1 Division of Nephrology, Department of Medicine, Mount Sinai School of Medicine, New York, New York, 10029; and 2 Division of Extramural Activities, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recombinant protein, designated hUAT,
the human homologue of the rat urate transporter/channel (UAT),
functions as a highly selective urate channel in lipid bilayers.
Functional analysis indicates that hUAT activity, like UAT, is
selectively blocked by oxonate from its cytosolic side, whereas
pyrazinoate and adenosine selectively block from the channel's
extracellular face. Importantly, hUAT is a galectin, a protein with two
-galactoside binding domains that bind lactose. Lactose
significantly increased hUAT open probability but only when added to
the channel's extracellular side. This effect on open probability was
mimicked by glucose, but not ribose, suggesting a role for
extracellular glucose in regulating hUAT channel activity. These
functional observations support a four-transmembrane-domain structural
model of hUAT, as previously predicted from the primary structure of
UAT. hUAT and UAT, however, are not functionally identical: hUAT has a
significantly lower single-channel conductance and open probability is
voltage independent. These differences suggest that evolutionary
changes in specific amino acids in these highly homologous proteins are
functionally relevant in defining these biophysical properties.
pyrazinoate; lactose; oxonate; glucose; adenosine; glycophorin A
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
HUMANS, AS WELL AS BIRDS, reptiles, and some nonhuman primates lack functional uricase and, as a consequence, urate is the end product of the intracellular degradation of the purines adenine and guanine (3). Subsequent to its metabolic production by the enzyme xanthine oxidase (5, 40, 78), urate effluxes from cells by an unknown mechanism to enter the extracellular compartment. In the absence of degradation of urate to allantoin by hepatic uricase in these species, maintenance of urate homeostasis is entirely dependent on elimination of urate from the body by the kidneys (3) and, to a much lesser extent, the gastrointestinal tract (70, 71). Considering the very limited solubility of urate (80) within cells, plasma, and urine, it is apparent that the avoidance of urate crystallization in any of these compartments in humans is even more critically dependent on the efflux and excretory transporters than in species that have hepatic uricase to metabolize urate to the water-soluble compound allantoin (12, 25, 50). Although there are limited data on the mechanism(s) responsible for elimination of urate by the intestinal epithelium (70, 71), the handling (filtration, reabsorption, and secretion) and mechanisms of urate transport have been extensively evaluated in the kidney (3). Renal transport has been ascribed to both an electroneutral urate-anion exchanger (9, 20, 21, 28-30, 61) and an electrogenic urate uniporter (1, 2, 33, 61) in a number of species, including humans (61).
We recently cloned a cDNA from a rat renal expression library that encodes a 322-amino acid protein, prepared recombinant protein from the cDNA, and demonstrated that this protein functions as a highly selective, voltage-sensitive 10-pS urate transporter/channel in planar lipid bilayers (38). This protein, designated UAT, displays a number of characteristics (36) that suggest that this channel is the transporter responsible for urate efflux from systemic cells as well as the molecular representation of the rat renal electrogenic urate transporter. Moreover, recent studies in which UAT has been expressed as a chimeric protein documented that UAT is an integral plasma membrane protein with intracellular termini in a variety of renal and nonrenal epithelial cells derived from a number of species (59). However, it is noteworthy that UAT belongs to a family of proteins, the galectins, that have all been presumed to be soluble, cytoplasmic, or secreted proteins (4, 7, 10, 15-17, 22, 26, 48, 56). Equally importantly, galectins have been assigned multiple functions (4, 7, 10, 15-17, 22, 26, 48, 56), but none have ever been proposed to serve a transport function. The demonstration that UAT functions as a channel in synthetic lipid bilayers (36, 38) and resides as an integral plasma membrane protein in living cells (59) thus represents both a unique function and previously undescribed subcellular localization for a galectin.
Subsequent to our publication on the cloning of UAT (38), galectin 9 was reported in rats (76, 77), mice (76, 77) and humans (51, 52, 75). It is of note that the cDNAs for rat, mouse and human galectin 9 are, respectively, 99, 89, and 73% identical to UAT, and the translated proteins, like other members of the galectin family, are considered to be soluble, cytoplasmic, or secreted proteins (51, 75-77). Although a functional role has not been assigned to rat galectin 9 (76, 77), mouse galectin 9 has been proposed to serve a role in thymocyte-epithelial interactions (76, 77), whereas human galectin 9 is believed to participate in cellular interactions of the immune system (75) and in eosinophil chemoattraction (51). In view of the high degree of homology between UAT (accession no. U67958) and human galectin 9/ecalectin (accession nos. Z49107, AB006782, and AB005894), we recently generated galectin 9 cDNA by RT-PCR from RNA of human white blood cells, prepared recombinant protein, and performed studies to evaluate the possibility that the apparent human homologue of UAT might serve a transport function (44). These studies demonstrated that recombinant human galectin 9, a 323-amino acid protein that is identical to accession no. AB006782 (minus the 32-amino acid insertion specific to the intestinal isoform of galectin 9), both functions as a highly selective urate channel in synthetic lipid bilayers and represents an integral plasma membrane protein with cytoplasmic NH2 and COOH termini in epithelium-derived cells (44). These observations led us to propose that the human homologue of UAT is also likely to represent the urate channel in plasma membranes of systemic cells and the electrogenic renal urate transporter in humans (44).
