J. Biol. Chem., Vol. 262, Issue 12, 5616-5621, Apr, 1987
Nuclear magnetic resonance investigation of cadmium 113 substituted pea and lentil lectins
L Bhattacharyya, PS Marchetti, PD Ellis and CF Brewer
The lentil (LcH) and pea (PSA) lectins, which are members of the class of
D-glucose/D-mannose binding lectins, are Ca2+ X Mn2+ metalloproteins that
require the metal ions for their saccharide binding and biological
activities. We have prepared a variety of Cd2+ derivatives of PSA and LcH,
with Cd2+ in either the transition metal (S1) or calcium (S2) sites, or in
both. Thus, Cd2+ X Zn2+, Cd2+ X Mn2+, and Ca2+ X Cd2+ derivatives were
prepared, in addition to the Cd2+ X Cd2+ derivatives which we have recently
reported. This is the first report of stable mixed metal Cd2+ complexes of
lectins. The physical and saccharide binding properties of the Cd2+
derivatives of both lectins were characterized by a variety of
physiochemical techniques and found to be the same as those of the
corresponding native proteins. 113Cd NMR spectra of mono- and disubstituted
113Cd2+ complexes of LcH and PSA were recorded and compared with 113Cd NMR
data for concanavalin A (ConA) (Palmer, A.R., Bailey, D.B., Behnke, W.D.,
Cardin, A.D., Yang, P.P., and Ellis, P.D. (1980) Biochemistry 19,
5063-5070). The data for the PSA and LcH derivatives were found to be very
similar, indicating close homology of their metal ion binding sites. 113Cd
resonances at 44.6 ppm and -129.4 ppm for 113Cd2+ X 113Cd2+ X LcH, and at
46.6 and - 130.4 for the corresponding PSA derivative, are chemical shifts
very similar to those observed for 113Cd2+ X 113Cd2+ X ConA. Assignment of
the resonances to the transition metal (S1) and calcium (S2) sites were
unambiguous since the Ca2+ X 113Cd2+ and 113Cd2+ X Zn2+ derivatives of both
lectins showed single resonances characteristic of the S1 and S2 sites,
respectively. The results indicate that, unlike ConA, 113Cd2+ binds tightly
to PSA and LcH. Binding of monosaccharide to both lectins induce small (2
ppm) upfield shifts in their S2 113Cd resonances, in contrast to the larger
shift (8 ppm) observed in ConA. The 113Cd2+ X Mn2+ complexes of PSA and LcH
fail to show a 113Cd resonance characteristic of these derivatives, which
provides evidence for the close proximity of the metal ions in the two
proteins. The present findings indicate that the coordinating ligand atoms
to the metal ions at the S1 and S2 sites in LcH, PSA, and ConA are the
same.
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