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Endocrinology Vol. 141, No. 12 4606-4612
Copyright © 2000 by The Endocrine Society

ARTICLES

The Role of Selenocysteine 133 in Catalysis by the Human Type 2 Iodothyronine Deiodinase1

Christoph Buettner, John W. Harney and P. Reed Larsen

Thyroid Division, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Dr. P. Reed Larsen, Thyroid Division, Brigham and Women’s Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115. E-mail: larsen{at}rascal.med.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human type 2 iodothyronine deiodinase (NOREF>hD2) catalyzes the activation of T4 to T3. D2, like types 1 and 3 deiodinases, contains selenocysteine (Sec) in the highly conserved active center at position 133. To evaluate the contribution of Sec133 to the catalytic properties of hD2, we generated mutants in which cysteine (Cys) or alanine (Ala) replaced Sec133. The Km (T4) of Cys133 D2 was 2.1 µM, strikingly higher than that of native D2 (1.4 nM). In contrast, the relative turnover number was 10-fold lower for Cys133D2, illustrating the greater potency of Se than S in supporting catalysis. The AlaD2 mutant was inactive. Studies in intact cells transiently expressing the native or Cys133D2 enzyme exhibited saturation kinetics expected from the Km as measured under in vitro conditions, indicating rapid equilibration of extracellular and intracellular T4. Blockade of the NTCP, OATP1–3, and LST-1 transporters with 10 mM sodium taurocholate did not alter the deiodination rate of T4 by Cys133D2 transiently expressed in intact cells, suggesting that intracellular transport of T4 is not rate limiting. These results illustrate that selenium plays a critical role in deiodination catalyzed by hD2.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TYPE 2 DEIODINASE (NOREF>D2) catalyzes the activation of T4 to T3 and is important in the local generation of T3, particularly in the brain, where thyroid hormone has important regulatory effects (reviewed in Refs. 1 and 2). Human D2 (NOREF>hD2) shows considerable similarity to the other two human selenodeiodinases, type 1 and 3 iodothyronine deiodinases (NOREF>D1 and D3, respectively) (3, 4). These three enzymes constitute a family of oxidoreductases all of which contain one (NOREF>D1 and D3) or two (NOREF>D2) selenocysteine residues encoded by UGA (uridine, guanine, adenine) codons. Successful insertion of selenocysteine at UGA codons in eukaryotes requires the presence of a specific stem-loop structure, the selenocysteine insertion sequence (NOREF>SECIS) element, in the 3'-untranslated region of the messenger RNA (5, 6). SECIS elements have now been identified in all selenoprotein messenger RNAs, including the human, mouse, and chicken D2 complementary DNAs (NOREF>cDNAs) (7, 8, 9). In human D2 the first selenocysteine, Sec133, resides in a region of almost complete identity among the three deiodinases (3, 4, 10). The corresponding Sec in D1 has been shown to be important for efficient catalytic activity (11, 12). Its mutation to leucine eliminates deiodinase function, and its mutation to cysteine, in effect exchanging sulfur (S) for selenium (Se), raises the apparent Km 2- to 3-fold, but reduces the turnover number approximately 100-fold. The Sec residues in the active center of the deiodinases are thought to function as the iodide acceptor (13). Mutation of the 3'-UGA (codon 266) of hD2 to UAA, an unambivalent stop codon, leads to the expression of a protein that lacks Sec266 and the last seven amino acids. The kinetic characteristics of this truncated protein are indistinguishable from those of the wild-type hD2 enzyme (14). Thus, neither the Sec266 in D2 nor the seven amino acids encoded by nucleotides 3' to this UGA are required for normal enzymatic function by D2. The following studies were performed to evaluate the role of Sec133 in hD2 function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mutagenesis
The construction of the mutant cDNAs was performed in two phases. The parent constructs were those prepared earlier in which hD2 cDNAs with sequences encoding a methionine and six histidine (His) residues were placed amino-terminal to the initiator methionine (15). These contained substitutions encoding either Cys (TGC) or Ala (GCA) in place of the TGA. A 459-bp ACC-1 fragment containing the sequences from codon 1 to codon 273 of hD2 (excluding the His residues) was then exchanged with the wild-type ACC-1 fragment of hD2-selP (Fig. 1Go) (4). The selenoprotein P element in hD2-selP was then removed by excision of the XbaI fragment (Fig. 1Go). The mutations were verified by manual and automated sequencing. The wild-type hD2 Genethon clone Z44085 was provided by Drs. Valerie A. Galton and Donald L. St. Germain (Dartmouth Medical School, Hanover, NH).



