From the Departments of Chemistry, Biochemistry & Molecular Biology, and Pediatrics and the Committee of Developmental Biology, The University of Chicago, Chicago, Illinois 60637
Received for publication, July 5, 2002, and in revised form, October 22, 2002
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ABSTRACT |
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PAPS synthetase (SK) catalyzes the two sequential
reactions of phosphoadenosine phosphosulfate (PAPS) synthesis. A
functional motif in the kinase domain of mouse SK, designated the
BM-motif (86
LDGDNhRxhh(N/S)(K/R)97), was
defined in the course of identifying the brachymorphic (bm) defect.
Sequence comparison and the secondary structure predicted for APS
kinase suggest that the BM-motif consists of a DGD-turn sequence
flanked by other conserved residues. Mutational analysis of the
DGD-turn revealed that a flexible and neutral amino acid is preferred
at residue 88, that negatively charged residues are strictly required
at positions 87 and 89, and that the active site is rigid. The
reduction in kinase activity for all DGD-turn mutants, except G88A, was
much less severe than the reduction in overall activity, indicating
that the BM-motif may also be playing a role in adenosine
phosphosulfate (APS) channeling. Two switch mutations, LD86DL
and DN89ND, designed to test the positional constraints of
Asp87 and Asp89, exhibited complete loss
of both kinase and overall activities, while LD86DL also exhibited a
significant (60%) loss of reverse sulfurylase activity, suggesting
that this peptide region is interacting with the sulfurylase domain as
well as functioning in the kinase reaction. Other residues targeted for
mutational analysis were the highly conserved flanking
Asn90, Arg92, and Lys97. N90A
resulted in a partial (30%) loss in kinase and overall activities,
R92A exhibited total loss of kinase and overall activities, and K97A
had no effect on any of the three activities. The complexity of the
bifunctional SK in catalyzing the kinase reaction and channeling APS is
illustrated by the strict requirements of this novel structural motif
in the kinase active site.
Sulfation is the second most common chemical modification
of biomolecules, and in higher organisms all sulfation of
carbohydrates, lipids, and protein substrates is mediated through the
universal sulfate donor, phosphoadenosine phosphosulfate
(PAPS)1 (1). The synthesis of
PAPS is catalyzed by the bifunctional enzyme PAPS synthetase in higher
organisms. PAPS synthetase consists of two activities, ATP sulfurylase,
which catalyzes synthesis of adenosine phosphosulfate (APS) from ATP
and SO
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(Eq. 1)
An animal model with reduced sulfation, the brachymorphic (bm)
mouse, was initially identified by Sugahara and Schwartz (2) to possess
a defective PAPS synthesis pathway (3, 4). Murine brachymorphism is a
severe growth disorder, affecting the skeletal elements as well as
certain physiological processes like blood clotting (5) and
liver-mediated detoxification (6). Biochemical analysis showed that
brachymorphic cartilage contains normal levels of glycosaminoglycans
but has disaccharides that are significantly under-sulfated. The
reduced incorporation of sulfate into brachymorphic cartilage is
associated with limited PAPS availability largely due to a reduction in
APS-kinase activity (2, 4).
The initial cloning of a bifunctional PAPS synthetase (SK1) did not identify the underlying etiology of the brachymorphic phenotype, thus prompting the subsequent elucidation of a second isoform (SK2) encoded by a separate gene (7). Sequence analysis of wild-type and bm SK2 cDNAs revealed that a G to A mutation in the bm allele changes a glycine to an arginine residue (G79R) in the APS-kinase domain of the bifunctional enzyme. Bacterially expressed recombinant normal SK2 protein catalyzes both the ATP sulfurylase and APS kinase reactions and synthesizes PAPS comparably to SK1. In contrast, recombinant SK2 protein containing the G79R mutation had no APS-kinase activity while maintaining its ATP sulfurylase activity. This study provided the first indication that the peptide region in the vicinity of residue 79 in SK2 may also be important for PAPS synthesis.
