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J Biol Chem, Vol. 273, Issue 14, 8454-8458, April 3, 1998
,From the Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104 and the § Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
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ABSTRACT |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Type A Pasteurella multocida, a prevalent animal pathogen, employs a hyaluronan [HA] polysaccharide capsule to avoid host defenses. We utilized transposon insertional mutagenesis to identify the P. multocida HA synthase, the enzyme that polymerizes HA. A DNA fragment from a wild-type genomic library could direct HA production in vivo in Escherichia coli, a bacterium that normally does not produce HA. Analysis of truncated plasmids derived from the original clone indicated that an open reading frame encoding a 972-residue protein was responsible for HA polymerization. This identification was confirmed by expression cloning in E. coli; we observed HA capsule formation in vivo and detected activity in membrane preparations in vitro. The polypeptide size was verified by photoaffinity labeling of the native P. multocida HA synthase with azido-UDP sugar analogs. Overall, the P. multocida sequence is not very similar to the other known HA synthases from streptococci, PBCV-1 virus, or vertebrates. Instead, a portion of the central region of the new enzyme is more homologous to the amino termini of other bacterial glycosyltransferases that produce different capsular polysaccharides or lipopolysaccharides. In summary, we have discovered a unique HA synthase that differs in sequence and predicted topology from the other known enzymes.
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INTRODUCTION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Certain bacterial pathogens, namely Gram-negative Carter type A
Pasteurella multocida and Gram-positive Groups A and C
streptococci, produce extracellular capsules composed of
HA1 (1, 2). HA, a linear
polysaccharide consisting of alternating 
1,4-GlcA and 1,3-GlcNAc,
is also a component of many vertebrate tissues (3). The HA capsules of
these bacteria are virulence factors that allow the microbes to be more
successful pathogens by rendering host defenses, including
complement-mediated killing and phagocytosis, less effective (4, 5).
Furthermore, HA is not immunogenic because the polymer is a normal
component of the host body (6). The capsules of other bacteria that are composed of different polysaccharides, however, are usually major targets of the immune response. The antibodies generated against capsular polymers are often responsible for clearance of microorganisms and long term immunity. Therefore, the HA capsule serves P. multocida and Groups A and C streptococci as molecular
camouflage.
Various HAS enzymes have been described in the last 5 years (reviewed in Ref. 7). HasA from Streptococcus pyogenes was the first HAS to be definitively identified (8). This integral membrane protein utilizes intracellular UDP-GlcA and UDP-GlcNAc as substrates. The nascent HA chain is extruded through the membrane to form the extracellular capsule. DG42, a Xenopus protein with a previously unknown function that had similarity to HasA (9), was shown to be a HA synthase (10, 11). Several groups identified human and murine homologs of DG42, named HAS1, HAS2, and HAS3, that directed HA synthesis (7); these mammalian enzymes are similar at the amino acid level, but they reside on different chromosomes.
Recently a viral HAS, an ORF called A98R, with 28-30% identity to the streptococcal and vertebrate enzymes was discovered (12). PBCV-1 (Paramecium bursaria Chlorella virus) produces authentic HA polysaccharide shortly after infection of its Chlorella-like green algae host (12). A98R is the first virally encoded enzyme reported to produce a carbohydrate polymer. This finding is also the initial discovery of HA outside of animals and their bacterial pathogens.
Carter type A P. multocida, the causative agent of fowl cholera, is responsible for great economic losses in the U. S. poultry industry. A capsule mutant did not thrive in the bloodstream of turkeys after intravenous injection in comparison with the encapsulated parental strain (13). This spontaneously arising mutant strain was also 105-fold less virulent than wild type, but the nature of the genetic defect was not known (13). Wild-type cells treated with hyaluronidase became complement-sensitive (14) and were more readily phagocytosed (4) in comparison with untreated microbes. Here we report the identification of a unique HAS from P. multocida with a primary structure that does not strongly resemble the enzymes from Streptococcus, PBCV-1 virus, or higher animals.
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EXPERIMENTAL PROCEDURES |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Tn916 Insertional Mutagenesis and Probe Generation-- Tn916 was used to disrupt and to tag the HA biosynthesis locus. The Tn element on a nonreplicating plasmid, pAM150 (provided by D. B. Clewell), was introduced into the wild-type encapsulated P. multocida strain P-1059 (ATCC number 15742) by electroporation (15). Altered colony morphology was initially screened by visual examination with oblique lighting. Wild-type P-1059 forms large mucoid ("wet" appearance) colonies that appear iridescent (red and green coloration). Smaller, "drier" colonies lacking iridescence were chosen and streaked out. India ink staining and light microscopy were used as a secondary screen to assess the state of encapsulation. The position of the Tn elements in the mutant chromosome was mapped by Southern analysis (15, 16).
