From the Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
The two hyaluronan synthases (HASs)
from Streptococcus pyogenes (spHAS) and Streptococcus
equisimilis (seHAS) were expressed in Escherichia
coli as recombinant proteins containing His6 tails. Both enzymes were expressed as major membrane proteins, accounting for
~5-8% of the total membrane protein. Using nickel chelate affinity
chromatography, the HASs were purified to homogeneity from
n-dodecyl Since the discovery of
HA1 over 60 years ago (1),
this saccharide polymer, which contains repeating disaccharide units of GlcUA Although cell-free HA biosynthesis was achieved 40 years ago (17) and
HAS activity was successfully detergent-solubilized in the 1980s from
the plasma membranes of both eukaryotes (18, 19) and bacteria (20, 21),
a functional enzyme-encoding gene or cDNA was not cloned until
1993, when we reported on the Group A HAS, spHAS (22-24). At the same
time, others reported that a 52-kDa protein from Group C
Streptococcus equisimilis was the HAS since an antibody to
this protein appeared to inhibit HA biosynthesis (25). However, this
52-kDa protein did not have HAS activity, and the cloned gene (26) had
no homology to the Group A HAS (23). When a bona fide HAS
from Group C S. equisimilis was cloned (27), this seHAS
protein showed 72% identity to spHAS and no similarity to the
previously reported Group C gene (26). Although the 52-kDa protein is
not a HAS, several studies used antiserum to this unknown 52-kDa
protein and erroneously reported on putative HASs (reviewed in Ref.
28).
The HAS reaction is membrane-associated and combines the sugar
nucleotides UDP-GlcNAc and UDP-GlcUA in an alternating fashion to
polymerize HA in the presence of Mg2+ at neutral pH. The
enzyme produces a linear nonsulfated polymer with an
Mr > 5 × 106. With the
discovery of this new family of HAS isozymes (28), there is a clear
need for the purification and characterization of an active enzyme.
Although there have been numerous attempts to solubilize, identify, and
purify HAS from strains of streptococci that produce HA as well as HAS
from eukaryotic cells (17-19, 21, 25, 29, 30), this has not yet been
accomplished. In this study, we have detergent-solubilized and purified
to homogeneity the active recombinant spHAS and seHAS enzymes
expressed in Escherichia coli. We have also determined that
these purified HASs, in the absence of other streptococcal proteins,
are lipid-dependent enzymes that are strongly stimulated by
CL. A preliminary report describing some of these findings was
published earlier (31).
Materials, Strains, and Plasmids--
Reagents were supplied by
Sigma unless stated otherwise. Media components were from Difco. Mucoid
Group A Streptococcus pyogenes strain S43/192/4 and Group C
S. equisimilis strain D181 were obtained from the
Rockefeller University collection. The E. coli host strain SURETM cells were from Stratagene. The HAS open reading
frames from S. pyogenes (23, 24) and S. equisimilis (27) were inserted into the pKK223-3 vector (Amersham
Pharmacia Biotech) and cloned into E. coli
SURETM cells by the method of DeAngelis and Weigel (24).
The pKK223-3 vector contains the strong tac promoter that
can be regulated by the lac repressor and induced with
isopropyl- HAS Extraction and Purification--
The thawed membrane pellets
were solubilized using 9.8 mM DDM (0.5%, w/v) in
extraction buffer (50 mM sodium and potassium phosphate, pH
7.0, 150 mM NaCl, 10 mM MgCl2, 1.0 mM HAS Activity--
HAS activity was determined in 100 µl of 25 mM sodium and potassium phosphate, pH 7.0, containing 50 mM NaCl, 20 mM MgCl2, 1 mM dithiothreitol, 0.1 mM EGTA, 15-20%
glycerol, 1 mM UDP-GlcUA (Fluka), and 0.8 µM
UDP-[14C]GlcUA (267 mCi/mmol; ICN). When assaying spHAS,
1.5 mM UDP-GlcNAc was used, whereas 1.0 mM
UDP-GlcNAc was used to assay seHAS. DDM (0.98 mM) was also
present in assays with the purified HAS. Depending on the type of
experiment, various concentrations of CL were also added to the assay
buffer. To initiate the enzyme reaction, 3 µg of whole membrane
protein or 0.3-0.5 µg of pure HAS was added, and the mixtures were
gently mixed in a Micromixer at 30 °C for 1 h. The reactions
were terminated and analyzed as described (34).
