On the Stringent Requirement of Mannosyl Substitution in
Mannooligosaccharides for the Recognition by Garlic (Allium
sativum) Lectin
A SURFACE PLASMON RESONANCE STUDY*
Kiran
Bachhawat
§,
Celestine J.
Thomas
,
B.
Amutha¶,
M. V.
Krishnasastry
,
M. I.
Khan¶, and
Avadhesha
Surolia
**
From the
Molecular Biophysics Unit, Indian Institute
of Science, Bangalore 560012, the ¶ Division of Biochemical
Sciences, National Chemical Laboratory, Pune 411008, the
National Center for Cell Science, Ganeshkhind, Pune 411007, and
the ** Jawaharlal Nehru Center for Advanced Scientific Research, Jakkur,
Bangalore 560064, India
Received for publication, October 18, 2000, and in revised form, November 13, 2000
 |
ABSTRACT |
The kinetics of the binding of
mannooligosaccharides to the heterodimeric lectin from garlic bulbs was
studied using surface plasmon resonance. The interaction of the bound
lectin immobilized on the sensor chip with a selected group of high
mannose oligosaccharides was monitored in real time with the change in
response units. This investigation corroborates our earlier study about
the special preference of garlic lectin for terminal
-1,2-linked
mannose residues. An increase in binding propensity can be directly
correlated to the addition of
-1,2-linked mannose to the
mannooligosaccharide at its nonreducing end. Mannononase
glycopeptide (Man9GlcNAc2Asn), the
highest oligomer studied, exhibited the greatest binding affinity (Ka = 1.2 × 106
M
1 at 25 °C). An analysis of
these data reveals that the
-1,2-linked terminal mannose on the
-1,6 arm is the critical determinant in the recognition of
mannooligosaccharides by the lectin. The association
(k1) and dissociation rate constants
(k
1) for the binding of
Man9GlcNAc2Asn to Allium sativum
agglutinin I are 6.1 × 104
M
1 s
1
and 4.9 × 10
2
s
1, respectively, at 25 °C. Whereas
k1 increases progressively from Man3 to Man7 derivatives, and more dramatically
so for Man8 and Man9 derivatives,
k
1 decreases relatively much less
gradually from Man3 to Man9 structures. An
unprecedented increase in the association rate constant for interaction
with Allium sativum agglutinin I with the structure of the
oligosaccharide ligand constitutes a significant finding in
protein-sugar recognition.
 |
INTRODUCTION |
The structurally and evolutionarily related monocot
mannose-binding proteins comprise a superfamily of mannose-specific
lectins. Amaryllidaceae, Alliaceae, Araceae, Orchidaceae, Iridaceae,
and Liliaceae families have been shown to possess these bulb
lectins (1). Among the unique features that set them apart from the Glc/Man/Gal-specific family of dicotyledonous legume lectins and the
C-type mannose-binding animal lectins is their high degree of
stereospecificity for mannose, so much so that they show no binding
propensity even for its epimer, glucose, or the conformationally related analog, L-fucose. Their classification into the
mannose-specific lectin family is corroborated by determination of the
crystal structures of snowdrop (Galanthus nivalis) (2),
daffodil (Narcissus pseudonarcissus) (3), bluebell
(Scilla campanulata) (4), amaryllis (Hippeastrum
hybrid) (5), and garlic (Allium sativum) lectin (6),
representatives of the family of bulb lectins. Their subunits have been
observed to possess a novel 3-fold symmetry having three four-stranded
antiparallel
-sheets arranged as three sides of a triangular prism,
forming a 12-stranded
-barrel referred to as the
-prism II fold.
These 12 strands are positioned perpendicular to the plane of symmetry,
unlike the other known all-
-folds:
-prism I (e.g.
Jacalin; (7)) and the
-trefoil (e.g. amaranthin; (8))
fold (6). The central region in the
-barrel is stacked with
conserved hydrophobic side chains, which stabilize the subunit.