The present studies were conducted to evaluate the functional
characteristics of the human urate transporter/channel, designated hUAT, to model its transmembrane organization and to assess the potential role in channel function of the two -galactoside binding sites within hUAT, the signature amino acid sequence of a galectin. These studies demonstrate that hUAT channel activity displays a number
of characteristics that suggest that the topologies of hUAT and UAT are
quite similar. In contrast, single-channel conductance and voltage
sensitivity of open probability of hUAT differ significantly from that
of UAT, presumably as a consequence of some evolutionary divergence in
critical amino acids in the respective sequences. Furthermore, these
studies provide evidence to suggest that binding of the
-galactoside
-lactose to hUAT is not simply a confirmation that this protein contains the signature sequences for galectins but
rather that such binding significantly influences hUAT channel activity. Finally, evidence is provided that glucose, the
physiologically more relevant sugar, similarly has a significant
modulating effect on hUAT channel activity.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of Recombinant Protein
As previously reported, recombinant protein was made from cDNA prepared by RT-PCR of RNA that was harvested from human white blood cells (44). In brief, the full length of the coding sequence of hUAT in pBluescript was amplified by PCR using a sense primer with an XhoI site immediately 5' to the start codon (5'-GCCTCGAGATGGCCTTCAGCGGTTCCCAG-3') and an antisense primer with a HindIII site just 3' to the stop codon (5'-GCAAGCTTCTATGTCTGCACATGGGTCGC-3'). The purified PCR product was subcloned into XhoI- and HindIII-digested pRSETA (Invitrogen, San Diego, CA) for subsequent production of a fusion protein with a six-histidine metal-chelating domain 5' to the coding region of UAT. pRSETA-hUAT was isolated (Qiagen Plasmid Maxi kit, Qiagen, Chatsworth, CA) and used to transform BL21(DE3)pLysE cells (Novagen, Madison, WI). Colonies of BL21(DE3)pLysE cells containing pRSETA-hUAT were grown until the optical density reached 0.6-0.7. Thereafter, isopropyl-1-thio-Functional Evaluation of Recombinant hUAT
Formation of proteoliposomes. A 1:1 (wt/wt) mixture of bovine brain phosphatidylethanolamine (PE) and phosphatidylserine (PS; Avanti Polar Lipids, Birmingham, AL), each at a concentration of 10 mg/ml, were evaporated to dryness under a stream of nitrogen. The resultant pellet was suspended in 25 µl of 220 mM Cs2SO4 and 10 mM HEPES-NaOH at pH 7.4, after which 2 µl of recombinant hUAT protein were added. Proteoliposomes were formed by sonicating the suspension for 30 s at 80 kHz in a bath sonicator (Laboratory Supplies, Hicksville, NY) (36-38, 44). Fresh proteoliposomes were prepared for each experiment.
Lipid bilayer chamber, formation of lipid bilayer, and channel
reconstitution.
The lipid bilayer system was identical to that previously reported
(36-38, 44). In all experiments, both chambers of the Plexiglas apparatus were filled with 1 ml of a solution containing 2.5 mM urate, 220 mM Cs2SO4, and 0.25 mM
CaCl2 that was buffered to pH 7.4 with 10 mM HEPES-NaOH.
Subsequently, a 50-µm hole in a Teflon film (type C-20, 12.5 µm
thick, DuPont Electronics, Wilmington, DE) that had been tightly fitted
between the two wells of the chamber was painted with lipids using a
club-shaped glass rod. The lipids used to paint the bilayer were
identical to those used to make the proteoliposomes (a 1:1 mixture of
PE and PS, each at 10 mg/ml) but, after drying under nitrogen, the
lipids were dissolved in n-decane (Sigma, St. Louis, MO) at
a concentration approximating 50 mg lipid/ml. Junction potentials were
corrected with the zero-adjust system of the patch-clamp amplifier
(Axopatch 200B, Axon Instruments, Burlingame, CA). The cis
chamber is defined as the chamber connected to the voltage-holding
electrode; all voltages are referenced to the trans (ground)
chamber. Voltage was generated, clamped at different voltages (100 to
+100 mV), and controlled with the patch-clamp amplifier. When a stable
resistance of at least 100 G
and a noise level of <0.1 pA were
maintained, the experiments were initiated by addition of 5 µl of the
hUAT-containing proteoliposomes to the trans chamber. The
solution in the trans chamber was stirred until the
proteoliposomes fused with the bilayer.
Functional analysis of the channel.
In each experiment, the activity of the channel was initially assessed
in the presence of symmetrical solutions of 2.5 mM urate in 220 mM
Cs2SO4, 0.25 mM CaCl2, and 10 mM
HEPES-NaOH at pH 7.4 in the cis and trans
chambers. Thereafter, the channel was reexamined in the
symmetrical 2.5 mM urate solutions, but after the cis or
trans chamber was pulsed with microliter volumes of one of
the following reagents to achieve progressively increased concentrations in the bath: 2.5 mM -lactose monohydrate (Sigma), 1.0 M D(+)-glucose (Sigma), 1.0 M D(
)-ribose
(Sigma), 2.5 mM oxonate (Sigma), 2.5 mM pyrazinoate (PZA; Aldrich
Chemical, Milwaukee, WI), or 1 mM adenosine (Sigma). All reagents were
prepared in 220 mM Cs2SO4 and 10 mM HEPES-NaOH
buffered to pH 7.4. In some experiments, channel activity was
reexamined after the solution in the trans and/or cis
chamber was replaced with reagent-free fresh urate solution.
Data collection and analysis. Current output of the patch clamp was filtered at 10 kHz through an eight-pole filter (Bessel filter, model 902, Frequency Devices, Haverhill, MA) that was digitized at 5 kHz (Digi Data 1200 series Interface, Axon Instruments). Data were analyzed with commercial software (pCLAMP, version 8.0, Axon Instruments) after additional digitized filtering at not less than 1 kHz.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Characteristics of the Reconstituted Channel
Figure 1 demonstrates single-channel activity of hUAT (evidenced by clear transitions between open and closed states) in the presence of symmetrical urate solutions after fusion of the hUAT-containing proteoliposomes with the lipid bilayer. Single-channel activity was evident in most experiments; however, multiple channels and apparent substate conductances were also observed. As is evident from the traces (Fig. 1, A and B), the open probability of hUAT is independent of voltage and, in this experiment, the slope conductance was 2 pS (Fig. 1C). As previously reported, the mean single-channel slope conductance of hUAT, calculated by linear regression analysis, was 4.0 ± 0.4 pS (n = 11), rectification was not obvious in symmetrical urate solutions, and the channel was highly selective to urate (44). These combined findings indicate that two important properties of hUAT, its single-channel conductance and its voltage insensitivity, are distinctly different from those previously described for rat UAT (36, 38). Moreover, in contrast to rat UAT, hUAT channel activity generally displayed run-down.
