Journal of Nrurochemisrry, 1976. Vol. 27. pp. 173-177. Pergamon Press. Printed tn Great Britain.
MECHANISM OF INTERACTION OF MYELIN BASIC PROTEIN AND S-100 PROTEIN: METAL BINDING AND FLUORESCENCE STUDIES1y2 A. S. PERUMAL, S. P. MAHADIKand M. M. RAPPORT Divison of Neuroscience, N.Y. State Psychiatric Institute and Department of Biochemistry, Columbia Univ College of Physicians and Surgeons, New York, NY 10032, U.S.A. (Received 4 November 1975. Accepted 7 January 1976)
Abstract-It has been reported that myelin basic protein (MBP) forms a specific complex with S-100 protein in the presence of either C a t + or M n 2 + , as detected by immunoelectrophoresis. We have now studied the binding of C a t + and M n 2 + to these two proteins. We find that M B P binds 1 mol of Mn*+/mol of protein, and this binding produces an increment in its fluorescence, indicating a conformational change. C a 2 + does not bind to M B P nor does it affect the fluorescence of MBP. S-100 protein, as has been reported, binds about 10mol of Caz+/mol and this binding produces a conformational change. S-100 protein also has 25 binding sites for Mn", but this binding does not alter fluorescence and does not appear to affect conformation. Competitive binding experiments demonstrate that the binding sites of S-100 protein for C a t + and M n Z +are independent. The alteration of electrophoretic migration in gels of S-100 protein produced by CaZi and of M B P produced by M n 2 + are in accord with the observations based on fluorescence. M n 2 + does not affect the electrophoretic mobility of S-100. These results indicate that the formation of the complex between MBP and S-100 protein in the presence of either Ca2' or M n 2 + is due to the conformational change induced by these ions in S-100 protein, MBP, or both.
ALTHOUGHmany studies of S-100, an acidic, soluble Ammonium sulfate, Tris and glycine were of ultra-pure protein specific for nervous tissue, have been carried grade (Schwartz-Mann Chemicals, Orangeburg, N.Y.) out, its function is still unknown. CALISSANO et al. Acrylamide, bis-acrylamide and TEMED were obtained (1969) demonstrated that C a 2 + binds to S-100 protein from Eastman Kodak Co., Rochester, N.Y. All other and induces a conformational change and CALISSANOchemicals were of analytical grade. & BANGHAM (1971) have shown that when S-100 proMyelin basic protein tein is bound to artificial lipid membranes, Ca2+ inMyelin basic protein was isolated from bovine brain duces permeability to cations. MAHADIKet al. (1974) according to the procedure of OSHIRO& EYLAR(1970). have shown by immunoelectrophoresis that S- 100 forms a complex with myelin basic protein, and this S-100 protein complex is stabilized in the presence of either C a 2 + S-100 protein was isolated by a 3-step purification from or Mn". the 100,000 g supernatant of a bovine brain homogenate. In this paper we are reporting studies of the bind- These steps involve precipitation between 60 and 100% ing of Ca2+ and Mn2+ to myelin basic protein and saturation of ammonium sulfate at pH 4.0, ion exchange to S-100 protein and the alterations in conformation chromatography on DEAE cellulose. eluting with a pH that take place in the presence of these ions. Alter- gradient at p H 5 and finally obtaining the fastest running ations in conformation are detected from changes in fraction on preparative polyacrylamide gel electrophoresis fluorescence. Both proteins exhibit this fluorescence at p H 8.6. The final preparation migrated as a single band which is attributable to the one tryptophane residue in 7.5% polyacrylamide gel at pH 8.3 and showed a single line of precipitate with rabbit antiserum to S-100 protein. per mol that each contains. MATERIALS AND METHODS
It was stored as a powder at -20°C. The preparation in Tris buffer (50 mM, pH 8.6) gave a molar extinction of 8000 at 280nm (assuming a mol. wt of 22,000 daltons).
Chemicals
Calcium-45 (1 6.6 mCi/mg) and Manganese-54 (carrierfree) were obtained from New England Nuclear, Chicago.
Fluorescence measurements
These were made at ambient temperature in a 1 cm quartz cell with an Aminco-Bowman spectrophotofluorimeter equipped with a high stability xenon lamp. Studies of the effect of metal concentration o n fluorescence (fluorescence titrations) were made with 120 pg of protein in 2 ml of Tris-HCl buffer (20 mM, pH 8.3) adding the salts (CaCI,, MgCIZ, KCI) in portions of 2 pl. pH was adjusted by adding 2 p1 portions of NaOH. Primary (incident) and secondary (emitting) monochromators were set at 290 and
' A preliminary report of this work was presented at the Fourth Annual meeting of the Society for Neuroscience, St. Louis, M O October 1974. This work was supported in part by a grant from the National Multiple Sclerosis Society. Ahhrruiations used: MBP: Myelin Basic Protein; EDTA: Ethylene-diamine tetra acetic acid. 173
A.
174
s. PFRLXIAL. S. P. MAHADIKand
360 nm respectively. A tryptophane standard & a s used to check instrument stability.