Amino acid sequence alignment of various monofunctional ATP sulfurylase
and APS kinase enzymes from lower organisms and plants and bifunctional
enzymes from animals revealed the presence of several highly conserved
regions (8), e.g. the ATP binding (P)-loop (9), phosphoryl
transfer (FISP) (10), phosphodiester-cleavage (11), and pyrophosphate
binding (PP)-loop (11), all of which we have shown by mutational
analysis to confer molecular binding or enzymatic activity to SK1.
Similarly, sequence conservation around the bm mutation site
(Gly79) is very high; however, no function had been
established for this region. Based on the sequence alignment shown in
Fig. 1, the proposed consensus
sequence of the region around Gly79, called the BM-motif,
is LDGDNhRxhh(N/S)(R/K), where
h is a hydrophobic residue and x is a
non-conserved residue. The available monofunctional fungal APS kinase
structure (12) shows that the homologous region in the fungal enzyme
consists of a -strand (residues 56-60) and an
-helix (residues
62-68), with Asp61 present between these two peptide
structures. Although MacRae et al. were unable to
crystallize the APS kinase (12) with bound ATP or APS, comparisons with
substrate-bound structures of guanylate kinase (13) or the
6-phosphofructo-2-kinase domain of the bifunctional enzyme
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (14) suggest
that Asp61 could be binding to the magnesium of MgATP.
Thus, to explore the functional significance of the BM-motif in the
bifunctional PAPS synthetases, we carried out conformational and
mutational analyses of the conserved residues, addressing:
i) the importance of the BM-motif glycine, and
ii) the role of other highly conserved residues around the
BM-motif glycine.
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MATERIALS AND METHODS |
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Generation of Mutant Constructs-- Our previous extensive mutational analyses (9, 11) were performed on the SK1 isoform protein, thus SK1 was used for functional analysis of the BM-motif (residues Leu86-Lys97). Site-directed mutations in SK1 cDNA were generated by insertion of double-stranded oligonucleotides. Briefly, in a ProEx plasmid obtained from a ProEx/SK1 construct, two unique restriction enzyme sites, Bsm1 and Bsu36I, flanking the BM-motif were used to remove the normal cDNA sequence and to ligate in the mutated BM-motif. The mutational inserts altering the BM-motif also contained an engineered translationally silent restriction site, AvrII, which is not present in the original ProEx/SK1 cDNA construct. Making use of the AvrII site, the colonies containing the mutated BM-motif constructs were identified by double digestion of plasmid DNA with AvrII and XhoI; positive clones were verified by DNA sequencing.
Expression and Purification of Protein--
BL21 DE3 (codon
plus) from cells transformed with normal or mutated plasmid constructs
were grown in LB/ampicillin/chloamphenicol liquid culture at 37 °C
to an A600 of 1.0. Following induction with 0.1 M isopropyl-thio--D-galactoside for 4 h, the cultures were centrifuged at 9000 × g for 8 min, and the pellet was resuspended in 4 volumes of 50 mM
Tris-HCl, 5 mM
-mercaptoethanol, 1 mM
phenylmethylsulfonyl fluoride (freshly prepared), pH 8.5 at
4 °C. The suspension was sonicated, the cellular debris removed by
ultracentrifugation at 15,000 × g, the supernatant
loaded onto a nickel-nitrilotriacetic acid-agarose resin column. The
column was then washed with 7 column volumes of Buffer A (20 mM Tris-HCl, 100 mM KCl, 5 mM
-mercaptoethanol, 20 mM imidazole, 10% glycerol, pH 8.5 at 4 °C), 7 column volumes of Buffer B (20 mM Tris-HCl,
1 M KCl, 5 mM
-mercaptoethanol, 10%
glycerol, pH 8.5 at 4 °C), and again with 7 column volumes of Buffer
A. Protein was eluted with buffer C (20 mM Tris-HCl, 100 mM KCl, 200 mM imidazole, 5 mM
-mercaptoethanol, 10% glycerol, pH 8.5 at 4 °C). The eluted
protein was dialyzed against 20 mM Tris-HCl, 1 mM EDTA, 1 mM dithiothreitol, pH 7.8, buffer.
Protein concentration was determined using the Bradford assay.
