SOLUTION PROPERTIES OF p NERVE GROWTH FACTOR PROTEIN AND SOME OF ITS DERIVATIVES P-F. PIGNATTI', M. E. BAKER'and E. M. SHOOTER Departments of Genetics and Biochemistry and the Lt. Joseph P. Kennedy, Jr. Laboratories for Molecular Medicine, Stanford University School of Medicine, Stanford, CA 94305, U.S.A. (Received 20 September 1974. Aceepted 14 Jaiiuary 1975)

Abstract-The molecular weight of 8 nerve growth factor protein determined by sedimentation equilibrium in sodium acetate buffer, pH 4.0, and at protein concentrations around 0.5 m g / d agrees with the value obtained from the amino acid sequence and confirms the dimeric character of the protein under these conditions. At pH values of 5.0 or greater, p nerve growth factor protein shows either partial dissociation into monomers or aggregation to higher polymers or both phenomena. The extent of dissociation or aggregation depends on buffer type and pH and is most pronounced at alkaline pH. The variation of molecular weight of 8 nerve growth factor with solvent conditions is similar to that of insulin or proinsulin. Removal of either the two COOH-terminal arginine residues or the two NH,-terminal octapeptide sequences from the protein has no effect on its solution properties at acid pH, the protein remaining a dimer. Species such as 2.5 S nerve growth factor or cyanogen bromide cleaved nerve growth factor which are partically deficient in COOH-terminal arginine residues and/or NH,-octapeptide or nonapeptide sequences are also dimers at pH 4.0. The protein derivative which lacks the two NH,-terminal octapeptide sequence does not, like 8-nerve growth factor, display dissociation or aggregation behavior at neutral pH, indicating that these sequences are involved in monomer-monomer interactions.

RECENTadvances in our knowledge concerning the been estimated to be 24,000 by gel filtration or elecstructure and mechanism of action of the nerve trophoresis in the presence of sodium dodecyl sulfate growth factor (NGF)' have only served to emphasize (GGREENE et a/., 1971). The molecular weight deterthe original premise that NGF is essential for the mined from the amino acid sequence is 26,500 normal development of certain of the neurons in sym- (STRAUSS et al., 1975). In denaturing solvents, the pathetic and sensory ganglia (LEVI-MONTALCINI,molecular weight is reduced to approx l2,ooO (GREEN 1966). While an extensive chemical characterization et a/., 1971). The two species, B'NGF and B'NGF of the NGF proteins has been carried out over the differ only in the absence of one of the two COOHpast few years, rather less has been done with respect terminal arginine residues in BzNGF and the existto their physical properties. The principal NGF pro- ence of the /?'NGF species supports the idea that tein of the mouse submaxillary gland, BNGF, is BNGF is a dimer in solution (MOOREet a/., 1974). An alternative method for isolating NGF activity extracted from the gland in the high mol. complex 7 s NGF (VARON et al., 1967) and may be isolated from 7 s NGF prior to its purification results in a from the latter by dissociation at mild acid or alkaline different preparation called 2.5s NGF (BOCCHMI& pH followed by ion exchange chromatography ANGELETTI, 1969; ANGELEITI et a/., 1971) which is a (VARONet a/., 1968). This BNGF preparation com- limited proteolytic product of PNGF. The 2.5s NGF prises a major basic protein (B'NGF) and a second protein contains two related but non-identical chains, minor component of slightly lower isoelectric point A and B, the latter differing from the former only et a/., 1971; MOOREet a/.. 1974). in the absence of the NH2-terminal octapeptide (F'NGF) (GREENE & BRADSHAW, 1971; ANGELETI~, The sedimentation coefficient of these proteins is 2.5 sequence (ANGELETTI & BRADSHAW, 1973; ANGELETTI, HERMODS (VARONet a/., 1968) and their molecular weight has MERCANTI SON & BRADSHAW.1973). In addition about 25% of the chains lack the COOH-terminal arginine residue ' Present address: Istituto di Genetica dell'Universita, (ANGELETTI et a/., 1973). The moiecufar weight of Via Sant'Epifanio 14. 27100 Pavia, Italy. NGF from the sequence data is 25.800. The ' Present address: The Salk Institute for Biological 2.5s molecular weights of 2.5s NGF measured in solution Studies, San Diego. CA 92112. U.S.A. are signficantly higher than this value. They range Abbreviations used: NGF, nerve growth factor; P'NGF. the NGF species with arginine residues at the COOH- from 28,000 determined by gel filtration (ZANINIet termini of both chains; P'NGF, the NGF species with an a/., 1968) to 32.000 calculated from the sedimentation arginine residue at the COOH-terminus of only one chain; and diffusion coefficientsof the protein ( B o c c ~ m&~ 1969).Sedimentation equilibrium analyses jx,.NGF, BNGF crosslinked with dimethyl suberimidate. ANGELETTI,