M. M. RAPPORT
cations were included both in the gel and in the electrode buffer at the same concentration.
Binding studies Equilibrium dialysis was carried out in Pyrex tubes (14mm id.) using 1'4in. dialysis casing (Arthur H. Thomas, Phila., Pa.) previously heattreated in EDTA. To the protein in Tris-HCI buffer (50mu. pH 8.3) various amounts of either 4sCa2' or 54Mn'' wcre added in addition to other salts as required. After bringing the final vol to 0.2 ml, dialysis was carried out against 5.0 ml of Tris-HC1 buffer, pH 8.3. at room temperature (24-28'C) for 24 h, although equilibriuni was usually attained by 20 h. The quantity of bound ion was calculated from determinations of radioactivity of samples from inside and outside the bag. In experiments with 4'Ca'' . 0.5 ml of TS-1 (Research Products Internat.. Inc.. Elk Grovc. Ill.) was added to the sample folloRed by scintillation fluid and counting was done in a p-scintillation counter (Packard model 3320). In experiments with '4Mn2 ' the samples were placed in 10 x 75 mm. disposable tubes and counted in a y-scintillation counter (Packard model 3001). Acrylamide gel efecrrophoresis Gel electrophoresis of S-100 protein was run in 7.5', gel according to CALISSASOP I of. (1969) at pH 8.6 using Tris-glycine buffer. Gels for myelin basic protein were prepared from a gel mixture containing 60 mM-K-acetate buffer pH 4.2. 7.S0, acrylamide, 0 . 2 " ~bis-acrylamide. 0.5"" T E M E D and 0.14", ammonium persulfate. Electrode buffer was 17.5 mM-Kacetate (pH4.2). Cytochrome C in a parallel gel served as a marker. The gels were prerun at 3 mA:gel for 15 min and, aftcr applying the samples. the electrophoresis was carried out at 1 ma;'gel for 15 h at which point the cytochrome C reached about 1 cm from the cathodic end of the gel. All the gels were stained with Amido Black (0.50, Amido Black in l00/, meth:inol-7", acetic acid) for 1 hr and destained using a diffusion destainer. When the effect of cations on the electrophoretic behavior of S-100 and myelin bdsic protein was studied the
RESULTS
Fluorescence studies with S-100 protein
Ca2' enhances the fluorescence of S-100 (CALISet a[., 1969) and our results confirmed this. A stable interaction between MBP and S-100 protein is observed at 10 mM-Ca2+,and at this concentration the fluorescence of S-100 protein was maximal. In contrast, Mn'+ did not affect the fluorescence of S-100 protein. SANO
Fluorescencr studies with M B P
Mn2' enhanced the fluorescence of MBP whereas Ca2 + , Mgz+ and K + did not have any effect (Fig. 1). This enhancement was reversible since addition of EDTA caused the fluorescence to return to its initial value. With MBP at 60pg/ml (pH 8.3) the enhancement by Mn'+ was detectable at 1 mM, rose rapidly with increasing concentration of M n 2 + and was maximal at 5 mM, producing an increase in fluorescence of about 70%. Complex formation between MBP and '3-100 protein (with MBP at a concentration of about 750pg/ml and S-100 protein at 250 pg/ml) required a minimal concentration of 1 mM-Mn". It is difficult to relate these numbers because S-100 protein has such a large number of binding sites for M n 2 + (see below), but our studies showed that as the concentration of MBP was increased. the effect of Mn2+ on the conformational change could be seen at lower concentrations of Mn" (Fig. 2). Since CALISSANO et al. (1969) had found that K + interferes with the change in conformation of S-100 protein induced by Ca", we studied the effect of K +
r
130r
Mg
KCI
0
2
4
6
8
1
0
Conc, of Divalent ion (rnM)
2
4
6
8
+ Mn
+ Co + Mn
1
0
Conc, of Mn2+(mM)
FIG I . Effect of various metal ions on the fluorescence of MBP. MBP (60/cg/ml) in 20mM-Tris. HC1 buffer (pH 8.3). KCI. when present. at 6 0 m ~ Fluorescence . i n arbitrary units. Left panel: M n Z + enhances fluorescence of MBP; Ca' and Mg' do not. K' inhibits this enhancement to some degree. Right panel: Mn" enhances the fluorescence even in the presence of Ca2' and Mgz+. When C a Z + and KCI are present together. fluorescence enhancement is markedly inhibited. C a Z + and M n Z C at I0 mM. +
Mechanism of myelin basic protein-S-100
protein interaction
175
not enhance MBP fluorescence at pH 4.7 whereas the full effect was seen at pH values of 7 and above. If the dissociation of a single group were responsible, the pH-fluorescence curve indicates that it would have a pK value of 5.1. It should be recalled that MBP-S-100 protein interaction occurs in the pH range of 6.68.6, and in this range the full effect of M n Z + on MBP conformation is found. Binding studies wirh M B P
I 0.2
I 0.4
I 0.6
I 0.8
The results of 3 equilibrium dialysis experiments with s4Mn2+ at 2 concentrations of MBP (Fig. 4) showed that MBP has only 1 binding site for M n 2 + and this site has an association constant of 1.25 mM. Similar results were obtained when equilibrium was reached starting with M n 2 + either inside or outside the dialysis bag. MBP was found not to bind Ca'+.
