Department of Life Sciences, National Tsing-Hua University,Hsin-Chu 30043, Taiwan
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Abstract |
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Keywords: 113Cd-NMR/calcium binding/-carboxyglutamic acid/circular dichroism/conantokin-T/conantokin-G/free energy perturbation (FEP)
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Introduction |
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Materials and methods |
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Crude con-G and con-T were purchased from SynPep Co. (Dublin, CA). Conantokin peptide analogues were synthesized on a MillGen/Biosearch Model 9500 automatic peptide synthesizer, using standard F-moc chemistry on a PAL resin support (PerSeptive BioSystems, Inc., Framingham, MA). Fmoc--carboxy-Glu(OtBu)2-OH was purchased from AnaSpec, Inc. (San Jose, CA). All other standard protected Fmoc-amino acids were obtained from either PerSeptive BioSystems, Inc. (Framingham, MA) or Advanced ChemTech (Louisville, KY). Cleavage and deprotection were affected by treatment with TFA (5% phenol)/thioanisole/H2O/ethanedithiol (35/2/2/1, v/v/v/v) at room temperature for 4 h and the products were precipitated with t-butyl ether (King et al., 1990
). Crude peptides were purified by reverse-phase HPLC using a Vydac C18 semi-preparative column (300 Å, 10 mmx25 cm) with a linear gradient of 1040% acetonitrile in the presence of 0.1% trifluoroacetic acid at a flow rate of 2.0 ml/min and monitored at 220 nm. The molecular weight of the purified peptides was confirmed by ESI-Mass spectroscopy.
Circular dichroism (CD) measurements
Circular dichroism spectra were run on an Aviv model 62A DS spectropolarimeter. The wavelength was checked by a two-point calibration with d-10-camphorsulfonic acid (Chen and Yang, 1977). All measurements reported were carried out in an aqueous solution of 10 mM HEPES buffer pH 7.4 at 25°C using a 1 mm path-length cuvette. Each reported spectrum is an average of three scans measured with a 0.5 nm step size. For con-T and its analogues, the concentrations of stock solutions were determined by using the absorption of tyrosine at 275.5 nm in 6 M guanidine hydrochloride (Brandts and Kaplan, 1973
). For Ca2+-free CD measurements, peptides were treated with chelex-100 (Sigma) for 1 h and passed through a 0.45 µm filter (Lida, WI) to remove the trace metal ion contaminants. The mean residue ellipticity at 222 nm, in deg·cm2/dmol, was calculated as
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where is the ellipticity observed at 222 nm, l is the path length of the cell, c is the concentration of sample (moles/liter) and n is the number of peptide bonds in the sequence. The fraction of helix was obtained from the relationship
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where []obs is the mean residue ellipticity observed at 222 nm. [
]max, the maximal mean residue ellipticity value for chain length n was estimated using the relationship
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with []
= 40 000 deg·cm2/dmol (Gans et al., 1991
).
113Cd-NMR spectroscopy
Cadmium-113 NMR experiments were performed on a Bruker DMX 600 spectrometer, equipped with a 10 mm broadband probe. All spectra were recorded at a frequency of 133.07 MHz. Isotopically enriched 113CdCl2 (96%) was purchased from ISOTEC, Inc. (Miamisburg, OH). A 200 mM stock solution of 113CdCl2 was used for the titration of the samples. All 113Cd-NMR samples were prepared in 10 mM HEPES buffer containing 10% D2O and the final pH was adjusted to 6.8 (±0.2). The 113Cd-NMR acquisition parameters were as follows: a 90° flip angle, acquisition time 0.06 s, relaxation delay 1.0 or 5.0 s, sweep width 67 kHz, 8 K data points and 20 00030 000 scans. All experiments were carried out at 298 K. All 113Cd spectra were referenced to external 0.1 M 113Cd(ClO4)2 in D2O, pH 1.0 (Armitage and Otvos, 1982).
