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Evidence for heterogeneity in the association properties of the gamma protein of mouse 7S nerve growth factor protein
Neuroscience Letters, 2 (1976) 289--294 © Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
289
EVIDENCE FOR HETEROGENEITY IN THE ASSOCIATION PROPERTIES OF THE GAMMA PROTEIN OF MOUSE 7S NERVE GROWTH FACTOR PROTEIN
MICHAEL BAKER*
Department of Genetics and Biochemistry, Lt. Joseph P. Kennedy, Jr., Laboratories for Molecular Medicine, Stanford University School of Medicine, Stanford, Calif. 94305 (U.S.A.) (Received May 3rd, 1976) (Accepted May 18th, 1976)
SUMMARY
The mouse 7S nerve growth factor 7 subunit, which is homogeneous with respect to molecular weight and heterogeneous with respect to charge, has been found to display heterogeneous self-association behavior at pH values 4 and 7.4.
The nerve growth factor (NGF) which is isolated at neutral pH from mouse submaxillary glands, has an s20,w of 7S (7S NGF) [29] and a molecular weight of 137,000 [4]. This protein stimulates the fiber outgrowth of sympathetic and embryonic sensory ganglia and is an arginine esteropeptidase [15,16]. Outside of the pH range 5--8, 7S NGF may be reversibly dissociated into three subunits called a, ~, and 7, each with s20,w values of about 2.5S [27,30]. The NGF activity is found only in the ~ subunit (~NGF), which is the same protein as 2.5S NGF [1,8,21]. The esteropeptidase activity resides in the 7 subunit [ 15,16] which, when isolated from 7S NGF, exists as three electrophoretically distinct species, with isoelectric points between 5.2 and 5.8 [ 31], all with the same enzymatic activity. Since the 7 subunit migrates as a single band on SDS gels, the three species appear to have essentially the same molecular weight [25], although their polypeptide chain composition differs [23]. The molecular weight of the 7 protein has been studied by sedimentation equilibrium in the analytical ultracentrifuge between pH values 4 and 10. Significant self-association occurs at all pH values [5]. The extrapolated monomer molecular weight is about 18,000 [5].
*Present address, for correspondence: Dept. of Medicine, M-013, University of California, San Diego, La Jolla, Calif. 92093, U.S.A.
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I wish to report the results of experiments which show that there is heterogeneity among the different 7 species in association behavior. The details of the sedimentation equilibrium technique, using Rayleigh interference optics, and tbe data analysis are reported elsewhere [4,5]. However, I want to emphasize t w o aspects of Chervenka's long column modification [10] of the Yphantis technique [ 33], which bear on the accuracy of the data reported here. The long column length reduces: (1) the density of fringes, giving greater accuracy in the measurement of the coordinates along the fringes, and (2) the required rotor speed to achieve a depleted meniscus, which results in less cell distortion and pressure effects. Using this technique, the molecular weight of the 7 protein was studied at different rotor speeds at pH values 4 and 7.4. At both pH values, the molecular weight depends on the rotor speed as well as protein concentration. The results of one of three experiments done at pH 4 in sodium acetate buffer are shown in Fig. 1. It is clear that Mw is a function of rotor speed. Since the 7 protein used in these studies was pure, as judged by polyacrylamide gel electrophoresis, I propose that the effect of rotor speed is due to heterogeneity in the association behavior of the different 7 protein species. This effect could also be due to (1) the formation of aggregates due to instability of the 7 protein during the experiment, or (2) the pressure variation along the cell [17,18]. The first possibility was eliminated because the 7 protein is stable at pH 7.4 for several days in the ultracentrifuge [5] and it is isolated and stored at pH 4 without any apparent changes [15,16,27,30]. Furthermore, I have observed a dependence of the molecular weight on rotor speed at pH 4 using 7 protein which was incubated with phenylmethylsulfonylfluoride [12] so that its esterase activity was inhibited. The second possibility, that the pressure changes along the cell could change the partial specific volume of 7 and therefore its measured molecular weight must also be considered. This possibility appears unlikely due to the low