I 1.0
Binding studies wirh S-100 protein
Conc. of Mn2+ (mM) FIG.2. At higher concentrations of MBP the enhancement of MBP fluorescence is seen with lower concentrations of M n 2 + . Closed circles, MBP, 0.72 mg/ml; Open circles, 0.31 mg/ml. Fluorescence in arbitrary units. pH 8.3.
and other ions on the enhancement of MBP fluorescence by Mn2+. K + at 6 0 m had ~ only a little effect and neither Ca2+ nor Mg2+ had any effect. However when both K + and C a 2 + were present together, they interfered, reducing by 50% the enhancement of MBP fluorescence by Mn2+. This result is consistent with the observation of MAKADIKet al. (1974) that with Mn2 at 1 mM, K c at 80 mM did not affect the formation of the MBP-S-100 protein complex. The enhancement of MBP fluorescence by Mn2+ was dependent on pH (Fig. 3): at 4 m ~ Mn2' , did +
Equilibrium dialysis studies of the binding of Ca2+ to S-100 protein showed that 10mol of Ca2+ were bound per mol of the protein, confirming the findings reported by CALISSANO et al. (1969). When similar studies were carried out using 0.1-10mM 54Mn2+, S-100 was found to have 25 binding sites per mol as revealed by double reciprocal plot (Fig. 5). M n 2 + did not affect the fluorescence of S-100. Since S-100 protein binds both Ca2+ and Mn2+, studies were carried out t o determine whether, at concentrations where these ions affect the complex formation between MBP and S-100, they compete for sites on the S-100 protein. We chose a C a 2 + concentration of 0.4 mM; at this concentration about 3 mol of Caz+ are bound per mol of S-100 which is sufficient to produce the conformational change (CALISSANO et al., 1969). The results (Table 1) show that at this concentration neither M n 2 + (0.1 and 1.OmM)
200 -
I
I60 -
120-
F 80 -
40 4 -
1 /conc. Mn2+(mM) 5
6
7
8
9
10
II
12
PH FIG. 3. Enhancement of MBP fluorescence by M n Z + is pH dependent. MBP, 80 pg/ml. Closed circles, MBP + M n 2 + (4 mM); Open circles, MBP alone.
FIG.4. Reciprocal plot of Mn' binding to MBP, showing that 1 mole of metal is bound per mol of protein. Molecular weight of MBP taken as 18,000. Open triangles, MBP at 0.8 mg/ml; closed circles 1.0 rng/ml. V = mol of metal bound per mol of protein determined by equilibrium dialysis. +
A. S PERLMAL.. S. P. MAHADIK and M. M. RAPPORT
176
2
1 -
v
I
1 /conc. Mnzf(mM) FIG.5. Reciprocal plot of hln'. binding to S-100 protein showing that 25 rnol of metal are bound per rnol of protein. Closed circles and open triangles: S-100 at 0.8 mg'ml. (2 experiments). Open circles, S-100 at 1.0mg ml. V = mol of metal bound per rnol of protein determined by equilibrium dialysis, assuming mol. wt of 22.ooO daltons for S-100 protein.
its 25 binding sites. With MBP, M n 2 + at 0.05mM slowed the migration and this effect was much greater at 0.1 mM (Fig. 6, left panel). As the concentration of Mn2' \vas further increased to 0.25 and 0.5mM the retardation increased slightly and multiple bands appeared (gels D and E). At 5.0 mM-Mn2+, the retardation i n migration of MBP was reduced and the separation into multiple bands was much less distinct (gel F). The identical pattern was retained at 10mwMn". The mechanism for the change that occurs at the higher concentrations of M n 2 + is not evident. but i t is not due to the higher concentration of divalent ions as can be seen from the lack of such an effect in the presence of 5 mM-Ca2' (gel G). Since Ca" does not bind to MBP, the explanation must be related to the binding of M n 2 + to MBP in some way that remains to be determined. DISCUSSION
Since the formation of a complex between MBP and S-100 protein requires Ca2+ or Mn2+ ions, we have examined the effect of these ions on protein conformation. MBP binds M n 2 + but not C a 2 + whereas nor Mg2+(0.5 mM) nor K ' (12.5 mM) affected the bindS-100 protein binds both Mil2+ and Ca". The binding of C a 2 + . Ferrous ion (0.5 mM) enhanced Ca" ing of Mn" induces a conformational change in binding somewhat. Also. with "Mn" at 0.1 mM. a MBP as evidenced by the increase in MBP fluoresconcentration at which about 0.8 rnol of Mn'* is cence. Since only one M n 2 + ion is bound per rnol bound. C a 2 + (0.05 and 0.1 mM) did not affect the of MBP. it is probable that this binding occurs in binding of Mn' the vicinity of the single tryptophane residue. Recently EPANDet al. (1974) demonstrated that MBP Gel electrophoresis (A-1 protein) has a folded conformation rather than In the presence of Ca". the migration of S-100 & THOMPSON, 1969; C k o ~ a random structure (EYLAR protein was retarded; the protein also showed five & EIXSTHN. 