, D. M., Shipolini, R. and Vernon, C. A. (1968) Biochem. J. 108, 157-158 12 Angeletti, P. U., Calissaro, P., Chen, J. S. and Levi-Montalcini, R. (1967) Biochim. Biophys. Acta 147, 180-182 13 Angeletti, R. H. (1968) J. Chromatogr. 37, 62-69 14 Pearce, F. L., Banks, B. E. C., Banthorpe, D. V., Berry, A. R., Davies, H. ft. S. and Vernon, C. A. (1972) Nerve Growth Factor and its Antiserum, pp. 3-18, Athlone Press, London 15 Banks, B. E. C., Banthorpe, D. V., Charlwood, K. A., Pearce, F. L., Vernon, C. A. and Edwards, D. C. (1973) Nature 246, 503-504 16 Spire, R. G. (1970) Annu. Rev. Biochem. 39, 599-638 17 Hjerten, S., Jerstedt, S. and Tiselius, A. (1965) Anal. Biochem. 11, 219-232 18 Clarke, J. T. (1964) Ann. N.Y. Acad. Sci. 121, 428-433 19 Bolton, C. H., Clamp, J. R. and Hough, L. (1965) Biochem. J. 96, 5c 20 Andrews, P. (1965) Biochem. J. 96, 595-606 21 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406--4412 22 Pearce, F. L., Banthorpe, D. V., Cook, J. M. and Vernon, C. A. (1973) Eur. J. Biochem. 32. 569575
