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• W2022646939 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 fromn-dodecyl β-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 K m for UDP-GlcUA, but decreased the K m for UDP-GlcNAc and gave an overall stimulation of V max. 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. 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 fromn-dodecyl β-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 K m for UDP-GlcUA, but decreased the K m for UDP-GlcNAc and gave an overall stimulation of V max. 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. Since the discovery of HA 1The abbreviations used are: HA, hyaluronan or hyaluronic acid; HAS, hyaluronan synthase; spHAS, S. pyogenes hyaluronan synthase; seHAS, S. equisimilishyaluronan synthase; CL, cardiolipin; DDM, n-dodecyl β-d-maltoside; PAGE, polyacrylamide gel electrophoresis; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight. over 60 years ago (1Meyer K. Palmer J.W. J. Biol. Chem. 1934; 107: 629-634Abstract Full Text PDF Google Scholar), this saccharide polymer, which contains repeating disaccharide units of GlcUAβ(1,3)GlcNAcβ(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 (2Evered D. Whelan J. CIBA Found. Symp. 1989; 143: 1-288Google Scholar, 3Turley E.A. Bowman P. Kytryk M.A. J. Cell Sci. 1985; 78: 133-145PubMed Google Scholar) development (4Toole B.P. Hay E.D. Proteoglycans and Hyaluronan in Morphogenesis and Differentiation in Cell Biology of the Extracellular Matrix. Plenum Press, New York1991: 305-341Google Scholar, 5Spicer A.P. McDonald J.A J. Biol. Chem. 1998; 273: 1923-1932Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 6Knudson C.B. Knudson W. FASEB J. 1993; 7: 1233-1242Crossref PubMed Scopus (601) Google Scholar), differentiation (7Kujawa M.J. Caplan A.I. Dev. Biol. 1986; 114: 504-518Crossref PubMed Scopus (81) Google Scholar, 8Kujawa M.J. Pechak D.J. Fizman M.Y. Caplan A.I. Dev. Biol. 1986; 113: 10-16Crossref PubMed Scopus (90) Google Scholar, 9Kujawa M.J. Tepperman K. Dev. Biol. 1983; 99: 277-286Crossref PubMed Scopus (69) Google Scholar), phagocytosis (6Knudson C.B. Knudson W. FASEB J. 1993; 7: 1233-1242Crossref PubMed Scopus (601) Google Scholar), and proteoglycan synthesis (2Evered D. Whelan J. CIBA Found. Symp. 1989; 143: 1-288Google Scholar, 4Toole B.P. Hay E.D. Proteoglycans and Hyaluronan in Morphogenesis and Differentiation in Cell Biology of the Extracellular Matrix. Plenum Press, New York1991: 305-341Google Scholar). As well as being a major structural component of the matrix, HA has wound healing, pharmaceutical, and analgesic effects (10Balazs E.A. Denlinger J.L. J. Rheumatol. 1993; 20: 3-9Google Scholar, 11Gressner A.M. Bachem M.G. Digestion. 1995; 56: 335-346Crossref PubMed Scopus (222) Google Scholar, 12Goa K.L. Benfield P. Drugs. 1994; 47: 536-566Crossref PubMed Scopus (388) Google Scholar, 13West D.C. Hampson I.N. Arnold F. Kumars S. Science. 1985; 228: 1324-1326Crossref PubMed Scopus (979) Google Scholar, 14Abatangelo G. Martinelli M. Vecchia P. J. Surg. Res. 1983; 35: 410-416Abstract Full Text PDF PubMed Scopus (113) Google Scholar) and is also being used as a vehicle for drug delivery (15Illum L. Forraj N.F. Fisher A.N. Gill I. Miglietta M. Benedetti L.M. J. Controlled Release. 1994; 29: 133-141Crossref Scopus (98) Google Scholar, 16Jubbell J.A. J. Controlled Release. 1996; 39: 305-313Crossref Scopus (125) Google Scholar). Although cell-free HA biosynthesis was achieved 40 years ago (17Markovitz A. Cifonelli J.A. Dorfman A. J. Biol. Chem. 1959; 234: 2343-2350Abstract Full Text PDF PubMed Google Scholar) and HAS activity was successfully detergent-solubilized in the 1980s from the plasma membranes of both eukaryotes (18Mian N. Biochem. J. 1986; 237: 343-357Crossref PubMed Scopus (36) Google Scholar, 19Ng K.F. Swartz N.B. J. Biol. Chem. 1989; 264: 11776-11783Abstract Full Text PDF PubMed Google Scholar) and bacteria (20Stoolmiller A.C. Dorfman A. J. Biol. Chem. 1969; 244: 236-246Abstract Full Text PDF PubMed Google Scholar, 21Triscott M.X. van de Rijn I. J. Biol. Chem. 1986; 261: 6004-6009Abstract Full Text PDF PubMed Google Scholar), a functional enzyme-encoding gene or cDNA was not cloned until 1993, when we reported on the Group A HAS, spHAS (22DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 14568-14571Abstract Full Text PDF PubMed Google Scholar, 23DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 19181-19184Abstract Full Text PDF PubMed Google Scholar, 24DeAngelis P.L. Weigel P.H. Biochemistry. 