The effect of glycophorin A on oxidation of globoside by galactose oxidase

The interaction of galactose oxidase with native and desialylated glycophorin A was studies by oxidizing human erythrocytes and globoside/phospholipid vesicles with the enzyme. Oxidation of the glycolipid was improved in the presence of vesicle-incorporationted glycophorin A. Although galactose oxidase is a very basic protein, it was not adsorbed on native human erythrocytes. Instead, neuraminidase-treated cells bound a substantial amount of galactose oxidase, but the enzyme seemed to be released into the buffer when desialylated glycoproteins had been oxidized.Abbreviation PBS 0.01 M sodium phosphate-0.15 M NaCl, pH 7.4     

A coupled enzyme assay for measurement of sialidase activity.

A multi-coupled enzyme assay system for determining sialidase activity is described. Enzymes, substrates and chromogens are reacted in situ and determined spectrophotometrically in ELISA microtiter plates. Sialidase is assayed by the extent of desialylated galactose on an appropriate sialoglycoconjugate (fetuin), which is otherwise unavailable for oxidation by galactose oxidase. The oxidation is monitored by the coupling of H2O2 released to a third enzyme, peroxidase. The rate of change of absorbance at 405 nm, resulting from the oxidized chromogen is a measure of the reaction rate of the coupled enzyme system. A similar system can be used for determining galactose oxidase in solution, or on blots using galactose as substrate. Due to the small-scale single-step measurement, the described assay is a sensitive, convenient, and inexpensive alternative to the classic colorimetric determination.     

Fluorometric determination of mucin-type glycoproteins by the galactose oxidase-peroxidase method

We developed a convenient and specific method for the determination of mucin-type glycoproteins using galactose oxidase and horseradish peroxidase on the basis of the contents of galactosyl and N-acetylgalactosaminyl residues in glycoproteins. Galactose and galactosamine residues released from glycoproteins after hydrolysis were oxidized with galactose oxidase and subsequently the resultant hydrogen peroxide was determined by a combination of horseradish peroxidase and 3-(p-hydroxyphenyl) propionic acid as a fluorogenic substrate. The contents of galactose/galactosamine residues in N- and O-glycans, as determined by the galactose oxidase-peroxidase method, were in good agreement with those described in the previous reports. We applied the present method to determine mucin-type glycoproteins secreted from rat gastric mucosa by stimulation with misoprostol, a prostaglandin E(1) analogue in vivo. Thus, the galactose oxidase-peroxidase method is useful for the determination of mucin-type glycoproteins in biological materials.     

Galactosyl transferase of a Golgi fraction from cultured neoplastic mast cells

Suspension cultures of neoplastic mouse mast cells were used to obtain large quantities of a homogeneous cell population as starting material for cell fractionation. A Golgi fraction was prepared by slight modification of established techniques and identified by electron microscopy. Assay of galactosyl transferase activity using ovalbumin, desialylated degalactosylated orosomucoid, and N-acetylglucosamine as galactose acceptors showed that the Golgi fraction was enriched in specific activity over the homogenate. The Golgi galactosyl transferase was examined in detail. Acceptor concentrations for optimal galactose incorporation were determined, and substrate inhibition effects were shown with higher concentrations of all three acceptors. Manganese was shown to be necessary for galactose incorporation. A higher concentration of manganese afforded some protection from substrate inhibition by acceptors, but at the same time was itself inhibitory. All three acceptors competed with one another for galactose incorporation, indicating that a single enzyme catalyzed the transfer of galactose for all acceptors.     

Serum inhibitors of desialylated glycoprotein binding to hepatocyte membranes.

Identification of the material present in human serum which is responsible for inhibition of binding of desialylated glycoproteins to rat hepatocyte membranes was accomplished by means of affinity chromatography using Sephadex to which the galactose-specific lectin, Ricinus Communis Agglutinin (RCAI) was covalently bound. RCAI-Sephadex was capable of extraction of virtually all of the inhibitory activity from cirrhotic serum. The RCA I-bound inhibitory activity could be eluted with 0.05 M D-galactose. The D-galactose eluate when subjected to radioimmunoelectrophoresis against a number of specific antibodies to human serum glycoproteins produced arcs corresponding to alpha 1-acid glycoprotein, alpha2-macroglobulin, IgG, IgA, and IgM. In another experiment putative terminal galactosyl groups of desialylated glycoproteins in the D-galactose eluate from cirrhotic serum exposed to RCAI-Sephadex were labelled with tritiated borohydride after treatment with galactose oxidase. Subsequent gel electrophoresis showed peaks of radioactivity throughout the area of the gel corresponding to protein molecular weights of the 19 S, 7 S, and 4 S classes. It thus appears that a heterogeneous population of desialylated serum glycoproteins accounts for the inhibition of binding of desialylated glycoprotein to the hepatocyte membrane and that these desialylated glycoproteins are present in small amounts in normal human serum and in greatly increased quantities in serum from patients with cirrhosis.     