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  		Figure 1. Schematic diagrams of hD2 cDNA constructs. The native hD2 cDNA is displayed in A. Indicated are the start codon ATG, the two TGA codons within the coding region (indicated by a bold line), and the SECIS element in the 3'-untranslated region. B, The chimeric construct hD2Sel P, in which the wild-type hD2 coding sequence is inserted 5' to the potent selenoprotein P SECIS element. In the Sec133Cys and Sec133Ala mutants, the SECIS element is deleted causing termination of translation at the TGA codon at position 262. Restriction enzyme sites used for subcloning are indicated.

 
Antibody and Western analysis
Polyclonal antibodies were raised in rabbits by conjugation of the peptide sequence RSKSTRGEWRRMLTS (amino acids 50–64, designated AB 24924) and SRSKSTRGEWRRMLTSEGLRC (amino acids 49–69, AB 85254) with KLM by Research Genetics, Inc. (Huntsville, AL) (15). These were immunopurified with the Sulfolink Kit (Pierce Chemical Co., Rockford, IL) according to the manufacturer’s instructions.

HEK-293 cells were transiently transfected with calcium phosphate/DNA precipitate and harvested after 48 h. Cells from two 60-mm plates were sonicated in 250 µl PE [0.1 M NaP04 (pH 6.9) and 1 mM EDTA] containing 0.25 M sucrose and 10 mM dithiothreitol. Typically, 200 µg protein were dissolved in 5 x SDS-PAGE sample buffer [0.3125 M Tris-HCl, 4% ß-mercaptoethanol, 50% glycerol, and 0.5 mg/ml bromophenyl blue (pH 8.3)] and heated for 5 min at 100 C. Samples were applied to SDS-PAGE gels as described by Laemmli, using 10% polyacrylamide (acrylamide-bis, 37.5:1) in the running gel. Gels were run at 15 mA for 15 h, then electrotransferred onto Immobilon (Millipore Corp., Bedford, MA) in 20% methanol, 25 mM Tris-HCl (pH 8.3), and 192 mM glycine at 100 mA for 16 h at 4 C. Membranes were blocked with 5% (wt/vol) nonfat milk in TBS-Tween [20 mM Tris-HCl (pH 7.6), 140 mM NaCl, and 0.1% Tween-20], and these incubated with the indicated antibodies at 1:200 dilutions in 1.25% (wt/vol) nonfat milk in TBS-Tween, followed by an incubation with peroxidase-conjugated secondary antibody (NEN Life Science Products, Boston, MA). Reaction products were visualized by reaction with ECL (Amersham Pharmacia Biotech, Arlington Heights, IL) and exposure to X-Omat film (Eastman Kodak, Rochester, NY).

DNA transfections
All constructs were cotransfected with plasmid pTKGH, a thymidine kinase promoter-directed human GH-expressing plasmid, into HEK-293 cells by calcium phosphate precipitation. Transfection efficiencies were monitored by assay of human GH in the medium (16).

Assay of 5'-deiodinase activity in sonicates (in vitro) and in intact transfected cells (in vivo)
Cell sonicates were assayed in duplicate. Purification of [125I]T4 or [125I]rT3 was performed on LH-20 columns just before use. It had less than 1% contamination with I-. D2 assays contained 10–150 µg cell sonicate, 125I-labeled T4, 2 nM T4, and 20 mM dithiothreitol in a final volume of 300 µl PE. Incubation was performed for 60 min at 37 C, and 125I was separated from T4 by trichloroacetic acid precipitation (17). For determination of the Km for T4 or rT3 of transiently expressed hD2 or the Sec133CysD2 mutant, varying concentrations of unlabeled T4 (1, 1.5, 3, and 10 nM) or rT3 (1, 1.5, 2.5, and 7.5 nM) for the wild-type and T4 (1, 1.5, 3, and 10 µM) or rT3 (1, 1.5, 2.5, and 7.5 µM) for the Sec133CysD2 mutant were used in PE buffer containing 20 mM DTT and 125I T4 or rT3, respectively. Activity was expressed as picomoles of substrate deiodinated per min/mg protein. Kinetic constants were determined by linear regression analysis of double reciprocal plots. Results are reported as the mean of values derived from at least two separate experiments. There was no significant deiodination by cells transfected with vector alone.