Bulbs of garlic are known to accumulate two types of mannose-binding
lectins: the heterodimeric A. sativum agglutinin I
(ASAI)1 and the homodimeric
ASAII (9). Interaction of ASAI and ASAII with mannooligosaccharides and
glycoproteins has been investigated extensively by an enzyme-linked
lectin adsorbent assay (10). Both of these lectins exhibit identical
sugar specificities, although subtle differences between them cannot be
ruled out in view of the qualitative nature of enzyme-linked lectin
adsorbent assay. Enzyme-linked lectin adsorbent assay implicated
-1,2-linked mannose at the nonreducing end to determine the affinity
for the higher order structures of mannose, evidenced by a sequential
increase in potency with an increase in the number of
-1,2-linked
mannose residues (10). In enzyme-linked lectin adsorbent assay, the recognition and adsorption properties of lectins with the specific sugar residues of glycoproteins are used for the assay purpose; therefore the method fails to provide information on the affinities of
interaction or the elementary steps involved therein. On the other
hand, kinetic studies using stopped flow and fluorescence titrations of
the interaction of lectins with its ligands require large amounts of
both the lectin and the saccharides as well as the labeling of the
latter with a fluorophore or a chromophore. Therefore, in this
study, the more sensitive, label-free, and fast response surface
plasmon resonance (SPR) method took precedence as a means to delineate
the affinities and the kinetic parameters for the interaction of ASAI
with its complementary mannooligosaccharide ligands (11-13).
Barre et al. (1) used SPR to determine the carbohydrate
specificity of a few monocot mannose-binding lectins. More recently, Van Damme et al. (14) characterized the carbohydrate binding propensity for Crocus vernus agglutinin using a
biosensor. Both these investigations were carried out with large
molecular weight glycoproteins, which are easier to monitor but fail to
reveal the subtle features of carbohydrate moieties involved in these recognitions. In contrast, the study reported here with the individual purified mannooligosaccharide helps to unravel, in detail, the specificity invoked by garlic lectin for the recognition of its complementary ligands.
 |
EXPERIMENTAL PROCEDURES |
Materials--
All the chemicals used, namely the components for
phosphate-buffered saline buffer (pH 7.4), as well as
N-ethyl-N'-(dimethylaminopropyl)carbodiimide hydrochloride and N-hydrosuccinamide, were of the highest
purity available. Certified grade CM5 sensor chips were purchased from Amersham Pharmacia Biotech.
Saccharides--
Mannose, methyl-
-mannose,
Man3GlcNAc2,
Man5GlcNAc2,
Man6GlcNAc2,
Man7GlcNAc2 (I and II), and
Man8GlcNAc2 were obtained from Sigma.
Man3 and Man5 were procured from Dextra
Laboratories. Man9GlcNAc2Asn was prepared in
the laboratory from the Pronase digestion of soybean agglutinin (15,
16). Man5GlcNAc2Asn was obtained as a byproduct of the above preparation (15). Man6GlcNAc2Asn
and Man5GlcNAc (Isosep, Tullinge, Sweden) were kind
gifts from Prof. C. F. Brewer, Albert Einstein College of
Medicine, New York and Prof. C. G. Gahmberg, Department of
Biochemistry, University of Helsinki, Finland, respectively.
Protein Purification--
ASAI was isolated and purified to
homogeneity as described previously (10). The protein samples were
prepared as required by the nature of the experiments described below.
Neutral Sugar Estimation--
Concentrations of all the
saccharides were determined by measuring their neutral sugar content by
the phenol-sulfuric acid method (17). Mannose was used as the standard.
BIAcore Biosensor Assays--
Biospecific interaction analysis
was performed using a BIAcore 2000TM (Pharmacia Biosensor AB, Uppsala,
Sweden) biosensor system based on the principle of SPR. Nearly 1500 response units (RUs) of ASAI (0.1 mg/ml in 5 mM NaAc (pH
4.5)) were coupled to a certified grade CM5 chip at a flow rate of 1 µl/min for 50 min using the amine coupling kit
(N-ethyl-N'-(dimethylaminopropyl)carbodiimide hydrochloride, N-hydrosuccinamide) supplied by the
manufacturer. Here, coupling of an RU corresponds to ~1
pg/mm2 of immobilized protein. The unreacted species on the
surface of the chip was blocked with ethanolamine. All measurements
were done using 20 mM phosphate-buffered saline (pH
7.4). Prior to injection, protein samples were dialyzed extensively
against the same buffer to avoid buffer mismatch. For the
determination of association rate constants, the solution of the
ligands was passed over the chip (1-80 µM) at a flow
rate of 5 µl/min. The dissociation rate constants were determined by
passing buffer subsequently at a flow rate of 5 µl/min. The surface
was regenerated by a 10-s pulse of 500 mM Me
Man flowing
at 50 µl/min.
