A fluorometric method for monitoring the enzymic hydrolysis of terminal galactose from GM1-ganglioside.

A fluorometric method for monitoring the enzymic hydrolysis of the terminal galactose from GM₁-ganglioside has been developed. The released galactose is oxidized with galactose dehydrogenase and NAD and the fluorescence of the product NADH measured. This method can detect as little as 0.1 nmol of galactose. β-Galactosidase from the gastropod Turbo cornutus was employed for the hydrolysis reaction. The rate of GM₁-ganglioside hydrolysis is linearly proportional to incubation time for 30 min under the assay conditions employed. In addition to galactose, the other product of hydrolysis, GM₂-ganglioside, is identified by thin-layer chromatography. This procedure provides a convenient and specific method for measuring the release of galactose from GM₁-ganglioside.     

Substrate specificity of small-intestinal lactase. Assessment of the role of the substrate hydroxyl groups.

Lactase-phlorizin hydrolase is a disaccharidase present in the small intestine of mammals. This enzyme has two active sites, one being responsible for the hydrolysis of lactose. Lactase activity is thought to be selective towards glycosides with a hydrophilic aglycon. In this work, we report a systematic study on the importance of each hydroxyl group in the substrate molecule for lactase activity. For this purpose, all of the monodeoxy derivatives of methyl beta-lactoside and other lactose analogues are studied as lactase substrates. With respect to the galactose moiety, it is shown here that HO-3' and HO-2' are necessary for hydrolysis of the substrates by lactase. Using these chemically modified substrates, it has been confirmed that lactase does not behave as a typical beta-galactosidase, since it does not show an absolute selectivity with respect to substitution and stereochemistry at C4' in the galactose moiety of the substrate. However, the glucose moiety, in particular the HO-6, appears to be important for substrate hydrolysis, although none of the hydroxyl groups seemed to be essential. In order to differentiate both activities of the enzyme, a new assay for the phlorizin-hydrolase activity has also been developed.     

Preparative scale isolation of galactosylhydroxylysine.

A method is described for the isolation and purification of gram quantities of the hydroxylysine-monosaccharide from commercially available marine sponge. The procedure utilized alkaline hydrolysis followed by purification by ionexchange chromatography and gel filtration. Compositional analysis indicated that the final product contained only galactose, hydroxylysine, and HCl which were present in equimolar quantities and comprised 94% of the dry weight. This preparation has been utilized as a substrate for the assay of UDP-glucose:collagen glucosyltransferase (EC 2.4.1.66) of human platelets.     

Relationship between two types of mouse sperm surface sites that mediate binding of sperm to the zona pellucida

There are at least two binding sites for the mouse egg zona pellucida on the surface of mouse sperm: a site with galactosyltransferase (GT) activity inhibitable by uridine-5'-diphosphate-dialdehyde (UDPd) and alpha-lactalbumin, and a trypsin inhibitor-sensitive (TI) site that hydrolyzes guanidinobenzoate (GB) esters. Characterization of GT activity gave the Km for UDP galactose as 37 microM with N-acetylglucosamine as galactose acceptor, and Vmax as 0.37 pmol/min/10(6) sperm. UDP galactose from 12.5-100 microM inhibited sperm binding to zona-intact eggs in a concentration-dependent manner with close correlation to GT activity (r = 0.95). To assess the independence and spatial relationship of the two types of site, cross-perturbation studies were performed. p-Nitrophenyl-GB, a low molecular mass inhibitor specific for the TI site, had no effect on the enzyme activity of the GT site. Conversely, UDPd, a specific inhibitor of GT, had no effect on GB hydrolysis. Weak inhibitions were found when soybean trypsin inhibitor (SBTI) was included with the GT assay and when GB hydrolysis was assayed in the presence of alpha-lactalbumin or asialo-agalacto-(alpha 1-acid glycoprotein). Acid-solubilized zona protein (ASZP) weakly inhibited the GT reaction, while stronger inhibition was seen with chymotrypsin-solubilized zona protein (CSZP). ASZP inhibited sperm binding to zonae with the same concentration dependence associated with inhibition of GB hydrolysis, but the inhibition of GT enzyme activity was on the same order as that found with SBTI, indicating that ASZP was only binding to the TI site under enzyme assay conditions. The results support the hypothesis that the two types of site are independent in binding their specific zona ligands, but are close enough for steric perturbation of the enzyme activity of one site by macromolecules bound to the other. The different interactions of solubilized zona preparations with the GT site under enzyme assay conditions are an indication that conditions which favor the enzyme activity of the site may interfere with the physiological binding functions of the site.     

