Site-directed mutation of conserved cysteine residues does not inactivate the Streptococcus pyogenes hyaluronan synthase.

Hyaluronan synthase (HAS), the enzyme responsible for the production of hyaluronic acid (HA), is a well-conserved membrane-bound protein in both prokaryotes and eukaryotes. This enzyme performs at least six discrete functions in producing a heterodisaccharide polymer of several million molecular weight and extruding it from the cell. Among the conserved motifs and domains within the Class I HAS family are four cysteine residues. Cysteines in many proteins are important in establishing and maintaining tertiary structure or in the coordination of catalytic functions. In the present study we utilized a combination of site-directed mutagenesis, chemical labeling, and kinetic analyses to determine the importance of specific Cys residues for catalysis and structure of the HA synthase from Streptococcus pyogenes (spHAS). The enzyme activity of spHAS was partially inhibited by cysteine-reactive chemical reagents such as N-ethylmaleimide. Quantitation of the number of Cys residues modified by these reagents, using MALDI-TOF mass spectrometry, demonstrated that there are no stable disulfide bonds in spHAS. The six Cys residues of spHAS were then mutated, individually and in various combinations, to serine or alanine. The single Cys-mutants were all kinetically similar to the wild-type enzyme in terms of their V(max) and K(m) values for HA synthesis. The Cys-null mutant, in which all Cys residues were mutated to alanine, retained approximately 66% of wild-type activity, demonstrating that despite their high degree of conservation within the HAS family, Cys residues are not absolutely necessary for HA biosynthesis by the spHAS enzyme.

Selection and implementation for coagulation instruments/reagents in a multiple hospital/clinic network.

Selection, standardization, and implementation of instrumentation and reagents throughout a health care facility network can often be a difficult process. However, in today's ever-changing health care setting, it is often mandated. The Veteran's Integrated Systems Network 16 (VISN 16) was faced with such a task early in 1999, with the targeted area being its coagulation laboratories. The plan outlined in this paper was drafted to help facilitate the selection, standardization and implementation of coagulation systems for 17 health care facilities that make up the VISN 16 network. The VISN, encompassing 170,000 square miles, has 10 tertiary care hospitals, six of which have close relationships with affiliate universities. There are 299,733 patients enrolled in this health delivery system. The facilities range from large institutions performing both tertiary and outpatient care to small outpatient clinics. Because of the plan's detailed, comprehensive content, which included analyses of a large number of performance parameters as well as cost-efficiency, the selection process was carried out using a checklist that could be helpful to other organizations selecting equipment and reagents for coagulation studies. An implementation process was devised, resulting in coagulation standardization across the Integrated Health Network.

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.

Kinetic characterization 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. The accompanying paper has described the purification and lipid dependence of both HASs, their preference for cardiolipin, and their stability during storage (Tlapak-Simmons, V. L., Baggenstoss, B. A., Clyne, T., and Weigel, P. H. (1999) J. Biol. Chem. 274, 4239-4245). Kinetic characterization of the enzymes in isolated membranes gave Km values for UDP-GlcUA of 40 +/- 4 microM for spHAS and 51 +/- 5 microM for seHAS. In both cases, the Vmax profiles at various concentrations of UDP-GlcNAc were hyperbolic, with no evidence of cooperativity. In contrast, membrane-bound spHAS, but not seHAS, showed sigmoidal behavior as the UDP-GlcNAc concentration was increased, with a Hill number of approximately 2, indicating significant cooperativity. The Hill number for UDP-GlcNAc utilization by seHAS was 1, confirming the lack of cooperativity for UDP-GlcNAc in this enzyme. The Km values for UDP-GlcNAc were 60 +/- 7 microM for seHAS and 149 +/- 3 microM for spHAS in the isolated membranes. The kinetic characteristics of the two affinity-purified HAS enzymes were assessed in the presence of cardiolipin after 8-9 days of storage at -80 degreesC without cardiolipin. With increasing storage time, the enzymes showed a gradual increase in their Km values for both substrates and a decrease in Vmax. Even in the presence of cardiolipin, the detergent-solubilized, purified HASs had substantially higher Km values for both substrates than the membrane-bound enzymes. The KUDP-GlcUA for purified spHAS and seHAS increased 2-4-fold. The KUDP-GlcNAc for spHAS and seHAS increased 4- and 5-fold, respectively. Despite the higher Km values, the Vmax values for the purified HASs were only approximately 50% lower than those for the membrane-bound enzymes. Significantly, purified spHAS displayed the same cooperative interaction with UDP-GlcNAc (nH approximately 2), whereas purified seHAS showed no cooperativity.