molecular weight of the ~ protein, the low rotor speeds used in these Fig. 1. a: sedimentation equilibrium of 7 protein at different rotor speeds. The v protein was isolated from purified 7S N G F [29 ] by the procedure of Smith et al. [27 ]. Sedimentation equilibrium experiments were performed using a Spinco Model E ultracentrifuge equipped with interference optics in a synthetic boundary cell having a 12 m m light path and fitted with sapphire windows. The cell'ssample sector was loaded with 0.03 ml of FC43 solution, then 0.05 ml "of dialyzed protein solution (0.8 mg/ml) added. The reference sector was filled with 0.01 ml of FC43 solution and 0.40 ml of the dialysate. The photographic plates were read on a Gaertner microcornparator. Three fringes were read and averaged per interference pattern. Equilibrium was ensured by the lack of change in the o b s e r v e d p a t t e r n over a 24-h period. C o n d i t i o n s : e, w = 20,000; o, w = 24,000; D, w = 28,000. All runs in s o d i u m a c e t a t e buffer. I = 0.5, p H 4.0 at 25°C + 0.1 M NaCI, t e m p e r a t u r e a b o u t 20°C. b: dependence o f m o l e c u l a r weight o f ~ p r o t e i n o n c o n c e n t r a t i o n . The m o l e c u l a r weight o f ~ was d e t e r m i n e d f r o m the slope o f Fig. l a using a least squares fit o f five c o n s e c u t i v e p o i n t s [4,5 ]. T h e increase in fringe h e i g h t was c o n v e r t e d t o p r o t e i n conc e n t r a t i o n using t h e f a c t o r 4.1 f r i n g e s / m g / m l [3 ].
292
experiments, and the distance from the axis of rotation where the measurements were made (Fig. la) [17,18]. Therefore, I conclude that the dependence of molecular weight on rotor speed is best accounted for by heterogeneity in the associative properties of the different 7 subunit species. It is interesting that chymotrypsinogen and the different chymotrypsin species, which have about the same molecular weight and different polypeptide chain compositions also show heterogeneous self-association behavior [2,19, 32]. It may be that the different 7 protein species also develop by proteolytic cleavage from a precursor ~-protein. The results presented here together with those recently obtained by other investigators [7,13,20,28] provide a basis to suggest a possible role for the gamma subunit of 7S NGF in the response of tissues to ~NGF. Lembach [20] reported recently that the epidermal growth factor (EGF)-binding arginine esterase (which shares antigenic determinants and substrate specificity with the gamma subunit [28] ) can increase the ability of EGF to stimulate the growth of cultured human fibroblasts. This occurs even though the EGFbinding arginine esterase is by itself inactive [20]. It has also been established that, in addition to sympathetic and embryonic sensory ganglia, nerve cells in the central nervous system [7,13] as well as in non-neuronal tissues [13] such as heart, spleen, uterus, and kidney have receptors for ~NGF. The heterogeneity observed in the structure and the association properties of the gamma subunit could thus possibly provide a certain tissue or cell-type specificity for ~NGF [6], similar to that found for a number of isozymes [11,22,33]. One possible mechanism of action for the gamma subunit would be to alter the mobility of the ~NGF receptor in the plasma membrane, as proteases have been shown to do with other receptors [9,26]. Such an altered mobility would be expected to affect the receptor's binding capacity to ~NGF, since this hormone-receptor interaction is characterized b y a negative cooperativity [ 14] resulting from an interaction between the receptors [ 24]. ACKNOWLEDGEMENTS
I am grateful to Professor Eric Shooter for his help during this study. This research was supported by U.S. Public Health Service Research Grant from the National Institute of Neurological Diseases and Stroke (NS 04270). REFERENCES 1 Angeletti, R.H. and Bradshaw, R.A., Nerve growth factor from mouse submaxillary gland -- amino acid sequence, Proc. nat. Acad. Sci. (Wash.), 68 (1971) 2417--2420. 2 Aune, K.S. and Timasheff, S.N., Dimerization of ~-chymotrypsin. I. p H dependence in the acid region, Biochemistry, 10 (1971) 1609--1617. 3 Babul, J. and Stellwagen, E., Measurement of protein concentration with interference optics, Analyt. Biochem., 28 (1969) 216--221. 4 Baker, M.E., Molecular weight and structure of 7S nerve growth factor protein, J. biol. Chem., 250 (1975) 1714--1717.