Data Analysis--
Association (k1) and
dissociation (k-1) rate constants were obtained
by nonlinear fitting of the primary sensorgram data using the BIA
evaluation software version 3.0. The dissociation rate constant
is derived using the equation,
|
(Eq. 1)
|
where Rt is the response at time t
and Rt0 is the amplitude of the initial
response. The association rate constant
k1 can be derived using Equation 2, from the
measured k
1 values,
|
(Eq. 2)
|
where Rmax is the maximum response and
C is the concentration of the analyte (ligand) in the
solution. Ka
(k1/k
1) is
the association constant. The parameters obtained from the binding
interaction of the ligands with the protein on the surface of the chip
were also plotted as per Scatchard analysis (18-20).
Isothermal Titration Calorimetry--
Isothermal titration
microcalorimetry was performed using an OMEGA microcalorimeter
from Microcal Inc. (Northampton, MA). In individual titrations,
injections of 5-6 µl of carbohydrate solution were made by a
computer-controlled 250-µl syringe at an interval of 3 min into the
lectin solution dissolved in the same buffer as the saccharide, while
stirring at 396 rpm. Control experiments were performed by identical
injections of saccharide into the cell containing only the buffer. The
experimental data were fitted to a theoretical titration curve using
the software provided by Microcal. All experiments performed were
within c values of 1 < c < 200, where
c = Ka × Mt(0), Mt(0) is the initial macromolecule concentration,
and Ka is the binding constant (21). The
thermodynamic parameters were calculated from the basic equations of
thermodynamics,
|
(Eq. 3)
|
where
G,
H, and
S are
the changes in free energy, enthalpy, and entropy of
binding, respectively, T is the absolute temperature, and
R = 1.98 cal mol
1
K
1.
 |
RESULTS |
Documented in Fig. 1 and Table
I are the various mannose-containing
carbohydrate ligands that were tested for their binding propensity to
ASAI. The selection of the sugars was based on our previous study,
wherein higher oligomers of mannose were implicated in binding to ASAI.
Binding of saccharides shorter than mannotriose could not be
analyzed, because no appreciable change in RUs was observed upon their
flow over the immobilized ASAI on the sensor chip, consistent with the
sensitivity and specifications of the instrument. The use of a
certified grade sensor chip in this study allowed satisfactory
recording of RU changes corresponding to oligosaccharides having
molecular masses greater than 400 Da. RU changes observed with
the mannobioses (molecular mass 360 Da) provided sensorgrams that were
not consistently reproducible.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 1.
Structures of
mannooligosaccharides. (1), Man3;
(2), Man3GlcNAc2; (3),
Man5; (4), Man5 GlcNAc;
(5), Man5GlcNAc2; (6),
Man5 GlcNAc2Asn; (7),
Man6GlcNAc2; (7'),
Man6GlcNAc2Asn; (8),
Man7GlcNAc2(I); (9),
Man7GlcNAc2(II); (10),
Man8GlcNAc2; and (11),
Man9GlcNAc2Asn.
|
|
A representative sensorgram for the interaction of varying amounts (5, 10, 20, 30, 40, 50, 60, 70, and 80 µM) of trimannoside (Man
1-3(Man
1-6)Man) (1) passed over ASAI immobilized on a certified grade CM5 chip is shown in Fig.
2A. The curves, fitted by mass
transport limited analyses at 25 °C, yield values for
k1 and k-1 of 77.3 M
1 s
1
and 0.61 s
1, respectively. An equal
distribution of residuals for both phases of the reaction support the
monoexponential nature of interaction (data not shown). The ratio
k1/k
1
provides an estimate of Ka of 127 M
1 at 25 °C (Table I). These
parameters evaluated from SPR data are in good agreement with those
obtained from isothermal titration calorimetric (ITC) measurements
(Fig. 3). The binding constant obtained
for the binding of (1) to ASAI at 25 °C is 144 M
1, and the
H°ITC and
G°ITC
are
20.2 and
11.5 kJ mol
1, respectively.
In addition, Scatchard analysis of the SPR data yielded
Ka, which is in accordance with the ratio
k1/k-1 as well as the
values of KbITC (Fig. 2A,
inset) (22, 23). An agreement between the SPR and ITC data
substantiate the monophasic nature of the binding interaction. ITC
experiments with the other mannooligosaccharides were not done due to
paucity of the saccharides.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 2.