Fluorescence Substrate Binding Experiments-- The concentration of protein used in fluorescence experiments was ~200 µg/ml. To eliminate condensation of moisture on the fluorescence sample cell, the experiments were performed at 10 °C. Tryptophan fluorescence emission was recorded in the 305-400 nm range with an excitation wavelength of 295 nm. Substrate binding experiments were performed at the peak emission wavelength for each substrate (342 nm for LAPSK2 and 338 nm for BM-LAPSK2). Buffer fluorescence was subtracted from sample fluorescence before determining binding constants.
Enzyme Assays--
The purified recombinant protein was assayed
for reverse ATP sulfurylase, APS kinase, and overall PAPS synthetase
activities using protocols as described earlier (9). All assays were
performed at least three times; data are presented as the average of
individual results. For low activity mutants, a higher stock
concentration of protein (400 µg/ml) was tested to differentiate the
specific activities.
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RESULTS |
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Structural and Substrate Binding Analysis-- To investigate the impact of the bm mutation on structural and substrate-binding properties of the kinase domain, fluorescence intensity experiments, which are highly sensitive to the molecular environment of tryptophan residues and allow conformational changes due to substrate binding to be detected directly, were performed. However, since there are 11 and 10 tryptophans in SK1 and SK2, respectively, and two/three substrate binding pockets, it is possible that an increase in fluorescence in one portion of the protein may mask a decrease in fluorescence from another portion resulting in no net change in signal, or that the obtained fluorescence signal may be difficult to de-convolute for the bifunctional protein. Thus, the expressed kinase domains of SK1 (LAPSK1, with two tryptophans) and SK2 (LAPSK2, with only one tryptophan), as well as BM-LAPSK2, the kinase domain of SK2 with bm mutation (G79R), were used for these studies.
Buffer-subtracted fluorescence of the three proteins is shown in Fig.
2. Expectedly, LAPSK1, which contains two
tryptophans, exhibited higher fluorescence intensity compared with
LAPSK2, which contains only one. However, it was surprising that
BM-LAPSK2 exhibited 60% loss of fluorescence intensity compared with
LAPSK2, thus indicating that i) Trp47 in LAPSK2 is
conformationally coupled to Gly79 and ii) there is
significant structural change in the kinase domain due to the bm
mutation. To assess the effect of the bm mutation on substrate binding,
the binding constants for ATP and APS were determined for LAPSK2 and
BM-LAPSK2. From the binding curves shown for LAPSK2 (Fig.
3, A and B)
KD(ATP) was determined to be 0.3 µM and KD(APS) was 0.13 µM., while for BM-LAPSK2 (Fig. 3, C and
D) KD(ATP) was 28.5 µM and KD(APS) was 5.4 µM, suggesting that BM-LAPSK2 binds both ATP and APS
poorly. Thus, a decrease in substrate binding caused by the bm mutation may account for the loss of kinase and overall activities in
BM-SK2.
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DGD-turn Mutants-- To explore the structural and functional contribution of the BM-motif (residues 86-97) to SK1 activity, we undertook site-specific mutagenesis studies targeting Gly88, the flanking aspartates Asp87 and Asp89 of the DGD-turn, and the highly conserved residues flanking the DGD-turn in addition to two residue-order switch mutants. Mutations of the DGD-turn residues included D87A, D87R, D87E, G88A, G88R, G88D, D89A, D89R, and D89E, designed to investigate structural aspects (i.e. glycine as a flexible amino acid) as well as charge requirements (i.e. negative charge due to aspartic acid residues). Activity data for wild-type SK1 and the mutant enzymes are presented in Tables I and II.
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Although G88A exhibited reverse sulfurylase activity comparable to wild-type, overall (75%) and kinase (69%) residual activities were reduced, suggesting that a flexible amino acid at residue 88 is required for optimal kinase activity. G88R, a mutation analogous to that in the brachymorphic mouse, was designed to test whether SK1 behaves similarly to SK2. As observed for the naturally occurring bm mutation in SK2, the G88R mutation in SK1 leads to complete loss of overall (0.5%) and kinase (4.6%) activities. The relative overall activity of G88D, designed to test the negative charge requirement for the turn region, was reduced to 0.2% and kinase activity to 4.4%. In reverse sulfurylase assays, both G88R and G88D showed activities comparable to wild type.