293
5 Baker, M.E., Molecular weight of the At subunit of mouse 7S nerve growth factor, Biochim. biophys. Acta (Amst.), 379 (1975) 592--597. 6 Baker, M.E., Proposed role of the a subunit of 7S NGF: implications for NGF activity, Physiol. Chem. Phys., 8 (1976) in press. 7 Bjorklund, A. and Stenevi, V., Nerve growth factor: stimulation of regeneration growth of central non-adlenergic neurons, Science, 175 (1972) 1251--1253. 8 Bocchini, V. and Angeletti, P.U., The nerve growth factor: purification as a 3 0 , 0 0 0 molecular weight protein, Proc. nat. Acad. Sci. (Wash.), 64 (1969) 787--794. 9 Burger, M.M., A difference in the architecture of the surface membrane of normal and virally transformed cells, Proc. nat. Acad. Sci. (Wash.), 62 (1969) 994--1001. 10 Chervenka, C.H., Long-column meniscus depletion sedimentation equilibrium technique for the analytical ultracentrifuge, Analyt. Biochem., 34 (1970) 24--29. 11 Everse, J. and Kaplan, N.O., Mechanisms of action and biological functions of various dehydrogenase isozymes. In C.L. Markert (Ed.), Isozymes. II. Physiological Function, Academic Press, New York, 1975, pp. 29--43. 12 Fahrney, D.E. and Gold, A.M., Sulfonyl fluorides as inhibitors of esterases, I. rates of reaction with acetylcholinesterase, ~-chymotrypsin, and trypsin, J. amer. Chem. Soc., 85 (1963) 997--1000. 13 Frazier, W.A., Boyd, L.F., Szutowicz, A., Pulliam, M.W. and Bradshaw, R.A., Specific binding sites for ,2Si.nerve growth factor in peripheral tissues and brain, Biochem. biophys. Res. Commun., 57 (1974) 1096--1103. 14 Frazier, W.A., Boyd, L.F. and Bradshaw, R.A., Properties of the specific binding of '2SI-nervegrowth factor to responsive peripheral neurons, J. biol. Chem., 249 (1974) 5513--5519. 15 Greene, L.A., Shooter, E.M. and Varon, S., Enzymatic activities of mouse nerve growth factor and its subunits, Proc. nat. Acad. Sci. (Wash.), 60 (1968) 1383--1388. 16 Greene, L.A., Shooter, E.M. and Varon, S., Subunit interaction and enzymatic activity of mouse 7S nerve growth factor, Biochemistry, 8 (1969) 3735--3741. 17 Harrington, W.F., The effects of pressure in ultracentrifugation of interacting systems, Fractions, (1975) 10--18. 18 Kegeles, G., Kaplan, S. and Rhodes, L., The effects of pressure in high-speed ultracentrifugation of chemically reacting systems, Ann. N.Y. Acad. Sei., 164 (1969) 183--191. 19 LaBar, F.E., A procedure for molecular weight measurements: application to chymotrypsinogen A, Proc. nat. Acad. Sci. (Wash.), 54 (1965) 31--36. 20 Lembach, K.J., Induction of human fibroblast proliferation by epidermal growth factor (EGF): enhancement by an EGF-binding arginine esterase and by ascorbate, Proc. nat. Acad. Sci. (Wash.), 73 (1976) 183--187. 21 Levi-Montalcini, R., The nerve growth factor, its mode of action on sensory and sympathetic nerve cells, Harvey Lect. Ser., 60 (1966) 217--259. 22 Markert, C.L., Biology of isozymes. In C.L. Markert (Ed.), Isozymes. I. Molecular Structure, Academic Press, New York, 1975, pp. 1--9. 23 Piltch, A., Structural Studies-on Mouse Nerve Growth Factor Subunits. Ph.D. Thesis, Stanford University, Stanford, Calif., 1972. 24 Pulliam, M.W., Boyd, L.F., Bagkin, N.C. and Bradshaw, R.A., Specific binding of covalently cross-linked mouse nerve growth factor to responsive peripheral neurons, Biochem. biophys. Res. Commun., 67 (1975) 1281--1289. 25 Shooter, E.M., Some aspects of gene expression in the nervous system. In F.O. Schmitt (Ed.), The Neurosciences: Second Study Program, The Rockefeller University Press, New York, 1970, pp. 812--827. 26 Singer, S.J. and Nieolson, G.L., The fluid mosaic model of the structure of cell membranes, Science, 175 (1972) 720--731.
