Sialidase assay by luminescence in the low picomole-range of sialic acid. Its application to the measurement of this activity in influenza virus

A new procedure for a sialidase assay, by bioluminescence, has been developed. The substrate, N- acetylneuraminyllactose (sialyllactose), hydrolysed by the sialidase activity, releases lactose. This lactose is hydrolysed with beta-galactosidase. The released galactose is oxidized with galactose dehydrogenase and NAD. The NADH produced in the last step is measured by a luminescence system, coupling two enzymes, NAD(P)H dehydrogenase (FMN) and luciferase. This microassay, which is specific, rapid, simple and ultra-sensitive, is a measure for amounts as little as (at least) 5 pmol of N-acetylneuraminic acid (corresponding to 0.15 ng of the released sialic acid). It uses commercialized reagents (non-radioisotopic) and avoids interferences common in other procedures. This method has been used for measuring sialidase activity directly on intact virus, avoiding inconvenient modifications produced in the extraction of the enzyme. The specific activity of sialidase of influenza virus X31 (H3N2), determined by this procedure, is 0.65 U/mg of total virus protein.     

A fluorometric procedure for measuring the neuraminidase activity: its application to the determination of this activity in influenza and parainfluenza viruses

A fluorometric procedure for quantitating the amount of N-acetylneuraminic acid enzymatically released by the neuraminidase activity from N-acetylneuraminyl-lactose (sialyl-lactose) has been developed. The liberated lactose is hydrolyzed with beta-galactosidase, and the released galactose is oxidized with galactose dehydrogenase and NAD+; finally, the NADH produced is measured by fluorometry (excitation at 340 nm and analysis of emitted light at 465 nm). The fluorometric assay is about 10-fold more sensitive than the spectrophotometric procedure that measures NADH at 340 nm. It readily measures amounts as little as 2 nmol of sialic acid, and does not require the use of radioactive isotopes. Interferences due to sucrose or other substances, which cause errors in some cases with the use of the periodate-thiobarbiturate method for neuraminidase activity determination, are avoided. The procedure reported here provides a sensitive, rapid, and relatively simple method (feasible with commercialized reagents) for measuring the neuraminidase activity not only in purified samples from different sources but also directly in biological materials such as viruses. The technique has been tested with some viruses recently isolated belonging to Orthomyxoviridae or Paramyxoviridae families, known to be rich in neuraminidase. Reciprocally, this method can also be employed for determining the sialic acid concentration in acylneuraminyl-lactose-containing compounds when using purified neuraminidase for hydrolysis.     

Lactobacillus casei 64H Contains a Phosphoenolpyruvate-Dependent Phosphotransferase System for Uptake of Galactose, as Confirmed by Analysis of ptsH and Different gal Mutants

Galactose metabolism in Lactobacillus casei 64H was analyzed by genetic and biochemical methods. Mutants with defects in ptsH, galK, or the tagatose 6-phosphate pathway were isolated either by positive selection using 2-deoxyglucose or 2-deoxygalactose or by an enrichment procedure with streptozotocin. ptsH mutations abolish growth on lactose, cellobiose, N-acetylglucosamine, mannose, fructose, mannitol, glucitol, and ribitol, while growth on galactose continues at a reduced rate. Growth on galactose is also reduced, but not abolished, in galK mutants. A mutation in galK in combination with a mutation in the tagatose 6-phosphate pathway results in sensitivity to galactose and lactose, while a galK mutation in combination with a mutation in ptsH completely abolishes galactose metabolism. Transport assays, in vitro phosphorylation assays, and thin-layer chromatography of intermediates of galactose metabolism also indicate the functioning of a permease/Leloir pathway and a phosphoenolpyruvate-dependent phosphotransferase system (PTS)/tagatose 6-phosphate pathway. The galactose-PTS is induced by growth on either galactose or lactose, but the induction kinetics for the two substrates are different.     