i ss

156

M . E. BAKER and E. M. SHOOTER P-F. PIGNATTI.

gave ValUeS Of 30.000 (&XCHIXI & AWCELERI.1969) calculated by equation 2 of YPHANTIS(1964) using a value and 28.800 in aqueous solvents a t p H 5.0 a n d 14.500 of i. for PNCF of 0.725 ml g- ' determined from its amino in 6 M guanidine hydrochloride indicating that acid composition (VARON& SHOOTER.1970) according to COHN& EDSALL(1943). Correct alignment of the optical 2.5s NGF is also a dimer (ANGELETTI et af.. 1971). system and functioning of the ultracentrifuge wdS checked In the present study the molecular weight of BNGF by measuring the molecular weight of chymotrypsinogen has been determined by sedimentation equilibrium A and ribonucleax A. With chymotrypsinogen A, three over a wide range of pH. In addition the molecular meniscus depletion experiments carried out at protein conweight of a number of partial a n d more complete centrations of 0.46mg/ml in 0.01 M sodium acetate buffer proteolytic products of BNGF have been measured. containing 0.1 M NaCI, pH 4.0. gave molecular weights of This data indicates that the high values for the mole- 24.400, 24,300 (I I T ) and 24.600 (21°C) respectively for a cular weight of B and 2.5s NGF arise from p H 6 = 0.721. which agree well with the figure of 25.650 from dependent aggregation and that the terminal residues the sequence data (BROWN & HARTLEY,1966). The have only a small effect on the solution properties ribonuclease A sample was treated as follows to remove dimers and other aggregates (FRUCHTER & CRESTFIELD, of the protein. 1965). A solution of ribonuclease A at a concentration of I mg/ml in 0.1 M NaCl was adjusted to pH 2.1 with 1 N HCI MATERIALS AND METHODS and heated at 50°C for 1 1 min. It was then placed in the cup of a Bio-Fiber 50 minibeaker b/HFA 1/20 (Biorad Nerw yrowrh foctor prorriiis Laboratories, Richmond, CA) and 0.1 M phosphate buffer The 7 s N G F complex was isolated according to the containing 0.1 M NaCI. pH 6.8, allowed to flow through the procedure of VARONet 01. (1967) and used without further fibers at 6 ml/min for 6 h at 23°C. The final protein conDurification. BNGF was isolated from 7s N G F a s de- centration was determined from the extinction coefficient scribed bv GREENEer al. (1971), concentrated by pressure at 277.5 nm ( E ~ , , . = ~ 9.8 x 10' M - ' cm-', SELA & dialysis against 02% acetic acid to approx I mg/ml and ANFINSEN,1957). For the sedimentation analysis of stored at -20°C. 2.5s NGF was prepared according to ribonuclease A. 0.24 ml of the protein sample and 0.25 ml the procedure of BOCCHINI& ANGELETTI (1969) and bis of the buffer solution were filled into the two sectors of desarginine"' BNGF by the method of MOORE ef al. the cell. Sedimentation to equilibrium was achieved at (1974). The BNGF species lacking the octapeptide 36,000 rev./min in 24 h. A straight line was observed for sequences at the NH,-termini of the chains, bis des(l-8) the plot of the log fringe displacement versus rz throughout PNGF, was prepared by incubating PNGF in the original the cell. A molecular weight of 13,600 was obtained from supernatant of the mouse submaxillary gland after adjust- this data using i. = 0.695 which may be compared with ment to p H 4.9 (in the ratio of PNGF to supernatant pro- the value of 13,600 using the same ir in the experiments tein of 5 : I ) for 24 h at 37T, followed by isolation of the reported by BALDWIN (1957) and of 13,680 from the NGF protein using the final CM-cellulose column pro- sequence data (SMYTH et al., 1963). cedure of the 2.5s NGF preparation. Another derivative Isoekcrric focusing in affylamide gel. The PNGF and of BNGF partially (72%) cleaved by cyanogen bromide at its derivative proteins were examined for purity by isoelecmethionine9 was prepared as described by MOBLEY(1974). tric focusing in 7-1/2% acrylamide gel in a pH 3-10 graThe species BxrNGF in which the two peptide chains dient (pH 3 1 0 Ampholine, LKB Produckter) following the are covalently crosslinked with dimethylsuberimidate was procedure of GREENE et al. (1971).The extent of proteolysis made according to the procedure described by STACH& at the COOH-termini was determined from these analyses SHOOTER(1974). All these derivatives of BNGF were con- as outlined by MWRErl al. (1974), and the extent of cleacentrated and stored in the same way as the parent protein. vage at both COOH and NH,-termini by terminating the isoelectric focusing analyses in 8 M urea (MOORErr al., Other proreins 1974) after W V / h when all four types of chain are Chymotrypsinogen A and ribonuclease A were obtained resolved. from Worthington Biochemicals. All chemicals were reagent grade and were used without further purification. RESULTS Sedimentatiori equilibrium analyses