Theoretical calculation and computer modeling
The coordinates representing the solution structure of con-G and con-T were taken from the Protein Data Bank (1AWY and 1ONT for con-G and con-T, respectively). Molecular simulations were performed using ENZYMIX developed by Warshel (Aqvist et al., 1993). In the simulation, the peptides were solvated by the surface constraint all-atom solvent (SCASS) water model (King and Warshel, 1989
) with a radius of 20 Å, outside which a continuum solvent model was used. Long-range electrostatic interaction was treated by the local reaction field method (Lee and Warshel, 1992
) in order to avoid errors introduced by the usual cut-off approach. The parameters for Ca2+ were calibrated against its solvation free energy. The calculated solvation free energy was 379.0 kcal/mol, which is quite close to the experimental value of 380.8 kcal/mol (Burgess, 1978
). The relative binding free energies of different probable metal binding sites of con-G and con-T were calculated by the free energy perturbation (FEP) method (Aqvist et al., 1993
). A typical mapping potential,
m used in the FEP calculations is written as
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where A and
B are the potentials of conantokin with a calcium ion binding to site A and site B, respectively. The mapping parameter
is set to be in the range 0 to 1. By changing the mapping parameter
from 0 to 1, we could calculate the difference in the binding free energy between these two sites (A and B) (Aqvist et al., 1993
). A typical simulation time for a complete FEP calculation is around 200 ps. Convergence of FEP calculations was determined by comparing the free energies obtained from the forward and backward mapping. All calculations were performed on IBM 395 and Digital Alpha 433 workstations.
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Results and discussion |
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The effect(s) of Ca2+ on the backbone conformation of conantokins and their analogous peptides were monitored using CD spectra. The CD spectra of con-G and con-EG both in the presence and absence of Ca2+ are shown in Figure 1A. The apo form of both peptides (con-G and con-EG) exhibited a disordered conformation, as indicated by the absence of the negative ellipticity minimum in the region 205230. Upon titrating con-G with Ca2+ the ellipticity values at 208 and 222 nm decreased, indicating a structural transition from random coil to helix. The changes in ellipticity values (at 208 and 222 nm) continued until a Ca2+ concentration of 4.5 mM was reached. Beyond this concentration of Ca2+ there seemed to be no apparent change in the conformation of con-G. Analysis revealed that con-G adopts an approximately 57% helical conformation. On the other hand, titration of con-EG with Ca2+ (up to 8 mM) did not seem to alter the conformation of the peptide. This indicates that the
-carboxyglutamate side chains (as in con-G) play a critical role in binding of Ca2+ while a similar negatively charged glutamic acid (as in con-EG) may not be able to fulfil the requirement for metal ion chelation. Figure 1B
shows the CD spectra of con-T, con-ET1, con-ET2, con-ET3 and con-ET in the absence of Ca2+. All these peptides exhibit double minima at 208 and 222 nm in the far UV region of the CD spectrum, indicating that the major secondary structural element in these peptides is
-helix. However, the percentage of residues that adopt a helical conformation decreases in the order con-T > con-ET1 > con-ET2 > con-ET3 > con-ET. It is apparent, in comparison with the corresponding amino acid sequences, that the Glu substitutions diminished the
-helical content of con-T. This effect seems to be in proportion to the number of Glu residues present in the sequence. We attribute this result to three possible reasons: (i) the ability of Gla residues to form salt bridges is better than that of Glu residue; (ii) Gla residues may have higher helix propensity than Glu; and (iii) Gla may be more effective in stabilizing the helical macrodipole due to its high negative charge (Lin et al., 1997
). However, the presence of calcium produced different effects in the secondary structural interactions of these con-T peptide analogues. On a par with con-EG, con-ET also showed no changes in the backbone conformation, even in the presence of Ca2+, which again indicates that Gla residues play an important role in chelating Ca2+. Although con-ET exhibited the double minima at 208 and 222 nm, signifying the presence of helical segment in the peptide, the percentage of helix was only 20% (both in the presence and absence of Ca2+). Con-ET3, which retains a single Gla residue at position 14, behaves similarly to con-ET upon Ca2+ addition. In contrast, the loss of helicity caused by the substitution of Glu residues in con-ET1 and con-ET2 was restored to some extent by the addition of Ca2+. The percentage of helix in each of these peptides, both in the presence and absence of calcium, is listed in Table I