294
27 Smith, A.P., Varon, S. and Shooter, E.M., Multiple forms of the nerve growth factor protein and its subunits, Biochemistry, 7 (1968) 3259--3268. 28 Taylor, J.M., Mitchell, W.M. and Cohen, S., Characterization of the binding protein for epidermal growth factor, J. biol. Chem., 249 (1974) 2188--2194. 29 Varon, S., Nomura, J. and Shooter, E.M., The isolation of the mouse nerve growth factor protein in a high molecular weight form, Biochemistry, 6 (1967) 2202--2209. 30 Varon, S., Nomura, J. and Shooter, E.M., Reversible dissociation of the mouse nerve growth factor protein into different subunits, Biochemistry, 7 (1968) 1296--1303. 31 Varon, S. and Shooter, E.M., The nerve growth factor proteins of the mouse submaxillary gland. In R.E. Bowman and S.P. Datta (Eds.), Biochemistry of Brain and Behavior, Plenum Press, New York, 1970, pp. 41--64. 32 Wilcox, P.E., Chymotrypsinogens-chymotrypsins. In G.E. Perlmann and L. Lorand (Eds.), Methods in Enzymology, Vol. 19, Academic Press, New York, 1970, pp. 64 108. 33 Yphantis, D.A., Equilibrium ultracentrifugation of dilute solutions, Biochemistry, 3 (1964) 297--317.
289
EVIDENCE FOR HETEROGENEITY IN THE ASSOCIATION PROPERTIES OF THE GAMMA PROTEIN OF MOUSE 7S NERVE GROWTH FACTOR PROTEIN
MICHAEL BAKER*
Department of Genetics and Biochemistry, Lt. Joseph P. Kennedy, Jr., Laboratories for Molecular Medicine, Stanford University School of Medicine, Stanford, Calif. 94305 (U.S.A.) (Received May 3rd, 1976) (Accepted May 18th, 1976)
SUMMARY
The mouse 7S nerve growth factor 7 subunit, which is homogeneous with respect to molecular weight and heterogeneous with respect to charge, has been found to display heterogeneous self-association behavior at pH values 4 and 7.4.
The nerve growth factor (NGF) which is isolated at neutral pH from mouse submaxillary glands, has an s20,w of 7S (7S NGF) [29] and a molecular weight of 137,000 [4]. This protein stimulates the fiber outgrowth of sympathetic and embryonic sensory ganglia and is an arginine esteropeptidase [15,16]. Outside of the pH range 5--8, 7S NGF may be reversibly dissociated into three subunits called a, ~, and 7, each with s20,w values of about 2.5S [27,30]. The NGF activity is found only in the ~ subunit (~NGF), which is the same protein as 2.5S NGF [1,8,21]. The esteropeptidase activity resides in the 7 subunit [ 15,16] which, when isolated from 7S NGF, exists as three electrophoretically distinct species, with isoelectric points between 5.2 and 5.8 [ 31], all with the same enzymatic activity. Since the 7 subunit migrates as a single band on SDS gels, the three species appear to have essentially the same molecular weight [25], although their polypeptide chain composition differs [23]. The molecular weight of the 7 protein has been studied by sedimentation equilibrium in the analytical ultracentrifuge between pH values 4 and 10. Significant self-association occurs at all pH values [5]. The extrapolated monomer molecular weight is about 18,000 [5].