T k molecular weight of BNGF and its variation with p H . T h e values of the molecular weight of P N G F

Molecular weights were determined by the meniscus depletion sedimentation equilibrium method of YPHANTIS a t different p H values are given in Table 1. Only a t (1964) in a Beckman Model E ultracentrifuge equipped p H 4.0 was a completely linear plot of the log of the with interference optics. The PNGF or its derivative pro- fringe displacement vs rz obtained (Fig. l a ) a n d with teins were diluted to a concentration of approx 0 6 mgjml it a unique value of molecular weight. T h e actual and dialyzed overnight at 4 T against at least two changes value of 26,500 was in excellent agreement with the of buffer. The concentration of the buffers were 0.1 M to molecular weight determined from the amino acid which 0 1 M NaCl (or 0.2 M NaCl for one experiment) was sequence. T h e Same result was obtained a t two differalso added. The concentration of the protein samples was ent speeds (32,000 and 36,000rev./min) and in the determined from the known extinction coefficient of PNGF presence of 0.1 M or 0.2 M NaCl. At the higher pH = 16.1) and varied from 0.40 to at 280nm [E:&&'" of 5.0 and 5.8 in acetate buffer the fringe displacement 055 mg/ml. Sedimentation was carried out in a 12 mm double sector graphs showed slight but significant curvature (Fig. Ib, c) and a corresponding range of molecular weight cell using @13ml of protein solution at 36,Wrev./min (30,000rev./min for one experiment) a t 25°C for 24 h. A (Table 1). At both pH values, the range was t o both control set of water interference fringes was obtained higher and lower values compared to the actual molebefore the cell was dismantled. Molecular weights were cular weight of PNGF. T h e data obtained at p H 7.6

Characterization of PNGF

TABLE1. THEVARIATION IN THE MOLECULAR WEIGHT PNGF AS A FUNCTION OF pH A N D OF BUFFER TYPE

OF

Molecular weight Average Range1

pH and buffer*

4.0, Acetate 26,500 k 300 5.0. Acetate 5.8, Acetate 7.6. Tris CI10.0, Carbonate 10.0 Ethanolamine Cl10.0 Ethanolamine Cl26,000 dialysed to 4.0 acetate