Escherichia coli Heat-Labile Enterotoxin Binds to Glycosylated Proteins with Lactose by Amino Carbonyl Reaction

The binding of Escherichia coli heat-labile enterotoxin (LT) type I to glycosylated proteins with lactose (Galβ1-4Glc) by amino carbonyl reaction was studied by the Western blot assay and by the microtiter well binding assay. LT bound to a lactose-α-lactalbumin amino carbonyl product (Lac-LA), whereas cholera toxin did not. The binding ability of Lac-LA was abolished by β-galactosidase treatment, indicating that the terminal galactose is essential for the binding of LT. The binding of LT to Lac-LA was inhibited by galactose and lactose, and most effectively inhibited by lactulose (Galβ1-4Fru), which is a structural analog of the Amadori rearrangement product of the amino carbonyl reaction between lactose and an ε-amino group of a lysine residue (lactuloselysine). The results suggest that LT recognizes the portion of lactuloselysine in Lac-LA. LT also bound to a melibiose (Galα1-6Glc)-α-lactalbumin amino carbonyl product (Mel-LA), but the binding ability of Mel-LA was weaker than that of Lac-LA, suggesting that the β1-4 linked terminal galactose is dispensable but preferable for the binding. Furthermore, LT bound to the amino carbonyl products of lactose with β-lactoglobulin, caseins, bovine serum albumin, and ovalbumin. These results indicate that LT binds to the amino carbonyl products between proteins and sugars containing the terminal galactose, such as lactose.     

A quantitation of the factors which affect the hydrolase and transgalactosylase activities of beta-galactosidase (E. coli) on lactose.

A study was implemented to quantitate the hydrolase and transgalactosylase activities of beta-galactosidase (E. coli) with lactose as the substrate and to investigate various factors which affect these activities. At low lactose concentrations the rate of galactose production was equal to the rate of glucose production. The rate of galactose production relative to glucose, however, dropped dramatically at lactose concentrations higher than 0.05 M and production of trisaccharides and tetrasaccharides began (galactose/glucose ratios of about 2:1 and 3:1, respectively, were found for these two types of oligosaccharides). At least five different trissacharides were formed and their patterns of formation showed that they probably utilized both lactose and allolactose as galactosyl acceptors. Allolactose was produced in amounts proportional to glucose at all lactose concentrations (ratios of allolactose/glucose were about 0.88). Analyses of various data, including a reaction analyzed at very early times, showed that the major means of production of allolactose (and the only means initially) was the direct enzymatic transfer of galactose from the 4 position to the 6 position of the glucose moiety of lactose without prior release of glucose from the enzyme. It was shown, however, that allolactose could also be formed in significant quantities by the transfer of galactose to the 6 position of free glucose, and also by hydrolysis of preformed trisaccharide. A mechanism which fits the initial velocity data was proposed in which the steps involving the formation of an enzyme-gallactose-glucose complex, the formation and breakage of allolactose on the enzyme, and the release of glucose all seem to be of roughly equal magnitude and rate determining. Various factors affected the amounts of transgalactosylase and hydrolase activities occurring. At high pH values (greater than 7.8) the transgalactosylase/hydrolyase activity ratio increased dramatically while it decreased at low pH values (less than 6.0). At mid pH values the ratio was essentially constant. The absence of Mg2+ caused a large decrease in the transgalactosylase/hydrolase activity ratio while the absence of all but traces of Na+ or K+ had no effect. The anomeric configuration of lactose altered the transgalactosylase/hydrolase activity ratios, alpha-Lactose resulted in a decrease of allolactose production (transgalactosylase activity) relative to hydrolase activities (glucose production) while beta-lactose had the opposite effect.     

Phosphorylation state of HPr determines the level of expression and the extent of phosphorylation of the lactose transport protein of Streptococcus thermophilus

The lactose transport protein (LacS) of Streptococcus thermophilus is composed of a translocator domain and a regulatory domain that is phosphorylated by HPr(His approximately P), the general energy coupling protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). Lactose transport is affected by the phosphorylation state of HPr through changes in the activity of the LacS protein as well as expression of the lacS gene. To address whether or not CcpA-HPr(Ser-P)-mediated catabolite control is involved, the levels of LacS were determined under conditions in which the cellular phosphorylation state of HPr greatly differed. It appears that HPr(Ser-P) is mainly present in the exponential phase of growth, whereas HPr(His approximately P) dominates in the stationary phase. The transition from HPr(Ser-P) to HPr(His approximately P) parallels an increase in LacS level, a drop in lactose and an increase in galactose concentration in the growth medium. Because the K(m)(out) for lactose is higher than that for galactose, the lactose transport capacity decreases as lactose concentration decreases and galactose accumulates in the medium. Our data indicate that S. thermophilus compensates for the diminished transport capacity by synthesizing more LacS and phosphorylating the protein, which results in increased transport activity. The link between transport capacity and lacS expression levels and LacS phosphorylation are discussed.     