1970). If the tryptophane residue were distinct bands (Fig. 6. right panel) at both 0.5 and buried within the fold and then released by unfolding 5.0 mM, confirming the earlier report of CALISSAYO ei as a consequence of M n 2 + binding, the enhanced a/. (1969). In contrast. Mn" at 0 . 5 m ~(gel D) did fluorescence would be accounted for. That the confornot show any retardation although a second minor mation of MBP is altered in the presence of M n 2 + component appeared. At 5.0mM-Mn" (gel E) the is also indicated by the change in its electrophoretic two bands were seen but the migration was appreciamobility. bly retarded. This retardation probably resulted from With regard to S-100 protein, our experiments conthe neutralization of negative charges in the S-100 firm the observations of CAL~SSANO et a/. (1969) that protein through binding of Mn" to most or all of S-100 protein contains about 10 binding sites for Ca' +. and that binding of Ca2 enhances the fluoresTABLE 1. OTHER DIVALEST 1135sDO ?OT COMPETE WITH THE cence of the protein as a result of its change in conforBIUDI% OF EITHER Ca'* OR Mn'* TO s-100 P R O T E I ~ mation. We have found that S-100 protein contains Mol of metal about 25 binding sites for M n 2 + , but the binding of bound per rnol of Mn'- does not alter the fluorescence and therefore protein Other Conc does not presumably affect the protein conformation. Mn'Bound metal Ion added (mM.l) Ca' These observations are consistent with the effects 2.86 Ca' ' (0.4 mM) none observed of these ions on electrophoretic mobility : 2.52 Mn" 0.1 in the presence of Ca" S-100 protein showed multi2.78 1 .o ple bands indicating several conformational states as 2.60 Mg' + 0.5 reported by CALISSANO c't ul. (1969) whereas no such Fe' + 3.30 0.5 K' 2.63 12.5 effect was seen with M n 2 + .In the absence of a conforMn2' (0.1 mM) none 0.67 mational change, it is not clear how the binding of Ca'+ 0.73 0.05 Mn" affects the properties of S-100 protein, but it 0.10 0.73 is worth noting that RNA polymerases, some of Binding assays were carried out by equilibriom dialqsis which are Mn2 dependent, have been reported to (see Methods). be activated by S-100 protein (MIANIv t a/. 1973). I.
+
-
+
FIG.6 . Effect of M n Z +and Ca2+on electrophoretic mobilities of MBP and S-100 protein in polyacrylamide gels. Protein and metal ion mixed at pH 7.5. Left panel: Electrophoresis at pH 4.2. Protein migration from anode to cathode. All gels contained MBP, 57 pg with the following additions: A, none. B to F, MnZ+0 . 0 5 m ~ 0.1 , mM, 0.25mM, OSmM, 5.0mM. G , Ca2+, 5.0mM. Right panel: Electrophoresis at pH 8.3. Protein migration from cathode to anode. All gels contained S-100 protein, 25 pg with the following additions: A, none. B and C, Caz+, O S r n M , 5.0mM. D and E, Mn2+, 0.5mM. 5.0 mM.
NC-176
Mechanism of myelin basic protein-S-100 It seems most probable in view of these conformational changes induced in MBP by M n 2 + and in S-100 protein by Ca2+ that the specific complex formation between these two proteins results from conformational changes that take place either in MBP due to Mn2+ or in S-100 due to Caz+ or both. How these changes affect the formation of the complex under physiological conditions remains to be determined, but in view of the heterogeneity of s-100 prort d.,1971) further studies are warteins (COMBOS ranted of the specificity of complex formation between MBP and various other acidic brain proteins, such as GP-350, GFA protein and C M SO-C, a protein recently described by HAGLIDet al. (1975). REFERENCES P , MOORFB. W. & FRIESFN A. (1969) BioCALISSANO c l i m i s t r y 8. 43 18-4326.
protein interaction
177
CALISSANO P. & BANGHAM A. D. (1971) Biaclipm. hiophys. Rrs. Conzmun. 43. 504-509. CHOA L. P. & EINSTEINE. R. (1970) J . Neurochenl, 17. 1121-1 132. EPANDR. M., MOSCARELLO M. A,, ZIERENBERC B. & VAIL W. J. (1974) Biochemistry 13. 1264-1267. EYLARE. H. & THOMPSON M. (1969) Archs Biochem. Bioplzys. 129. 468-479. GOMROS G.. ZANETTAJ. P., MANDELP. & VINCENDON G. (1971) Biochemir 53. 645-655. HAGLIDK. G., RONNsACK L. & STAVRON D. (1975) J . Nrurochem. 24. 1053-1057. MAHADIKS. P., GRAFL. & RAPPORTM. M. (1974) Frdn. Proc. Fedn Am. Socs e.up. Biol. 33, 777. MIANIN., MICHETTIF., DE RENZISG. & CANIGLIA A. (1973) Experientiu 29. 1499-1501. OSHIRO Y. & EYLARE. H. (1970) Archs Biochem. Biophys. 138. 392-396.