Homogeneous bioluminescence assay for galactosuria: interference and kinetic analysis

Elevated galactose concentration in urine is an important clinical symptom of galactosemia and other metabolic disorders. A quantitative assay for galactose using firefly luciferase bioluminescence is presented. The assay couples the galactokinase and firefly luciferase reactions. A higher concentration of galactose present in the sample produces a faster decrease in ATP concentration, which is monitored by firefly luciferase bioluminescence. The kinetic assay is modeled and analyzed. The interference between the two reactions, the interference of certain sugars and other components in the urine, the specificity, and the optimal pH for galactokinase were studied. Calibration curves were constructed and compared with a conventional spectrophotometric assay for galactose. The bioluminescence assay is relatively fast and specific for galactose with a linear range from 1 to 20 mM galactose. The effect of other galactose metabolites (galactonate and galactitol) has also been studied.     

Galactomannans and a galactoglucomannan in legume seed endosperms: Structural requirements for β-mannanase hydrolysis

A series of galactomannans with varying degrees of galactose substitution have been extracted from the endosperms of legume seeds with water and alkali and the amount of substitution required for water solubility has been determined. Some were heterogeneous with respect to the degree of galactose substitution. The structural requirements for hydrolysis by plant β-mannanase have been studied using the relative rates and extents of hydrolysis of these galactomannans. A more detailed examination of the products of hydrolysis of carob galactomannan has been made. At least two contiguous anhydromannose units appear to be needed for scission. This is similar to the requirement for hydrolysis by microbial enzymes. Judas tree (Cercis siliquastrum) endosperm contained a polysaccharide with a unique composition for a legume seed reserve. Gel chromatography and electrophoresis on cellulose acetate indicated homogeneity. Hydrolysis with a mixture of β-mannanase and α-galactosidase gave a glucose-mannose disaccharide and acetolysis gave a galactose-mannose. These results, as well as the pattern of hydrolysis by β-mannanase were consistent with a galactoglucomannan structure.     

Reconstitution of the binding protein-dependent galactose transport of Salmonella typhimurium in proteoliposomes.

Binding protein-dependent transport systems mediate the accumulation of diverse substrates in bacteria. The binding protein-dependent galactose transport of Salmonella typhimurium has been reconstituted in proteoliposomes. The proteoliposomes were made with proteins solubilized and renatured from inclusion bodies produced by a bacterial strain containing a plasmid with the mgl (methylgalactose permease) operon of Salmonella typhimurium. Galactose transport is dependent both on the addition of the purified galactose binding protein to the transport assay, and on ATP. The interaction between the liganded galactose binding protein and proteoliposomes displays Michaelis type kinetics with a Km of around 15 microM. Galactose transport is coupled to ATP hydrolysis with a stoichiometry (ATP/galactose) of 2.5:1. Galactose transport in proteoliposomes is not significantly inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone, but is inhibited by 0.5 mM vanadate. The present reconstitution of galactose transport in proteoliposomes suggests that the MglA, MglC and MglE proteins have been solubilized and renatured in an active form from the inclusion bodies of the mgl hyperproducing strain.     

Determination of human serum galactosyltransferase using a kinetic spectrophotometric technic

A kinetic spectrophotometric method in which galactose transfer is coupled to the production of NADH, has been adapted to the assay of galactosyltransferase activity in human serum. Under the described conditions, the rate of NADH production is linear with regard to enzyme concentration, and directly depends upon the various biochemical factors which control galactosyltransferase activity.     

A colorimetric procedure for measuring the enzymatic hydrolysis of terminal galactose from GM ganglioside

The enzymatic hydrolysis of the terminal galactose from GM₁-ganglioside is monitored by a colorimetric procedure. The NADH generated from the oxidation of released galactose with NAD and galactose dehydrogenase is employed to reduce p-iodonitrotetrazolium and the absorbance of the product, p-iodonitrotetrazolium formazan, is measured. The method can detect as little as 0.5 nmol of galactose. Hydrolysis of GM₁-ganglioside is accomplished using β-galactosidase from the marine gastropod Turbo cornutus. The enzymatic release of galactose is maximal at pH 3.5, and the reaction rate is linearly proportional to incubation time for 30 min, under the conditions employed. The presence of GM₂-ganglioside in the reaction mixture, after hydrolysis has occurred, is demonstrated by thin-layer chromatography.     