The active streptococcal hyaluronan synthases (HASs) contain a single HAS monomer and multiple cardiolipin molecules.

The functional sizes of the two streptococcal hyaluronan synthases (HASs) were determined by radiation inactivation analysis of isolated membranes. The native enzymes in membranes from Group A Streptococcus pyogenes HAS and Group C Streptococcus equisimilis HAS were compared with the recombinant proteins expressed in Escherichia coli membranes. Based on their amino acid sequences, the masses of these four proteins as monomers are approximately 48 kDa. In all cases, loss of enzyme activity was a simple single exponential function with increasing radiation dose. The functional sizes calculated from these data were identical for the four HASs at approximately 64 kDa. In contrast, the sizes of the proteins estimated by the loss of antibody reactivity on Western blots were essentially identical at 41 kDa for the four HAS species, consistently lower than the functional size by approximately 23 kDa. Matrix-assisted laser desorption time of flight mass spectrometry analysis of purified S. pyogenes HAS-H6 and S. equisimilis HAS-H6 gave masses that differed by <0.07% from the predicted monomer sizes, which confirms that neither protein is posttranslationally modified or covalently attached to another protein. Ongoing studies indicate that the purified HAS enzymes require cardiolipin (CL) for maximal activity and stability. When irradiated membranes were detergent solubilized and the extracts were incubated with exogenous CL, the residual level of HAS activity increased. Consequently, the calculated functional size decreased by approximately 23 kDa to the expected size of the HAS monomer. The approximately 23-kDa larger size of the functional HAS enzyme, compared with the HAS monomer, is due, therefore, to CL molecules. We propose that the active streptococcal HA synthases are monomers in complex with approximately 16 CL molecules.

Obfuscation of allosteric structure-function relationships by enthalpy-entropy compensation.

The pH and temperature dependence of the allosteric properties of phosphofructokinase (PFK) from Bacillus stearothermophilus have been studied from 5 to 9 and 6 to 40 degrees C, respectively. Throughout this pH and temperature range the allosteric ligands MgADP and phospho(enol)pyruvate (PEP) have no effect on kcat. The dissociation constants of the substrate, fructose 6-phosphate, and the allosteric ligands, as well as the absolute value of the coupling free energies between these ligands, all increase when the pH is raised, indicating that the inhibition by PEP and the activation by MgADP increase despite each ligand's somewhat lower affinity. However, the constituent coupling enthalpies and entropies substantially diminish in absolute value as pH is increased, suggesting that the magnitudes of molecular perturbations engendered by the binding of allosteric ligands do not correlate with the magnitudes of the functional consequences of those perturbations. Temperature and pH exert their influence on the observed allosteric behavior by changing the relative contributions made by the largely compensating DeltaH and TDeltaS terms to the coupling free energy.

Adaptation of a functional assay for protein S to a Cobas Fara II centrifugal analyzer.