|
Effect of -Lactose on Activity of hUAT
|
|
Effect of D(+)-Glucose, But Not
D()-Ribose, on Activity of hUAT
|
Local Block of Homology to Glycophorin A Within hUAT
On the basis of the data obtained after addition of
|
Effect of Oxonate on the Activity of hUAT
Oxonate, a specific inhibitor of the enzyme uricase (14), both inhibits electrogenic urate transport in rat and rabbit renal cortical membrane vesicles (1, 2, 33) and blocks the activity of recombinant rat UAT that is reconstituted in the lipid bilayer system (36). As in other studies, multiple conductance states were detected in the absence of oxonate (Fig. 6, A and B). Oxonate concentrations up to 188 µM in the trans chamber failed to influence hUAT activity (Fig. 6, A and B). In contrast, addition of increasing concentrations of oxonate to the cis chamber, to a concentration of 138 ± 30.5 µM (n = 7), progressively decreased the number of conductance states and ultimately virtually abolished channel activity (Fig. 6, A and B). As depicted (Fig. 6C), the effect of oxonate on the open probability of hUAT (in the presence or absence of lactose) was quite similar to its effect on the activity of UAT (36). This oxonate-induced block of hUAT was reversible in that channel activity was fully restored after the oxonate-containing solution in the cis chamber was replaced with a fresh oxonate-free urate solution (not depicted). Of note, the effect of oxonate on both rat UAT (36) and hUAT activity is restricted in that the oxonate-induced block is only observed when the cytoplasmic face of the channel is exposed to the reagent. As previously proposed with UAT (36), the distinct asymmetrical effect of oxonate implies that this compound interacts with a specific domain in hUAT that is consistently localized on the cis face of the channel. It is of note that in our bilayer system the cis side of the chamber is exposed to changes in voltage, simulating an intracellular compartment; the cis chamber is connected to the voltage-holding electrode with all voltages referenced to the trans (ground) side. Insofar as the cis chamber represents an intracellular compartment, the domain in hUAT that interacts with oxonate, like the domain in UAT (36), must then reside on the cytoplasmic face of the channel. In this context, because lactose only influences hUAT when added to the opposite chamber of the bilayer (Fig. 3), the two
|
Local Block of Homology to Uricase Within hUAT
Because oxonate is a competitive inhibitor of uricase (14), and oxonate blocks hUAT channel activity (Fig. 6), the amino acid sequence of the human homologue was evaluated to determine whether it contains a block of homology to the substrate binding site in uricase. Importantly, the Q228 of Aspergillus uricase, which is critical to substrate binding (11) (presumably to oxonate as well as urate), is conserved within a 12-amino acid domain of porcine uricase, Aspergillus uricase, hUAT, rat UAT, and the intestinal isoform of galectin 9 (Fig. 7A). As depicted, alignment of a 12-amino acid block of porcine uricase (residues 231-242) and Aspergillus uricase (residues 224-235) with residues 158-169 of hUAT reveals that hUAT has 50% homology to both porcine and Aspergillus uricase (Fig. 7A). Alignment of residues 157-168 of rat UAT with residues 158-169 of hUAT reveals that this block of amino acids is highly conserved with 92% homology between the rat and human sequences (Fig. 7B). It is of note that a sequence for a gastrointestinal isoform of human galectin 9, which is inserted immediately after residue 148 of galectin 9, has been deposited in GenBank (accession no. AB006782). This domain in hUAT appears to be in part duplicated insofar as alignment of amino acids 4-15 of the 32-amino acid isoform sequence also has a high degree of homology to uricase, having 50 and 67% homology to porcine and Aspergillus uricase, respectively (Fig. 7).
|
Effect of PZA on Activity of hUAT
PZA, a potent inhibitor of urate transport in intact kidneys of multiple species (3) and an inhibitor of electrogenic urate transport in rat and rabbit membrane vesicles (1, 2, 33), also blocks channel activity of recombinant rat UAT (36). Comparable to observations made with recombinant rat UAT (36), despite the raising of the PZA concentration to 150 µM in the cis chamber, PZA failed to alter channel activity of hUAT (Fig. 8, A and B). Similar to the effect of PZA on rat UAT (36), PZA induced a dose-dependent block of hUAT activity (Fig. 8, A and B) and a reduction in open probability (Fig. 8C) when added to the trans chamber (in the presence or absence of lactose). The open probability of the channel was profoundly reduced at a concentration of 87.5 ± 43.3 µM (n = 5). This block was completely reversed after the PZA-containing solution was replaced with a fresh PZA-free urate solution (not shown). Because PZA and oxonate only effectively block channel activity when in contact with the trans and cis faces of the channel, respectively, the specific domains that bind these substrates in hUAT, as in UAT (36), must be located on opposite faces of the channel. Insofar as oxonate binds to a site on the cytoplasmic face of the channel, then the domain that binds PZA in hUAT, as in the case of the domain in UAT (36), must reside within an extracellular portion of the channel.
|
Effect of Adenosine on Activity of hUAT
As demonstrated in Fig. 9, hUAT, like UAT, contains a local block of amino acids that has 73 and 45% homology to the adenosine A1 (60, 73) and A3 (64) receptors, respectively. Importantly, this block of amino acids is identical to or homologous with the specific residues in the A1 receptor, P249, H251, and N254, that bind adenosine and xanthine (32, 57). These amino acids align with amino acids P127, H129, and D132 of hUAT. Because functional studies with rat recombinant protein documented that adenosine is a potent blocker of UAT channel activity (36), the possibility was evaluated that adenosine would also interact with hUAT. As depicted in Fig. 10, adenosine essentially eliminated channel activity when the concentration of adenosine in the trans chamber reached 14.2 ± 5.8 µM, producing a profound decrease in open probability (n = 3). As was the case with rat UAT, adenosine failed to block channel activity when a comparable concentration was achieved in the cis chamber (Fig. 10, A and B). This unilateral effect of adenosine on hUAT clearly implies that the block of homology to the adenosine receptors in hUAT is functional and, as in the case of UAT (36), is only exposed when adenosine is applied to the extracellular (trans) face of the channel.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present studies demonstrate that the human homologue of the urate transporter/channel, hUAT, (44) has characteristics that are both similar to and different from the highly homologous rat protein, rat UAT (36, 38). Three reagents that were previously documented to block rat UAT channel activity, oxonate, PZA, and adenosine (36), have been shown to similarly block hUAT channel activity (Figs. 5, 7, and 9). Moreover, with both the human and rat channels, each of these substrates only blocks channel activity when a specific side of the channel is exposed to the compound (Figs. 5, 7 and 9). There are, however, two important differences in the biophysical properties of these channels. First, the mean single-channel conductance of hUAT approximates one-half that of rat UAT (4.0 ± 0.4 vs. 9.5 ± 0.47 pS). Second, the open probability of hUAT is voltage independent (Fig. 1), whereas that of rat UAT is consistently voltage dependent (36, 38).