MECHANISM OF INTERACTION OF MYELIN BASIC PROTEIN AND S-100 PROTEIN: METAL BINDING AND FLUORESCENCE STUDIES1y2 A. S. PERUMAL, S. P. MAHADIKand M. M. RAPPORT Divison of Neuroscience, N.Y. State Psychiatric Institute and Department of Biochemistry, Columbia Univ College of Physicians and Surgeons, New York, NY 10032, U.S.A. (Received 4 November 1975. Accepted 7 January 1976)
Abstract-It has been reported that myelin basic protein (MBP) forms a specific complex with S-100 protein in the presence of either C a t + or M n 2 + , as detected by immunoelectrophoresis. We have now studied the binding of C a t + and M n 2 + to these two proteins. We find that M B P binds 1 mol of Mn*+/mol of protein, and this binding produces an increment in its fluorescence, indicating a conformational change. C a 2 + does not bind to M B P nor does it affect the fluorescence of MBP. S-100 protein, as has been reported, binds about 10mol of Caz+/mol and this binding produces a conformational change. S-100 protein also has 25 binding sites for Mn", but this binding does not alter fluorescence and does not appear to affect conformation. Competitive binding experiments demonstrate that the binding sites of S-100 protein for C a t + and M n Z +are independent. The alteration of electrophoretic migration in gels of S-100 protein produced by CaZi and of M B P produced by M n 2 + are in accord with the observations based on fluorescence. M n 2 + does not affect the electrophoretic mobility of S-100. These results indicate that the formation of the complex between MBP and S-100 protein in the presence of either Ca2' or M n 2 + is due to the conformational change induced by these ions in S-100 protein, MBP, or both.
ALTHOUGHmany studies of S-100, an acidic, soluble Ammonium sulfate, Tris and glycine were of ultra-pure protein specific for nervous tissue, have been carried grade (Schwartz-Mann Chemicals, Orangeburg, N.Y.) out, its function is still unknown. CALISSANO et al. Acrylamide, bis-acrylamide and TEMED were obtained (1969) demonstrated that C a 2 + binds to S-100 protein from Eastman Kodak Co., Rochester, N.Y. All other and induces a conformational change and CALISSANOchemicals were of analytical grade. & BANGHAM (1971) have shown that when S-100 proMyelin basic protein tein is bound to artificial lipid membranes, Ca2+ inMyelin basic protein was isolated from bovine brain duces permeability to cations. MAHADIKet al. (1974) according to the procedure of OSHIRO& EYLAR(1970). have shown by immunoelectrophoresis that S- 100 forms a complex with myelin basic protein, and this S-100 protein complex is stabilized in the presence of either C a 2 + S-100 protein was isolated by a 3-step purification from or Mn". the 100,000 g supernatant of a bovine brain homogenate. In this paper we are reporting studies of the bind- These steps involve precipitation between 60 and 100% ing of Ca2+ and Mn2+ to myelin basic protein and saturation of ammonium sulfate at pH 4.0, ion exchange to S-100 protein and the alterations in conformation chromatography on DEAE cellulose. eluting with a pH that take place in the presence of these ions. Alter- gradient at p H 5 and finally obtaining the fastest running ations in conformation are detected from changes in fraction on preparative polyacrylamide gel electrophoresis fluorescence. Both proteins exhibit this fluorescence at p H 8.6. The final preparation migrated as a single band which is attributable to the one tryptophane residue in 7.5% polyacrylamide gel at pH 8.3 and showed a single line of precipitate with rabbit antiserum to S-100 protein. per mol that each contains. MATERIALS AND METHODS
It was stored as a powder at -20°C. The preparation in Tris buffer (50 mM, pH 8.6) gave a molar extinction of 8000 at 280nm (assuming a mol. wt of 22,000 daltons).
Chemicals
Calcium-45 (1 6.6 mCi/mg) and Manganese-54 (carrierfree) were obtained from New England Nuclear, Chicago.
Fluorescence measurements
These were made at ambient temperature in a 1 cm quartz cell with an Aminco-Bowman spectrophotofluorimeter equipped with a high stability xenon lamp. Studies of the effect of metal concentration o n fluorescence (fluorescence titrations) were made with 120 pg of protein in 2 ml of Tris-HCl buffer (20 mM, pH 8.3) adding the salts (CaCI,, MgCIZ, KCI) in portions of 2 pl. pH was adjusted by adding 2 p1 portions of NaOH. Primary (incident) and secondary (emitting) monochromators were set at 290 and
' A preliminary report of this work was presented at the Fourth Annual meeting of the Society for Neuroscience, St. Louis, M O October 1974. This work was supported in part by a grant from the National Multiple Sclerosis Society. Ahhrruiations used: MBP: Myelin Basic Protein; EDTA: Ethylene-diamine tetra acetic acid. 173
A.
174
s. PFRLXIAL. S. P. MAHADIKand
360 nm respectively. A tryptophane standard & a s used to check instrument stability.
M. M. RAPPORT
cations were included both in the gel and in the electrode buffer at the same concentration.