A, representative sensorgram depicting
interaction of increasing amounts of trimannoside to the immobilized
ASAI at 25 °C. The sugar, ranging in concentration from 5 µM (bottom trace) to 80 µM
(top trace), was injected for 600 s at a flow rate of 5 µl/min. The dissociation reaction was recorded by flowing buffer at 5 µl/min. The surface of the chip was regenerated by a 10-s pulse of
Me- -mannopyranoside. Inset, Scatchard analysis of the
sensorgram. B, the sensorgram depicts the binding of the
higher order structures of mannose,
Man7GlcNAc2(I) (8),
Man8GlcNAc2 (10), and
Man9GlcNAc2Asn (11) to ASAI
immobilized on a CM5 sensor chip.
|
|

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 3.
a, an isothermal calorimetric titration
of 5.2-µl aliquots of 112.5 mM trimannoside into 7.5 mM ASAI at 25 °C. b, the integrated curve
shows experimental points (solid squares) and the best fit (solid
line) of the curve.
|
|
Among the mannooligosaccharides both (1) and mannopentaose
(3), as well as their reducing end sugar derivatives, i.e. Man3GlcNAc2 (2),
Man5GlcNAc (4),
Man5GlcNAc2 (5), and
Man5GlcNAc2Asn (6), show very slow
association and fast dissociation rate constants (Table I). Extension
of (3) with two
-1,2-linked mannosyl residues at its
nonreducing end increases k1 in a profound
manner. Further extensions as in Man8GlcNAc2
(10) and Man9GlcNAc2Asn
(11) do so even more dramatically (Fig. 2B).
Although k-1 also decreases progressively for
the extended structures as compared with Man3 and
Man5, these changes are relatively less pronounced compared
with the effects on the association rate constants (Table I).
 |
DISCUSSION |
The binding kinetics for ASAI with the set of selected
mannooligosaccharides (Fig. 1) is seen to follow a single-step
mechanism. The k1 values observed here are in
the range of 0.077 × 103
M
1 s
1
(for (1)) to 6.1 × 104
M
1 s
1
(for (11)). These second order rate constants are
significantly lower than diffusion-controlled reactions (22, 24).
Binding for such reactions is thought to involve an intermediate
PLi, which subsequently isomerizes to the final
complex PL*.
In such a situation,
|
(Eq. 4)
|
where K
1 = k
1/k1.
The agreement between kinetically determined values of association
constants
(k1/k
1) and
those determined by the Scatchard analyses of the SPR data suggest that
the association and dissociation reactions are monoexponential in
nature and describe faithfully the overall energetics of the system
(Fig. 2A, inset). Moreover, the values of
Ka determined by SPR data are close to those
estimated from ITC studies, thus ruling out an appreciable contribution
from any unobserved binding process to the overall energetics of the
system. These results thus imply a single step bimolecular association
reaction between these saccharides and the lectin.
The "core" structure of the mannooligosaccharide recognized by the
ASAI carbohydrate recognition domain appears to be the trimannoside
(1) common to all the N-linked glycans
(Ka = 127 M
1
at 25 °C), which is only a marginally poorer ligand than
(3) (Ka = 169 M
1 at 25 °C). The equivalence
of the observed binding parameters for the interaction of compounds
(1)-(6) suggests that the Man3
structure constitutes the minimal epitope recognized by the lectin and
that its substitution at the reducing end with GlcNAc2 Asn
does not alter either the mechanism or the extent of binding.
As listed in Table I, the binding affinity for the mannose-containing
oligosaccharides increases with increasing chain length, with a
significant jump observed when extended with
-1,2-linked mannose
residues at their nonreducing ends. These observations corroborate our
previous study on the elucidation of the carbohydrate specificity of
ASAI (10).