The concordance of findings observed with recombinant rat
(36) and human homologues of UAT relative to the
inhibitory effects of oxonate, PZA, and adenosine on channel activity
(Figs. 5, 7 and 9), in conjunction with the identical sidedness of
effects of the respective substrates, implies that the topologies of
the human and rat transporters are similar. We previously proposed a
molecular model for rat UAT that incorporated intracellular NH2 and COOH termini and four transmembrane domains
(36). This model, including the specific amino acid
residues that represent the four transmembrane -helices
(36), was based on electrophysiological studies in lipid
bilayers that revealed the sidedness of effects of these same three
reagents, the location of local blocks of homology to the
A1/A3 receptors and uricase within UAT, the
hydrophobicity profile of UAT, and the detection of hydrophobic
segments (long enough to span the membrane) with significant homology
to transmembrane domain 2 in urate/xanthine permease
(19), the
-helix documented to form transmembrane
domain E in bacterial rhodopsin (58), and a
portion of the
-helix reported to form transmembrane domain IX of subunit 1 of cytochrome c oxidase
(74). By incorporating all of this information, the
hydrophobic segments with homology to urate/xanthine permease,
bacterial rhodopsin, and cytochrome c oxidase were modeled
as transmembrane domains 1, 2, and 3,
respectively, in rat UAT (36).
Confirmation that rat and human UAT are transmembrane proteins has been
obtained in surface biotinylation studies of renal and nonrenal
epithelia-derived cells transfected with the cDNA of rat and human UAT
(44, 59). Moreover, recent evidence in support of the
above-described model was obtained with immunofluorescent and confocal
microscopy of nonpermeabilized and permeabilized epithelial cells
subsequent to transfection with NH2 or COOH FLAG-tagged UAT
cDNAs; the NH2 and COOH termini of both rat and human UAT were observed to reside on the intracellular side of the plasma membrane (44, 59). Additional strong support for this
model is provided by the present studies. First, the high degree of homology that has been detected within amino acids 18-33 of both hUAT and UAT to the dimerization domain within the single transmembrane -helix of GpA (Fig. 5) (41-43, 46, 47, 62, 63, 66)
supports our previous molecular model in which amino acids 15-35
of UAT were designated as transmembrane domain 1 (36). Second, we previously proposed that UAT contains two
large extracellular domains, one located between transmembrane
domains 1 and 2 and the second located between
transmembrane domains 3 and 4 (Fig. 2).
Importantly, hUAT contains two
-galactoside binding sites, one
encompassed by residues 61-96 within the first putative
extracellular domain and the second incorporated by residues
235-271 within the second putative extracellular domain. Of note,
the specific amino acids involved in each of these sites are 100%
conserved in human and rat UAT (the latter within residues 60-95
and 234-270). The present finding that
-lactose only influences
hUAT channel activity when in contact with the extracellular face of
the channel (Fig. 3) is thus consistent with our model. The combination
of findings of intracellular locations of the NH2 and COOH
termini of hUAT and UAT (44, 59) and extracellular locations of the two
-galactoside binding sites could be consistent with two transmembrane
-helices. However, the unilateral
intracellular block of channel activity induced by the uricase
inhibitor oxonate (Fig. 6) and the high degree of likelihood that this
substrate interacts with amino acids 158-169 of the uricase-like
domain in hUAT (Fig. 7) require that this site is exposed to the
intracellular face of the channel. Because the uricase-like domain is
located between the two
-galactoside binding sites, hUAT therefore
must contain at least four rather than two transmembrane
-helices (Fig. 2), a model entirely compatible with our previously proposed molecular model of the rat urate transporter/channel (36).
We previously suggested that a local block of homology to uricase within UAT (36) is most likely responsible for the functional similarities between the electrogenic urate transporter and uricase (1, 2, 33, 34, 37) and the ability of our polyclonal antibody to porcine uricase to select the UAT clone from the rat cDNA library (38), react with recombinant UAT (38), block electrogenic urate transport in membrane vesicles prepared from rat kidney (34), and selectively block UAT channel activity from the cytoplasmic side of the channel (36). It was also suggested that the oxonate-induced block of UAT activity was most likely consequent to its interaction with the uricase-like domain in UAT (36). In previously aligning a local block of homology in rat UAT to the substrate binding site in uricase, Q156 in rat UAT was aligned with Q228 in Aspergillus uricase (36), the amino acid that has been identified by X-ray crystallography as being critically important in formation of the substrate-uricase complex (11). However, on the basis of additional sequence data, specifically hUAT (44), the human (51, 75), mouse, and rat sequences of galectin 9 (76), the pig sequence for a urate transporter/channel (72), and the intestinal isoforms of human (accession no. AB006782), mouse, and rat galectin 9 (76) and pig urate transporter/channel (72), an adjustment has been made in this alignment. We now assign the substrate binding glutamine as Q161 in rat UAT as it is this glutamine, rather than Q156, that is conserved in the rat, human, pig, and mouse sequences (Fig. 7, A and B). In addition to conservation of this glutamine in the four mammalian species, it is evident that there is also an extremely high degree of homology within the block of amino acids that is located just proximal to the uricase-like domain in the intestinal isoform of these same species (Fig. 7B).
Despite the high degree of homology (Fig.
11) between and apparent similarities
in the topology of the human and rat homologues of UAT, evidence has
been obtained that two important biophysical properties of these
proteins differ (voltage sensitivity of the open probability and
single-channel conductance). The voltage sensitivity of channels has
been ascribed to the presence of charged residues, specifically, basic
residues localized in critical points in the structure of the channel
(8). These residues are presumed to sense the electrical
field across the membrane (8) and produce a significant
change in the conformation of the protein that affects the open
probability of the channel. We previously reported (36) that rat UAT contains two putative -sheets (residues 96-104 and 111-119) linked by six amino acids (105-110)
that carry a net positive charge (R106, E108, and K110) in a domain
located between transmembrane
-helices 1 and 2 (Fig. 2). We proposed that this segment of the protein could act as a
mobile domain (69), interact with the amphiphilic
-helices forming the UAT pore, result in presentation of this
charged region to the cytoplasmic side of the channel
(23), and thereby function as the voltage sensor. It is of
note that in contrast to rat UAT, the six amino acids (106-111) that link the two putative
-sheets in
hUAT (residues 97-105 and 112-120) carry a net neutral charge
(S107, D109, and K111) (Fig. 2). Insofar as this region does contain
the voltage sensor in the channel, the evolutionary modification in the
first of these three amino acids may then be responsible for the lack of voltage sensitivity of the open probability of hUAT. Clearly, confirmation of this proposal will require an analysis of the voltage
sensitivity of recombinant rat UAT subsequent to an experimentally induced mutation of amino acid 106 from arginine to serine.