SDS-PAGE and Western Blotting--
Samples were prepared for
SDS-PAGE as described by Laemmli (35). Briefly, the samples were
incubated with buffer containing 10 mM dithiothreitol and
2% (w/v) SDS for 2 h at 42 °C before loading onto 1-mm 12%
(w/v) polyacrylamide gels. The gels were electrophoresed using a
Bio-Rad Mini-PROTEAN II at 25 mA until the dye reached the running gel
interface, at which point, the current was held constant at 40 mA. The
gels were stained with Coomassie Blue R-250 for 30 min and destained
with 25% methanol and 10% acetic acid. After SDS-PAGE, proteins were
transferred to nitrocellulose (0.1 µm; Schleicher & Schuell) using a
Bio-Rad Mini-Transblot device at 90 V for 2 h in buffer with 20%
methanol and 0.01% (w/v) SDS as described by Towbin et al.
(36). The blots were blocked, incubated with a specific polyclonal
antibody to the spHAS sequence Glu147-Thr161,
and developed as described previously (24).
spHAS and seHAS, containing C-terminal His6 tails,
were affinity-purified using nickel chelate chromatography. Conditions were chosen so that at least 95% of each protein was solubilized from
isolated membranes and recovered in the DDM detergent extracts. Overall, only a 15-17-fold purification was needed to obtain
homogeneous preparations (Table I) since
at least 5-8% of the total E. coli membrane protein can be
the recombinant HAS (27). Based on SDS-PAGE followed by Coomassie Blue
staining (Fig. 1) or Western analysis (data not shown), the affinity-purified spHAS and seHAS were >99% pure. Both spHAS and seHAS migrated anonymously fast on SDS-PAGE and
give relative molecular masses of 42-43 kDa, although the calculated
molecular masses based on amino acid sequence were 48,677 and 48,600 Da, respectively, for the two enzymes containing a His6
tail. These predicted molecular masses were, in fact, found to be
accurate within 0.07% by MALDI-TOF mass spectrometry (34). Despite
their almost identical masses, we observed that seHAS consistently
migrated slightly slower than spHAS, giving a greater apparent
molecular mass of ~l kDa (Fig. 1, lanes 8 and
9).
-D-maltoside extracts. High levels
of HAS activity could be achieved only if the purified enzymes were
supplemented with either bovine or E. coli cardiolipin
(CL), although bovine CL gave consistently greater activity. Mass
spectroscopic analysis revealed that the fatty acid compositions of
these two CL preparations did not overlap. The two HAS enzymes showed
similar but distinct activation profiles with the 10 other lipids
tested. For example, phosphatidic acid and phosphatidylethanolamine
stimulated seHAS, but not spHAS. Phosphatidylserine stimulated both
enzymes. spHAS appears to be more CL-specific than seHAS, although both
purified enzymes still contain endogenous CL that can not easily be
removed. Both seHAS and spHAS were inhibited by phosphatidylcholine,
sphingomyelin, and sulfatides and were not substantially stimulated by
cerebrosides, phosphatidylglycerol, or phosphatidylinositol. With both
HASs, CL increased the Km for UDP-GlcUA, but
decreased the Km for UDP-GlcNAc and gave an overall
stimulation of Vmax. A kinetic characterization
of the two membrane-bound and purified HASs is presented in the
accompanying paper (Tlapak-Simmons, V. L., Baggenstoss, B. A., Kumari, K., Heldermon, C., and Weigel, P. H. (1999)
J. Biol. Chem. 274, 4246-4253). Both purified HASs became inactive after storage for ~5 days at 4 °C. Both purified enzymes also lost activity over 4-5 days when stored at -80 °C in
the presence of CL, but reached a level of activity that then slowly
decreased over a period of months. Although the purified enzymes stored
in the absence of CL at
80 °C were much less active, the enzymes
retained this same low level of activity for at least 5 weeks. When
both spHAS and seHAS were stored without CL at
80 °C, even after 2 months, they could be stimulated by the addition of bovine CL to
~60% of the initial activity of the freshly purified enzyme.