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.     

The use of HPLC-pulsed amperometry for the characterization and assay of glycosidases and glycosyltransferases.

A sensitive and reproducible high performance chromatographic procedure is described for the assay of jack bean beta-galactosidase in which the reaction products are separated on a Dionex AS6 ion exchange column under alkaline conditions and detected by triple-pulsed amperometry. Quantition of the enzyme-released galactose is accomplished by using either fucose or lactose, the substrate, as an internal standard. The validity of the procedure as a general method for the assay and kinetic characterization of exoglycosidases was demonstrated by performing parallel measurements of galactose using an established coupled-enzyme assay, and using these values to calculate Km and Vmax values against lactose. Additional data are presented which establish the applicability of using a similar HPLC approach for the assay of glycosyltransferases.     

Metabolic control of lactose entry in Escherichia coli.

A general method has been developed for determining the rate of entry of lactose into cells of Escherichia coli that contain beta-galactosidase. Lactose entry is measured by either the glucose or galactose released after lactose hydrolysis. Since lactose is hydrolyzed by beta-galactosidase as soon as it enters the cell, this assay measures the activity of the lactose transport system with respect to the translocation step. Using assays of glucose release, lactose entry was studied in strain GN2, which does not phosphorylate glucose. Lactose entry was stimulated 3-fold when cells were also presented with readily metabolizable substrates. Entry of omicron-nitrophenyl-beta-D-galactopyranoside (ONPG) was only slightly elevated (1.5-fold) under the same conditions. The effects of arsenate treatment and anaerobiosis suggest that lactose entry may be limited by the need for reextrusion of protons which enter during H+/sugar cotransport. Entry of omicron-nitrophenyl-beta-D-galactopyranoside is less dependent on the need for proton reextrusion, probably because the stoichiometry of H+/substrate cotransport is greater for lactose than for ONPG.     

Exopolysaccharide Colanic Acid and Its Occurrence in the Enterobacteriaceae

A study of strains from the genera Salmonella, Escherichia, and Aerobacter has shown that under appropriate conditions many strains produce an exopolysaccharide slime of identical composition, which has been identified as colanic acid on the basis of its chemical composition and its sensitivity to certain bacteriophage-induced depolymerase enzymes. Chemical analysis shows that the polysaccharide contains O-acetyl groups in addition to the sugars glucose, galactose, fucose, and glucuronic acid. Mild acid hydrolysis has led to the isolation of a β-glucosylfucose in addition to glucuronic acid containing oligosaccharides. Many strains were found to synthesize colanic acid under normal conditions of growth or under conditions favoring polysaccharide synthesis, whereas others only synthesized colanic acid when the control mechanism was derepressed by p-fluorophenylalanine.     

On the role of superoxide radical in the mechanism of action of galactose oxidase

The inhibition of galactose oxidase by superoxide dismutase is a function of the method of assay, nature of substrate, and composition of incubation and assay mixtures, as well as the concentration of dismutase. A reasonable level of inhibition is attained only when superoxide dismutase is present prior to the onset of catalysis although this effect is not observed under all conditions tried. Peroxidase activates galactose oxidase and blocks its interaction with either superoxide dismutase or catalase. These results further obscure the possible role of superoxide radical in the galactose oxidase reaction.     

A single-cell assay of beta-galactosidase activity in Saccharomyces cerevisiae

A novel assay of single-cell exogenous beta-galactosidase activity in Saccharomyces cerevisiae has been developed. Intracellular fluorescence due to the hydrolysis of resorufin-beta-D-galactopyranoside attains a steady state between production of resorufin and its subsequent leakage from the cell. The cells are permeabilized with Triton X-100, and the assay is performed at 0 degrees C. These conditions were chosen to minimize intercellular fluorescence communication. Free resorufin in the extracellular space is bound by bovine serum albumin to prevent its uptake by cells. Two regimes of fluorescence accumulation are observed, one limited by the rate of diffusion of substrate into the cell, and one limited by the rate of enzymatic cleavage of the substrate. A quantitative correlation between fluorescence and beta-galactosidase activity is obtained under optimized assay conditions.     

Studies on the chemical composition of lipopolysaccharide from Neisseria meningitidis group B.