|
The second significant difference between the biophysical properties of
the rat and human homologues of UAT is the conductance of the
respective channels. The single-channel conductance of hUAT that has
been measured in prior (44) and present studies (Figs. 1
and 3) in the absence of -lactose approximates one-half of that
observed with rat UAT (36, 38). Although the actual basis
for this difference is not known, we assume that the conductance difference reflects evolutionary changes in some amino acids in the
region of the rat and human channels that are critical to the
conformation of the pore. It is of interest that there appears to be a
clustering of nonhomologous amino acids in the human and rat sequences
within putative transmembrane
-helices 2 and 3 and, probably most significantly, in the block of cytoplasmic amino
acids that connect these
-helices (Figs. 2 and 11). Importantly, within the hairpin turn between these transmembrane helices, the human
channel contains three prolines (P150, P154, and P157), whereas rat UAT
has only one (P153) (Fig. 11). Because prolines are known to induce
turns in transmembrane segments and result in the formation of helical
hairpins (53, 55), the increased number of prolines in the
hairpin turn of hUAT may alter the conformation of this domain and
result in increased packing of the transmembrane
-helices. Insofar
as transmembrane
-helices 2 and 3 participate in formation of the channel pore, closer packing of these
-helices may decrease the size of the channel pore and thereby reduce channel conductance in hUAT relative to that of rat UAT. Alternatively, the
difference between the initial amino acid of the dimerization motif in
the first transmembrane
-helix of human and rat UAT (Fig. 5) may
influence packing of the transmembrane domains and thereby affect
conductance. Induced mutations in hUAT to recapitulate the amino acids
found in rat UAT in the first transmembrane
-helix and/or the domain
between transmembrane
-helices 2 and 3 will be
required to assess the possibility that the difference in conductance in the rat and human homologues is consequent to naturally evolved divergences in their primary structure.
Lactose, a -galactoside, has previously only been utilized as a tool
to isolate and purify galectins (4, 7, 10, 15-17, 22, 26,
48, 51, 56, 76). The ability to use this reagent for
purification purposes is consequent to the fact that lactose binds to
the highly conserved
-galactoside binding domains within galectins
(7). Because various
-galactosides are found on glycolipids and glycoproteins on cell surfaces and extracellular matrix, some of which have also been shown to bind to the
-galactoside binding sites in galectins (39), it has
been suggested that secreted galectins could function as biologically
significant ligands that play a role in cell migration, cell
proliferation, immune function, and adhesion (4, 7, 10,
15-17, 22, 26, 48, 56). To date, however, there is no
direct evidence to support these proposals.
In contrast to the absence of functional data relative to lactose or
the -galactoside binding sites in galectins, the present study
indicates that lactose, presumably by binding to the
-galactoside binding domains in hUAT, regulates the activity of hUAT by
significantly augmenting its conductance and open probability (Fig. 3).
Although
-lactose per se would not be relevant to physiological
function in vivo, it is important to note that both of lactose's
component sugars, galactose and glucose, interact with the
-galactoside binding domain in galectins (7), and
therefore these sugars could regulate channel activity in vivo. The
functional consequence of an increase in conductance and open
probability of hUAT would be an increase in urate flux. On the basis of
membrane potential and the likely prevailing electrochemical gradient
for urate, this would represent an increase in urate efflux from
systemic cells (inducing hyperuricemia) and an increase in urate
excretion consequent to an increase in the rate of urate secretion in
the renal proximal tubule and intestine (inducing hypouricemia). In this context, it is of interest that elevation of blood galactose levels in galactosemic patients (13) and rather modest
elevations in blood glucose levels in diabetic patients
(79) are associated with hyperuricemia. However, during
periods of poor metabolic control hypouricemia and hyperuricosuria
are evident in diabetic patients (18, 49). Of note, both
are corrected when blood glucose is normalized (18).
Consistent with the likelihood that increased proximal tubular fluid
glucose concentration also has a direct effect on urate flux is the
observation that the infusion of glucose induces an increase in urate
clearance in humans that significantly exceeds that induced by mannitol
at comparable osmolar clearances (54, 67, 68). Reports of
this (18, 49, 54, 67, 68, 79), in conjunction with the
observed effect of glucose on hUAT channel activity at concentrations
comparable to physiological (5 mM) and pathological (20 mM) plasma
levels in humans (Fig. 4), suggest that extracellular glucose may
interact with urate transporter/channels that reside in systemic cells and the renal proximal tubule (27) and thereby exert a
direct regulatory effect on the activity of this channel.
In summary, the present studies have provided evidence that recombinant
protein that was prepared from the cDNA of hUAT has topological
characteristics that are comparable to those of the rat homologue UAT.
However, these proteins are not functionally identical: differences in
their biophysical properties suggest that evolutionary changes in
specific amino acids in these two highly homologous proteins are
functionally relevant in defining these properties. Finally, the
present data suggest that an interaction of selective sugars with the
-galactoside binding domains in hUAT may be responsible, at least in
part, for regulating hUAT channel activity in vivo.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-52785 (R. G. Abramson) and DK-57867 (M. S. Lipkowitz).
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: R. G. Abramson, Div. of Nephrology, Box 1243, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029 (E-mail: ruth.abramson{at}mssm.edu).
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.
First published February 20, 2002;10.1152/ajprenal.00333.2001
Received 1 November 2001; accepted in final form 12 February 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abramson, RG,
King VF,
Reif MC,
Leal-Pinto E,
and
Baruch SB.
Urate uptake in membrane vesicles of rat renal cortex: effect of copper.
Am J Physiol Renal Fluid Electrolyte Physiol
242:
F158-F170,
1982
2.
Abramson, RG,
and
Lipkowitz MS.
Carrier-mediated concentrative urate transport in rat renal membrane vesicles.
Am J Physiol Renal Fluid Electrolyte Physiol
248:
F574-F584,
1985
3.
Abramson, RG,
and
Lipkowitz MS.