An adaptation of the manual Staclot protein S functional assay (American Bioproducts, Parsippany, NJ) for the Cobas FARA II centrifugal analyzer is described. This automated method is based on determining the clotting time by measuring the change in turbidity of the specimen as clotting progresses. The accurate and precise pipetting of the analyzer combined with its enhanced data acquisition and rapid parallel processing features result in a markedly improved procedure compared to the less accurate and reproducible manual method. The assay is linear over the range of protein S concentrations encountered clinically (0% to 150%); has a limit of detection of 3% protein S; within and between day precision (CV) at a level of 50% protein S is 4.7 and 12.6%, respectively; and results agree closely with those obtained on the STA automated hemostasis analyzer (slope = 0.98, intercept 3.92, and r = 0.98). Protein S values obtained with this assay for specimens from 20 normal males and 20 normal females were 115% +/- 22.31% (mean +/- standard deviation [SD]) and 98% +/- 18.2%, respectively. In 14 patients on chronic stable treatment with Coumadin (DuPont Pharmaceuticals, Wilmington, DE) the mean functional protein S activity was 16.2% and the SD 11.7%. The method for determination of protein S activity on the centrifugal analyzer yields results comparable to data reported with a variety of other techniques.

Heparin-induced increase in the international normalized ratio. Responses of 10 commercial thromboplastin reagents.

A clinician's concern about an erratic response to oral anticoagulation in a patient treated concurrently with heparin and warfarin led the authors to investigate the effect of heparin on INR values obtained with various commercial thromboplastin reagents. Studies conducted with pooled normal plasma and with pooled plasma from patients treated long-term with oral anticoagulants demonstrated a wide range of sensitivities to heparin of these reagents as characterized by prolongation of INR values. Innovin was unaffected by concentrations of heparin as high as 1 U/mL. In contrast, Ortho thromboplastin showed the greatest increase in INR values over the range of heparin concentrations studied. Three other thromboplastins including Neoplastine CI, Dade thromboplastin, and Simplastin A demonstrated only limited sensitivity to heparin. Prolongation of the INR by heparin was reversed by protamine in a dose-related manner and also by preincubation of the plasma with heparinase. Some patients treated with warfarin while on the authors' institutional protocol for heparin had plasma concentrations greater than 0.8 U/mL. When thromboplastin reagents sensitive to heparin were used with such specimens, the INR values obtained were falsely elevated. The authors suggest that reagents insensitive to heparin be employed to avoid this difficulty.

Comparison of the inhibition by phospho(enol)pyruvate and phosphoglycolate of phosphofructokinase from B. stearothermophilus.

A comparison between the inhibition by phospho(enol)pyruvate (PEP) versus the inhibition by phosphoglycolate (PG) of phosphofructokinase (PFK) from Bacillus stearothermophilus is presented. Both inhibitors act by decreasing the apparent affinity displayed by the enzyme for its substrate fructose 6-phosphate (Fru-6-P) while having little effect on Vmax. However, the two ligands differ in both their affinity for the enzyme and their effectiveness at antagonizing the subsequent binding of Fru-6-P. Although PG binds with approximately 10-fold lower affinity, it antagonizes the binding of Fru-6-P 3.5-fold more strongly than does PEP. Moreover, the enthalpy and entropy contributions to the coupling free energy between inhibitor and Fru-6-P, from which these antagonisms derive, reveal even greater differences between the ligands. These data indicate, therefore, that the changes in the structure of PFK from B. stearothermophilus that result from PG binding, which have been determined by X-ray crystallography (T. Schirmer and P. R. Evans, 1990 Nature 343, 140-145), may not be comparable to those that result from PEP binding and consequently do not represent the generic "T-state," as has been presumed.

Temperature-induced inversion of allosteric phenomena.

Two instances, involving the enzymes carbamoyl-phosphate synthetase from Escherichia coli and phosphofructokinase from Bacillus stearothermophilus, respectively, are described in which increasing temperature alone causes the actions of an allosteric ligand to change from inhibition to activation. In neither case are these effects due to a change in the activation energy of the enzyme catalyzed reaction induced by the allosteric ligand. Rather, they are due to temperature-dependent changes in the extent to which the binding of allosteric ligand modifies the affinity of the enzyme for substrate. The data can be readily explained by an analysis of the apparent delta H and delta S components of the coupling free energy, which quantitatively describe the actions of allosteric ligands that act in this manner. These observations underscore the shortcomings of expecting to explain the actions of an allosteric ligand solely by the structural perturbations that accompany the binding of an allosteric ligand such as those often revealed by x-ray crystallography.