INTRODUCTION
Top
Abstract
Introduction
References
(1,3)GlcNAc
(1,4), has been shown to have numerous biological functions. For example, HA provides the viscous lubrication of synovial
fluid in joints and provides cartilage with its viscoelastic properties. HA is involved in a wide variety of cellular functions and
behaviors, including cell migration (2, 3) development (4-6),
differentiation (7-9), phagocytosis (6), and proteoglycan synthesis
(2, 4). As well as being a major structural component of the matrix, HA
has wound healing, pharmaceutical, and analgesic effects (10-14) and
is also being used as a vehicle for drug delivery (15, 16).
EXPERIMENTAL PROCEDURES
-D-thiogalactoside. To facilitate purification
of spHAS and seHAS, a C-terminal fusion of 6 His residues was
introduced into each construct. This modification does not
significantly alter enzyme activity (24). Membranes from S. pyogenes and S. equisimilis were obtained as described in the accompanying paper (32). Lipids were obtained in sealed vials
from Matreya, Inc. (Pleasant Gap, PA).
-mercaptoethanol, 20% glycerol, 0.5 µg/ml
leupeptin, 0.7 µg/ml pepstatin, and 46 µg/ml phenylmethylsulfonyl
fluoride) at 4 °C for 2 h with gentle mixing in a Micromixer
(Taitec). Insoluble membrane components were sedimented by
centrifugation at 100,000 × g for 1 h, and imidazole was then added to the supernatant to a final concentration of
20 mM to minimize nonspecific binding of E. coli
proteins to the Ni2+-nitrilotriacetic acid resin (QIAGEN
Inc.). The final extract was applied directly to a mini-spin column
(Bio-Rad) containing Ni2+-nitrilotriacetic acid resin,
which had been equilibrated with extraction buffer lacking
MgCl2. The enzyme was incubated with the resin for 90 min
at 4 °C with constant mixing. After incubation, the flow-through
fraction from the Ni2+-nitrilotriacetic acid resin was
passed over the resin four times (on ice). The resin was then washed
with 10 volumes of extraction buffer, and the HAS was eluted with 25 mM sodium and potassium phosphate, pH 7.0, 50 mM NaCl, 1.0 mM dithiothreitol, 20% (v/v) glycerol, 0.98 mM DDM, 0.5 µg/ml leupeptin, 0.7 µg/ml
pepstatin, 46 µg/ml phenylmethylsulfonyl fluoride, and various
concentrations of histidine. For certain experiments, this elution
buffer also contained CL. spHAS and seHAS were solubilized, purified,
and stored with or without the addition of bovine heart or E. coli CL depending on the type of experiment. Exposure to, or the
presence of, histidine did not affect the activity of either HAS.
Protein concentrations were determined with the Coomassie protein assay reagent (Pierce) using bovine serum albumin as the standard (33).
RESULTS
Purification of spHAS and seHAS
View larger version (97K):
[in a new window]
Fig. 1.