. These results suggest that the possible Ca2+ binding site is located between Gla10 and Gla14. For convenience, we will denote the metal binding site located between Glai and Glaj as [ij]. The Gla to Glu replacements in con-ET1 or con-ET2 only affected [34] and left [1014] unperturbed. Thus, the Ca2+ binding at the [1014] site can still stabilize the helical conformation and increase the helical content of both peptides. The Ca2+ binding cannot provide further induction of helicity in con-T as its helical content is already very high. In contrast, the [1014] site in con-ET3 and con-ET was perturbed by the Glu substitutions and both these peptides lost their metal binding properties. This might be the reason for the absence of any conformational change in con-ET3 and con-ET in the presence of Ca2+. The Glu substitution in con-ET not only decreased helical content (compared with con-T), but also diminished its NMDA antagonist properties (Lin et al., 1997
). Similar cases are also seen in Glu-substituted conantokin analogues, which yield pharmacologically inert peptides (Chandler et al., 1993
). The abnormal prothrombin, which lacks post-translationally modified glutamic acids, lacks calcium binding and membrane binding properties (Esmon et al., 1975
; Ratcliffe et al., 1993
). These findings, along with our CD data, strongly suggest that the additional
-carboxyl group on glutamic acid is functionally important for forming the metal complex.
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The above mentioned CD experiments give a clue regarding the potential metal binding site(s), but the mode of binding of Ca2+ to the peptides remains unclear. In order to elucidate the mode of Ca2+ binding of -carboxyglutamic acids in conantokins, we carried out 113Cd-NMR experiments on these peptides. 113Cd-NMR is a useful tool for the study of various metalloproteins, including a range of calcium-binding proteins (Armitage and Boulanger, 1983
). It is common to replace the native diamagnetic metal by the isotopically enriched 113Cd to study the metal coordination sites, since diamagnetic metal ions such as Zn2+, Mg2+ and Ca2+ are not directly detectable by NMR spectroscopy (Armitage and Otvos, 1982
). The similarity of the ionic radii of Cd2+ (0.97 Å) and Ca2+ (0.99 Å) makes Cd2+ a fairly good substitute for calcium in calcium binding proteins (Forsén et al., 1980
; Ohki et al., 1993
). In addition, 113Cd chemical shifts are very sensitive to the number and geometry of the ligands within the coordination sphere (Armitage and Boulanger, 1983
). Figure 2
shows the 113Cd NMR spectra of Cd2+-saturated con-G and con-EG. Four distinct resonances, besides the free 113Cd peak (~6.2 p.p.m.), were observed for con-G, while only the free 113Cd peak was found for con-EG. The existence of a large free 113Cd peak at the 5:1 ion to peptide ratio might imply that binding affinities of Cd2+ are low. It is consistent with the potentiometric measurements that the average dissociation constant (Kd) value of Ca2+ binding sites in con-G is about 2.8 mM (Prorok et al., 1996
). Although much tighter binding has been observed for Mg2+ and Zn2+ with Kd values ranged from 0.2 to 311 µM (Prorok and Castellino, 1998
), Cd2+ may resembles Ca2+ in weak binding due to the similarities between the two ions. The chemical shifts of all four bound cadmium resonances (8.3, 20.3, 74.4 and 77.5 p.p.m.) were in the same range as found in other 113Cd-substituted metalloproteins (Armitage and Boulanger, 1983
). This indicates that the oxygen atoms of the peptide and water are involved in binding to Ca2+. Based on the theoretical calculation, it has been previously proposed that the number of Ca2+ binding sites is four for con-G (Rigby et al., 1997b
). But the Scatchard analysis suggested that the best models for con-G were two or three Ca2+ binding sites (Prorok et al., 1996
). However, our 113Cd-NMR data provided the direct experimental evidence to support the existence of four Cd2+ binding sites. This result enables us to make a reasonable assumption that con-G contains four distinct Ca2+ binding sites. Figure 3
shows the 113Cd-NMR spectra of con-T, -ET1, -ET2 and -ET. Only one resonance is observed, apart from the free 113Cd, in the spectrum of con-T. In line with our previous analysis, this single resonance implies the presence of only one metal binding site for con-T. Single Glu substitution at position 3 and double Glu substitutions at positions 3 and 4 in con-ET1 and con-ET2, respectively, did not eliminate this resonance. This finding reveals that the binding site is located at Gla10 and Gla14. This is further supported by the substitutions of all Gla residues with Glu in con-ET, which led to the absence of this resonance.