24.600-28.100 22.5W27.100 22,8W26,700 27,7W36,000 14,400-25,800

~

* Buffers were 0.1 M and contained

0.1 M NaCl.

t Derived from the slope of the plot of log of the fringe displacement vs rz over intervals of protein column usually covering from 0-250.50 of the column height. in TrisHCl buffer was similar to that at the slightly acidic pH values (Table 1) and the fringe displacement graph was again curved (not shown). Even more pronounced curvature of the fringe displacement graph was evident at pH values greater than 9.0 and at pH 10.0 in carbonate buffer, for example, (Fig. Id) molecular weights as high as 36,000 were noted (Table 1). Aggregation of BNGF is clearly occurring under

I57

hese conditions. The limiting value of the molecular veight in the low protein concentration part of the yadient approached that of BNGF itself. In contrast, while the fringe displacement plot was again curved n an ethanolamine buffer at pH 10.0 (Fig. le), the :orresponding molecular weight values all fell at or xlow that of PNGF (Table 1) suggesting dissociation ather than aggreagation, of the dimer BNGF mole:ule. Subsequent dialysis of ethanolamine treated 3NGF against acetate buffer, pH4.0, resulted in a Edimentation profile (Fig. If) and molecular weight Table 1) indistinguishable from that of BNGF :xposed only to the acetate buffer, pH 4.0. The same result was also obtained when BNGF exposed to carbonate buffer was returned to pH 4.0. The molecular properties of modified PNGFs. Since :he molecular weight of BNGF measured at p H 4 0 igreed with that determined from the amino acid equence, the effects of the partial and complete proteolytic cleavage on BNGF were examined at this pH. [n the 2% NGF preparation about 25% of the pep1ride chains lack the COOH-terminal residues and SO% lack the NH,-terminal octapeptide sequence. Its molecular weight at pH 4.0 was found to be 26.000 'Table 2) in very close agreement also with the value

2.3629

2.40741

>

1.7693

1.7976

f

1.4725

1.4949

I

I

I

1

n

+

9

1.1758 0.8789 0.8830 49.52 49.84 50.16 50.49 50.81 51.13 49.46 49.75 50.05 50.34 50.63 50.92

,

2.30281 2.03131

n

+ > u

1.7855

0

1.4918

s"

1.1980

I

I

1

(dl

1.2167 k,op'

-

I

i

0.9 4 5 2 1 1 I " I 0.9043 49.76 50.00 50.23 50.47 50.70 50.94 49.52 49.84 50.16 50.49 50.81 51.13 2.3627

CI

+

>

1.7618

9

1.4748

1.4614

1.1832 0.8917 0.8605 49.26 49.58 49.90 50.22 5054 60.84 49.08 49.46 49.84 50.22 50.60 50.98

FIG. 1. Sedimentation equilibrium data for PNGF as a function of pH. Each graph IS the plot of the log of the fringe displacement (Ay= y, - yo) vs rz at equilibrium determined under the conditions described in Materials and Methods. (a) PNGF in 0.1 M sodium acetate buffer. pH 4.0: (b) in 0-1M sodium acetate buffer. pH 5.0; (c) in 0.1 M sodium acetate buffer. pH 5.8: (d) in 0.1 M sodium carbonate buffer, pH 10.0; (e) in 01 M ethanolamine chloride buffer. pH 10.0 and (0 in 0.1 M sodium acetate buffer. pH 40 after exposure to the conditions given in (e).All the buffers also contained 01 M NaCI.