N-terminal to the bm glycine, an aspartate analogous to residue Asp87 is strictly conserved among all the monofunctional APS kinases and bifunctional PAPS synthetases (Fig. 1A). Based on the APS kinase crystal structure from Penicillium chrysogenum, MacRae et al. (12) predicted that a negative charge presented by Asp87 may help bind Mg2+, which in turn presents a positive charge to ATP/APS. Removal of the negative charge by mutating Asp87 to alanine resulted in decreased overall (0.6%) and kinase (6.7%) activities. Likewise, D87R, with the charge reversed, had an activity profile comparable D87A. D87E, which retains the negative charge but extends the side chain by one C-C bond (1.5 Å), was designed to test the tightness and the charge requirements of the substrate binding pocket. Unexpectedly, the relative overall activity for the charge-conserved mutation, D87E, was observed to be only 2.2%, while, there was a slight improvement in kinase activity (18%) compared with D87A or D87R. None of the Asp87 mutants exhibited any loss of reverse sulfurylase activity.
Asp89, the residue C-terminal to the BM-motif glycine, is
located at the N terminus of the -helix that follows the DGD-turn, based on the crystal structure of APS kinase from P. chrysogenum (12). Although the D89A and D89R mutant proteins both
exhibited reverse sulfurylase activity comparable to that of wild-type
SK1, they show <0.5% residual overall and <5% kinase activities,
suggesting that the negative charge contributed by Asp89 is
also essential for a functional APS kinase. Designed to assess the
structural flexibility and residue stringency for this position, the
D89E mutant showed an improvement in overall (9%) and kinase (23%)
activities relative to D89A and D89R, but still indicates a size
restriction in this position.
Residue-order Switch Mutants-- To determine whether there is functional restriction on the location of the aspartates, a set of residue-order switch mutations, LD86DL and DN89ND, were designed in which the critical aspartates were moved one residue further away from the DGD-turn on either the N-terminal or C-terminal side of the central glycine. The residual overall (4.9%) and kinase (5.3%) activities of the LD86DL protein suggest that not only is the presence of aspartate necessary for activity, but that the active site is rigid and cannot easily adapt to an altered chemical and electrostatic environment by a shift in the position of the acidic residues. Surprisingly, LD86DL, although mutated in the kinase domain, also had a 60% loss of reverse sulfurylase activity, suggesting involvement of these residues in both the sulfurylase and kinase activities. When residues Asp89 and Asn90 were interchanged in DN89ND, complete loss of kinase (1.5%) and overall (0.9%) activities was observed, again indicating that the BM-motif is very rigid and that this alteration is detrimental to both exogenous and endogenous APS utilization. In contrast to LD86DL, the DN89ND protein maintained reverse sulfurylase activity comparable to wild type.
Conserved Flanking Residue Mutations--
The third set of mutants
included N90A, N90Q, R92A, and K97A, which are highly conserved and are
located near the C terminus of the DGD-turn (Fig. 1). Despite
the strong conservation of Asn90 and its proximity to the
DGD-loop, deletion of the side chain amide functionality in N90A
resulted in 67% residual overall and kinase activities, N90Q tested
whether substituting another amide-presenting side chain residue could
prevent losses in overall or kinase activity. Interestingly, the
residual overall (64%) and kinase (69%) activities were similar to
those for N90A with retention of reverse sulfurylase activity
comparable to that of wild-type SK1. In contrast, R92A exhibited
complete loss of overall (1.1%) and kinase (0.9%) activities, while
reverse sulfurylase activity was normal. Lastly, Lys97,
which is also highly conserved and present in the loop following the
-helix (residue 88-94), exhibited all three activities comparable to those of wild-type SK1, suggesting that Lys97 is not
essential in SK function.