In vivo deiodinase activity was assayed as described with the following modifications (18). Pairs of 60-mm plates of the indicated cell types were independently transfected with either the hD2 or the Cys133D2 mutant. One day after transfection, cell monolayers were washed twice with sterile PBS and then cultured for up to an additional 24 h in serum-free DMEM to which was added [125I]T4 (10,000 cpm/100 µl) plus the indicated concentrations of unlabeled T4 with or without various inhibitors. Two hundred-microliter aliquots of medium were removed from each plate 1 h after the addition of radiolabeled compounds (basal samples) and at additional time points during the next 24 h. Immediately after harvesting, 100 µl horse serum were added to each aliquot, and protein was precipitated by the addition of 100 µl 50% trichloroacetic acid followed by centrifugation at 12,000 x g for 5 min in a microcentrifuge. The 125I- was determined by counting 200 µl of the supernatant in a {gamma}-scintillation counter. The 125I- generated was then calculated as the fraction of the total counts present in the 200-µl aliquot of medium minus the fraction of 125I- present in the basal (time zero) sample and the nonspecific deiodination in control cells transfected with vector alone. This amounted to less than 5% of the total counts.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Kinetic studies of mutant D2 enzymes in vitro
To define the role of Sec133 in hD2 for the kinetic properties of the enzyme we substituted either Ala or Cys at this position. Alanine was chosen because it has no active side-chain to participate in any reaction and does not disturb the secondary structure, but fills physical space. The substitution of Cys for Sec133 replaces the selenium of Sec with sulfur. As estimated by Western blotting using polyclonal hD2 antibodies, the Sec133Ala and Sec133Cys mutant hD2 proteins (hereafter termed AlaD2 and CysD2) were transiently expressed in approximately 100-fold greater amounts than the native D2 protein (Fig. 2AGo). Despite expression in large amounts, the AlaD2 mutant had no deiodinase activity (Fig. 2BGo). On the other hand, the fractional deiodination of 2 nM T4 by the CysD2 mutant was significantly lower than was that by native D2 despite its much greater expression (Fig. 2Go). Note that in addition to approximately 100-fold lower native D2 than CysD2 expression in the HEK-293 cell sonicate by Western blotting (Fig. 2AGo and see below), only 10 µg wild-type D2 sonicate were used in the assay, as opposed to 100 µg for the CysD2 mutant (Fig. 2BGo). Thus, selenium (Se) or sulfur (S) is essential to D2 function, but Se is far superior to S.



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  		Figure 2. Quantitation of protein synthesized and enzyme activities of wild-type, Sec133Cys, and Sec133Ala hD2 mutants. A, Wild-type protein, the Sec133Cys and Sec133Ala mutants, and control vector were transiently expressed in HEK 293 cells. Crude cell lysates were analyzed by Western using a polyclonal antiserum (AB 24924) directed against the amino-terminal half of hD2. The Sec133Ala and Sec133Cys mutant D2 proteins are expressed in equal amounts, approximately 100-fold higher than wild-type D2. The band at 20 kDa is nonspecific. B, One hundred micrograms of the Sec133Ala-, Sec133Cys-, and vector-transfected sonicates and 10 µg of the wild-type D2 sonicates were assayed for D2 activity. The substrate concentration was 2 nM T4; the incubation time was 1 h.

 
Michaelis-Menton kinetic analyses of the CysD2 protein showed an apparent Km for T4 of 2.1 µM and for rT3 of 4.6 µM. These Km values are approximately 1000-fold higher than those for native hD2 (4). The mean maximum velocity (Vmax) values of the CysD2 mutant were 25 pmol/min·mg protein for T4 and 9.3 pmol/min·mg protein for rT3. We also analyzed the sensitivity of the CysD2 mutant to inhibition by gold thioglucose (GTG; Fig. 3Go). The apparent Ki is 120 µM, and the inhibition of deiodination is noncompetitive.