Among the higher oligomers of mannose, the binding affinities for
Man7 GlcNAc2 (I) (8),
(10), and (11) are appreciably higher than those
of the above compounds, indicating that the extension of the
Man5 structure with
-1,2-mannosyl residues at its
nonreducing end increases the binding potencies. However, a mere
extension with a single mannosyl residue in the
-1,2 linkage at the
-1,3 branch, as in Man6 GlcNAc2
(7) or Man6 GlcNAc2 Asn
(7'), not only is inadequate but also perhaps compromises the binding. Additionally, these data suggest that a mere extension of
the Man5 structure by an
-1,2Man, although necessary, is
not sufficient to drive the reaction. The high affinities of
(8), (10), and (11) suggest that the
substitution of the
-1,6 branch by two
-1,2-linked mannosyl
residues is apparently necessary for the moderate to strong binding to
ASAI. Absence of this branch in (7), (7'), and
Man7 GlcNAc2 (II) (9), therefore,
make them nonbinders. A significant, 500-fold increase in the binding
potency over manno pentaose is observed with (8) as the
carbohydrate ligand (8.4×104
M
1 at 25 °C), which has two
-1,2-linked mannosyl residues, one on the
-1,3 arm and the
other on the
-1,6 arm. This suggests that for the combining site of
ASAI to establish stronger bonding contacts, substitution with
-1,2-linked mannose residues on both the arms is sufficient.
Moreover, extensions on the
-1,3 arm are recognized, subsequent to
recognition of the
-1,2-linked mannose on the
-1,6 arm. These
binding characteristics thereby implicate the
-1,2-linked mannose on
the
-1,6arm to play a crucial role in lodging the complementary
mannooligosaccharide in the ASAI combining site. Moreover, although
(7) and (8) are structurally similar, an
additional
-1,2-linked terminal mannose residue on the
-1,6 arm
of (8) confers on it an exceedingly high binding affinity.
This provides further insight into the mode of recognition of
carbohydrate ligands by ASAI, where in the Man
(1-2) Man at the
-1,6 arm seems to drive the binding interaction further.
In general, the binding constant (Ka) is
determined by the ratio of k1 and
k-1, and its value can increase with either an
enhancement in the former or a decrease in the latter. For most
enzyme-substrate interactions and also for the lectin-sugar interactions studied so far, it is the decrease in the dissociation rate constants that has been shown to be responsible for the increased binding affinities (25). In these studies on ASAI-manno oligosaccharide interaction, the occurrence of both, i.e. an increase in
k1 and decrease in k-1,
is implicated for the observed enhancement in affinities, although to a
large extent it is the relatively more dramatic increase in
k1 that imparts to (10) and
(11) their highest affinities. Thus, one of the interesting
observations of this study is the significant increase in the
association rate constants with the change in the structure of the
manno oligosaccharide ligand.
In conclusion, this study provides a molecular basis for explaining the
exquisite specificity of ASAI for the high mannose oligosaccharides by
demonstrating a stringent requirement for
-1,2-mannosyl substitution
at their
-1,6 arms. Additionally, ASAI-manno oligosaccharide
interactions constitute a unique example where the association rate
constants vary dramatically by a factor of about 800 between the
weakest and the strongest ligand.
 |
FOOTNOTES |
*
This work was supported by grants from the Department of
Science and Technology and the Department of Biotechnology, Government of India (to A. S.). The BIACoreTM facility was funded by the
Department of Biotechnology, Government of India for program support to
the Indian Institute of Science, Bangalore in the area of Drug and Molecular Design.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
A Senior Research Fellow supported by the University Grants Commission.
**
To whom correspondence should be addressed. Tel.: 91-80-3092389;
Fax: 91-80-3600535/3600683; E-mail: surolia@mbu.iisc.
ernet.in.
Published, JBC Papers in Press, November 13, 2000, DOI 10.1074/jbc.M009533200
 |
ABBREVIATIONS |
The abbreviations used are:
ASA, Allium sativum agglutinin;
SPR, surface plasmon resonance;
RU, response unit;
ITC, isothermal titration calorimetric.
 |
REFERENCES |
1.
|
Barre, A.,
Van Damme, E. J. M.,
Peumans, W. J.,
and Rouge, P.
(1996)
Plant Physiol.
112,
1531-1540[Abstract/Free Full Text]
|
2.
|
Hester, G.,
Kaku, H.,
Goldstein, I. J.,
and Wright, C. S.
(1995)
Nat. Struct. Biol.
2,
472-479[Medline]
[Order article via Infotrieve]
|
3.
|
Sauerborn, M. K.,
Wright, L. M.,
Reynolds, C. D.,
Grossman, J. G.,
and Rizkallah, P. J.
(1999)
J. Mol. Biol.
290,
185-199[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Wood, S. D.,
Wright, L. M.,
Reynolds, C. D.,
Rizkallah, P. J.,
Allen, A. K.,
Peumans, W. J.,
and Van Damme, E. J. M.
(1999)
Acta Crystallogr. Sect. D Biol. Crystallogr.