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DISCUSSION |
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The BM-motif, which is comprised of a DGD-turn and flanking conserved residues, was initially identified through the mutation discovered in SK2 cDNA cloned from the brachymorphic mouse (7). Sequence comparisons show that the BM-motif is highly conserved among several bifunctional PAPS synthetases and monofunctional APS kinases. Analysis of the BM-motif by tryptophan fluorescence experiments provides the first evidence that the region in SK2 containing the bm mutation (G79R) is important for the structure of the protein and that alteration in conformation associated with the bm mutation accounts for the loss of kinase activity. Furthermore, substrate binding fluorescence experiments show that LAPSK2 exhibits tight binding to APS and ATP, while the bm mutation in LAPSK2 leads to significant reductions in ATP (100-fold) and APS (50-fold) binding, indicating that Gly79 is located in the vicinity of both the ATP and APS binding sites and that the peptide region around Gly79 is a critical component of the PAPS synthetase active site(s).
A systematic mutagenesis analysis of the BM-motif showed that, as
observed in mutant SK2 from the brachymorphic mouse, changing the SK1
DGD-turn glycine to arginine (G88R) produces a nearly inactive kinase
and subsequent loss of overall activity (7). Substitution of a positive
charge at this position could result in displacement of the
MgATP-magnesium or misalignment of the -phosphate of ATP, leading to
either weaker or complete loss of ATP binding and thereby causing the
observed decreases in kinase and overall activities. Even recombinant
enzyme with a G88A substitution, which replaces a neutral, flexible
amino acid with a neutral, good
-helix-forming residue, exhibited
some reduction (30%) in overall and kinase activities, possibly due to
an increase in rigidity of the active site via N-terminal extension of
the
-helix that follows the DGD-turn, thus causing misalignment of
critical aspartates around Gly88. Similar structural
consequences may induce the activity loss observed for the G88R and
G88D mutant enzymes, as both arginine and aspartic acid residues have
higher
-helix propensities than glycine. Furthermore, while the
presence of negatively charged residues is a requirement for DGD-turn
function, the number and spacing of acidics is critically important
since the G88D mutant exhibited a severe loss (0.2%) of overall activity.
The results of multiple mutations at Asp87 and
Asp89 strongly suggest an absolute requirement for both
acidics. Furthermore, the low overall activity observed for glutamic
acid substitutions at Asp87 and Asp89 indicate
that the active site is not very flexible and that the correctly sized
residue side chains are important for internal transfer of APS. These
possibilities are partly supported by the findings of MacRae
et al. (12), which predicted a role for Asp87 in
ATP binding, while no such role was predicted for Asp89.
However, structural comparison of P. chrysogenum APS kinase with substrate-bound structures of guanylate kinase (13) and 6-phosophofructo-2-kinase (14) suggest that APS is placed between the
P-loop, FISP-motif, and the -helix C-terminal of the DGD-loop. Furthermore, during the course of our functional analysis of the BM-motif, MacRae et al. crystallized P. chrysogenum ATP sulfurylase in the presence of APS (15), and
showed that APS was bound to the sulfurylase active site as well as to
the C-terminal kinase-like regulatory domain. As we predicted, this
crystal structure shows residue Asp434, analogous to
Asp89 in SK1, interacting with the 3'-OH of APS, thereby
aiding in proper alignment of APS for phosphoryl transfer. Thus,
Asp89 may be participating in internal transfer and
reactive alignment of endogenously generated APS in the kinase active
site, whereas Asp87 may be involved in the ATP binding and
phosphoryl transfer step of the kinase reaction.
As mentioned, monofunctional ATP sulfurylases of P. chrysogenum and Saccharomyces cerevisiae contain inactive C-terminal APS-kinase-like regulatory domains (Fig. 1B); however, the kinase DGD-loop is replaced by LGD-loop in these sulfurylase regulatory domains. In addition to the presence of a leucine at residue 432 (analogous to Asp87 in SK1), the P-loop in the kinase-like domain is changed from GLSASGKS to GYMNSGKD. Independent studies of bifunctional SK1 (9) and monofunctional APS kinase (16) have shown that a single mutation in the key residues of the P-loop is sufficient to inactivate the kinase. Loss of kinase activity in our D87A mutant supports the hypothesis put forward by MacRae et al. (15, 16) that lack of APS kinase activity in the ATP sulfurylase regulatory domain is due both to the altered P-loop and the absence of an aspartate at residue 432.