A lipopolysaccharide was isolated from Neisseria meningitidis group B by phenol/water extraction and purified by differential ultracentrifugation. This preparation exhibited endotoxic properties as shown by the limulus-lysate assay. Mild acid hydrolysis of the lipopolysaccharides yielded a lipid A fraction and a polysaccharide fraction. The lipid A fraction contained fatty acids, phosphorus and glucosamine. Analysis of the polysaccharide fraction revealed the presence of glucose, galactose, glucosamine, 2-keto-3-deoxyoctonic acid and phosphorus. There was no heptose.     

Synthesis and characterization of ubiquitin ethyl ester, a new substrate for ubiquitin carboxyl-terminal hydrolase

A new substrate for ubiquitin carboxyl-terminal hydrolase, the carboxyl-terminal ethyl ester of ubiquitin, has been synthesized by a trypsin-catalyzed transpeptidation. In the presence of 1.6 M glycylglycine ethyl ester, trypsin removes the carboxyl-terminal glycylglycine of ubiquitin and replaces it with the dipeptide ester. The equilibrium mixture under these conditions contains 30% ubiquitin ethyl ester and 70% hydrolysis product, the 74-residue fragment of ubiquitin. Ubiquitin ethyl ester can be purified by gel filtration and ion-exchange chromatography. The structure of this product has been verified by identification of the products of base hydrolysis, tryptic cleavage in aqueous solution, and peptide mapping. When ubiquitin ethyl ester is incubated with purified ubiquitin carboxyl-terminal hydrolase, specific cleavage of the ester linkage is observed. A rapid, sensitive assay is described utilizing high-performance liquid chromatography. By use of this assay, it has been shown that ubiquitin carboxyl-terminal hydrolase is inactivated in the absence of thiols. Optimal protective effects are seen with 10 mM dithiothreitol. The rate of catalysis is maximal at pH 8.5, with evidence for catalytically important groups with pK values of 5.2, 7.6, and 9.5. These findings are consistent with the participation of a thiol group in the active site. Native ubiquitin is a competitive inhibitor of ubiquitin ethyl ester hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubility and selective crystallization of lactose from solutions of its hydrolysis products glucose and galactose

A high degree of conversion is desired when lactose is hydrolyzed to glucose and galactose. This produces, however, a high concentration of galactose, which is inhibitory for the enzyme catalyst (beta-galactosidase). The inhibition can be reduced by limiting the conversion per pass over the enzyme (e.g. to ca. 50%), separating unconverted lactose from the reactor effluent, and recycling it to the reactor inlet. (This allows the overall conversion to be raised to ca. 80-90%). The solubilities of lactose, glucose, and galactose have been determined at various temperatures and for sugar mixtures having different concentrations and degrees of hydrolysis. Various cooling crystallizations have defined convenient and simple processes for the selective separation of lactose from its hydrolysis products.     

Structural features of the sulphated polysaccharide from a green seaweed, Cladophora socialis.

A sulphated heteropolysaccharide, [alpha]27D + 59.9 degrees, has been isolated from a green seaweed, Cladophora socialis, by extraction with dilute acid and purified by fractional precipitation. The polymer is composed of galactose (58.3%), arabinose (31.8%), xylose (10.6%) and sulphate (16.9%). The results of methylation analysis, periodate oxidation and partial acid hydrolysis studies indicate that the polymer is a branched one and is composed of 1,3-linked galactose and 1,4-linked arabinose units. Xylose is present at the non-reducing end position of the branches. Both arabinose and galactose carry branches. Desulphation and subsequent analysis of the polymer show that some of the arabinose units carry sulphate groups at C-3 and some of the galactose units carry the sulphate groups at C-4 and some at C-4 and C-6 as well.     

Galactose catabolism in Caulobacter crescentus.

Caulobacter crescentus wild-type strain CB13 is unable to utilize galactose as the sole carbon source unless derivatives of cyclic AMP are present. Spontaneous mutants have been isolated which are able to grow on galactose in the absence of exogenous cyclic nucleotides. These mutants and the wild-type strain were used to determine the pathway of galactose catabolism in this organism. It is shown here that C. crescentus catabolizes galactose by the Entner-Duodoroff pathway. Galactose is initially converted to galactonate by galactose dehydrogenase and then 2-keto-3-deoxy-6-phosphogalactonate aldolase catalyzes the hydrolysis of 2-keto-3-deoxy-6-phosphogalactonic acid to yield triose phosphate and pyruvate. Two enzymes of galactose catabolism, galactose dehydrogenase and 2-keto-3-deoxy-6-phosphogalactonate aldolase, were shown to be inducible and independently regulated. Furthermore, galactose uptake was observed to be regulated independently of the galactose catabolic enzymes.