Structure-based exploration of the ganglioside GM1 binding sites of Escherichia coli heat-labile enterotoxin and cholera toxin for the discovery of receptor antagonists

Ganglioside GM1 is the natural receptor for cholera toxin (CT) and heat-labile enterotoxin (LT), which are the causative agents of cholera and traveler's diarrhea, respectively. This observation suggests that small molecules interfering with this recognition process may prevent entry of the toxins into intestinal cells, thereby averting their devastating effects. Here, the terminal sugar of ganglioside GM1, galactose, was chosen as a lead in designing such receptor antagonists. Guided by the experimentally determined binding mode of galactose, we selected a "substructure" for searching the Available Chemicals Database, which led to the purchase of 35 galactose derivatives. Initial screening of these compounds in an LT ELISA revealed that 22 of them have a higher affinity for LT than galactose itself. A structurally diverse subset of these galactose derivatives was selected for determination of IC50 values in the LT ELISA and IC50 values in a CT assay, as well as for the determination of Kd's using the intrinsic fluorescence of LT. The best receptor antagonist found in this study was m-nitrophenyl alpha-galactoside with an IC50 of 0.6 (2) mM in the LT ELISA and 0.72 (4) mM in the CT assay, 100-fold lower than both IC50 values of galactose. Careful analysis of our binding data and comparison with crystal structures led to the derivation of correlations between the structure and affinity of the galactose derivatives. These characteristics will be used in the design of a second round of LT and CT receptor antagonists.     

Utilization of spline functions for smoothing fermentation data and for estimation of specific rates

A response surface method of smoothing fermentation data with spline functions is presented. The available electron balance is used to optimally select the values of the smoothing parameters associated with the spline functions. The method is applied to six sets of anaerobic fermentation data in which pure and mixed cultures are grown in batch followed by fed batch culture. Lactobacillus bulgaricus and Streptococcus thermophilus are cultured on 3% dry milk. Measured concentrations of biomass, lactose, galactose, lactic acid, and other acid products are smoothed using spline functions. Values of specific growth rate, specific lactose consumption rate, specific galactose formation rate, and specific acid product formation rate are estimated and the consistency of the results is examined using the available electron balance. The results show that the method works reasonably well, but that an upper bound should be used for the smoothing parameters to obtain accurate estimates of the derivative quantities.     

Interaction between ricin hemagglutinin and its ligands, galactose and lactose. Microcalorimetry and equilibrium dialysis]

The interaction of Ricinus communis hemagglutinin with galactose and lactose has been studied by means of microcalorimetry, equilibrium dialysis and analytical ultracentrifugation. A first class of beta-galactoside-binding sites involves two similar and independent sites of which affinity constants are 2600 M-1 for galactose and 26700 M-1 for lactose at 25 degrees C. The binding of one galactose or one lactose molecule leads to enthalpy changes of--12.3 Kcal and--11 Kcal, respectively. Considering the negative entropy changes of the association, and as for ricin, the binding of galactosides with hemagglutinin is driven by favorable enthalpic contributions. In presence of high lactose concentrations, a second endothermic step of the calorimetric titration curve was observed. This result and the biphasic nature of Scatchard plots of equilibrium dialysis suggest the existence of a second class of binding sites on the lectin molecule. As for ricin, the interaction between these secondary sites and lactose would be entropically driven.     

Influence of the lactose plasmid on the metabolism of galactose by Streptococcus lactis.