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The CD and NMR data presented here indicate only one calcium-binding site exists in con-T. However, there are two possible locations, [34] and [1014], available for calcium binding. Despite the [34] site containing a neighboring Gla pair, which has been suggested to be important in calcium binding (Prorok et al., 1996; Rigby et al., 1997a
), our experimental data seem to favor the [1014] site. In order to rationalize the experimental results concerning metal binding, we carried out FEP simulation which could be used to calculate the relative binding free energies of these two sites ([34] and [1014]). It should be mentioned that it is usually difficult to parameterize transitional metal ions in classical molecular simulation due to their large orbital polarizability. Therefore we used the Ca2+ ion rather than the Cd2+ ion for molecular simulations. Our results show that the binding free energy associated with the Ca2+ ion binding at the [1014] location is lower than that of [34] by 10.0 kcal/mol. This result is in accordance with our experimental finding that [1014] appears to be the binding site in con-T. In the case of con-G, there are five possible calcium-binding sites, i.e. [34], [37], [47], [710] and [1014]. Our FEP calculations indicate that [47] is the least favorable for calcium binding among these possible binding sites (data not shown), which may be due to the steric effect. We propose a `four-site' binding model for con-G based on both theoretical and experimental considerations. Figure 4
depicts the surface charge representation of our models for con-G and con-T. Calcium ions were docked to the four favorable binding sites in con-G and a unique binding site in con-T. Electrostatic potentials have been mapped onto the surfaces of the helix. However, 113Cd-NMR experiments only provide evidence for the existence of four distinct binding sites. It is possible that there are alternate binding sites which might not all be occupied simultaneously. For example, the Cd2+ binding sites might also occur in the following `three-site' equilibrium binding mode:
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where [37]-[710]-[1014] and [47]-[710]-[1014] describe the three binding sites of Cd2+. Thus, further experiments are needed to exclude other possibilities.
It is interesting to note that [34] is found to be a favorable binding site in the case of con-G, but not so in con-T. This disparity could be due to the difference(s) in the residues occupied at position 7 in con-G and con-T. Lysine, a positively charged residue, is present at position 7 in the amino acid sequence of con-T, whereas a negatively charged Gla residue occupies the same position in con-G. In order to study the effect(s) of amino acid substitutions at position 7 on calcium binding of the [34] site, we performed FEP calculations on several mutants of con-G and con-T. Comparison of the relative binding free energies indicates that the binding strength of the [34] site increased (relative to [1014]) when Lys7 of con-T was mutated to other residues such as Gly, Asn, Ala and Asp. In the case of con-G, the binding strength of the [34] site was greatly diminished when Gla7 was mutated to a positively charged Lys. This indicates that the presence of a positively charged residue at position 7 has significant effect on the calcium binding of conantokins by lowering the electron charge density on Gla3 and Gla4.
It is pertinent to address the issue of the binding mode(s) of the -carboxyglutamic acid residues in conantokins. The experimental data does not unequivocally indicate whether the
-carboxyglutamic acid residues bind to the metal in unidentate or bidentate mode. Therefore, the results of molecular simulation, providing time-averaged pictures, can be utilized to probe into the
-carboxyglutamate binding mode(s). Figure 5
shows the snapshot of two possible binding modes of metal chelation in both con-T and con-G. The geometry of the Ca2+-loaded con-T complex shows a bidentate metal binding, in which both oxygens of the carboxyl group participate in metal coordination. In contrast, con-G appears to exhibit unidentate binding in [10-14]. These different binding properties may be one of the reasons for the varied binding affinities of con-G and con-T.