Binding studies Equilibrium dialysis was carried out in Pyrex tubes (14mm id.) using 1'4in. dialysis casing (Arthur H. Thomas, Phila., Pa.) previously heattreated in EDTA. To the protein in Tris-HCI buffer (50mu. pH 8.3) various amounts of either 4sCa2' or 54Mn'' wcre added in addition to other salts as required. After bringing the final vol to 0.2 ml, dialysis was carried out against 5.0 ml of Tris-HC1 buffer, pH 8.3. at room temperature (24-28'C) for 24 h, although equilibriuni was usually attained by 20 h. The quantity of bound ion was calculated from determinations of radioactivity of samples from inside and outside the bag. In experiments with 4'Ca'' . 0.5 ml of TS-1 (Research Products Internat.. Inc.. Elk Grovc. Ill.) was added to the sample folloRed by scintillation fluid and counting was done in a p-scintillation counter (Packard model 3320). In experiments with '4Mn2 ' the samples were placed in 10 x 75 mm. disposable tubes and counted in a y-scintillation counter (Packard model 3001). Acrylamide gel efecrrophoresis Gel electrophoresis of S-100 protein was run in 7.5', gel according to CALISSASOP I of. (1969) at pH 8.6 using Tris-glycine buffer. Gels for myelin basic protein were prepared from a gel mixture containing 60 mM-K-acetate buffer pH 4.2. 7.S0, acrylamide, 0 . 2 " ~bis-acrylamide. 0.5"" T E M E D and 0.14", ammonium persulfate. Electrode buffer was 17.5 mM-Kacetate (pH4.2). Cytochrome C in a parallel gel served as a marker. The gels were prerun at 3 mA:gel for 15 min and, aftcr applying the samples. the electrophoresis was carried out at 1 ma;'gel for 15 h at which point the cytochrome C reached about 1 cm from the cathodic end of the gel. All the gels were stained with Amido Black (0.50, Amido Black in l00/, meth:inol-7", acetic acid) for 1 hr and destained using a diffusion destainer. When the effect of cations on the electrophoretic behavior of S-100 and myelin bdsic protein was studied the
RESULTS
Fluorescence studies with S-100 protein
Ca2' enhances the fluorescence of S-100 (CALISet a[., 1969) and our results confirmed this. A stable interaction between MBP and S-100 protein is observed at 10 mM-Ca2+,and at this concentration the fluorescence of S-100 protein was maximal. In contrast, Mn'+ did not affect the fluorescence of S-100 protein. SANO
Fluorescencr studies with M B P
Mn2' enhanced the fluorescence of MBP whereas Ca2 + , Mgz+ and K + did not have any effect (Fig. 1). This enhancement was reversible since addition of EDTA caused the fluorescence to return to its initial value. With MBP at 60pg/ml (pH 8.3) the enhancement by Mn'+ was detectable at 1 mM, rose rapidly with increasing concentration of M n 2 + and was maximal at 5 mM, producing an increase in fluorescence of about 70%. Complex formation between MBP and '3-100 protein (with MBP at a concentration of about 750pg/ml and S-100 protein at 250 pg/ml) required a minimal concentration of 1 mM-Mn". It is difficult to relate these numbers because S-100 protein has such a large number of binding sites for M n 2 + (see below), but our studies showed that as the concentration of MBP was increased. the effect of Mn2+ on the conformational change could be seen at lower concentrations of Mn" (Fig. 2). Since CALISSANO et al. (1969) had found that K + interferes with the change in conformation of S-100 protein induced by Ca", we studied the effect of K +
r
130r
Mg
KCI
0
2
4
6
8
1
0
Conc, of Divalent ion (rnM)
2
4
6
8
+ Mn
+ Co + Mn
1
0
Conc, of Mn2+(mM)
FIG I . Effect of various metal ions on the fluorescence of MBP. MBP (60/cg/ml) in 20mM-Tris. HC1 buffer (pH 8.3). KCI. when present. at 6 0 m ~ Fluorescence . i n arbitrary units. Left panel: M n Z + enhances fluorescence of MBP; Ca' and Mg' do not. K' inhibits this enhancement to some degree. Right panel: Mn" enhances the fluorescence even in the presence of Ca2' and Mgz+. When C a Z + and KCI are present together. fluorescence enhancement is markedly inhibited. C a Z + and M n Z C at I0 mM. +
Mechanism of myelin basic protein-S-100
protein interaction
175
not enhance MBP fluorescence at pH 4.7 whereas the full effect was seen at pH values of 7 and above. If the dissociation of a single group were responsible, the pH-fluorescence curve indicates that it would have a pK value of 5.1. It should be recalled that MBP-S-100 protein interaction occurs in the pH range of 6.68.6, and in this range the full effect of M n Z + on MBP conformation is found. Binding studies wirh M B P
I 0.2
I 0.4
I 0.6
I 0.8
The results of 3 equilibrium dialysis experiments with s4Mn2+ at 2 concentrations of MBP (Fig. 4) showed that MBP has only 1 binding site for M n 2 + and this site has an association constant of 1.25 mM. Similar results were obtained when equilibrium was reached starting with M n 2 + either inside or outside the dialysis bag. MBP was found not to bind Ca'+.