Oxidation of glycolipids in liposomes by galactose oxidase

Small unilamellar phosphatidylcholine vesicles containing globo-series glycolipids were labeled by the galactose oxidase/NaB[3H]4 procedure. The major glycolipid of human red cells, globoside, was the best substrate for galactose oxidase both in vesicles and in tetrahydrofuran-containing buffer. The oxidation rates of membrane-bound ceramide trihexoside and Forssman glycolipid were one-fourth and one-tenth, respectively, of the oxidation rate of globoside. Membrane-bound ceramide dihexoside was not a substrate for galactose oxidase, although it was readily oxidized in tetrahydrofuran-containing buffer. Soluble sialoglycoproteins and membrane-incorporated glycophorin A stimulated the oxidation of globoside-containing vesicles, whereas membrane-bound GD1a ganglioside had no effect on globoside oxidation.     

Specificity of sialytransferase: structure of alpha1-acid glycoprotein sialylated in vitro. A new methodology for the determination of the structure of the linkage formed by glycosyltransferase action on galactosyl-oligosaccharide-protein acceptors.

The terminal galactosyl units of desialylated alpha1-acid glycoprotein were selectively labeled with tritium by a galactose oxidase/NaB3H4 procedure. The 3H-labeled glycoprotein was effective as an acceptor in sialytransferase reactions catalyzed by rat liver microsomes in vitro with unlabeled CMP-N-acetyl-neuramininic acid as sialic acid donor. Permethylation/hydrolysis of glycopeptides derived from the resialylated 3H-labeled glycoprotein yielded radioactive 2,3,4-trimethylgalactose indicating that rat liver microsomes are capable of transferring sialic acid to position C-6 of the terminal galactosyl units of desialylated alpha1-acid glycoprotein. No indication was obtained for transfer of sialic acid to other positions. This result is discussed in view of the multiplicity of positions of attachment of sialic acid to galactosyl residues in native alpha1-acid glycoprotein.     

External labeling of human erythrocyte glycoproteins. Studies with galactose oxidase and fluorography.

Glycoproteins of the human erythrocyte membrane were labeled with tritiated sodium borohydride after oxidation of terminal galactosyl and N-acetylgalactosaminyl residues with galactose oxidase. After separation of the polypeptides on polyacrylamide slab gels, a scintillator was introduced into the gel, and the radioactive proteins were visualed by autoradiography (fluorography). The following results were obtained. (a) The erythrocyte membrane contains at least 20 glycoproteins, many of which are minor components. (b) The carbohydrate of all the labeled glycoproteins is exposed only to the outside, since no additional glycoproteins can be labeled in isolated unsealed ghosts. (c) The membrane contains two major groups of glycoproteins. The first group of proteins contains sialic acids linked to the penultimate galactosyl/N-acetylgalactosaminyl residues, which are efficiently labeled only after pretreatment with neuraminidase. The second group has terminal galactosyl/N-acetylgalactosaminyl residues which can be easily labeled without neuraminidase treatment. The glycoproteins from fetal erythrocytes all belong to the first group, whereas only five glycoproteins of erythrocytes from adults belong. (d) Trypsin cleaves the proteins containing sialic acids, and fragments containing carbohydrate remain tightly bound and exposed in the membrane. (e) Pronase cleaves Band 3 in addition to the sialic acid containing glycoproteins, but most of the glycoproteins still remain unmodified in the membrane. (f) No difference is seen between membrane glycoproteins from cells of different ABH blood groups.     

The requirement for cell surface galactosyl residues during oxidative activation of normal and chronic lymphocytic leukemia lymphocytes.