Evolution of the uric acid transport mechanisms in vertebrate kidney.
In: Basic Principles in Transport, edited by Kinne RKH. Basel: Karger, 1990, p. 115-153.
4.
Albrandt, K,
Orida NK,
and
Liu FT.
An IgE-binding protein with a distinctive repetitive sequence and homology with an IgG receptor.
Proc Natl Acad Sci USA
84:
6859-6863,
1987[Abstract].
5.
Al-Khalidi, UAS,
and
Chaglassian TH.
The species distribution of xanthine oxidase.
Biochem J
97:
318-320,
1965[ISI].
6.
Barondes, SH,
Castronovo V,
Cooper DN,
Cummings RD,
Drickamer K,
Feizi T,
Gitt MA,
Hirabayashi J,
Hughes C,
Kasai K,
Barondes SH,
Castronovo V,
Cooper DNW,
Cummings RD,
Drickamer K,
Feizi T,
Gitt MA,
Hirabayashi J,
Hughes C,
Kasai K,
Leffler H,
Liu F-T,
Lotan R,
Mercurio AM,
Monsigny M,
Pillai S,
Poirer F,
Raz A,
Rigby PWJ,
Rini JM,
and
Wang JL.
Galectins: a family of animal beta-galactoside-binding lectins.
Cell
76:
597-598,
1994[ISI][Medline].
7.
Barondes, SH,
Cooper DN,
Gitt MA,
Leffler H.,
and
Galectins
Structure and function of a large family of animal lectins.
J Biol Chem
269:
20807-20810,
1994
8.
Bezanilla, F.
The voltage sensor in voltage-dependent ion channels.
Physiol Rev
80:
555-592,
2000
9.
Blomstedt, JW,
and
Aronson PS.
pH gradient-stimulated transport of urate and p-aminohippurate in dog renal microvillus membrane vesicles.
J Clin Invest
65:
931-934,
1980[ISI][Medline].
10.
Clerch, LB,
Whitney P,
Hass M,
Brew K,
Miller T,
Werner R,
and
Massaro D.
Sequence of a full-length cDNA for rat lung beta-galactoside-binding protein: primary and secondary structure of the lectin.
Biochem
27:
692-699,
1988[ISI][Medline].
11.
Colloc'h, N,
El Hajji M,
Bachet B,
L'Hermite G,
Schiltz M,
Prangé T,
Castro B,
and
Mornon JP.
Crystal structure of the protein drug urate oxidase-inhibitor complex at 2.05 Å resolution.
Nature Structural Biol
4:
947-952,
1997[ISI][Medline].
12.
De Duve, C,
and
Baudhuin P.
Peroxisomes (microbodies and related particles).
Physiol Rev
46:
323-357,
1966
13.
Forster, J,
Schuchmann L,
Hans C,
Niederhoff H,
Kunzer W,
and
Keppler D.
Increased serum urate in galactosemia patients after a galactose load: a possible role of nucleotide deficiency in galactosemic liver injury.
Klin Wochenschr
53:
1169-1170,
1975[ISI][Medline].
14.
Fridovich, I.
The competitive inhibition of uricase by oxonate and by related derivatives of s-triazines.
J Biol Chem
240:
2491-2494,
1965
15.
Gitt, MA,
Colnot C,
Poirier F,
Nani KJ,
Barondes SH,
and
Leffler H.
Galectin-4 and galectin-6 are two closely related lectins expressed in mouse gastrointestinal tract.
J Biol Chem
273:
2954-2960,
1998
16.
Gitt, MA,
Massa SM,
Leffler H,
and
Barondes SH.
Isolation and expression of a gene encoding L-14-II, a new human soluble lactose-binding lectin.
J Biol Chem
267:
10601-10606,
1992
17.
Gitt, MA,
Wiser MF,
Leffler H,
Herrmann J,
Xia YR,
Massa SM,
Cooper DN,
Lusis AJ,
and
Barondes SH.
Sequence and mapping of galectin-5, a beta-galactoside-binding lectin, found in rat erythrocytes.
J Biol Chem
270:
5032-5038,
1995
18.
Gonzalez-Sicilia, L,
Garcia-Estan J,
Martinez-Blazquez A,
Fernandez-Pardo J,
Quiles JL,
and
Hernandez J.
Renal metabolism of uric acid in type I insulin-dependent diabetic patients: relation to metabolic compensation.
Horm Metab Res
29:
520-523,
1997[ISI][Medline].
19.
Gorfinkiel, L,
Diallinas G,
and
Scazzocchio C.
Sequence and regulation of the uapA gene encoding a uric acid-xanthine permease in the fungus Aspergillus nidulans.
J Biol Chem
268:
23376-23381,
1993
20.
Guggino, SE,
and
Aronson PS.
Paradoxical effects of pyrazinoate and nicotinate on urate transport in dog renal microvillus membranes.
J Clin Invest
76:
543-547,
1985[ISI][Medline].
21.
Guggino, SE,
Martin GJ,
and
Aronson PS.
Specificity and modes of the anion exchanger in dog renal microvillus membranes.
Am J Physiol Renal Fluid Electrolyte Physiol
244:
F612-F621,
1983
22.
Hadari, YR,
Paz K,
Dekel R,
Mestrovic T,
Accili D,
and
Zick Y.
Galectin-8. A new rat lectin, related to galectin-4.
J Biol Chem
270:
3447-3453,
1995
23.
Hille, B.
Ionic Channels of Excitable Membranes. Sunderland, MA: Sinauer, 1992.
24.
Holzinger, A,
Phillips KS,
and
Weaver TE.
Single-step purification/solubilization of recombinant proteins: application to surfactant protein B.
Biotechniques
20:
804-808,
1996[ISI][Medline].
25.
Hruban, Z,
and
Recheigl M.
Microbodies and related particles: morphology, biochemistry, and physiology.
In: International Review of Cytology, edited by Bourne GH,
and Danielli JF.. New York: Academic, 1969, suppl. 1, p. 20-39.
26.
Hughes, RC.
Secretion of the galectin family of mammalian carbohydrate-binding proteins.
Biochim Biophys Acta
1473:
172-185,
1999[ISI][Medline].
27.
Hyink, DP,
Rappoport JZ,
Wilson PD,
and
Abramson RG.
Expression of the urate transporter/channel is developmentally regulated in human kidneys.
Am J Physiol Renal Physiol
281:
F875-F886,
2001
28.