SDS-PAGE analysis of purified streptococcal
HAS. Samples at several stages of purification of seHAS were
analyzed by SDS-PAGE using a 12% (w/v) gel and stained with Coomassie
Brilliant Blue. Lane 1, whole membranes; lane 2,
residual nonextracted membrane proteins; lane 3, DDM extract
after centrifugation; lane 4, flow-through fraction from the
Ni2+-nitrilotriacetic acid column; lane 5, first
column wash; lane 6, third column wash; lane 7,
fifth column wash; lane 8, eluted seHAS; lane 9,
eluted spHAS from a parallel purification.
We recently found that the activity of recombinant spHAS or seHAS is stimulated when exogenous CL is added to purified E. coli membranes (34). Triscott and van de Rijn (21) also found that digitonin-solubilized seHAS was stimulated by CL. Based on these results, we investigated the influence of CL and other lipids on HAS activity as well as on HAS purification and stability during storage.
When either spHAS (Fig. 2) or seHAS (Fig. 3) was purified under CL-deficient conditions, the re-addition of CL caused a large increase in enzyme activity. As the CL concentration was increased, the specific activity of both enzymes also increased. In both cases, there was an apparent critical concentration of ~0.5 mM CL required before HAS activity was stimulated. The stimulation of HAS activity with CL occurred almost immediately, in <1 min (data not shown). With spHAS, bovine or E. coli CL gave similar results (Fig. 2), although bovine CL consistently resulted in a significantly higher specific activity: a 11.5-fold stimulation compared with a 9-fold increase at 3 mM. The seHAS enzyme behaved in a qualitatively similar manner (Fig. 3), although the difference between bovine and E. coli CL was greater, and the overall stimulation of HAS activity was less than that found for spHAS. Consistent with this result, the residual HAS activity without exogenous CL was always considerably higher (e.g. ~7-fold) for seHAS compared with spHAS.
|
|
The difference in the ability of the bovine and E. coli CL preparations to stimulate either seHAS or spHAS was unexpected, especially since the enzyme was expressed in and purified from E. coli, which is known to contain CL (37). To assess the molecular basis for this different ability to stimulate, we examined the composition of these lipids using MALDI-TOF mass spectrometry (Fig. 4). Interestingly, the two CL preparations were quite different and, in fact, did not share any of the same CL species. Since CL contains four fatty acyl chains and there are dozens of different possible fatty acids that can be linked at each position, there are many possible CL isoforms. Bovine CL contained essentially one species (>90%) with the major mass peak at 1447.93, which was not present in the bacterial CL. E. coli CL was much more complex, containing at least seven different CL species ranging in mass from 1335.91 to 1432.09. These seven species constitute a series with each member differing in mass by 14 or one -CH2 group.
|
A variety of other phospholipids were also tested for their ability to stimulate HAS activity. Freshly purified spHAS (Fig. 5) and seHAS (Fig. 6) were both stimulated to the greatest extent by bovine CL and to the next greatest extent by phosphatidylserine. spHAS showed the greater specificity for CL since none of the other 10 lipids tested gave a specific activity of >30% of that with bovine CL (Fig. 5). The only other lipids to give a moderate stimulation of spHAS activity were lysolecithin, cerebrosides, and phosphatidylserine. Phosphatidylserine was the second best activating lipid, with a 2.5-fold stimulation of activity. The basal activity of purified spHAS was actually inhibited in the presence of phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, sphingomyelin, and sulfatides (Fig. 5).
|
|
The results with seHAS were qualitatively similar, but quantitatively different (Fig. 6). The basal activity of purified seHAS was also inhibited by phosphatidylcholine, sphingomyelin, and sulfatides. Both enzymes were essentially inactive in the presence of this latter anionic lipid. Like spHAS, seHAS was moderately stimulated by lysolecithin and cerebrosides, but unlike spHAS, seHAS was also weakly stimulated by phosphatidylinositol and phosphatidylethanolamine. Perhaps the biggest difference between the two enzymes was the effect of phosphatidic acid, which stimulated seHAS >4-fold (Fig. 6), but inhibited spHAS (Fig. 5). We conclude from these data that seHAS and spHAS are very sensitive to the phospholipids in their environment and are essentially dependent on CL.