*Present address, for correspondence: Dept. of Medicine, M-013, University of California, San Diego, La Jolla, Calif. 92093, U.S.A.
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291
I wish to report the results of experiments which show that there is heterogeneity among the different 7 species in association behavior. The details of the sedimentation equilibrium technique, using Rayleigh interference optics, and tbe data analysis are reported elsewhere [4,5]. However, I want to emphasize t w o aspects of Chervenka's long column modification [10] of the Yphantis technique [ 33], which bear on the accuracy of the data reported here. The long column length reduces: (1) the density of fringes, giving greater accuracy in the measurement of the coordinates along the fringes, and (2) the required rotor speed to achieve a depleted meniscus, which results in less cell distortion and pressure effects. Using this technique, the molecular weight of the 7 protein was studied at different rotor speeds at pH values 4 and 7.4. At both pH values, the molecular weight depends on the rotor speed as well as protein concentration. The results of one of three experiments done at pH 4 in sodium acetate buffer are shown in Fig. 1. It is clear that Mw is a function of rotor speed. Since the 7 protein used in these studies was pure, as judged by polyacrylamide gel electrophoresis, I propose that the effect of rotor speed is due to heterogeneity in the association behavior of the different 7 protein species. This effect could also be due to (1) the formation of aggregates due to instability of the 7 protein during the experiment, or (2) the pressure variation along the cell [17,18]. The first possibility was eliminated because the 7 protein is stable at pH 7.4 for several days in the ultracentrifuge [5] and it is isolated and stored at pH 4 without any apparent changes [15,16,27,30]. Furthermore, I have observed a dependence of the molecular weight on rotor speed at pH 4 using 7 protein which was incubated with phenylmethylsulfonylfluoride [12] so that its esterase activity was inhibited. The second possibility, that the pressure changes along the cell could change the partial specific volume of 7 and therefore its measured molecular weight must also be considered. This possibility appears unlikely due to the low molecular weight of the ~ protein, the low rotor speeds used in these Fig. 1. a: sedimentation equilibrium of 7 protein at different rotor speeds. The v protein was isolated from purified 7S N G F [29 ] by the procedure of Smith et al. [27 ]. Sedimentation equilibrium experiments were performed using a Spinco Model E ultracentrifuge equipped with interference optics in a synthetic boundary cell having a 12 m m light path and fitted with sapphire windows. The cell'ssample sector was loaded with 0.03 ml of FC43 solution, then 0.05 ml "of dialyzed protein solution (0.8 mg/ml) added. The reference sector was filled with 0.01 ml of FC43 solution and 0.40 ml of the dialysate. The photographic plates were read on a Gaertner microcornparator. Three fringes were read and averaged per interference pattern. Equilibrium was ensured by the lack of change in the o b s e r v e d p a t t e r n over a 24-h period. C o n d i t i o n s : e, w = 20,000; o, w = 24,000; D, w = 28,000. All runs in s o d i u m a c e t a t e buffer. I = 0.5, p H 4.0 at 25°C + 0.1 M NaCI, t e m p e r a t u r e a b o u t 20°C. b: dependence o f m o l e c u l a r weight o f ~ p r o t e i n o n c o n c e n t r a t i o n . The m o l e c u l a r weight o f ~ was d e t e r m i n e d f r o m the slope o f Fig. l a using a least squares fit o f five c o n s e c u t i v e p o i n t s [4,5 ]. T h e increase in fringe h e i g h t was c o n v e r t e d t o p r o t e i n conc e n t r a t i o n using t h e f a c t o r 4.1 f r i n g e s / m g / m l [3 ].
