Purification and lipid dependence of the recombinant hyaluronan synthases from Streptococcus pyogenes and Streptococcus equisimilis.

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 approximately 5-8% of the total membrane protein. Using nickel chelate affinity chromatography, the HASs were purified to homogeneity from n-dodecyl beta-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 approximately 5 days at 4 degreesC. Both purified enzymes also lost activity over 4-5 days when stored at -80 degreesC 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 degreesC 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 degreesC, even after 2 months, they could be stimulated by the addition of bovine CL to approximately 60% of the initial activity of the freshly purified enzyme.

Since the discovery of HA 1 over 60 years ago (1), this saccharide polymer, which contains repeating disaccharide units of GlcUA␤ (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)(8)(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).
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 enzymeencoding gene or cDNA was not cloned until 1993, when we reported on the Group A HAS, spHAS (22)(23)(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 Mg 2ϩ at neutral pH. The enzyme produces a linear nonsulfated polymer with an M r Ͼ 5 ϫ 10 6 . 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 (23,24) and S. equisimilis (27) were inserted into the pKK223-3 vector (Amersham Pharmacia Biotech) and cloned into E. coli SURE TM 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-␤-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).
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 MgCl 2 , 1.0 mM ␤-mercaptoethanol, 20% glycerol, 0.5 g/ml leupeptin, 0.7 g/ml pepstatin, and 46 g/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 ϫ 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 Ni 2ϩ -nitrilotriacetic acid resin (QIAGEN Inc.). The final extract was applied directly to a mini-spin column (Bio-Rad) containing Ni 2ϩ -nitrilotriacetic acid resin, which had been equilibrated with extraction buffer lacking MgCl 2 . The enzyme was incubated with the resin for 90 min at 4°C with constant mixing. After incubation, the flowthrough fraction from the Ni 2ϩ -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 g/ml leupeptin, 0.7 g/ml pepstatin, 46 g/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).
HAS Activity-HAS activity was determined in 100 l of 25 mM sodium and potassium phosphate, pH 7.0, containing 50 mM NaCl, 20 mM MgCl 2 , 1 mM dithiothreitol, 0.1 mM EGTA, 15-20% glycerol, 1 mM UDP-GlcUA (Fluka), and 0.8 M UDP-[ 14 C]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 g of whole membrane protein or 0.3-0.5 g 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 m; 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 Glu 147 -Thr 161 , and developed as described previously (24).

RESULTS
spHAS and seHAS, containing C-terminal His 6 tails, were affinity-purified using nickel chelate chromatography. Condi-tions 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 Ͼ99% 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 His 6 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 ϳl kDa (Fig. 1, lanes 8 and 9).
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 ϳ0.5 mM CL required before HAS activity was stimulated. The stimulation of HAS activity with CL occurred almost immediately, in Ͻ1 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. ϳ7-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 (Ͼ90%) with the major mass peak at 1447.93, which was not present in the  (50). CL was dissolved in deionized water at a concentration of 10 pmol/l, and 1 l was spotted to the target followed by 1 l of matrix. The matrix compound was 6-aza-2-thiothymine dissolved at a concentration of 3 mg/ml in 50% acetonitrile containing 0.1% trifluoroacetic acid. The samples were then rapidly dried (Ͻ30 s) under vacuum. The MALDI-TOF mass spectrometer used was a Voyager Elite (Perceptive Biosystems, Framingham, MA) equipped with an N 2 laser (337 nm), located in the Oklahoma Laser Mass Spectrometry Facility. Samples were analyzed in the linear negative-ion mode with a delayed extraction of 150 ns and were subject to a 20-kV accelerating voltage.
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 -CH 2 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 Ͼ30% 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 Ͼ4-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 K m values for the sugar nucleotide substrates or that only increases V max . 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 K UDP-GlcNAc of both HASs by ϳ65%. Interestingly, the effect of CL on K UDP-GlcUA was in the opposite direction. With both enzymes, CL caused a 4 -10-fold increase in the K m 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  The addition of 2 mM bovine CL substantially increased the specific activity of purified spHAS and seHAS, respectively, to 20 and 35 nmol/g/h.
b The activity of the extracted enzymes increased after solubilization with DDM. Therefore, the percent yields at this step were Ͼ100%. V max 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 longterm 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 Ϫ80°C, enzyme activity was retained, but changed substantially. Most typically, at Ϫ80°C with CL, both enzymes lost ϳ20 -50% of their activity within 4 -5 days. Storage of purified seHAS or spHAS at Ϫ80°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 Ϫ80°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 ϳ70% 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 ϳ2-3 months (data not shown).
If the enzymes were stored at Ϫ80°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 Ϫ80°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.

TABLE II
Effect of cardiolipin on the kinetic properties of purified spHAS and seHAS The HASs were purified in the absence of CL and then assayed within 24 h for HAS activity in the presence or absence of CL as described under "Experimental Procedures" and in the accompanying paper (32). The second substrate concentration was held constant at 1.0 mM, except for the determination of K UDP-GlcNAc for spHAS, which was at 1.5 mM UDP-GlcUA. and seHAS (B) were purified in the absence of added CL, supplemented with nothing (Ⅺ, q, OE) or with 2 mM bovine (E) or E. coli (‚) CL, immediately aliquoted, and frozen at -80°C. Samples were removed on the indicated day, thawed on ice, and assayed for HAS activity as described under "Experimental Procedures" for the standard conditions. Samples that had been stored without CL were assayed in the absence of CL (Ⅺ) or in the presence of either 2 mM E. coli (OE) or bovine (q) CL. This CL stimulation of HAS activity after storage at Ϫ80°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 ϳ48 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 ϳ23 kDa. Based on mass spectrometry of the purified enzymes, neither spHAS nor seHAS is disulfide-bonded to other proteins or post-translation-  9. Model of the cardiolipin-HAS complex. The scheme depicts the close association of 16 CL molecules (red dots) with one HAS protein to create an active enzyme. The figure represents a hypothetical plane parallel to the plasma membrane (purple) and viewed from outside the cell so that the cytoplasm (white) is seen through the enzyme. The growing HA chain would be transferred through this pore-like opening. The membrane domains of the HAS are labeled as transmembrane (TMD) or membrane-associated (MAD) domains according to preliminary studies on the enzyme's topology (48). ally modified (34). Interestingly, this extra mass was identified as CL. Thus, the active enzyme is a complex of one HAS protein with ϳ16 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 porelike 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 ϳ75% 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 K UDP-GlcNAc , which offsets the increase in K UDP-GlcUA and results in substantially greater V max 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 Ϫ80°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 Ϫ80°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 Ϫ80°C is due to changes in both V max and K m . 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.