P-F. PIGNATTI. M. E. BAKERand E. M. SHOOTEK

15s TABLt

2. THEMOLECULAR WEIGHT OF A TIVES OF

NUMHER OF OERIVA-

BNGF

Molecular species

25s NGF bis Desarginine"' BNGF bls D ~ (1-8) s PNGF his Des(1-8) PNGF (pH 7.0)t bis Des (1-9) PNGF: BXLNGF

weight*

26.000 25,000 25.400 25,300 24.400 27,100

* Determined in 0.1 M sodium acetate buffer pH 4.0 containing 0 1 M NaCl unless indicated. t Determined in 0.1 M phosphate buffer pH 7.0 containing 01 M NaCI. A partial (72%)cyanogen bromide cleaved product. determined from the sequence data. The partial proteolysis of m G F seen in 2.5s NGF preparations can be carried to completion by the use of appropriate enzymes. Digestion of PNGF. for example. with carboxypeptidase B rapidly removes both COOH-terminal arpinine residues (MOORE et al., 1974). The product of this reaction, bis desarginine1I8 PNGF, also has a molecular weight close to that of the native BNGF (Table 2). Similarly, digestion of PNGF with the extract of the mouse submaxillary gland results in complete removal of the NH,-terminal octapeptide sequences from both chains as well as limited (45%) cleavage of the COOH-terminal arginine residues. The product, which is 1 6 1 7 amino acids shorter than BNGF, gave a molecular weight of 25,400 (Table 2) and the linearity of the fringe displacement plot emphasized both the homogeneity of this modified PNGF preparation and the specificity of the cleavage at the eighth peptide bond in the NGF chain. In contrast to PNGF, bis des(l-8)jNGF also gave a unique molecular value at neutral pH. The value obtained was 25,300 (Table 2), close to that observed at pH 4.0. and the narrow range of the molecular weight values seen across the cell (24,300-25,900) fell within the normal error for these determinations and precluded any significant dissociation or aggregation of the modified BNGF dimers. In line with these results for PNGF modified by complete specific proteolytic cleavage at the two ends of the peptide chains the molecular weights of another partially modified derivative also fell within the normal values for a PNGF dimer. Thus the preparation in which 72% of the chains were shortened by nine amino acids from the NH,-terminus by cyanogen bromide cleavage at methionineg gave a molecular weight of 24,400 (Table 2) which may be compared to the expected value of 25,000. Finally, the crosslinked PXLNGF also gave the dimer molecular weight at pH 4.0. Although the value of 27,100 (Table 2) is within experimental error of the actual molecular weight of BNGF, a higher molecular weight would be expected because of the addition to BNGF of a number of amidate groups. The extent of amidation in the crosslinking reaction is not yet known.

DISCUSSION

The data presented here show that there are limited solvent conditions under which the molecular weight of BNGF determined by sedimentation equilibrium agrees with that derived from the amino acid sequence data. Only at pH 4.0 was the fringe displacement graph linear throughout the cell and thc molecular weight value determined from its slope the same as the value from the sequence data. In the pH range 5.&7.6, the data suggests that the BNGF dimer is capable, at low protein concentration, of dissociating into monomers and at high protein concentration of aggregating into higher polymers. That aggregation can occur is confirmed by the results obtained in carbonate buffers at pH 10-0. It is of interest that the relatively extensive aggregation seen under these conditions is reversible when the pH is lowered to 40. It is surprising, in view of the known stability of the PNGF dimer that some degree of dissociation into monomers can be seen at mild acid or neutral pH. GREENE et al. (1971), for example, found that the dissociation of dimers into monomers in 1% sodium dodecyl sulfate at 37°C had a half life of 2 h. On the other hand, MCQRE & SHOOTER (1975) have found that hybrid PNGF dimers form readily at mild acid pH when PNGF and bis desarg"& PNGF dimers are mixed, indicating a mobile equilibrium between dimer and monomer under these conditions. The sedimentation equilibrium experiments reflect this equilibrium by the shift to lower than expected molecular weight in the low concentration part of the protein column. These observations explain the data in the literature. The sedimentation equilibrium studies of ANCELETTIet al. (1971) were carried out at pH 5.0 and at higher protein concentrations than used in this investigation (0.62-0.90 mg/ml). Under these conditions, the aggregation phenomena would be enhanced and molecular weight in the region of 28,OW29,000 would be anticipated from the data reported here. That this explanation is correct is evidenced by the fact that 2.5s NGF gave the correct sequence molecular weight when analysed at pH 4.0 and lower protein concentration. conditions under which PNGF also displays no aggregation. Both the gel filtration studies of ZANINIet al. (1968) and the sedimentation velocity-diffusion experiments of BocCHMI & ANGELETTI(1969) were also carried out at significantly higher protein concentrations than used here and the higher molecular weight values obtained (28,000-32,OOO) are consistent with a significant degree of aggregation. That the PNGF system is a relatively complex one in terms of dissociation and aggregation is emphasized by the apparent dissociation in ethanolamine buffer at pH 10.0 as opposed to aggregation in carbonate buffer at the same pH. The extent of either reaction is clearly determined by a variety of solvent conditions such as pH, salt concentration and type as well as protein concentration. Because of the demonstrated homology in the amino acid sequences of NGF and proinsulin (FRAZIER et al., J972) and of similarities in the topography