55,
1264-1272[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Chantalat, L.,
Wood, S. D.,
Rizkallah, P. J.,
and Reynolds, C. D.
(1996)
Acta Crystallogr. Sect. D Biol. Crystallogr.
52,
1146-1152[CrossRef]
|
6.
|
Chandra, N. R.,
Ramachandraiah, G.,
Bachhawat, K.,
Dam, T. K.,
Surolia, A.,
and Vijayan, M.
(1999)
J. Mol. Biol.
285,
1157-1168[CrossRef][Medline]
[Order article via Infotrieve]
|
7.
|
Shankarnarayanan, R.,
Sekar, K.,
Banerjee, R.,
Sharma, V.,
Surolia, A.,
and Vijayan, M.
(1996)
Nat. Struct. Biol.
3,
596-602[Medline]
[Order article via Infotrieve]
|
8.
|
Murzin, A. G.,
Lesk, A. M.,
and Chothia, C.
(1992)
J. Mol. Biol.
223,
531-543[Medline]
[Order article via Infotrieve]
|
9.
|
Van Damme, E. J. M.,
Smeets, K.,
Torrekens, S.,
Van Leuven, F.,
Goldstein, I. J.,
and Peumans, W. J.
(1992)
Eur. J. Biochem.
206,
413-420[Abstract]
|
10.
|
Dam, T. K.,
Bachhawat, K.,
Rani, P. G.,
and Surolia, A.
(1998)
J. Biol. Chem.
273,
5528-5535[Abstract/Free Full Text]
|
11.
|
Thomas, C. J.,
Surolia, N.,
and Surolia, A.
(1999)
J. Biol. Chem.
274,
29624-29627[Abstract/Free Full Text]
|
12.
|
Johnsson, B.,
Lofas, S.,
and Lindquist, G.
(1991)
Anal. Biochem.
198,
268-277[Medline]
[Order article via Infotrieve]
|
13.
|
Lee, R. T.,
Shinohara, Y.,
Hasegawa, Y.,
and Lee, Y. C.
(1999)
Biosci. Rep.
19,
283-292[Medline]
[Order article via Infotrieve]
|
14.
|
Van Damme, E. J. M.,
Astoul, C. H.,
Barre, A.,
Rouge, P.,
and Peumans, W. J.
(2000)
Eur. J. Biochem.
267,
5067-5077[Abstract/Free Full Text]
|
15.
|
Lis, H.,
and Sharon, N.
(1978)
J. Biol. Chem.
253,
3468-3476[Medline]
[Order article via Infotrieve]
|
16.
|
Mandal, D. K.,
and Brewer, C. F.
(1992)
Biochemistry
31,
2602-2609
|
17.
|
Dubois, M.,
Gilles, K. A.,
Hamilton, J. K.,
Rebers, P. A.,
and Smith, F.
(1956)
Anal. Chem.
28,
350-356
|
18.
|
Rao, J.,
Lin, Y.,
Bing, X.,
and Whitesides, G. M.
(1999)
J. Am. Chem. Soc.
121,
2629-2630[CrossRef]
|
19.
|
Scatchard, G.
(1949)
Ann. N. Y. Acad. Sci.
51,
660-672
|
20.
|
Patil, A. R.,
Thomas, C. J.,
and Surolia, A.
(2000)
J. Biol. Chem.
275,
24348-24356[Abstract/Free Full Text]
|
21.
|
Wiseman, T.,
Williston, S.,
Brandts, J. F.,
and Lin, L. N.
(1989)
Anal. Biochem.
179,
131-137[Medline]
[Order article via Infotrieve]
|
22.
|
Puri, K. D.,
Khan, M. I.,
Gupta, D.,
and Surolia, A.
(1993)
J. Biol. Chem.
268,
16378-16387[Abstract/Free Full Text]
|
23.
|
Gupta, D.,
Rao, N. V.,
Puri, K. D.,
Matta, K. L.,
and Surolia, A.
(1992)
J. Biol. Chem.
267,
8909-8918[Abstract/Free Full Text]
|
24.
|
Sastry, M. V. K.,
Swamy, M. J.,
and Surolia, A.
(1988)
J. Biol. Chem.
263,
14826-14831[Abstract/Free Full Text]
|
25.
|
Clegg, R. M.,
Loontiens, F. G.,
and Jovin, T. M.
(1977)
Biochemistry
16,
167-175[Medline]
[Order article via Infotrieve]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.