The large increase in synthase activity with CL could be due to a CL-dependent effect on the enzyme that decreases the Km values for the sugar nucleotide substrates or that only increases Vmax. To determine this, the kinetic profiles of both purified enzymes were measured in the absence and presence of bovine CL (Fig. 7 and Table II). In the absence of CL, both freshly purified enzymes had responses to increasing UDP-GlcUA similar to those shown in Figs. 2 and 4 for the membrane-bound enzymes, but they had very different responses to UDP-GlcNAc (Fig. 7). Detergent-solubilized, purified spHAS without CL showed the same sigmoidal response to increasing UDP-GlcNAc as observed for the membrane-bound enzyme (32). The Hill number in this case was 2.0, whereas the value for pure seHAS without CL was 1.1, indicating no cooperativity for UDP-GlcNAc utilization (Table II). The addition of CL decreased the KUDP-GlcNAc of both HASs by ~65%. Interestingly, the effect of CL on KUDP-GlcUA was in the opposite direction. With both enzymes, CL caused a 4-10-fold increase in the Km for UDP-GlcUA. Although CL had opposite effects on the HAS efficiency for utilizing each substrate, the overall effect on enzyme activity was highly favorable since CL increased the Vmax of both enzymes 3.6-6-fold. A more complete kinetic characterization of the HASs in the presence of CL is presented in the accompanying paper (32).
|
|
We also investigated conditions that would allow the long-term storage
of the purified HAS enzymes with good retention of activity. At
4 °C, with or without CL, the enzymes were inactive by day 5 (data
not shown). When affinity-purified spHAS (Fig. 8A) or seHAS (Fig.
8B) was stored for weeks with or without CL at 80 °C,
enzyme activity was retained, but changed substantially. Most
typically, at
80 °C with CL, both enzymes lost ~20-50% of their activity within 4-5 days. Storage of purified seHAS or spHAS at
80 °C with 2 mM bovine CL usually yielded a relatively
stable level of HAS activity from day 5 through at least day 34. For both enzymes, storage in the presence of bovine CL was better than with
E. coli CL.
|
Both enzymes consistently displayed an unusual biphasic, changing
activity pattern during the first week of storage at 80 °C. This
biphasic loss and then recovery of activity with storage time occurred
consistently in the presence of bovine CL, not with E. coli
CL. The increase noted around days 3 and 4 in Fig. 8 is reproducible,
although the time at which these changes occurred and the magnitude of
the activity change varied considerably among different enzyme
preparations. For example, in another experiment (data not shown),
enzyme activity declined ~70% over the first 5 days and then
rebounded by day 10 to a level of only 20% less activity than at day
0. Typically, either HAS then loses activity irreversibly with a
half-life of ~2-3 months (data not shown).
If the enzymes were stored at 80 °C without CL, they also had a
much lower, but fairly constant level of activity when assayed without
CL (Fig. 8). Interestingly, spHAS (Fig. 8A) or seHAS (Fig. 8B) stored at
80 °C in the absence of CL was capable of
being restored to a high level of activity by the subsequent addition of CL. The levels of enzyme activity for both spHAS and seHAS attained
by this protocol were essentially identical to the activity of the
enzymes that had been stored in the continuous presence of either
bovine or E. coli CL. This CL stimulation of HAS activity after storage at
80 °C without CL was observed for up to at least 1 month. This fortunate result means that purified seHAS or spHAS can
be stored for substantial periods in the absence of added lipids.