1994; 33: 9033-9039Crossref PubMed Scopus (97) Google Scholar). At the same time, others reported that a 52-kDa protein from Group CStreptococcus equisimilis was the HAS since an antibody to this protein appeared to inhibit HA biosynthesis (25Prehm P. Mausolf A. Biochem. J. 1986; 235: 887-889Crossref PubMed Scopus (33) Google Scholar). However, this 52-kDa protein did not have HAS activity, and the cloned gene (26Lansing M. Lellig S. Mausolf A. Martini I. Crescenzi F. Oregan M. Prehm P. Biochem. J. 1993; 289: 179-184Crossref PubMed Scopus (40) Google Scholar) had no homology to the Group A HAS (23DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 19181-19184Abstract Full Text PDF PubMed Google Scholar). When a bona fide HAS from Group C S. equisimilis was cloned (27Kumari K. Weigel P.H. J. Biol. Chem. 1997; 272: 32539-32546Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), this seHAS protein showed 72% identity to spHAS and no similarity to the previously reported Group C gene (26Lansing M. Lellig S. Mausolf A. Martini I. Crescenzi F. Oregan M. Prehm P. Biochem. J. 1993; 289: 179-184Crossref PubMed Scopus (40) Google Scholar). 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.28Weigel P.H. Hascall V.C. Tammi M. J. Biol. Chem. 1997; 272: 13997-14000Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar). 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 anM r > 5 × 106. With the discovery of this new family of HAS isozymes (28Weigel P.H. Hascall V.C. Tammi M. J. Biol. Chem. 1997; 272: 13997-14000Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar), 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 (17Markovitz A. Cifonelli J.A. Dorfman A. J. Biol. Chem. 1959; 234: 2343-2350Abstract Full Text PDF PubMed Google Scholar, 18Mian N. Biochem. J. 1986; 237: 343-357Crossref PubMed Scopus (36) Google Scholar, 19Ng K.F. Swartz N.B. J. Biol. Chem. 1989; 264: 11776-11783Abstract Full Text PDF PubMed Google Scholar, 21Triscott M.X. van de Rijn I. J. Biol. Chem. 1986; 261: 6004-6009Abstract Full Text PDF PubMed Google Scholar, 25Prehm P. Mausolf A. Biochem. J. 1986; 235: 887-889Crossref PubMed Scopus (33) Google Scholar, 29Philipson L.H. Schwartz N.B. J. Biol. Chem. 1984; 259: 5017-5023Abstract Full Text PDF PubMed Google Scholar, 30Prehm P. Biochem. J. 1983; 211: 181-189Crossref PubMed Scopus (91) Google Scholar), 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 (31Tlapak-Simmons V.L. Baggenstoss B.A. Weigel P.H. Glycobiology. 1997; 7: 1032Google Scholar). Reagents were supplied by Sigma unless stated otherwise. Media components were from Difco. Mucoid Group A Streptococcus pyogenes strain S43/192/4 and Group CS. 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 (23DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 19181-19184Abstract Full Text PDF PubMed Google Scholar, 24DeAngelis P.L. Weigel P.H. Biochemistry. 1994; 33: 9033-9039Crossref PubMed Scopus (97) Google Scholar) and S. equisimilis (27Kumari K. Weigel P.H. J. Biol. Chem. 1997; 272: 32539-32546Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) were inserted into the pKK223-3 vector (Amersham Pharmacia Biotech) and cloned into E. coliSURETM cells by the method of DeAngelis and Weigel (24DeAngelis P.L. Weigel P.H. Biochemistry. 1994; 33: 9033-9039Crossref PubMed Scopus (97) Google Scholar). The pKK223-3 vector contains the strong tac promoter that can be regulated by the lac repressor and induced with isopropyl-β-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 (24DeAngelis P.L. Weigel P.H. Biochemistry. 1994; 33: 9033-9039Crossref PubMed Scopus (97) Google Scholar). Membranes from S. pyogenes and S. equisimilis were obtained as described in the accompanying paper (32Tlapak-Simmons V.L. Baggenstoss B.A. Kumari K. Heldermon C. Weigel P.H. J. Biol. Chem. 1999; 274: 4246-4253Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Lipids were obtained in sealed vials from Matreya, Inc. (Pleasant Gap, PA). 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 β-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. coliproteins 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 (33Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217548) Google Scholar). 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 μmUDP-[14C]GlcUA (267 mCi/mmol; ICN). When assaying spHAS, 1.5 mm UDP-GlcNAc was used, whereas 1.0 mmUDP-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 (34Tlapak-Simmons V.L. Kempner E.S. Baggenstoss B.A. Weigel P.H. J. Biol. Chem. 1998; 273: 26100-26109Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Samples were prepared for SDS-PAGE as described by Laemmli (35Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar). 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. (36Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar). The blots were blocked, incubated with a specific polyclonal antibody to the spHAS sequence Glu147–Thr161, and developed as described previously (24DeAngelis P.L. Weigel P.H. Biochemistry. 1994; 33: 9033-9039Crossref PubMed Scopus (97) Google Scholar). 