Characterization of BNGF

I 59

R . H.. BRADSHAW. R . A. & WADE.R . D. (1971) of the NGF and insulin molecules (FRAZIER et al., ANGELETTI, Biochemistry 10. 4 6 H 6 9 . 1973) a comparison of their physical properties in R. H.. HERMODSON. M. A. & BRADSHAW.R . solution is also of interest. Most insulins and prginsu- ANGELETTI, A. (1973) Biochemistry 12. IW115. lins exist as dimers in solution but show aggregation ANGELETTI,R . H.. MERCANTI.D. & BRADMAW. R . A. under suitable conditions, the behavior of proinsulin (1973) Biocheniistry 12. 9 0 9 . being similar to that of insulin over a wide range BALDWIN.R. L. (1957) Biochrm. J . 65. 503 512. of pH values (FRANK& VEROS,1968; ZIMMERMAN et BOCCHINI.V. & ANGELEWI. P. U. (1969) Proc. nufn Arud. al., 1972; GRANTut al., 1972). Like BNGF, aggregaSci. U.S.A. 64. 787-794. tion is most pronounced at neutral or mild alkaline BROWN.J. R . & HARTLEY.B. S. (1966) Biochmi. J . 101. 214228. pH while the dimer is the predominant species at acid pH. One insulin. guinea-pig insulin, is found only in COHN,E. J. & EDSALL.J. T. (1943)in Proteins. Amino Acids and Peptides p. 375. Reinhold. New York. the monomer state and does not form either dimers or higher polymers (ZIMMERMANet al.. 1972). It there- FRANK.B. H. & VEROS.A. J. (1968) Biorhrm. hiopliys Res. Commun. 32, 1 5 5 1 6 0 . fore displays in exaggerated form the dissociation FRAZIER. W. A., ANCELETTI, R. H. & BRADSIUW.R . A. phenomenon seen with BNGF at mild acid or neutral (1972) Sciertce. N.Y. 176, 482488. pH. In general the solution properties of BNGF and FRAZIER.W. A.. ANGELEWI. R . H.. SHERMAN, R . & BRADthe insulins or proinsulins are similar. the differences SHAR'. R . A . (1973) Biochrmistrj~12. 3281~3293. in the extent of dissociation or aggregation probably FRAZIER.W. A., BOYD. L. F. & BRAIXHAW.R . A. (1973) Proc. m t i i . Acad. Sci. L . S . A . 70, 2931-2935. being determined by the amino acid sequences of the R. G. & CRESITIELD, A. M . (1965) J . h i d . C'licm. chains which in turn determine the non-polar interac- FRUCHTER, 10. 3868-3874. tions between them. It would be of interest to determine if PNGF, like insulin or proinsulin, forms hex- GRANT.P. T.. COOMBST. L. & FRANK.B. H. (1972) Biochem. J . 126, 433440. amers stabilized by zinc ions, especially in view of GREEN+ L. A., VARON,S.. PILICH.A. & SHOOTER, E. M . the recent observation that 7s NGF contains zinc (1971) Nrurohiology 1. 3748. (PATTISON& DUNN,1975). LEVI-MONTALCINI. R . (1966) Harory h c f . Scr. 60.217- 259. Partial or complete removal of either the NH,-ter- MOBLEY.W. C. (1974) Ph.D. Thesis. Stanford University. minal octapeptide sequences or the COOH-terminal Stanford, Calif. arginine residues has no effect on the dimeric charac- MORLI:Y.W. C., M(x)RI;. J. B.. JR.. k l i t N K I R . A. & SHOO?