I 1.0
Binding studies wirh S-100 protein
Conc. of Mn2+ (mM) FIG.2. At higher concentrations of MBP the enhancement of MBP fluorescence is seen with lower concentrations of M n 2 + . Closed circles, MBP, 0.72 mg/ml; Open circles, 0.31 mg/ml. Fluorescence in arbitrary units. pH 8.3.
and other ions on the enhancement of MBP fluorescence by Mn2+. K + at 6 0 m had ~ only a little effect and neither Ca2+ nor Mg2+ had any effect. However when both K + and C a 2 + were present together, they interfered, reducing by 50% the enhancement of MBP fluorescence by Mn2+. This result is consistent with the observation of MAKADIKet al. (1974) that with Mn2 at 1 mM, K c at 80 mM did not affect the formation of the MBP-S-100 protein complex. The enhancement of MBP fluorescence by Mn2+ was dependent on pH (Fig. 3): at 4 m ~ Mn2' , did +
Equilibrium dialysis studies of the binding of Ca2+ to S-100 protein showed that 10mol of Ca2+ were bound per mol of the protein, confirming the findings reported by CALISSANO et al. (1969). When similar studies were carried out using 0.1-10mM 54Mn2+, S-100 was found to have 25 binding sites per mol as revealed by double reciprocal plot (Fig. 5). M n 2 + did not affect the fluorescence of S-100. Since S-100 protein binds both Ca2+ and Mn2+, studies were carried out t o determine whether, at concentrations where these ions affect the complex formation between MBP and S-100, they compete for sites on the S-100 protein. We chose a C a 2 + concentration of 0.4 mM; at this concentration about 3 mol of Caz+ are bound per mol of S-100 which is sufficient to produce the conformational change (CALISSANO et al., 1969). The results (Table 1) show that at this concentration neither M n 2 + (0.1 and 1.OmM)
200 -
I
I60 -
120-
F 80 -
40 4 -
1 /conc. Mn2+(mM) 5
6
7
8
9
10
II
12
PH FIG. 3. Enhancement of MBP fluorescence by M n Z + is pH dependent. MBP, 80 pg/ml. Closed circles, MBP + M n 2 + (4 mM); Open circles, MBP alone.
FIG.4. Reciprocal plot of Mn' binding to MBP, showing that 1 mole of metal is bound per mol of protein. Molecular weight of MBP taken as 18,000. Open triangles, MBP at 0.8 mg/ml; closed circles 1.0 rng/ml. V = mol of metal bound per mol of protein determined by equilibrium dialysis. +
A. S PERLMAL.. S. P. MAHADIK and M. M. RAPPORT
176
2
1 -
v
I
1 /conc. Mnzf(mM) FIG.5. Reciprocal plot of hln'. binding to S-100 protein showing that 25 rnol of metal are bound per rnol of protein. Closed circles and open triangles: S-100 at 0.8 mg'ml. (2 experiments). Open circles, S-100 at 1.0mg ml. V = mol of metal bound per rnol of protein determined by equilibrium dialysis, assuming mol. wt of 22.ooO daltons for S-100 protein.
its 25 binding sites. With MBP, M n 2 + at 0.05mM slowed the migration and this effect was much greater at 0.1 mM (Fig. 6, left panel). As the concentration of Mn2' \vas further increased to 0.25 and 0.5mM the retardation increased slightly and multiple bands appeared (gels D and E). At 5.0 mM-Mn2+, the retardation i n migration of MBP was reduced and the separation into multiple bands was much less distinct (gel F). The identical pattern was retained at 10mwMn". The mechanism for the change that occurs at the higher concentrations of M n 2 + is not evident. but i t is not due to the higher concentration of divalent ions as can be seen from the lack of such an effect in the presence of 5 mM-Ca2' (gel G). Since Ca" does not bind to MBP, the explanation must be related to the binding of M n 2 + to MBP in some way that remains to be determined. DISCUSSION
Since the formation of a complex between MBP and S-100 protein requires Ca2+ or Mn2+ ions, we have examined the effect of these ions on protein conformation. MBP binds M n 2 + but not C a 2 + whereas nor Mg2+(0.5 mM) nor K ' (12.5 mM) affected the bindS-100 protein binds both Mil2+ and Ca". The binding of C a 2 + . Ferrous ion (0.5 mM) enhanced Ca" ing of Mn" induces a conformational change in binding somewhat. Also. with "Mn" at 0.1 mM. a MBP as evidenced by the increase in MBP fluoresconcentration at which about 0.8 rnol of Mn'* is cence. Since only one M n 2 + ion is bound per rnol bound. C a 2 + (0.05 and 0.1 mM) did not affect the of MBP. it is probable that this binding occurs in binding of Mn' the vicinity of the single tryptophane residue. Recently EPANDet al. (1974) demonstrated that MBP Gel electrophoresis (A-1 protein) has a folded conformation rather than In the presence of Ca". the migration of S-100 & THOMPSON, 1969; C k o ~ a random structure (EYLAR protein was retarded; the protein also showed five & EIXSTHN. 