Streptococcus lactis strain DR1251 was capable of growth on lactose and galactose with generation times, at 30 degrees C, of 42 and 52 min, respectively. Phosphoenolpyruvate-dependent phosphotransferase activity for lactose and galactose was induced during growth on either substrate. This activity had an apparent K(m) of 5 x 10(-5) M for lactose and 2 x 10(-2) M for galactose. beta-d-Phosphogalactoside galactohydrolase activity was synthesized constitutively by these cells. Strain DR1251 lost the ability to grow on lactose at a high frequency when incubated at 37 degrees C with glucose as the growth substrate. Loss of ability to metabolize lactose was accompanied by the loss of a 32-megadalton plasmid, pDR(1), and Lac(-) isolates did not revert to a Lac(+) phenotype. Lac(-) strains were able to grow on galactose but with a longer generation time. Galactose-grown Lac(-) strains were deficient in beta-d-phosphogalactoside galactohydrolase activity and phosphoenolpyruvate phosphotransferase activity for both lactose and galactose. There was also a shift from a predominantly homolactic to a heterolactic fermentation and a fivefold increase in galactokinase activity, relative to the Lac(+) parent strain grown on galactose. These results suggest that S. lactis strain DR1251 metabolizes galactose primarily via the tagatose-6-phosphate pathway, using a lactose phosphoenolpyruvate phosphotransferase activity to transport this substrate into the cell. Lac(-) derivatives of strain DR1251, deficient in the lactose phosphoenolpyruvate phosphotransferase activity, appeared to utilize galactose via the Leloir pathway.     

Characterization of a novel alpha1,2-fucosyltransferase of Escherichia coli O128:b12 and functional investigation of its common motif

The wbsJ gene from Escherichia coli O128:B12 encodes an alpha1,2-fucosyltransferase responsible for adding a fucose onto the galactose residue of the O-antigen repeating unit via an alpha1,2 linkage. The wbsJ gene was overexpressed in E. coli BL21 (DE3) as a fusion protein with glutathione S-transferase (GST) at its N-terminus. GST-WbsJ fusion protein was purified to homogeneity via GST affinity chromatography followed by size exclusion chromatography. The enzyme showed broad acceptor specificity with Galbeta1,3GalNAc (T antigen), Galbeta1,4Man and Galbeta1,4Glc (lactose) being better acceptors than Galbeta-O-Me and galactose. Galbeta1,4Fru (lactulose), a natural sugar, was furthermore found to be the best acceptor for GST-WbsJ with a reaction rate four times faster than that of lactose. Kinetic studies showed that GST-WbsJ has a higher affinity for lactose than lactulose with apparent Km values of 7.81 mM and 13.26 mM, respectively. However, the kcat/appKm value of lactose (6.36 M(-1) x min(-1)) is two times lower than that of lactulose (13.39 M(-1) x min(-1)). In addition, the alpha1,2-fucosyltransferase activity of GST-WbsJ was found to be independent of divalent metal ions such as Mn2+ or Mg2+. This activity was competitively inhibited by GDP with a Ki value of 1.41 mM. Site-directed mutagenesis and a GDP-bead binding assay were also performed to investigate the functions of the highly conserved motif H152xR154R155xD157. In contrast to alpha1,6-fucosyltransferases, none of the mutants of WbsJ within this motif exhibited a complete loss of enzyme activity. However, residues R154 and D157 were found to play critical roles in donor binding and enzyme activity. The results suggest that the common motif shared by both alpha1,2-fucosyltransferases and alpha1,6-fucosyltransferases have similar functions. Enzymatic synthesis of fucosylated sugars in milligram scale was successfully performed using Galbeta-O-Me and Galbeta1,4Glcbeta-N3 as acceptors.     

A new kinetic model of recombinant beta-galactosidase from Kluyveromyces lactis for both hydrolysis and transgalactosylation reactions

Previous models based on the Michaelis-Menten kinetic equation, that glucose was not used as an acceptor, did not explain our experimental data for lactose conversion by a recombinant beta-galactosidase from Kluyeromyces lactis. In order to create a new kinetic model based on the data, the effects of galactose and glucose on beta-galactosidase activity were investigated. Galactose acted as an inhibitor at low concentrations of galactose and lactose, but did not inhibit the activity of beta-galactosidase at high concentrations of galactose (above 50mM) and lactose (above 100mM). The addition of glucose at concentrations below 50mM resulted in an increased reaction rate. A new model of K. lactis beta-galactosidase for both hydrolysis and transgalactosylation reactions with glucose and lactose as acceptors was proposed. The proposed model was fitted well to the experimental data of the time-course reactions for lactose conversion by K. lactis beta-galactosidase at various concentrations of substrate.     