292
experiments, and the distance from the axis of rotation where the measurements were made (Fig. la) [17,18]. Therefore, I conclude that the dependence of molecular weight on rotor speed is best accounted for by heterogeneity in the associative properties of the different 7 subunit species. It is interesting that chymotrypsinogen and the different chymotrypsin species, which have about the same molecular weight and different polypeptide chain compositions also show heterogeneous self-association behavior [2,19, 32]. It may be that the different 7 protein species also develop by proteolytic cleavage from a precursor ~-protein. The results presented here together with those recently obtained by other investigators [7,13,20,28] provide a basis to suggest a possible role for the gamma subunit of 7S NGF in the response of tissues to ~NGF. Lembach [20] reported recently that the epidermal growth factor (EGF)-binding arginine esterase (which shares antigenic determinants and substrate specificity with the gamma subunit [28] ) can increase the ability of EGF to stimulate the growth of cultured human fibroblasts. This occurs even though the EGFbinding arginine esterase is by itself inactive [20]. It has also been established that, in addition to sympathetic and embryonic sensory ganglia, nerve cells in the central nervous system [7,13] as well as in non-neuronal tissues [13] such as heart, spleen, uterus, and kidney have receptors for ~NGF. The heterogeneity observed in the structure and the association properties of the gamma subunit could thus possibly provide a certain tissue or cell-type specificity for ~NGF [6], similar to that found for a number of isozymes [11,22,33]. One possible mechanism of action for the gamma subunit would be to alter the mobility of the ~NGF receptor in the plasma membrane, as proteases have been shown to do with other receptors [9,26]. Such an altered mobility would be expected to affect the receptor's binding capacity to ~NGF, since this hormone-receptor interaction is characterized b y a negative cooperativity [ 14] resulting from an interaction between the receptors [ 24]. ACKNOWLEDGEMENTS
I am grateful to Professor Eric Shooter for his help during this study. This research was supported by U.S. Public Health Service Research Grant from the National Institute of Neurological Diseases and Stroke (NS 04270). REFERENCES 1 Angeletti, R.H. and Bradshaw, R.A., Nerve growth factor from mouse submaxillary gland -- amino acid sequence, Proc. nat. Acad. Sci. (Wash.), 68 (1971) 2417--2420. 2 Aune, K.S. and Timasheff, S.N., Dimerization of ~-chymotrypsin. I. p H dependence in the acid region, Biochemistry, 10 (1971) 1609--1617. 3 Babul, J. and Stellwagen, E., Measurement of protein concentration with interference optics, Analyt. Biochem., 28 (1969) 216--221. 4 Baker, M.E., Molecular weight and structure of 7S nerve growth factor protein, J. biol. Chem., 250 (1975) 1714--1717.
293
5 Baker, M.E., Molecular weight of the At subunit of mouse 7S nerve growth factor, Biochim. biophys. Acta (Amst.), 379 (1975) 592--597. 6 Baker, M.E., Proposed role of the a subunit of 7S NGF: implications for NGF activity, Physiol. Chem. Phys., 8 (1976) in press. 7 Bjorklund, A. and Stenevi, V., Nerve growth factor: stimulation of regeneration growth of central non-adlenergic neurons, Science, 175 (1972) 1251--1253. 8 Bocchini, V. and Angeletti, P.U., The nerve growth factor: purification as a 3 0 , 0 0 0 molecular weight protein, Proc. nat. Acad. Sci. (Wash.), 64 (1969) 787--794. 9 Burger, M.M., A difference in the architecture of the surface membrane of normal and virally transformed cells, Proc. nat. Acad. Sci. (Wash.), 62 (1969) 994--1001. 10 Chervenka, C.H., Long-column meniscus depletion sedimentation equilibrium technique for the analytical ultracentrifuge, Analyt. Biochem., 34 (1970) 24--29. 