Galactose oxidase stimulated normal and leukemic lymphocytes to undergo DNA synthesis and cell division. Although the response of normal lymphocytes to galactose oxidase was enhanced with neuraminidase pretreatment, substantial activation of leukemic lymphocytes required pretreatment with neuraminidase. Leukemic lymphocytes exhibited maximal response to neuraminidase-galactose oxidase later than that observed in normal lymphocytes. Treatment of lymphocytes with trypsin diminished their response to galactose oxidase. When lymphocytes were pretreated with β-galactosidase to specifically remove cell surface galactosyl residues, the response to galactose oxidase was prevented. The response of normal and leukemic lymphocytes to sodium periodate was also reduced after treatment with galactose oxidase. These data support the concept that oxidation of cell surface galactosyl residues is critical during lymphocyte activation.     

Role of tryptophan in the spectral and catalytic properties of the copper enzyme, galactose oxidase.

Previous results indicate that a tryptophan residue(s) may interact with the sugar substrate and Cu(II) atom of galactose oxidase (Ettinger, M. J., and Kosman, D. J. (1974), Biochemistry 13, 1248). We now show that N-bromosuccinimide (NBS) reduces enzymatic activity to 2% as two tryptophans are oxidized; only four residues are easily oxidized in the holoenzyme. An enzymatic activity vs. number of residues oxidized profile suggests that this inactivation is probably associated with only one of the first 2 residues oxidized. There is no evidence for chain cleavage or modification of amino acids other than tryptophan. While substrate protection is not afforded by the sugar substrate, the activity-related tryptophan is placed within the active-site locus by spectral evidence. NBS oxidation of two tryptophans results in a marked diminution of the large copper optical-activity transition at 314 nm. Under some reaction conditions, a doubling of ellipticity in the 600-nm region of copper CD is also observed. The effects of the NBS oxidation on the CD spectra of galactose oxidase permit the assignment of the 314-nm CD band to a charge-transfer transition and the 229-nm extremum to a specific tryptophan contribution. The AZZ parameter from electron spin resonance spectra is also markedly reduced by the NBS oxidation. Moreover, while cyanide binds to the native enzyme without reducing the Cu(II) atom, cyanide rapidly reduces the Cu(II) atom to Cu(I) in the NBS-oxidized enzyme. These CD and ESR results are taken to suggest that one aspect of the inactivation by NBS oxidation may be a conversion of the pseudosquare planar copper complex in the native enzyme to a more distorted, towards tetrahedral, complex in the inactivated enzyme. Since the inactivation can be accomplished without affecting binding of the sugar substrate, tryptophan oxidation must affect catalysis per se.     

Plasmodium berghei: modification of sialic acid on red cells from infected mouse blood

The surface membrane glycoproteins of normal mouse erythrocytes can be labeled by oxidation with either periodate or galactose oxidase in the presence of neuraminidase, followed by reduction with NaB³H₄. Without neuraminidase there is little galactose oxidase-catalyzed labeling of protein. Analysis of labeled proteins by SDS-polyacrylamide gel electrophoresis showed that both methods labeled the same set of glycoproteins. Plasmodium berghei infection dramatically reduced the sialoglycoprotein labeling of red blood cells from infected blood using the periodate/NaB³H₄ method. Provided neuraminidase was present, labeling by the galactose oxidase method gave identical results to normal erythrocytes. We conclude that the glycoprotein sialic acid of uninfected as well as infected red cells is modified during infection such that it is refractory to periodate oxidation. Acylation of the exocyclic hydroxyls of sialic acid is suggested to account for this. Lectin binding and cell agglutination experiments using Limulin, soybean and wheatgerm lectins, and concanavalin A confirmed and extended these observations. The possible implications of these results with regard to anemia induced by malaria are briefly discussed.     

Effect of O-sulphate groups in lactose and N-acetylneuraminyl-lactose on their enzymic hydrolysis.