Additional conserved residues, C-terminal to the DGD-turn, also
contribute to PAPS synthetase activity. Based on the P. chrysogenum APS kinase structure, Asn90 is expected to
be present on the face opposite the substrate binding pocket,
Arg92 is on the substrate binding face of the -helix,
and Lys97 on the loop following the
-helix. Equal and
low (30%) residual activity for N90A and N90Q suggests that
Asn90 may be playing a structural role via interactions
with other residues present on the non-substrate binding face of the
-helix to allow the correct orientation of critical residues on the
substrate binding face. The presence of Arg92 on the
substrate binding face and a near-complete loss of the overall and
kinase activities for R92A suggest that it could be participating in
the binding of APS or in the alignment of Asp89 via a salt
bridge. In fact, the crystal structure of P. chrysogenum ATP
sulfurylase, which shows that one of the specific interactions between
APS and the allosteric domain is a bidentate salt linkage between the
guanidino head of Arg437 and the phosphosulfate moiety of
APS, confirms our prediction that Arg92 is binding to
APS.
In our previous study of the P-loop (9) we showed that mutation of the kinase domain residue Gly59 to alanine disrupted sulfurylase activity. Also, in our subsequent analysis of the PP-loop (11) which is located in the sulfurylase domain, we identified two residues, His506 and Asp523, that when mutated to alanine resulted in altered kinase activity. Similar cross-reactivity behavior was observed in the present study for LD86DL, a switch mutant designed to probe the strictness of the spatial constraint on the critical aspartate, Asp87, and the flexibility of the active site. Lastly, it is interesting to observe that in all of the DGD-turn mutants the loss in overall activity is greater than the loss in kinase activity, suggesting that the conversion of endogenous APS, an intermediate in the overall reaction to PAPS, is affected by the BM-motif mutations to a greater degree than is the conversion of exogenous APS, a substrate in the kinase reaction. Thus, in addition to participating in the kinase reaction, the BM-motif may be interacting with the sulfurylase domain and playing a role in the transfer of APS from the sulfurylase to the kinase active site. The hypothesis of less efficient utilization of endogenous APS in the bm mutant enzyme is consistent with results from our previous study where we demonstrated that SK2 isolated from brachymorphic mice exhibits a channeling defect (4). Further studies to understand the channeling of APS by the recombinant bm-like SK1 are in progress.
In summary, our past work sought to identify the components of the APS
kinase active site by analyzing the contribution of three functional
motifs: the P-loop (9), the FISP-motif (10) and the PAPS binding motif
(10). With the elucidation of the mutation in the bm mouse (7), we have
now identified a fourth component of the kinase active site, the
BM-motif. Our mutational analysis of the BM-motif shows the functional
importance of several highly conserved residues of this region in
enzyme catalysis, endogenous APS transfer, and in formation of a rigid
active site and as well provides a rationale for the lack of kinase
activity in the kinase-like regulatory domain of P. chrysogenum ATP sulfurylase.
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ACKNOWLEDGEMENTS |
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We thank Dr. Youli Wang for help in constructing mutant enzymes, James Mensch for helpful comments during the course of this study, and Daniel Vatner for help in sequence comparison.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant HD-17332 and a fellowship from the Markey Foundation (to B. S.).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.
To whom correspondence should be addressed: Dept. of Pediatrics,
University of Chicago, 5841 S. Maryland Ave., MC 5058, Chicago, IL
60637. Tel.: 773-702-6426; Fax: 773-702-9234; E-mail:
n-schwartz@uchicago.edu.
Published, JBC Papers in Press, October 31, 2002, DOI 10.1074/jbc.M206688200
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ABBREVIATIONS |
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The abbreviations used are: PAPS, 3'-phosphate 5'-phosphosulfate; APS, adenosine 5'-phosphosulfate; bm, brachymorphic; PP, pyrophosphate; SK1, mouse PAPS synthetase isoform 1; SK2, mouse PAPS synthetase isoform 2.
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REFERENCES |
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