Kinetics of the coupled reaction catalysed by a fusion protein of beta-galactosidase and galactose dehydrogenase.

The mechanistic implications of the kinetic behaviour of a fusion protein of beta-galactosidase and galactose dehydrogenase have been analysed in view of predictions based on experimentally determined kinetic parameter values for the galactosidase and dehydrogenase activities of the protein. The results show that the time course of galactonolactone formation from lactose in the coupled reaction catalysed by the fusion protein can be most satisfactorily accounted for in terms of a free-diffusion mechanism when consideration is given to the mutarotation of the reaction intermediate galactose. It is concluded that no tenable kinetic evidence is available to support the proposal that the fusion protein catalyses galactonolactone formation from lactose by a mechanism involving channelling of galactose.     

Substrate recognition at the cytoplasmic and extracellular binding site of the lactose transport protein of Streptococcus thermophilus

The lactose transport protein (LacS) of Streptococcus thermophilus catalyzes the uptake of lactose in an exchange reaction with intracellularly formed galactose. The interactions between the substrate and the cytoplasmic and extracellular binding site of LacS have been characterized by assaying binding and transport of a range of sugars in proteoliposomes, in which the purified protein was reconstituted with a unidirectional orientation. Specificity for galactoside binding is given by the spatial configuration of the C-2, C-3, C-4, and C-6 hydroxyl groups of the galactose moiety. Except for a C-4 methoxy substitution, replacement of the hydroxyl groups for bulkier groups is not tolerated at these positions. Large hydrophobic or hydrophilic substitutions on the galactose C-1 alpha or beta position did not impair transport. In fact, the hydrophobic groups increased the binding affinity but decreased transport rates compared with galactose. Binding and transport characteristics of deoxygalactosides from either side of the membrane showed that the cytoplasmic and extracellular binding site interact differently with galactose. Compared with galactose, the IC(50) values for 2-deoxy- and 6-deoxygalactose at the cytoplasmic binding site were increased 150- and 20-fold, respectively, whereas they were the same at the extracellular binding site. From these and other experiments, we conclude that the binding sites and translocation pathway of LacS are spacious along the C-1 to C-4 axis of the galactose moiety and are restricted along the C-2 to C-6 axis. The differences in affinity at the cytoplasmic and extracellular binding site ensure that the transport via LacS is highly asymmetrical for the two opposing directions of translocation.     

Galactose metabolism by Streptococcus mutans

The galK gene, encoding galactokinase of the Leloir pathway, was insertionally inactivated in Streptococcus mutans UA159. The galK knockout strain displayed only marginal growth on galactose, but growth on glucose or lactose was not affected. In strain UA159, the sugar phosphotransferase system (PTS) for lactose and the PTS for galactose were induced by growth in lactose and galactose, although galactose PTS activity was very low, suggesting that S. mutans does not have a galactose-specific PTS and that the lactose PTS may transport galactose, albeit poorly. To determine if the galactose growth defect of the galK mutant could be overcome by enhancing lactose PTS activity, the gene encoding a putative repressor of the operon for lactose PTS and phospho-beta-galactosidase, lacR, was insertionally inactivated. A galK and lacR mutant still could not grow on galactose, although the strain had constitutively elevated lactose PTS activity. The glucose PTS activity of lacR mutants grown in glucose was lower than in the wild-type strain, revealing an influence of LacR or the lactose PTS on the regulation of the glucose PTS. Mutation of the lacA gene of the tagatose pathway caused impaired growth in lactose and galactose, suggesting that galactose can only be efficiently utilized when both the Leloir and tagatose pathways are functional. A mutation of the permease in the multiple sugar metabolism operon did not affect growth on galactose. Thus, the galactose permease of S. mutans is not present in the gal, lac, or msm operons.     

Binding of Lactose and Galactose to Native and Iodinated Ricin D

The binding of lactose and galactose to native and iodinated ricin D was investigated by equilibrium dialysis and ultraviolet difference spectroscopy. The results provided direct evidence that native ricin D has two independent saccharide binding sites with different affinities, of which the high-affinity (HA-) binding site is able to bind with both lactose and galactose while the low-affinity (LA-) binding site binds only with lactose. In contrast, the iodinated ricin D possesses only one binding site both for lactose and galactose with high affinity.