The DNA sequences at the Tn-disrupted sites from several independently selected mutants were obtained by direct dideoxy sequence analysis of tagged chromosomal DNA (15). Briefly, a chimeric DNA fragment consisting of a 12-kb portion of the Tn916 element and a short region of the P. multocida DNA generated by HhaI digestion of mutant chromosomal DNA was purified by agarose gel electrophoresis (all of the wild-type P-1059 HhaI genomic fragments are
7 kb). The chimeric
fragment served as the template in cycle sequencing reactions using
33P terminators (Thermosequenase® system, Amersham) and a
Tn916 right arm terminus primer
(5'-GACCTTGATAAAGTGTGATAAGTCC-3'). The sequence data were used to
design PCR primers. Gel-purified PCR products were labeled with
digoxigenin utilizing the High Prime system (Boehringer Mannheim).
Isolation of a Functional HAS Locus--
A 
library of
Sau3A partially digested P-1059 DNA was made using
BamHI-cleaved Zap ExpressTM vector system (Stratagene). The plaque lifts were screened by hybridization (16) with
digoxigenin-labeled PCR product. Escherichia coli XLI-Blue
MRF' was co-infected with individual purified positive clones and
ExAssist helper phage to yield phagemids. The resulting phagemids were
transfected into E. coli XLOLR cells to recover the
plasmids.
0.1
µg/ml (20). Multiple determinations of HA levels were averaged. The HA concentration in bacterial cultures was normalized for differences in cell number by measuring the A600 value and
presenting the data as µg HA/ml /A600 of
bacteria. One plasmid, pPm7A, with a 5.8-kb insert conferred E. coli K5 with the ability to produce HA; no HA was produced by
cells with vector plasmid alone. A truncated derivative of pPm7A
containing a ~3.3-kb insert, called pPm
6e, could direct the
biosynthesis of HA when transformed into E. coli K5.
Therefore, the sequence of both strands of the pPm7A plasmid corresponding to the pPm6e DNA insert was determined. A single complete 972-residue ORF, which we called PmHAS, was found.
Expression of Recombinant P. multocida HAS--
The PmHAS ORF in
the pPm7A insert was amplified by 13 cycles of PCR (16) with
Taq polymerase (Fisher) and primers corresponding to the
sequence near the deduced amino and carboxyl termini (codons in capital
letters: sense,
5'-gcgaattcaaaggacagaaaATGAAcACATTATCACAAG-3', and antisense,
5'-gggaattctgcagttaTAGAGTTATACTATTAATAATGAAC-3'; start and stop codons, respectively, in bold). Codon 2 (T 
C) was
altered (italic lowercase letter) to increase protein production in
E. coli. The primers also contained EcoRI and
PstI restriction sites (underlined letters) to facilitate
cloning into the expression plasmid pKK223-3 (tac promoter;
Pharmacia). The resulting recombinant construct, pPmHAS, was
transformed into E. coli SURE cells (Stratagene), and this
strain was used as the source of membrane preparations for in
vitro HAS assays (21). Log phase cultures (LB broth, 30 °C)
were induced with 0.5 mM isopropylthiogalactoside for
3 h before harvest. The plasmid was also transformed into E. coli K5; the resulting strain was examined for the presence of
capsule by light microscopy and buoyant density centrifugation (19). The K5 bacterial cultures were not induced routinely because
isopropylthiogalactopyranoside addition did not increase HA levels in
LB or defined medium significantly.
Photoaffinity Labeling of Native P. multocida HAS-- The radiolabeled UDP sugar analogs, [32P]azido-UDP-GlcA (3 mCi/µMol) and [32P]azido-UDP-GlcNAc (2.5 mCi/µMol), were prepared and purified as described (Refs. 22 and 23, respectively). Membrane preparations from P. multocida P-1059 (24) in 50 mM Tris, 20 mM MgCl2, pH 7, were incubated with either probe (final concentration, 20 µM) for 30 s on ice before irradiation with ultraviolet light (254 nm, 90 s). The proteins were precipitated with 5% trichloroacetic acid before SDS-polyacrylamide gel electrophoresis analysis (16, 22). No radiolabel was incorporated if the irradiation step was omitted. As a specificity control, 10-fold molar excess of the normal UDP sugar was co-incubated with the probe and membranes. [32P]Azido-UDP-Glc (3 mCi/µMol; Ref. 22) was also used as another control.