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  		Figure 3. Lineweaver-Burk plot of T4 deiodination catalyzed by Sec133Cys D2 with or without GTG. A, Lineweaver-Burk plot of the 5'-deiodination of T4 in the absence and presence of GTG. Each data point is the average of closely agreeing duplicate determinations. B, Slope replot of the T4 deiodination rates in the presence and absence of GTG.

 
To quantitate the effects of the substitution of S for Se in D2 on its deiodination efficiency, it is necessary to know the concentration of enzyme protein. In previous studies with D1 and D3, it was possible to use a covalent label, N-bromoacetyl-[125I]iodothyronine (BrAcT3 or T4), to obtain an estimate of the amount of specific enzyme protein produced (11, 19). However, as neither BrAcT4 nor BrAcT3 reacts in specific and saturable fashion with hD2, it is not possible to use this approach to quantitate the transiently expressed D2 (4, 20). Accordingly, we performed semiquantitative Western blotting to obtain estimates of the relative amounts of Cys and native D2 protein in the cell sonicates. Figure 4Go shows the results of Western blots of HEK-293 sonicate previously transfected with either native or CysD2-encoding cDNA plasmids and incubated in selenium-supplemented medium. Comparison of the densities of the 32-kDa bands indicates that the amount of CysD2 is between 80- and 160-fold greater than that of native D2. Using an estimate that roughly 100-fold higher quantities of CysD2 protein are expressed relative to native D2, the relative turnover number for the wild-type protein is 810 fmol/min·OD unit compared with 83 for the CysD2 mutant (Table 1Go). The results of the kinetic analyses shown in Table 1Go were obtained using the same D2 sonicates analyzed by Western blotting in Fig. 4Go, accounting for the slight differences from the mean values reported above.



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  		Figure 4. Quantitation of the relative enzyme amounts by Western blot. The wild-type D2 and the Sec133Cys mutant were transiently expressed in HEK-293 cells, and the medium was supplemented with 100 nM selenium to achieve a high expression level of the enzyme. After 48 h, transfected cells were harvested, sonicated, and subjected to Western blot analysis with AB 85254. To compare the relative amount of enzyme expressed between wild-type and mutant D2, the mutant enzyme was diluted in cell lysate derived from control transfected HEK-293 cells as indicated. In the presence of selenium supplementation, the wild-type D2 enzyme was expressed approximately 100-fold less well than the Sec133Cys mutant.

 

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  		Table 1. Comparison of the kinetic properties of the Sec133Cys mutant with the native human type 2 iodothyronine deiodinase

 
Deiodination by wild-type and CysD2 mutants in intact cells
A recent report indicates that modest changes in deiodination kinetics resulting from mutations introduced into the active center of D1 were not apparent when these same constructs were studied under in vivo conditions simulated by transient expression of the mutated enzymes in whole cells (18). This was attributed to the fact that modest changes in enzyme kinetics are only apparent under the optimum maximum velocity conditions designed for in vitro analyses. Therefore, we tested whether the much higher in vitro Km of the CysD2 mutant would be reproduced under simulated in vivo conditions. When wild-type D2 was transiently expressed in HEK-293 cells, addition of increasing concentrations of unlabeled T4 resulted in a saturation of [125I]T4 deiodination, as reflected in a decrease in the release of 125I- into the medium (Fig. 5AGo). Saturation first appeared between medium T4 concentrations of 1 and 10 nM consistent with the in vitro Km of 2.1 nM. When the CysD2 mutant was studied in a similar manner, there was no reduction in 125I- release at a T4 concentration of 100 nM (Fig. 5BGo). A progressive decrease in the fractional deiodination of [125I]T4 occurred at higher T4 concentrations, which was reduced to near-background levels at 10 µM T4. These results indicated that the marked increase in the Km due to the replacement of Sec133 by Cys was reflected under both in vitro and in vivo conditions.