By UV-difference spectroscopic analysis we found that there is one tyrosyl residue at or near the HA-binding site in ricin D which may be involvled in binding with saccharide. This tyrosyl residue was not iodinated in the presence of lactose but was iodinated in the absence of lactose and was perturbed by an addition of lactose even after iodination.

From these results, it was inferred that the binding site abolished by the iodination is the LA-binding site and this may be due to the conformational alteration of the LA-binding site caused by the iodination of the tyrosyl residue(s) present near the LA-binding site.     

The frequency distribution of lactose malabsorption among adult populations from the Eastern and Western Egyptian Deserts

The study consisted of 172 subjects belonging to ethnic groups from Sinai in the Eastern Desert and the New Valley in the Western Desert, with respective mean ages of 36.7±2.0 and 26.6±1.0 years. Lactose absorption was assessed by measurement of urinary galactose in pooled 2-hr urine samples following ingestion of an oral lactose dose of 40 g. Mean 2-hr excretion values after ingestion were 32.3 mg galactose in the Sinai and 7.7 mg in the New Valley. In the evaluation of lactose malabsorption, a diagnosis of lactose malabsorption is based upon a cutoff point of 0.075 mg/mg urinary galactose:creatinine ratio. The overall prevalence rate in those populations is 34.3%. The proportion of lactose malabsorbers was 11.1% in Sinai and 51.0% in the New Valley. Highly significant differences (X²=29.5,P<0.0001) were found between the two ethnic groups with regard to the frequency distribution of lactose malabsorption. The existence of an east-west gradient of increasing frequencies of lactose malabsorption gene is suggested.     

Presence of galactose in precultures induces lacS and leads to short lag phase in lactose-grown Lactococcus lactis cultures

Lactose conversion by lactic acid bacteria is of high industrial relevance and consistent starter culture quality is of outmost importance. We observed that Lactococcus lactis using the high-affinity lactose-phosphotransferase system excreted galactose towards the end of the lactose consumption phase. The excreted galactose was re-consumed after lactose depletion. The lacS gene, known to encode a lactose permease with affinity for galactose, a putative galactose–lactose antiporter, was upregulated under the conditions studied. When transferring cells from anaerobic to respiration-permissive conditions, lactose-assimilating strains exhibited a long and non-reproducible lag phase. Through systematic preculture experiments, the presence of galactose in the precultures was correlated to short and reproducible lag phases in respiration-permissive main cultivations. For starter culture production, the presence of galactose during propagation of dairy strains can provide a physiological marker for short culture lag phase in lactose-grown cultures.

     

Coregulation of beta-galactoside uptake and hydrolysis by the hyperthermophilic bacterium Thermotoga neapolitana

Regulation of the beta-galactoside transport system in response to growth substrates in the extremely thermophilic anaerobic bacterium Thermotoga neapolitana was studied with the nonmetabolizable analog methyl-beta-D-thiogalactopyranoside (TMG) as the transport substrate. T. neapolitana cells grown on galactose or lactose accumulated TMG against a concentration gradient in an intracellular free sugar pool that was exchangeable with external galactose or lactose and showed induced levels of beta-galactosidase. Cells grown on glucose, maltose, or galactose plus glucose showed no capacity to accumulate TMG, though these cells carried out active transport of the nonmetabolizable glucose analog 2-deoxy-D-glucose. Glucose neither inhibited TMG uptake nor caused efflux of preaccumulated TMG; rather, glucose promoted TMG uptake by supplying metabolic energy. These data show that beta-D-galactosides are taken up by T. neapolitana via an active transport system that can be induced by galactose or lactose and repressed by glucose but which is not inhibited by glucose. Thus, the phenomenon of catabolite repression is present in T. neapolitana with respect to systems catalyzing both the transport and hydrolysis of beta-D-galactosides, but inducer exclusion and inducer expulsion, mechanisms that regulate permease activity, are not present. Regulation is manifest at the level of synthesis of the beta-galactoside transport system but not in the activity of the system.