![]() |
DISCUSSION |
---|
Although it may seem strange that some bacteria can make the same HA polymer that mammals make, these microorganisms have created a clever way to live a pathogenic lifestyle. Bacteria such as Group C S. equisimilis (a pathogen in animals and sometimes humans), Group A S. pyogenes (a human pathogen), and Pasteurella multocida (an animal pathogen) are able to hide from the immune systems of their host by surrounding themselves with a thick layer of HA. Since this HA barrier, or capsule, has the same structure as the HA in our tissues, the bacteria are not easily recognized by antibodies or attacked by phagocytes. Studies have confirmed that the HA capsule contributes in large part to the pathogenicity of these bacteria (38, 39).
The Group A (spHAS) and Group C (seHAS) enzymes are 72% identical and are the smallest members of the HAS family at 419 and 417 amino acids, respectively (27, 28). The HAS family is now also composed of three distinct mammalian HASs (5, 40-44): bacteriophage (45) and amphibian (46) HASs and a third bacterial enzyme that may represent a second type of HAS (47). Biochemical, genetic, and radiation inactivation studies with the Group A and Group C streptococcal HAS genes and proteins have allowed us to confirm (discussed below) that only the HAS gene product (the HAS protein) is required for HA biosynthesis.
The finding that HA synthesis requires only a single gene product was initially surprising since the HA biosynthesis process is multifunctional and very complex, requiring at least six distinct functions to synthesize HA (28), as shown schematically in Fig. 7 of the accompanying paper (32). Many investigators, for example, expected that at least two proteins would be involved to catalyze the two glycosyltransferase reactions. We expected the enzyme to be larger than ~48 kDa to perform these six functions. The streptococcal HAS is the first enzyme that makes a heteropolysaccharide polymer to be studied in a purified state.
The streptococcal HASs are very compact and tightly folded since, even in the presence of the strong detergent SDS, these proteins behave as though they are only 42 kDa rather than 48 kDa. Reducing agents do not change this behavior,2 which indicates that disulfide bonds are probably not present. Preliminary protein chemistry studies and mutagenesis studies, in which we have altered the 4 Cys residues of seHAS or the 6 Cys residues of spHAS, are also consistent with this conclusion (48).
The streptococcal and eukaryotic HASs are thought to be organized in the membrane in the same way (28). The majority of the protein, including both the amino and carboxyl termini, is inside the cell. Four of the streptococcal HAS membrane domains appear to pass through the membrane, giving only two small loops of the protein exposed to the extracellular side. Ongoing studies have indicated that another two or more regions of the protein, including portions of the large central domain (24), may associate with the membrane as amphipathic helices or reentrant loops that do not span the membrane.3
There is a paradox or dilemma, related to the HA transfer function, posed by this topology model and the finding that no protein other than the HAS is needed for HA biosynthesis, at least when the enzyme is solubilized in detergent. How can we account for the ability of this relatively small protein to transfer the growing HA chain, which has some hydrophobic character but which is mostly hydrophilic, across the hydophobic lipid barrier of the plasma membrane? Other membrane transporters that shepherd small molecules, like sugars, across membranes usually contain 12 or more transmembrane domains and create a pore (49). The HAS protein is not large enough and does not have the necessary number of transmembrane domains to form such a pore. So, how does it guide HA across the membrane?
One solution to this HA transfer dilemma would be if the HAS could only synthesize HA if it forms a dimer or larger oligomer, so that enough transmembrane domains from each HAS protein could be arranged to form a pore. To determine if the active HAS is a monomer or a larger oligomer, we recently used radiation inactivation analysis (34). Both active streptococcal HASs contained a single HAS protein, but the protein was associated with an additional mass of ~23 kDa. Based on mass spectrometry of the purified enzymes, neither spHAS nor seHAS is disulfide-bonded to other proteins or post-translationally modified (34). Interestingly, this extra mass was identified as CL. Thus, the active enzyme is a complex of one HAS protein with ~16 molecules of CL. Our interpretation of these results is depicted in Fig. 9. We propose that CL molecules help the enzyme to solve the HA transfer dilemma by creating a pore-like passage within the enzyme through which the growing HA chain passes. The lipid portion of CL could interact with the lipid bilayer and hydrophobic patches of the HA chain, whereas the acidic head groups of CL molecules could interact with the enzyme and hydrophilic portions of the HA chain.