1. Lactose 6''-O-sulphate, N-acetylneuraminyl-(alpha 2 leads to 3)-D-lactose 6''-O-sulphate, N-acetylneuraminyl ?-O-sulphate-(alpha 2 leads to 3)-D-lactose 6''0-O-sulphate, N-acetylneuraminyl ?-O-sulphate-(alpha 2 leads to 6)-D-lactose and N-acetylneuraminyl-(alpha 2 leads to 3)- and -(alpha 2 leads to 6))-lactose 6''-O-sulphate were prepared by chemical sulphation of lactose, N-acetylneuraminyl-lactose and tis isomers by using pyridine-SO3 reagent. 2. Significant kinetic differences were observed in the enzymic hydrolysis of the sulphated derivatives compared with unsubstituted substrates. 3. In the case of reactions catalysed by rat liver lysosomal and Clostridium perfringens neuraminidases (EC 3.2.1.18), the presence of an O-sulphate group in the N-acetylneuraminyl moiety affected the reaction by decreasing the Km and the Vmax, its presence in the galactosyl moiety affected the reaction by decreasing the Km and increasing the Vmax. and its presence in both N-acetylneuraminyl and galactosyl moieties decreased the Km and the Vmax. of the reaction. 4. Mixed-substrate reaction kinetic data indicated competition between the sulphated and unsubstituted substrates for the same active sites on the neuraminidase molecule. 5. Lactose 6''-O-sulphate neither behaved as a substrate nor acted as an inhibitor with respect to unsubstituted lactose and p-nitrophenyl beta-D-galactopyranoside when tested with lactase of suckling rat intestine and Escherichia coli beta-D-galactosidase (EC 3.2.1.23). 6. Preliminary investigation also indicated that, whereas glucose 6-O-sulphate and glucose 3-O-sulphate were were neither substrate nor inhibitor of glucose oxidase (EC 1.1.3.4), galactose 6-O-sulphate was oxidized half as fast as unsubstituted galactose by galactose dehydrogenase (EC 1.1.1.48).     

Enzymatic and chemical oxidation of gangliosides in cultured cells: effects of choleragen.

Cell surface glycolipids of normal human fibroblasts and NCTC2071 cells (transformed mouse fibroblasts) were labeled by incubating the intact cells with either galactose oxidase or sodium periodate, followed by reduction of the oxidized sugar residues with NaB3H4. In intact human fibroblasts, incorporation of 3H was increased with increasing time of exposure to galactose oxidase prior to treatment with NaB3H4. Following limited exposure to galactose oxidase, more label was incorporated into the larger glycolipids. Although labeling of the monosialoganglioside GM1 was maximal by 16 h, not all of the GM1 in the intact cells appeared to be accessible to galactose oxidase, since 10 to 12 times more GM1 was labeled when cells were disrupted before incubation with the enzyme. The human fibroblasts contained approximately 8 X 10(6) molecules of GM1 per cell. Maximal binding of choleragen (5 X 10(5) molecules of [125I]choleragen per cell) completely prevented cholevented oxidation of GM1 in intact fibroblasts by galactose oxidase but only partially protected the sialic acid moiety of GM1 from oxidation by periodate. Choleragen had little effect on the enzymatic or chemical oxidation of other glycolipids. NCTC 2071 cells do not contain endogenous GM1 but incorporate exogenous GM1 from the culture medium. When bound to NCTC 2071 cells, exogenous GM1 was protected by choleragen from oxidation by galactose oxidase or whether endogenous or taken up from the incubation medium, are, after interaction with choleragen, less accessible to oxidation by periodate or galactose oxidase.     

Alterations of red cell membranes from phenylhydrazine-treated rabbits

Reticulocytosis was induced in rabbits by two methods: phlebotomy and injection of phenylhydrazine. Normal erythrocytes, reticulocytes from bed rabbits, reticulocytes from phenylhydrazine-treated rabbits, and erythrocytes treated in vitro with phenylhydrazine were compared with respect to their plasma membrane labeling by galactose oxidase and NaB³H₄, and lactoperoxidase-catalyzed incorporation of ¹²⁵I. Normal erythrocyte membranes and membranes from reticulocytes of bled rabbits showed almost identical labeling patterns, the presence of 2–3 glycoproteins with moderate to low mobilities on dodecyl sulfate acrylamide gel electrophoresis. Labeling in the absence of enzyme was negligible. In contrast, the reticulocytes from phenylhydrazine-treated rabbits exhibited a large incorporation of tritium without prior treatment with galactose oxidase. Even after prereduction with unlabeled NaBH₄ to remove this nonspecific labeling, the labeled glycoprotein components found in normal erythrocytes were not detectable. Normal erythrocytes treated in vitro with phenylhydrazine, washed, and labeled with galactose oxidase had labeling patterns, including high nonspecific incorporation of ³H, similar to those observed with in vivo phenylhydrazine treatment.Solubilization of membranes with lithium diiososalicylate followed by partitioning with phenol showed that the same glycoproteins were presented in normal or phenylhydrazine membranes, although only the former extract was labeled by galactose oxidase. Individual carbohydrates from the membranes were analyzed by gas-liquid chromatography and, in the case of hexosamines, on the amino acid analyzer. The results of these analyses indicated a slight decline in galactose content with phenylhydrazine treatment. Reticulocyte membranes from bled rabbits also showed a decrease in galactose content, although it was less pronounced.Most of the label incorporated by nonspecific borohydride labeling of membranes from phenylhydrazine-treated animals was found associated with protein. The modified amino acids from labeled proteins are similar to those formed in reactions of oxidized lipids and proteins in model systems.     