Kahn, AM,
and
Aronson PS.
Urate transport via anion exchange in dog renal microvillus membrane vesicles.
Am J Physiol Renal Fluid Electrolyte Physiol
244:
F56-F63,
1983
29.
Kahn, AM,
Branham S,
and
Weinman EJ.
Mechanism of urate and p-aminohippurate transport in rat renal microvillus membrane vesicles.
Am J Physiol Renal Fluid Electrolyte Physiol
245:
F151-F158,
1983
30.
Kahn, AM,
Shelat H,
and
Weinman EJ.
Urate and p-aminohippurate transport in rat renal basolateral vesicles.
Am J Physiol Renal Fluid Electrolyte Physiol
249:
F654-F661,
1985
31.
Keleshian, AM,
Edeson RO,
Liu GJ,
and
Madsen BW.
Evidence for cooperativity between nicotinic acetylcholine receptors in patch clamp records.
Biophys J
78:
1-12,
2000
32.
Kim, J,
Wess J,
van Rhee AM,
Shöneberg T,
and
Jacobson KA.
Site-directed mutagenesis identifies residues involved in ligand recognition in the human A2A adenosine receptor.
J Biol Chem
270:
13987-13997,
1995
33.
Knorr, BA,
Beck JC,
and
Abramson RG.
Classical and channel-like urate transporters in rabbit renal brush border membranes.
Kidney Int
45:
727-736,
1994[ISI][Medline].
34.
Knorr, BA,
Lipkowitz MS,
Potter BJ,
Masur SK,
and
Abramson RG.
Isolation and immunolocalization of a rat renal cortical membrane urate transporter.
J Biol Chem
269:
6759-6764,
1994
35.
Krouse, ME,
and
Wine JJ.
Evidence that CFTR channels can regulate the open duration of other CFTR channels: cooperativity.
J Membr Biol
182:
223-232,
2001[ISI][Medline].
36.
Leal-Pinto, E,
Cohen BE,
and
Abramson RG.
Functional analysis and molecular modeling of a cloned urate transporter/channel.
J Membr Biol
169:
13-27,
1999[ISI][Medline].
37.
Leal-Pinto, E,
London RD,
Knorr BA,
and
Abramson RG.
Reconstitution of hepatic uricase in planar lipid bilayer reveals a functional organic anion channel.
J Membr Biol
146:
123-132,
1995[ISI][Medline].
38.
Leal-Pinto, E,
Tao W,
Rappaport J,
Richardson M,
Knorr BA,
and
Abramson RG.
Molecular cloning and functional reconstitution of a urate transporter/channel.
J Biol Chem
272:
617-625,
1997
39.
Leffler, H,
and
Barondes SH.
Specificity of binding of three soluble rat lung lectins to substituted and unsubstituted mammalian beta-galactosides.
J Biol Chem
261:
10119-10126,
1986
40.
Lehninger, AL,
Nelson DL,
and
Cox MM.
Biosynthesis of amino acids, nucleotide, and related molecules.
In: Principles of Biochemistry (2nd ed.). New York: Worth, 1993, p. 688-734.
41.
Lemmon, MA,
Flanagan JM,
Hunt JF,
Adair BD,
Bormann BJ,
Dempsey CE,
and
Engelman DM.
Glycophorin A dimerization is driven by specific interactions between transmembrane alpha-helices.
J Biol Chem
267:
7683-7689,
1992
42.
Lemmon, MA,
Flanagan JM,
Treutlein HR,
Zhang J,
and
Engelman DM.
Sequence specificity in the dimerization of transmembrane alpha-helices.
Biochemistry
31:
12719-12725,
1992[ISI][Medline].
43.
Lemmon, MA,
Treutlein HR,
Adams PD,
Brunger AT,
and
Engelman DM.
A dimerization motif for transmembrane alpha-helices.
Nat Struct Biol
1:
157-163,
1994[ISI][Medline].
44.
Lipkowitz, MS,
Leal-Pinto E,
Rappoport JZ,
Najfield V,
and
Abramson RG.
Functional reconstitution, membrane targeting, genomic structure and chromosomal localization of a human urate transporter.
J Clin Invest
107:
1103-1115,
2001
45.
Lobsanov, YD,
Gitt MA,
Leffler H,
Barondes SH,
and
Rini JM.
X-ray crystal structure of the human dimeric S-Lac lectin, L-14-II, in complex with lactose at 2.9-Å resolution.
J Biol Chem
268:
27034-27038,
1993
46.
MacKenzie, KR,
and
Engelman DM.
Structure-based prediction of the stability of transmembrane helix-helix interactions: the sequence dependence of glycophorin A dimerization.
Proc Natl Acad Sci U S A
95:
3583-3590,
1998
47.
MacKenzie, KR,
Prestegard JH,
and
Engelman DM.
A transmembrane helix dimer: structure and implications.
Science
276:
131-133,
1997
48.
Madsen, P,
Rasmussen HH,
Flint T,
Gromov P,
Kruse TA,
Honore B,
Vorum H,
and
Celis JE.
Cloning, expression, and chromosome mapping of human galectin-7.
J Biol Chem
270:
5823-5829,
1995
49.
Magoula, I,
Tsapas G,
Paletas K,
and
Mavromatidis K.
Insulin-dependent diabetes and renal hypouricemia.
Nephron
59:
21-26,
1991[ISI][Medline].
50.
Mahler, HR,
Baum HM,
and
Hubscher G.
Enzymatic oxidation of urate.
Science
124:
705-708,
1956[ISI].
51.
Matsumoto, R,
Matsumoto H,
Seki M,
Hata M,
Asano Y,
Kanegasaki S,
Stevens RL,
and
Hirashima M.
Human ecalectin, a variant of human galectin-9, is a novel eosinophil chemoattractant produced by T lymphocytes.
J Biol Chem
273:
16976-16984,
1998
52.
Matsushita, N,
Nishi N,
Seki M,
Matsumoto R,
Kuwabara I,
Liu FT,
Hata Y,
Nakamura T,
and
Hirashima M.
Requirement of divalent galactoside-binding activity of ecalectin/galectin-9 for eosinophil chemoattraction.
J Biol Chem
275:
8355-8360,
2000
53.
Monne, M,
Nilsson I,
Elofsson A,
and
von Heijne G.
Turns in transmembrane helices: determination of the minimal length of a "helical hairpin" and derivation of a fine-grained turn propensity scale.