1970). If the tryptophane residue were distinct bands (Fig. 6. right panel) at both 0.5 and buried within the fold and then released by unfolding 5.0 mM, confirming the earlier report of CALISSAYO ei as a consequence of M n 2 + binding, the enhanced a/. (1969). In contrast. Mn" at 0 . 5 m ~(gel D) did fluorescence would be accounted for. That the confornot show any retardation although a second minor mation of MBP is altered in the presence of M n 2 + component appeared. At 5.0mM-Mn" (gel E) the is also indicated by the change in its electrophoretic two bands were seen but the migration was appreciamobility. bly retarded. This retardation probably resulted from With regard to S-100 protein, our experiments conthe neutralization of negative charges in the S-100 firm the observations of CAL~SSANO et a/. (1969) that protein through binding of Mn" to most or all of S-100 protein contains about 10 binding sites for Ca' +. and that binding of Ca2 enhances the fluoresTABLE 1. OTHER DIVALEST 1135sDO ?OT COMPETE WITH THE cence of the protein as a result of its change in conforBIUDI% OF EITHER Ca'* OR Mn'* TO s-100 P R O T E I ~ mation. We have found that S-100 protein contains Mol of metal about 25 binding sites for M n 2 + , but the binding of bound per rnol of Mn'- does not alter the fluorescence and therefore protein Other Conc does not presumably affect the protein conformation. Mn'Bound metal Ion added (mM.l) Ca' These observations are consistent with the effects 2.86 Ca' ' (0.4 mM) none observed of these ions on electrophoretic mobility : 2.52 Mn" 0.1 in the presence of Ca" S-100 protein showed multi2.78 1 .o ple bands indicating several conformational states as 2.60 Mg' + 0.5 reported by CALISSANO c't ul. (1969) whereas no such Fe' + 3.30 0.5 K' 2.63 12.5 effect was seen with M n 2 + .In the absence of a conforMn2' (0.1 mM) none 0.67 mational change, it is not clear how the binding of Ca'+ 0.73 0.05 Mn" affects the properties of S-100 protein, but it 0.10 0.73 is worth noting that RNA polymerases, some of Binding assays were carried out by equilibriom dialqsis which are Mn2 dependent, have been reported to (see Methods). be activated by S-100 protein (MIANIv t a/. 1973). I.
+
-
+
FIG.6 . Effect of M n Z +and Ca2+on electrophoretic mobilities of MBP and S-100 protein in polyacrylamide gels. Protein and metal ion mixed at pH 7.5. Left panel: Electrophoresis at pH 4.2. Protein migration from anode to cathode. All gels contained MBP, 57 pg with the following additions: A, none. B to F, MnZ+0 . 0 5 m ~ 0.1 , mM, 0.25mM, OSmM, 5.0mM. G , Ca2+, 5.0mM. Right panel: Electrophoresis at pH 8.3. Protein migration from cathode to anode. All gels contained S-100 protein, 25 pg with the following additions: A, none. B and C, Caz+, O S r n M , 5.0mM. D and E, Mn2+, 0.5mM. 5.0 mM.
NC-176
Mechanism of myelin basic protein-S-100 It seems most probable in view of these conformational changes induced in MBP by M n 2 + and in S-100 protein by Ca2+ that the specific complex formation between these two proteins results from conformational changes that take place either in MBP due to Mn2+ or in S-100 due to Caz+ or both. How these changes affect the formation of the complex under physiological conditions remains to be determined, but in view of the heterogeneity of s-100 prort d.,1971) further studies are warteins (COMBOS ranted of the specificity of complex formation between MBP and various other acidic brain proteins, such as GP-350, GFA protein and C M SO-C, a protein recently described by HAGLIDet al. (1975). REFERENCES P , MOORFB. W. & FRIESFN A. (1969) BioCALISSANO c l i m i s t r y 8. 43 18-4326.
protein interaction
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CALISSANO P. & BANGHAM A. D. (1971) Biaclipm. hiophys. Rrs. Conzmun. 43. 504-509. CHOA L. P. & EINSTEINE. R. (1970) J . Neurochenl, 17. 1121-1 132. EPANDR. M., MOSCARELLO M. A,, ZIERENBERC B. & VAIL W. J. (1974) Biochemistry 13. 1264-1267. EYLARE. H. & THOMPSON M. (1969) Archs Biochem. Bioplzys. 129. 468-479. GOMROS G.. ZANETTAJ. P., MANDELP. & VINCENDON G. (1971) Biochemir 53. 645-655. HAGLIDK. G., RONNsACK L. & STAVRON D. (1975) J . Nrurochem. 24. 1053-1057. MAHADIKS. P., GRAFL. & RAPPORTM. M. (1974) Frdn. Proc. Fedn Am. Socs e.up. Biol. 33, 777. MIANIN., MICHETTIF., DE RENZISG. & CANIGLIA A. (1973) Experientiu 29. 1499-1501. OSHIRO Y. & EYLARE. H. (1970) Archs Biochem. Biophys. 138. 392-396.