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Based on the experimental and molecular dynamic studies, it is proposed that the unique Ca2+ binding site in con-T is located at Gla10 and Gla14 rather than between the consecutive Gla residues at positions 3 and 4. Although the presence of consecutive Gla residues has been implicated in the formation of a potential metal binding site (Prorok et al., 1996; Rigby et al., 1997a
), the results discussed here clearly indicate that the occurrence of consecutive Gla residues need not imply a preferential and strong metal binding site. In fact, it has been shown that in other Gla-containing proteins (Soriano-Garcia et al., 1992
; Banner et al., 1996
), the consecutive Gla residues are not the most preferred metal binding sites. For example, in the human blood coagulation factor VIIa (PDB entry code 1DAN), which contains 10 Gla residues, none of its three consecutive Gla pairs is the preferred binding site for Ca2+. In contrast to the one binding site in con-T, there are four binding sites in con-G. The proposed four binding sites, [34], [37], [710] and [1014], constitute a GlaCa2+ network along the helical axis. This GlaCa2+ network is comparable with the salt-bridge network in con-T (Lin et al., 1997
). Such a GlaCa2+ network is not possible in con-T due to the presence of Lys7. Lys7 in con-T greatly reduces the negative electrostatic potential in comparison with the same region in con-G. Substitution of Lys7 with Gla7 destroyed the salt bridge network and destabilized the helical conformation of this con-T analog in the absence of Ca2+ (unpublished data).
We have provided solid evidence, by employing a series of synthetic analogs, to support the `four-site' and `single-site' model for con-G and con-T, respectively. The key residue of the `four-site' binding model is Gla7 in con-G, which participates in the chelation of two metal ions. The lack of Gla7 disrupts the formation of a GlaCa2+ network in con-T; while the existence of Lys7 helps the formation of its salt-bridge network, which also stabilizes the helical conformation regardless of the presence of Ca2+. Interestingly, substitution at position 7 by Ala in con-G increased the inhibition of spermine-enhanced [3H]MK801 binding by about fourfold (Zhou et al., 1996; Blandl et al., 1998
). We have seen from the theoretical calculations that a Gla
Ala substitution at position 7 in con-G affected the metal binding site at the N-terminus. It is generally believed that the well-conserved N-terminal residues in both con-T and con-G are responsible for their NMDA antagonist activity. Thus, we suspect that the mutation of the residue at position 7 in con-G can allosterically affect the N-terminal region through the interruption of the GlaCa2+ network, which may provide more flexibility for the conserved N-terminal residues. This in turn facilitates the interaction of the conantokin analogue with the targeting site in the NMDA receptor. Structural studies of the mutants at position 7 of both con-G and con-T are required to completely understand the role of residue 7 in conantokins and such studies are now in progress.
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Acknowledgments |
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Notes |
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References |
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Armitage,I.M. and Otvos,J.D. (1982) In Berliner,L.J and Reuben,J. (eds), Biological Magnetic Resonance, Vol. 4. Plenum, New York, pp. 97144.
Armitage,I.M. and Boulanger,Y. (1983) In Pierre,L. (ed.) NMR of Newly Accessible Nuclei, Vol. 2. Academic Press, New York, pp. 337365.
Blandl,T., Zajicek,J., Prorok,M. and Castellino,F.J. (1997) Biochem. J., 328, 777783.[ISI][Medline]
Blandl,T., Prorok,M. and Castellino,F.J. (1998) FEBS Lett., 435, 257262.[ISI][Medline]
Banner,D.W., D'Arcy,A., Chene,C. Winkler,F.K., Guha,A., Konigsberg,W.H., Nemerson,Y. and Kirchhofer,D. (1996) Nature, 380, 4146.[ISI][Medline]
Brandts,J.F. and Kaplan,L. (1973) Biochemistry, 12, 20112014.[ISI][Medline]
Burgess,J. (1978) Metal Ions in Solution. John Wiley & Sons, New York.
Chandler,P., Pennington,M., Maccecchin,M.-L., Nashed,N.T. and Skolnick,P. (1993) J. Biol. Chem., 268, 17 17317 178.