J Mol Biol
293:
807-814,
1999[ISI][Medline].
54.
Moriwaki, Y,
Yamamoto T,
Takahashi S,
Suda M,
and
Higashino K.
Effect of glucose infusion on the renal transport of purine bases and oxypurinol.
Nephron
69:
424-427,
1995[ISI][Medline].
55.
Nilsson, I,
and
von Heijne G.
Breaking the camel's back: proline-induced turns in a model transmembrane helix.
J Mol Biol
284:
1185-1189,
1998[ISI][Medline].
56.
Oda, Y,
Herrmann J,
Gitt MA,
Turck CW,
Burlingame AL,
Barondes SH,
and
Leffler H.
Soluble lactose-binding lectin from rat intestine with two different carbohydrate-binding domains in the same peptide chain.
J Biol Chem
268:
5929-5939,
1993
57.
Olaf, ME,
Ren H,
Ostrowski J,
Jacobson KA,
and
Stiles JL.
Cloning, expression, and characterization of the unique bovine A1 adenosine receptor. Studies on the ligand binding site by site-directed mutagenesis.
J Biol Chem
267:
10764-10770,
1992
58.
Pebay-Peyroula, E,
Rummel G,
Rosenbusch JP,
and
Landau EM.
X-ray structure of bacteriorhodopsin at 2.5 angstroms from microcrystals grown in lipidic cubic phases.
Science
277:
1676-1682,
1997
59.
Rappoport, JZ,
Lipkowitz MS,
and
Abramson RG.
Localization and topology of a urate transporter/channel, a galectin, in epithelium-derived cells.
Am J Physiol Cell Physiol
281:
C1926-C1939,
2001
60.
Ren, H,
and
Stiles GL.
Characterization of the human A1 adenosine receptor gene. Evidence for alternative splicing.
J Biol Chem
269:
3104-3110,
1994
61.
Roch-Ramel, F,
Werner D,
and
Guisan B.
Urate transport in brush-border membrane of human kidney.
Am J Physiol Renal Fluid Electrolyte Physiol
266:
F797-F805,
1994
62.
Russ, WP,
and
Engelman DM.
The GxxxG motif: a framework for transmembrane helix-helix association.
J Mol Biol
296:
911-919,
2000[ISI][Medline].
63.
Russ, WP,
and
Engelman DM.
TOXCAT: a measure of transmembrane helix association in a biological membrane.
Proc Natl Acad Sci USA
96:
863-868,
1999
64.
Salvatore, CA,
Jacobson MA,
Taylor HE,
Linden J,
and
Johnson RG.
Molecular cloning and characterization of the human A3 adenosine receptor.
Proc Natl Acad Sci USA
90:
10365-10369,
1993[Abstract].
65.
Schuler, GD,
Altschul SF,
and
Lipman DJ.
A workbench for multiple alignment construction and analysis.
Proteins
9:
180-190,
1991[ISI][Medline].
66.
Senes, A,
Gerstein M,
and
Engelman DM.
Statistical analysis of amino acid patterns in transmembrane helices: the GxxxG motif occurs frequently and in association with beta-branched residues at neighboring positions.
J Mol Biol
296:
921-936,
2000[ISI][Medline].
67.
Skeith, MD,
Healey LA,
and
Cutler RE.
Effect of phloridzin on uric acid excretion in man.
Am J Physiol
219:
1080-1082,
1970
68.
Skeith, MD,
Healey LA,
and
Cutler RE.
Urate excretion during mannitol and glucose diuresis.
J Lab Clin Med
70:
213-220,
1967[ISI][Medline].
69.
Slatin, SL,
Qui XQ,
Jakes KS,
and
Finkelstein A.
Identification of a translocated protein segment in a voltage-dependent channel.
Nature
371:
158-161,
1994[ISI][Medline].
70.
Sorenson, LB.
The elimination of uric acid in man studied by means of C14-labeled uric acid.
Uricolysis Scand J Clin Lab Invest
12, Suppl54:
1-214,
1960.
71.
Sorenson, LB.
Extrarenal disposal of uric acid.
In: Uric Acid, Handbook of Experimental Pharmacology, edited by Kelley WN,
and Weiner IM.. Berlin: Springer-Verlag, 1978, p. 325-336.
72.
Spitzenberger, F,
Graessler J,
and
Schroeder HE.
Molecular and functional characterization of galectin 9 mRNA isoforms in porcine and human cells and tissues.
Biochimie
83:
851-862,
2001[ISI][Medline].
73.
Townsend-Nicholson, A,
and
Shine J.
Molecular cloning and characterisation of a human brain A1 adenosine receptor cDNA.
Brain Res Mol Brain Res
16:
365-370,
1992[ISI][Medline].
74.
Tsukihara, T,
Aoyama H,
Yamashita E,
Tomizaki T,
Yamaguchi H,
Shinzawa-Itoh K,
Nakashima R,
Yaono R,
and
Yoshikawa S.
The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 Å.
Science
272:
1136-1144,
1996[Abstract].
75.
Tureci, O,
Schmitt H,
Fadle N,
Pfreundschuh M,
and
Sahin U.
Molecular definition of a novel human galectin which is immunogenic in patients with Hodgkin's disease.
J Biol Chem
272:
6416-6422,
1997
76.
Wada, J,
and
Kanwar YS.
Identification and characterization of galectin-9, a novel beta-galactoside-binding mammalian lectin.
J Biol Chem
272:
6078-6086,
1997
77.
Wada, J,
Ota K,
Kumar A,
Wallner EI,
and
Kanwar YS.
Developmental regulation, expression, and apoptotic potential of galectin-9, a beta-galactoside binding lectin.
J Clin Invest
99:
2452-2461,
1997
78.
Westerfeld, WW,
and
Richert DA.
The xanthine oxidase activity of rat tissues.
Proc Soc Exp Biol Med
71:
181-184,
1949.
79.
Whitehead, TP,
Jungner I,
Robinson D,
Kolar W,
Pearl A,
and
Hale A.
Serum urate, serum glucose and diabetes.
Ann Clin Biochem
29:
159-161,
1992[ISI][Medline].
80.
Wilcox, WR,
Khalaf A,
Weinberger A,
Kippen I,
and
Klinenberg JR.
Solubility of uric acid and monosodium urate.
Mol Biol Eng
10:
522-531,
1972.