11 Everse, J. and Kaplan, N.O., Mechanisms of action and biological functions of various dehydrogenase isozymes. In C.L. Markert (Ed.), Isozymes. II. Physiological Function, Academic Press, New York, 1975, pp. 29--43. 12 Fahrney, D.E. and Gold, A.M., Sulfonyl fluorides as inhibitors of esterases, I. rates of reaction with acetylcholinesterase, ~-chymotrypsin, and trypsin, J. amer. Chem. Soc., 85 (1963) 997--1000. 13 Frazier, W.A., Boyd, L.F., Szutowicz, A., Pulliam, M.W. and Bradshaw, R.A., Specific binding sites for ,2Si.nerve growth factor in peripheral tissues and brain, Biochem. biophys. Res. Commun., 57 (1974) 1096--1103. 14 Frazier, W.A., Boyd, L.F. and Bradshaw, R.A., Properties of the specific binding of '2SI-nervegrowth factor to responsive peripheral neurons, J. biol. Chem., 249 (1974) 5513--5519. 15 Greene, L.A., Shooter, E.M. and Varon, S., Enzymatic activities of mouse nerve growth factor and its subunits, Proc. nat. Acad. Sci. (Wash.), 60 (1968) 1383--1388. 16 Greene, L.A., Shooter, E.M. and Varon, S., Subunit interaction and enzymatic activity of mouse 7S nerve growth factor, Biochemistry, 8 (1969) 3735--3741. 17 Harrington, W.F., The effects of pressure in ultracentrifugation of interacting systems, Fractions, (1975) 10--18. 18 Kegeles, G., Kaplan, S. and Rhodes, L., The effects of pressure in high-speed ultracentrifugation of chemically reacting systems, Ann. N.Y. Acad. Sei., 164 (1969) 183--191. 19 LaBar, F.E., A procedure for molecular weight measurements: application to chymotrypsinogen A, Proc. nat. Acad. Sci. (Wash.), 54 (1965) 31--36. 20 Lembach, K.J., Induction of human fibroblast proliferation by epidermal growth factor (EGF): enhancement by an EGF-binding arginine esterase and by ascorbate, Proc. nat. Acad. Sci. (Wash.), 73 (1976) 183--187. 21 Levi-Montalcini, R., The nerve growth factor, its mode of action on sensory and sympathetic nerve cells, Harvey Lect. Ser., 60 (1966) 217--259. 22 Markert, C.L., Biology of isozymes. In C.L. Markert (Ed.), Isozymes. I. Molecular Structure, Academic Press, New York, 1975, pp. 1--9. 23 Piltch, A., Structural Studies-on Mouse Nerve Growth Factor Subunits. Ph.D. Thesis, Stanford University, Stanford, Calif., 1972. 24 Pulliam, M.W., Boyd, L.F., Bagkin, N.C. and Bradshaw, R.A., Specific binding of covalently cross-linked mouse nerve growth factor to responsive peripheral neurons, Biochem. biophys. Res. Commun., 67 (1975) 1281--1289. 25 Shooter, E.M., Some aspects of gene expression in the nervous system. In F.O. Schmitt (Ed.), The Neurosciences: Second Study Program, The Rockefeller University Press, New York, 1970, pp. 812--827. 26 Singer, S.J. and Nieolson, G.L., The fluid mosaic model of the structure of cell membranes, Science, 175 (1972) 720--731.
294
27 Smith, A.P., Varon, S. and Shooter, E.M., Multiple forms of the nerve growth factor protein and its subunits, Biochemistry, 7 (1968) 3259--3268. 28 Taylor, J.M., Mitchell, W.M. and Cohen, S., Characterization of the binding protein for epidermal growth factor, J. biol. Chem., 249 (1974) 2188--2194. 29 Varon, S., Nomura, J. and Shooter, E.M., The isolation of the mouse nerve growth factor protein in a high molecular weight form, Biochemistry, 6 (1967) 2202--2209. 30 Varon, S., Nomura, J. and Shooter, E.M., Reversible dissociation of the mouse nerve growth factor protein into different subunits, Biochemistry, 7 (1968) 1296--1303. 31 Varon, S. and Shooter, E.M., The nerve growth factor proteins of the mouse submaxillary gland. In R.E. Bowman and S.P. Datta (Eds.), Biochemistry of Brain and Behavior, Plenum Press, New York, 1970, pp. 41--64. 32 Wilcox, P.E., Chymotrypsinogens-chymotrypsins. In G.E. Perlmann and L. Lorand (Eds.), Methods in Enzymology, Vol. 19, Academic Press, New York, 1970, pp. 64 108. 33 Yphantis, D.A., Equilibrium ultracentrifugation of dilute solutions, Biochemistry, 3 (1964) 297--317.