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  		Figure 5. In vivo deiodination at varying T4 concentrations. HEK-293 cells were transfected with the wild-type and the Sec133Cys mutant, and 24 h posttransfection in vivo deiodination was determined as described in Materials and Methods. The wild-type D2 (A) and the Sec133Cys (B) mutant both catalyzed T4 deiodination at linear rates for 10–20 h. Note the much higher fractional deiodination at 1 µM T4 by the Sec133Cys D2 mutant (B) as opposed to the wild-type D2 (A).

 
As the absolute rate of T4 deiodination in cells transiently expressing the CysD2 mutant is much higher than that in cells transiently expressing the wild-type hD2 (Fig. 5Go), we used this in vivo system to analyze the potential role of cellular transport as a rate-limiting step for intracellular T4 deiodination. To be deiodinated, T4 must enter the cell. The topology of the membrane-associated D2 enzyme is still unknown, but analysis of its protein sequence indicates that it contains a hydrophobic domain in the amino-terminal region (3, 4). Because of its similarity to D1 in this respect, it is likely that D2 also has a type 1 membrane topology with the catalytic center in the cytosol. Once T4 has reached the cell interior, the rate of its deiodination will depend on the enzyme concentration and the availability of a putative reducing cofactor.

The saturation of the fractional deiodination of T4 by the CysD2 mutant at high medium T4 concentrations indicated that cellular T4 uptake did not limit its deiodination. Recently, several organic anion transporters have been shown to facilitate thyroid hormone transport. These include the sodium taurocholate-cotransporting polypeptide NTCP; the organic anion transporters OATP-1, -2, and –3; and the liver-specific organic anion transporter, LST-1 (21, 22, 23, 24, 25). All of these share the capacity to transport taurocholate with apparent Km values ranging up to 35 µM. To determine whether one or several of these transporters could limit the equilibration of high medium T4 concentrations with the interior of the cell and, hence, its deiodination, we tested the in vivo deiodination rate in the presence of a large excess (10 mM) of sodium taurocholate. There was no significant reduction of the in vivo deiodination rate of 100 nM T4 in CysD2-transfected CV-1, COS-7 (Fig. 6Go, A and B), or HEK-293 cells (not shown) by taurocholate, suggesting that transport by taurocholate-saturable transporters is not rate limiting relative to deiodination in these cell lines.



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  		Figure 6. The in vivo deiodination by the Cys133D2 mutant is not inhibited by the addition of taurocholate. The Sec133D2 mutant was expressed in different cell lines, and in vivo deiodination was examined at 100 µM T4 with or without the addition of 10 mM taurocholate in CV-1 (A) or COS-7 (B) cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The aim of the present studies was to explore the role of Sec133 in hD2 by changing this residue to Ala or Cys using site-directed mutagenesis. As expected from earlier results with D1, removal of either an Se or S atom at this position eliminated catalysis (11). A surprising finding is the 1000-fold higher Km values for T4 and rT3 when S is substituted for Se. This is reflected in a much-reduced absolute rate of deiodination at 2 nM T4 despite roughly 100-fold higher CysD2 expression. However, if T4 is increased to micromolar concentrations, the limited expression of native D2 results in saturation of deiodination with a much lower Vmax than with CysD2. To obtain a valid experimental comparison between the efficiency of the transiently expressed mutant and native D2 enzymes in intact cells as opposed to cell sonicates, it would be necessary to reduce the expression of the CysD2 mutant approximately 100-fold/cell to equalize the amount of D2 expressed. However, the low activity of the CysD2 mutant would not allow accurate quantitation of deiodination in vivo at such low expression levels.

Our results support conclusions of earlier experiments in which a Sec to Cys change was introduced into the Rana catesbeiana D2 (26). The researchers found this mutant essentially inactive when expressed in Xenopus laevis oocytes. The absence of activity as opposed to markedly reduced catalysis with CysD2 in the present report presumably reflects the inefficiency of the Xenopus laevis expression system relative to that of the mammalian transient expression systems used here (7).

The 1000-fold increase in apparent Km for T4 and rT3 contrasts sharply with the difference found between the wild-type D1 and its Cys126-containing isoform. The Km for rT3 of the Cys mutant of D1 is increased only 3-fold (11). The change of Sec to Cys has been studied in other selenoproteins as well, and the change in Km is usually within 1 order of magnitude. For example, in human thioredoxin reductase, this exchange increases the Km from 0.55 to 1.06 µM (27). It suggests that in D2, Se may have more than its chemical role as a nucleophile, a function that is less efficiently filled by S in this enzyme compared with other selenoenzymes.