Chen,G.C. and Yang,J.T. (1977) Anal. Lett., 10, 11951207.[ISI]
Dowd,P., Hershline,R., Ham,S.W. and Naganathan,S. (1995) Science, 269, 16841691.[ISI][Medline]
Esmon,C.T., Suttie,J.W. and Jackson,C.M. (1975) J. Biol Chem., 250, 40954099.[Abstract]
Forsén,S., Thulin,E., Drakenberg,T., Krebs,J. and Seamon,K. (1980) FEBS Lett., 117,189193.
Gans,P.J., Lyu,P.C., Manning,M.C., Woody,R.W. and Kallenbach,N.R. (1991) Biopolymers, 31, 16051614.[ISI][Medline]
Hsu,K.S., Huang,C.C. and Lyu,P.C. (1996) Neuroscience Lett., 220, 113116.[ISI][Medline]
Huang,C.C., Lyu,P.C., Lin,C.H. and Hsu,K.S. (1997) Toxicon, 35, 355363.[ISI][Medline]
King,G. and Warshel,A. (1989) J. Chem. Phys., 91, 36473661.[ISI]
King,D., Fields,C. and Fields,G. (1990) Int. J. Pept. Protein Res., 36, 255266.[ISI][Medline]
Lee,F.S. and Warshel,A. (1992) J. Chem. Phys., 97, 31003107.[ISI]
Lin,C.H., Chen,K.S., Hsu,K.S., King,D.S. and Lyu,P.C. (1997) FEBS Lett., 407, 243248.[ISI][Medline]
Myers,R.A., McIntosh,J.M., Imperial,J., Williams,R.W., Oas,T., Haack,J.A., Hernandez,J.-F., River,J.E., Cruz,L.J. and Olivera,B.M. (1990) J. Toxicol. (Toxin Reviews), 9, 179202.
Myers,R.A., Cruz,L.J., River,J.E. and Oliver,B.M. (1993) Chem. Rev., 93, 19231936.[ISI]
Ohki,S.-Y., Iwamoto,U., Aimoto,S., Yazawa,M. and Hikichi,K. (1993) J. Biol. Chem., 268, 12 38812 392.
Olivera,B.M., McIntosh,J.M., Clark,C., Middlemas,D., Gray,W.R. and Cruz,L.J. (1985) Toxicon, 23, 277282.[ISI][Medline]
Olivera,B.M., Rivier,J., Clark,C., Ramilo,C.A., Corpuz,G.P., Abogadie,F.C., Mena,E.E., Woodward,S.R., Hillyard,D.R. and Cruz,L.J. (1990) Science, 249, 257263.[ISI][Medline]
Prorok,M. and Castellino,F.J. (1998) J. Biol. Chem., 273, 1957319578.
Prorok,M., Warder,S.E., Blandl,T. and Castellino,F.J. (1996) Biochemistry, 35, 1652816534.[ISI][Medline]
Ratcliffe,J.V, Furie,B. and Furie,B.C. (1993) J. Biol. Chem., 268, 24 33924 345.
Rigby,A.C., Baleja,J.D., Furie,B.C. and Furie,B. (1997a) Biochemistry, 36, 69066914.[ISI][Medline]
Rigby,A.C., Baleja,J.D., Li,L., Pedersen,L.G., Furie,B.C. and Furie,B. (1997b) Biochemistry, 36, 15 67715 684.
Skjærbæk,N., Nielsen,K.J., Lewis,R.J., Alewood,P. and Craik,D.J. (1997) J. Biol. Chem., 272, 22912299.
Skolnick,P., Boje,K., Miller,R., Pennington,M. and Maccecchini,M.-L. (1992) J. Neurochem., 59, 15161521.[ISI][Medline]
Soriano-Garcia,M., Padmanabhan,K., deVos,A.M. and Tulinsky,A. (1992) Biochemistry, 31, 25542566.[ISI][Medline]
Warder,S.E., Chen,Z., Zhu,Y., Porok,M., Castellino,F.J. and Ni,F. (1997) FEBS Lett., 411, 1926.[ISI][Medline]
Zhou,L.M., Szendrei,G.I., Fossom,L.H., Maccecchini,M.L., Skolnick,P., Otros,L.Jr. (1996) J. Neurochem., 66, 620628.[ISI][Medline]
Received December 17, 1998; revised April 7, 1999; accepted April 16, 1999.