|
Consistent with this radiation inactivation analysis, we found in the present study that either purified streptococcal HAS had very low activity in the absence of CL. When this phospholipid was added back, however, enzyme activity increased up to 10-fold. In particular, spHAS was very specifically activated and dependent on CL. The CL dependence of seHAS seemed less stringent since phosphatidic acid or phosphatidylserine was ~75% as effective as CL. Neither spHAS nor seHAS was effectively stimulated by cerebrosides, phosphatidylethanolamine, phosphatidylglycerol, or phosphatidylinositol, all of which are uncharged or cationic. Both enzymes were inhibited by phosphatidylcholine, also a cationic lipid. Cardiolipin, which is diphosphatidylglycerol, has a neutral head group and two negative phosphoryl groups. In general, the HAS-activating lipids are anionic, but not cationic. It may be relevant that the two most effective phospholipids, CL and phosphatidylserine, contain a single free -OH group. This hydroxyl may be important as a hydrogen bond acceptor or donor to the enzyme and/or the growing HA chain.
The biggest effect of CL on HAS activity appears to be the decrease in KUDP-GlcNAc, which offsets the increase in KUDP-GlcUA and results in substantially greater Vmax values for both enzymes.
Detailed conclusions about the lipid dependence must await further studies on the purified HASs, including the amount and type of endogenous lipids that may copurify with these enzymes. Preliminary results using MALDI-TOF mass spectrometry indicate that when the HAS enzymes are purified under CL-depleted conditions, they nonetheless contain detectable CL species. It is therefore likely that some of the CL associated with the active enzyme is very tightly bound and retained by the isolated protein, although loss of less tightly associated CL still results in enzyme inactivation. The rapid activation of purified enzyme in response to the re-addition of CL shows that HAS interaction with CL occurs readily.
When purified HAS is stored at 80 °C in the presence of CL, the
enzyme slowly loses activity irreversibly; further addition of fresh CL
does not rescue the enzyme. Both enzymes stored at
80 °C without
CL are still capable of being activated by CL, although the level of
enzyme activity also slowly declines. Apparently under any of these
conditions, the HAS enzyme undergoes an irreversible conformational
change and is inactivated with a half-life of several months. The
decline in enzyme activity with time of storage at
80 °C is due to
changes in both Vmax and Km.
Nonetheless, the ability to purify and store the streptococcal HASs in
the absence of added lipids will enable these enzymes to be studied in
detail and their mechanisms of action to be elucidated.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Anil Singh for technical support, Coy Heldermon and Dr. Kshama Kumari for helpful discussions, Debbie Blevins for help preparing the manuscript, and Dr. Paul DeAngelis for preparation of the spHAS-H6 construct.
![]() |
FOOTNOTES |
---|
* This work was supported in part by NIGMS Grant GM35978 from the National Institutes of Health. Work carried out in the Oklahoma Laser Mass Spectrometry Facility was supported by a National Science Foundation EPSCoR grant.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. Tel.: 405-271-1288;
Fax: 405-271-3092; E-mail: paul-weigel{at}ouhsc.edu.
The abbreviations used are:
HA, hyaluronan or
hyaluronic acid; HAS, hyaluronan synthase; spHAS, S.
pyogenes hyaluronan synthase; seHAS, S. equisimilis
hyaluronan synthase; CL, cardiolipin; DDM, n-dodecyl
-D-maltoside; PAGE, polyacrylamide gel electrophoresis; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight.
2 Tlapak-Simmons, V. L., Heldermon, C., Kempner, E. S., and Weigel, P. H. (1999) Biochem. Soc. Trans., in press.
3 C. Heldermon, P. L. DeAngelis, and P. H. Weigel, manuscript in preparation.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|