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RESULTS |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Production and Characterization of HA Capsule Mutants--
About
8 × 104 Tn-containing transformants produced by
several rounds of mutagenesis were screened for differences in colony morphology. By light microscopy with India Ink, the cells from small
noniridescent colonies (n = 4) possessed no detectable
capsule (acapsular), whereas the cells from medium-sized iridescent
colonies (n = 8) appeared to have a capsule of about
10-25% of the diameter of the wild type (microcapsular).
Interestingly, two of the acapsular mutants, named H and L, which had
Tn elements that mapped to the same HindIII or
BstXI genomic fragments (data not shown), reverted to
wild-type colony morphology at rates of ~10
3. The Tn
element in each revertant had excised from the original position and
reinserted at different, new locations as judged by Southern analysis;
on the other hand, all acapsular subclones retained the Tn element at
the original location (data not shown). No significant HAS activity was
detected in membrane preparations from mutant H cells, whereas
substantial HAS activity was obtained from wild-type P-1059 cells
(0.7 versus 120 pmol transfer GlcA/mg protein/h,
respectively). These findings suggested that the Tn elements in mutants
H and L were indeed responsible for disrupting the HA biosynthesis
locus.
Molecular Cloning of the P. multocida HAS Locus--
To bridge the
gap between the Tn insertion sites of two acapsular mutants, PCR using
the mutant L chromosomal DNA template was performed with a primer
derived from sequence at the mutant H disruption site, PmHF
(5'-CTCCAGCTGTAAATTAGAGATAAAG-3'), and a primer corresponding to the
left terminus of Tn916, TnL2
(5'-GCACATAGAATAAGGCTTTACGAGC-3'). A specific ~1-kb PCR product was
obtained; on the other hand, no product was formed if PmHR
(inverse complement of PmHF) or the Tn916 right arm primer
were substituted. The PCR product was used as a hybridization probe to
obtain a functional copy of the P. multocida HAS. Six
positively hybridizing plaques were found after screening
~104 plaques, and these phage were converted into
plasmids. One plasmid, pPm7A, was found that could direct E. coli K5 to produce HA in vivo (20 µg
HA/ml/A600 of bacteria). E. coli K5
with control plasmids did not produce HA (0.05 µg
HA/ml/A600). E. coli XLOLR or
E. coli EV5 cells (which lack UDP-Glc dehydrogenase
activity) do not produce HA even if they contain the pPm7A
plasmid (0.05 µg HA/ml/A600). This genetic
evidence implied that the insert of pPm7A does not encode a functional
UDP-Glc dehydrogenase enzyme.
6e, contained
a single complete ORF encoding a 972 residue protein (Fig.
1). Smaller plasmids with partial deletions of this ORF lost the ability to direct HA polymer production in E. coli K5. We hypothesized that this protein was the HA
synthase and called it PmHAS. The predicted Mr
is 111,923, and the calculated isoelectric point is 6.84.
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22-1010;
Fig. 2A). The most notable
sequence similarities are the DGSTD and DXDD motifs.
Unexpectedly, there was no significant overall similarity of PmHAS to
the streptococcal, viral, or vertebrate HASs (HasA smallest sum
probability 0.33). Only one short region of streptococcal HasA aligns
with PmHAS in a convincing fashion (Fig. 2A). A few segments
of the first half of PmHAS are also similar to portions of the
mammalian UDP-GalNAc:polypeptide GalNAc-transferase, an enzyme that
initiates O-glycosylation of mucin-type proteins (smallest
sum probability, ~103; Fig. 2B).
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52), a protein putatively
involved in the transport of capsular polysaccharide out of the
cell.
Photoaffinity Labeling of P. multocida Membranes-- The predicted size of 972 residues (112 kDa) for PmHAS was confirmed by photoaffinity labeling of membrane preparations from P. multocida P-1059. Both [32P]azido-UDP-GlcA and [32P]azido-UDP-GlcNAc probes photoincorporated into a ~110-kDa protein in an UV-dependent manner (Fig. 3A). Competition with the corresponding unlabeled natural UDP sugar precursors lowered the extent of probe photoincorporation. In parallel experiments, [32P]azido-UDP-Glc, an analog of the normal HA precursors, did not label this 110-kDa protein (data not shown). Furthermore, membranes derived from Tn mutants had either no or very low amounts of azido-UDP-GlcA photoincorporation into this protein (Fig. 3B).