It is well known that selenoprotein synthesis is a relatively inefficient process, because the components of the highly specialized translational machinery required are limited (12, 28, 29). The much greater efficiency of the D2 protein containing Sec as opposed to Cys is especially critical for an enzyme such as D2 for which the substrate concentration is so low. The free T4 concentration in human serum is about 2 x 10-11 M. Based on the comparative rates of T4 deiodination performed at T4 concentrations even 100-fold higher than this, much higher amounts of CysD2 than native D2 would be required to deiodinate the same quantity of T4 per unit time. This is in part due to the fact that the relative turnover number of the CysD2 mutant is approximately 10-fold lower than that of the wild-type isoform when studied at maximal velocity conditions (Table 1Go). A reduction in calculated turnover number is also found when Cys is substituted for Sec in D1 and in the E. coli formate dehydrogenase (12, 30).

To compare the relative amounts of D2 proteins expressed, we used Western blots and performed kinetic analysis on the same protein preparations. Although the Vmax of the native D2 in the protein sample used for Western analysis is 0.81 pmol T4/min·mg protein, 8-fold higher than that of hypothyroid rat pituitary tissue, the tissue and condition with the highest endogenous D2 activity (31), the signal is barely detectable. This may explain why detection of native D2 protein in tissue homogenates by Western blots has not yet been demonstrated (32).

The Ki for GTG is about 100-fold higher for the CysD2 mutant than for the native protein. This mirrors the change in sensitivity toward GTG of native vs. CysD1, which also resulted in a 100-fold higher Ki for GTG (11). We had initially interpreted the markedly higher Ki for GTG for D2 vs. D1 as indirect evidence suggesting that D2 did not contain selenocysteine (33). As revealed by the recent cloning of D2 from several species, this was incorrect. In fact, the mechanism by which GTG inhibits catalysis by both wild-type and Cys133D2 is noncompetitive, whereas it is a competitive inhibitor of both D1 and D3 (19, 20, 33). The fact that GTG blocks the binding of BrAcT3 or T4 to D1 and D3, as does substrate, implies that GTG is interacting directly with the active center of these enzymes (1, 19). In contrast, the noncompetitive inhibition of the native D2 and the CysD2 mutant makes it difficult to predict the mechanism (20). The interaction with gold may, in fact, be occurring with a Cys residue outside the catalytic center.

A recent finding by Croteau et al. indicated that minor changes in kinetic activities due to mutations in the active center of D1 were difficult to detect under in vivo conditions (18). A reassuring result of the present studies is that the large increase in the Km for T4 in vitro is mirrored by the apparent Km in vivo. This substantiates the inferences made as to the physiological advantages of Sec vs. Cys in the function of this enzyme based on in vitro kinetic analyses.

We used various cell lines transiently expressing the CysD2 mutant as an in vivo system to analyze whether transmembrane transport of T4 might be rate limiting for T4 deiodination. As the absolute amount of T4 deiodinated by transiently expressed CysD2 is about 2 orders of magnitude higher than that in cells transiently expressing native D2, we speculated that we might be able to reduce T4 deiodination by the use of high concentrations of taurocholate to block T4 uptake by systems cotransporting T4 and this organic anion (22, 23, 24, 25, 26). There was no effect of this agent. However, our studies do not address the role of the phenylalanine and tryptophan transporter 4F2hc-IU12, which has also been shown to transport thyroid hormone (34). These results argue that under these unphysiological conditions with high CysD2 mutant expression, deiodination is the slowest step in T4 activation. Alternatively, passive transfer of T4 may occur sufficiently rapidly under these conditions that adequate substrate is available for maximal deiodination rates to occur.


   Acknowledgments
 
The wild-type hD2 Genethon clone Z44085 was kindly provided by Drs. Valerie A. Galton and Donald L. St. Germain.


   Footnotes
 
1 This work was supported by NIH Grant R01-DK-36256. Back

Received May 1, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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