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 (27Kumari K. Weigel P.H. J. Biol. Chem. 1997; 272: 32539-32546Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). 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 His6tail. These predicted molecular masses were, in fact, found to be accurate within 0.07% by MALDI-TOF mass spectrometry (34Tlapak-Simmons V.L. Kempner E.S. Baggenstoss B.A. Weigel P.H. J. Biol. Chem. 1998; 273: 26100-26109Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). 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 and9).Table IPurification of spHAS and seHASTotal proteinSpecific activityaThe addition of 2 mm bovine CL substantially increased the specific activity of purified spHAS and seHAS, respectively, to 20 and 35 nmol/μg/h.Total activityYieldbThe activity of the extracted enzymes increased after solubilization with DDM. Therefore, the percent yields at this step were >100%.Purificationmgnmol/μg/hnmol UDP-GlcUA%-foldspHASMembranes20.900.3063501001.0DDM extract19.000.3871881131.25Eluted spHAS0.805.544377017.5seHASMembranes31.100.7924,4171001.0DDM extract30.300.9829,5901211.25Eluted seHAS1.5812.018,7907715.2a The addition of 2 mm bovine CL substantially increased the specific activity of purified spHAS and seHAS, respectively, to 20 and 35 nmol/μg/h.b The activity of the extracted enzymes increased after solubilization with DDM. Therefore, the percent yields at this step were >100%. Open table in a new tab We recently found that the activity of recombinant spHAS or seHAS is stimulated when exogenous CL is added to purified E. colimembranes (34Tlapak-Simmons V.L. Kempner E.S. Baggenstoss B.A. Weigel P.H. J. Biol. Chem. 1998; 273: 26100-26109Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Triscott and van de Rijn (21Triscott M.X. van de Rijn I. J. Biol. Chem. 1986; 261: 6004-6009Abstract Full Text PDF PubMed Google Scholar) 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.Figure 3Effect of cardiolipin on the activity of purified seHAS. seHAS was purified as described under “Experimental Procedures,” except that exogenous CL was omitted at each step. The freshly purified enzyme was then assayed in the presence of the indicated final concentrations of either bovine (■) orE. coli (▪) CL.View Large Image Figure ViewerDownload (PPT) 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 (37Dowhan W. Annu. Rev. Biochem. 1997; 66: 199-232Crossref PubMed Scopus (789) Google Scholar). 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).Figure 6Effect of various lipids on seHAS activity. Freshly purified seHAS, prepared in the absence of added CL, was assayed in the presence of the indicated lipids as described in the legend to Fig. 5.View Large Image Figure ViewerDownload (PPT) 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 theK 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 (32Tlapak-Simmons V.L. Baggenstoss B.A. Kumari K. Heldermon C. Weigel P.H. J. Biol. Chem. 1999; 274: 4246-4253Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). 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 ∼65%. Interestingly, the effect of CL onK UDP-GlcUA was in the opposite direction. With both enzymes, CL caused a 4–10-fold increase in theK 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 theV 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 (32Tlapak-Simmons V.L. Baggenstoss B.A. Kumari K. Heldermon C. Weigel P.H. J. Biol. Chem. 1999; 274: 4246-4253Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar).Table IIEffect of cardiolipin on the kinetic properties of purified spHAS and seHASUDP-GlcUAUDP-GlcNAcK mV maxK mV maxHill No.μmnmol/μg/hμmnmol/μg/hspHAS+0 mm CL11 ± 53.5 ± 1.11090 ± 1203.0 ± 1.32.0 ± 0.2+2.0 mm CL117 ± 2319.8 ± 3.3410 ± 11017.9 ± 3.91.9 ± 0.1seHAS+0 mm CL44 ± 178.8 ± 1.1950 ± 1709.4 ± 1.41.1 ± 0.1+2.0 mm CL173 ± 2231.7 ± 5.6315 ± 5234.0 ± 4.31.0 ± 0.0The 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 (32Tlapak-Simmons V.L. Baggenstoss B.A. Kumari K. Heldermon C. Weigel P.H. J. Biol. Chem. 1999; 274: 4246-4253Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The second substrate concentration was held constant at 1.0 mm, except for the determination ofK UDP-GlcNAc for spHAS, which was at 1.5 mm UDP-GlcUA. Open table in a new tab 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 (32Tlapak-Simmons V.L. Baggenstoss B.A. Kumari K. Heldermon C. Weigel P.H. J. Biol. Chem. 1999; 274: 4246-4253Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The second substrate concentration was held constant at 1.0 mm, except for the determination ofK UDP-GlcNAc for spHAS, which was at 1.5 mm UDP-GlcUA. 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.8 A) or seHAS (Fig.8 B) 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 withE. 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. coliCL. The increase noted around" @default.
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