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Heterologous Expression of P. multocida HAS--
Membranes derived
from E. coli SURE cells containing the pPmHAS plasmid, but
not samples from cells with the vector pKK223-3 alone,
synthesized HA in vitro when supplied with both UDP-GlcA and
UDP-GlcNAc (25 versus 1.5 pMol GlcA transfer/mg protein/h, respectively). No incorporation of [14C]GlcA was observed
if UDP-GlcNAc was omitted or if divalent metal ions were chelated with
EDTA. The HAS activity derived from recombinant HAS was similar to the
enzyme obtained from wild-type P. multocida membranes
because Mn2+ stimulated at least 10-fold more activity than
Mg2+ (24).
0.01 µg
HA/ml/A600). For comparison, wild-type P. multocida P-1059 grown in the same media produced 1,100 µg
HA/ml/A600. E. coli K5 with pPmHAS
produced such high levels of HA that the cells became encapsulated
(Fig. 4A). The radius of the
capsule of the recombinant strain was ~0.2-0.5 µm
(assuming a bacterial cell width of 0.5 µm). This capsule could be
removed by treatment with either ovine testicular hyaluronidase or
Streptomyces HA lyase (Fig. 4B). Neither the
native K5 host strain nor transformants containing pKK223-3 vector
possessed a readily observable capsule as determined by light
microscopy. K5 cells with pPmHAS were also deemed encapsulated by
buoyant density centrifugation. The recombinant cells floated on top of
the 58% Percoll cushion, whereas the vector control cells or
hyaluronidase-treated recombinant cells pelleted through the
Percoll cushion (not shown).
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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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We found that the PmHAS protein polymerizes authentic HA polysaccharide. The carbohydrate from recombinant E. coli was recognized by the cartilage HA-binding protein and was sensitive to HA lyase digestion; these reagents are regarded as specific for HA polysaccharide (20, 26). Both UDP-GlcA and UDP-GlcNAc were required for HA synthesis in vitro. Furthermore, azido-UDP-GlcA and azido-UDP-GlcNAc but not azido-UDP-Glc specifically photoincorporated into PmHAS. As in the case of streptococcal HasA (21) and Xenopus DG42 (11), it appears that one polypeptide species, PmHAS, transfers two distinct sugar groups to the nascent HA chain.
Many encapsulated Gram-negative bacteria, including E. coli, Neisseria meningitidis, and Hemophilus influenzae, possess clusters of genes responsible for capsule biosynthesis organized in operons (17, 18, 27). These operons often contain genes encoding (i) enzymes required for sugar nucleotide precursor synthesis, (ii) glycosyltransferases for polymerizing the exopolysaccharide, and (iii) proteins implicated in polysaccharide export. Thus far we have found that the Type A P. multocida HA capsule operon contains: a KfaA analog, a HA synthase, and a putative UDP-Glc dehydrogenase. The Tn916 elements in the P. multocida acapsular mutants H and L were not integrated directly in the HAS gene but rather were located in the KfaA homolog gene. The resulting disruption probably caused a polar mutation resulting in no expression of the downstream genes encoding the HAS or the UDP-Glc dehydrogenase. Potential ribosome-binding sites preceded these latter two ORFs, but there were no obvious promoter elements. The S. pyogenes HAS gene, hasA, is followed by an UDP-Glc dehydrogenase, hasB, and an UDP-Glc pyrophosphorylase, hasC; in this case, however, the promoter element is at the 5' end of hasA (28).
At the amino acid level, PmHAS is not as similar to the other cloned HASs as one would initially expect. Two potential short motifs, DGS(S/T) at residues 477-480 and DSD at residues 527-529 of PmHAS, are present in HasA. Another similar DGS-containing motif is found repeated at residues 196-198 of PmHAS. The DG of the first motif and the DSD are conserved in all HASs. On the other hand, several absolutely conserved motifs ((S/G)GPLXXY, GDDRXLTN, and LXQQXRWXKS(Y/F/W)(F/C)RE) found in all previously cloned HASs are absent from PmHAS (7, 12). Instead, a variety of bacterial glycosyltransferases align more closely with the sequence in the central portion of the P. multocida HAS protein. These enzymes, which have been either shown or predicted to transfer GlcNAc, galactose, or GalNAc groups, are roughly one-third the size of the P. multocida enzyme; it is interesting to note that their amino termini sequences align together with the middle of the PmHAS polypeptide (residues 430-540). Sections of the first 420 residues of PmHAS show some similarity to portions of the mammalian UDP-GalNAc:polypeptide GalNAc-transferase. These observations may be a reflection of a possible domain structure within PmHAS. The last ~340 residues of this new HAS are not very similar to other entries in the sequence data bases. Therefore, the P. multocida HAS may be unique or may be the prototype of a new class of HAS.