% E. M. (1974) in Mod. frohlcms in Pcdiutrrrh ter of the NGF protein at least as determined at acid 13. 1 12. pH, nor on its NGF activity in the iri ciwo assay W. C. & SIIOOTI.R, E. M . (1974) (MOOREet al., 1974). Removal of the octapeptide MOORE.J. B.. JR.. MORLI-Y. Biochemistry 13. X33 -X40. sequences does, however, have some minimum effect MOORE.J. B. JR. & S H O O ~ R E.. M . (197.5) ~ V c w o h i o k q y on the chain interactions because his des(l-X)bNGF. in press. unlike P N G F . displays no dissociation or aggregation PATTISON. S. E. & D ~ I N NM. . F. (19751 Biochmistry in at pH 7.0 (Table 2). It is not surprising that modificapress. tion of chain interaction has no effect on the NGF SELA.M . & ANFINSEN. C. B. (1957) Biorhim. hwphys. Arta activity of the derivative because both NGF dimers 24, 229--235. (STACH& SHOOTIR. 1974) and monomers (FRAZERSWYTH.D. G.. STFIN.W. H . & MOORI.. S. (1963) J . h i d Chem.. 238. 227 234. or a/.. 1973) are biologicd11y active. Thus. a displaceE. M. (1974) J . hiol. Chzm. 249. ment in the equilibrium between monomer and dimer STACH.R . & SHOOI~R. 66hH-6674. at the NGF concentrations which givc optimum reSTRAUS. D.. Dr JONO. W. W . W.. OHMS. J.. MOHI I Y. W C-.. sponse in the bioassay. i.e. 4 x 10- M. will have no SCIWNKER. A. & S H O I ~ RE.. M . (1975) N c w o h i f h q ) . effect in the bioresponse unless there is a large differin press. ence in the activities of the monomer and dimer spe- VARON.S., NOMIJRA. J. & SHOOTER,E. M . (1967) Biockvw istry 6, 2202-2209. cies. While the prcsent data define the solution properties of PNCF and some of its derivatives in VARON.S.. NOMURA.J. & SWTER, E. M . (1967) flrockmisrry 7. I 2 9 6 1303. the concentration range from approx 1W5to 5 x S. & S H O o n R . E. M. (1970) in fluxhemistry cf lo-' M it still remains to explore the monomcrdimer VAROK. Bruin arid Bzlturiour. (BOWWAS. R . E. & DATTA. S. P.. equilibrium at the much lower protein concentration eds.) pp. 41 M.Plenum Press. New York. u.hcre NGF exerts its specific biological effects. YPHASTIS. D. A . (1964) Bioc/itwii,\trv 3. 297 21' ZA\IW. A.. As(;rLi.rri. P. & LI \-I-MO\TAI c '\I. R (19hXt

'"

REFERENCES AXXLLTTI. R. H. & BRAUSHAW. R. A. 119714 Proc. num. .4cud. Sci. L.S.A. 68. 2417-2420.

Proc. iwfn. Acud. Sci. 1.S.A. 61. R35-UJ2. ZIMMERMAUS. A. E.. KFLLS.D 1. C. & YIP. C C. 119721 Riochem. hiophys. Res. Commuii. 46. 2127~213.7

Solution properties of beta nerve growth factor protein and some of its derivatives.

SOLUTION PROPERTIES OF p NERVE GROWTH FACTOR PROTEIN AND SOME OF ITS DERIVATIVES P-F. PIGNATTI', M. E. BAKER'and E. M. SHOOTER Departments of Genetics...
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