PmHAS is roughly twice the size of the streptococcal, viral, or vertebrate HASs (972 versus 417-588 residues, respectively; Ref. 7). Furthermore, the hydropathy plots of PmHAS and the other known HASs are dissimilar. Using the TMPRED program (29), PmHAS is predicted to have only two candidate transmembrane helices (centered on residues 170 and 510), and both termini of the protein may be located in the cytoplasm. Topologically, this speculation implies that perhaps one-third of the P. multocida polypeptide (~340 residues) is located outside of the cytoplasm. On the other hand, a different topology is predicted for the other class of HASs (7, 9). Reporter enzyme fusion analysis of streptococcal HasA concurs with these predictions; the topological arrangement appears to consist of (i) two transmembrane helices near the amino terminus, (ii) a putative cytoplasmic domain, followed by (iii) three membrane-associated regions at the carboxyl half of the protein (30). The connecting loops between membrane-associated regions are rather short (4-10 residues); therefore, the vast majority of the polypeptide chain is probably not extracellularly exposed. In the future, it will be interesting to elucidate if these two distinct structural classes of HASs utilize the same or different mechanisms to polymerize a product with the identical composition and glycosidic linkages.
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ACKNOWLEDGEMENTS |
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We thank Amy J. Padgett, Philip E. Pummill, and Hui Zhu, as well as Tiffany Farrell and Preeti Joseph (University of Oklahoma Health Sciences Center Summer Undergraduate Research Experience Program students) for technical assistance; Dr. Robert H. Broyles for use of the microscopy equipment; and Dr. Paul H. Weigel for helpful comments on the manuscript.
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FOOTNOTES |
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* This work was supported by National Research Initiative Grant for Sustaining Animal Health and Well-Being 94-37204-0929 from the U. S. Dept. of Agriculture and National Institutes of Health Grant RO1-GM56497 (to P.L.D.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF036004.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, University of Oklahoma Health Sciences Center,
940 Stanton L. Young Blvd., Oklahoma City, OK 73104. Tel.: 405-271-2227; Fax: 405-271-3092; E-mail: paul-deangelis{at}OUHSC.edu.
1 The abbreviations used are: HA, hyaluronan, hyaluronate or hyaluronic acid; HAS, HA synthase; Tn, transposon; GlcA, glucuronic acid; GlcNAc, N-acetylglucosamine; Glc, glucose; GalNAc, N-acetylgalactosamine; ORF, open reading frame; kb, kilobase(s); PCR, polymerase chain reaction.
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REFERENCES |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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 K. M. Townsend, J. D. Boyce, J. Y. Chung, A. J. Frost, and B. Adler Genetic Organization of Pasteurella multocida cap Loci and Development of a Multiplex Capsular PCR Typing System J. Clin. Microbiol., March 1, 2001; 39(3): 924 - 929. [Abstract] [Full Text]
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W. Jing and P. L. DeAngelis Dissection of the two transferase activities of the Pasteurella multocida hyaluronan synthase: two active sites exist in one polypeptide Glycobiology, September 1, 2000; 10(9): 883 - 889. [Abstract] [Full Text] [PDF]
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P. L. DeAngelis Molecular Directionality of Polysaccharide Polymerization by the Pasteurella multocida Hyaluronan Synthase J. Biol. Chem., September 10, 1999; 274(37): 26557 - 26562. [Abstract] [Full Text] [PDF]
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V. L. Tlapak-Simmons, B. A. Baggenstoss, T. Clyne, and P. H. Weigel Purification and Lipid Dependence of the Recombinant Hyaluronan Synthases from Streptococcus pyogenes and Streptococcus equisimilis J. Biol. Chem., February 12, 1999; 274(7): 4239 - 4245. [Abstract] [Full Text] [PDF]
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V. L. Tlapak-Simmons, B. A. Baggenstoss, K. Kumari, C. Heldermon, and P. H. Weigel Kinetic Characterization of the Recombinant Hyaluronan Synthases from Streptococcus pyogenes and Streptococcus equisimilis J. Biol. Chem., February 12, 1999; 274(7): 4246 - 4253. [Abstract] [Full Text] [PDF]
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C. Heldermon, P. L. DeAngelis, and P. H. Weigel Topological Organization of the Hyaluronan Synthase from Streptococcus pyogenes J. Biol. Chem., January 12, 2001; 276(3): 2037 - 2046. [Abstract] [Full Text] [PDF]
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lanes). To
assess the specificity of photoincorporation, a parallel sample was
treated identically except that the reaction mixtures included a
10-fold excess of unlabeled competitor (UDP-GlcNAc or UDP-GlcA,
respectively; + lane); the band intensities are reduced in
comparison with 