New technique for enzymic hydrolysis of glycosphingolipids

A method is described for the study of glycosyl ceramide glycosyl hydrolases. Problems arising from the limited solubility of glycosyl ceramides in aqueous media were overcome by coating the substrate on a filter paper disc that had been treated with phosphatidyl choline. A comparison between the disc method and conventional dispersion of the substrate by detergents was made with two enzymes, galactosylgalactosyl-glucosyl ceramide galactosyl hydrolase (trihexosyl ceramide galactosyl hydrolase) from lysosomes of human and rat small intestine and human spleen, and d-galactose oxidase. In both cases enzymatic activity was greater with the paper disc method than it was with substrates dispersed by detergents. The galactose liberated by the glycosyl hydrolase was determined as the trimethylsilyl derivative of the free sugar by gas-liquid chromatography.     

Enzymatically oxidized lactose and derivatives thereof as potential protein cross-linkers

The enzyme galactose oxidase [EC 1.1.3.9] was applied to convert lactose, lactylamine and lactobionic acid into their corresponding 6'-aldehyde compounds. The potential protein cross-linking ability of these oxidized lactose and derivatives thereof was investigated using n-butylamine as the model compound. First, oxidized lactose gave double Maillard reaction products that were stable under mild alkaline conditions. Second, reductive amination of lactose followed by enzymatic oxidation gave cross-links that were stable under both neutral and alkaline conditions. Third, stable cross-links were obtained through enzymatic oxidation and amidation of lactobionic acid.     

External labeling of galactose in surface membrane glycoproteins of the intact myelin sheath.

The molecular organization of surface galactose residues in glycoproteins of the intact myelin sheath was investigated using the enzymatic membrane probe, galactose oxidase. Rat spinal cords treated under physiological conditions with this nonpermanent probe were labeled specifically in galactose residues by reduction with tritiated sodium borohydride. The enzymatically modified proteins from isolated myelin were analyzed electrophoretically and their specific radioactivities determined. Results indicated tritium label associated with a surprising variety of high molecular weight proteins. The most extensively labeled peak corresponded to the major myelin glycoprotein as indicated by the coincidence of tritium label with that of [14C]fucose used as an internal marker for the glycoproteins. The radioactivity associated with this protein was 1.1 to 2.7 times higher after treatment with galactose oxidase when compared to reduction in the absence of the enzyme and 1.4 to 4.8 times higher when oxidized and reduced after prior treatment with neuraminidase. The results suggest a complex heterogeneity of minor glycoproteins associated with isolated myelin. It is concluded that from this complexity of glycoproteins, a major glycoprotein is at least partially localized on the external surface of either the intact myelin sheath or the closely associated oligodendroglial plasma membrane. Such a localization of this glycoprotein and the probable localization of the other glycoproteins enhances their potential role in specific interactions in the process of mpyelination or myelin maintenance.     

Loss of covalently labeled glycoproteins and glycolipids from the surface of newly transformed schistosomula of Schistosoma mansoni

Schistosomula of Schistosoma mansoni were labeled by oxidation with galactose oxidase or with periodate followed by reduction with NaB3H4 to study the loss of the surface membrane of these parasites in vitro. Grain counts of light microscope autoradiographs (LMARG) of radiolabeled schistosomula show that both galactose oxidase and periodate specifically label the surface of the organisms. Galactose oxidase labels 11 glycoproteins on the surface of skin and mechanical schistosomula, ranging in apparent molecular weight from 17,000 to greater than 105,000. These glycoproteins are lost from the surface of schistosomula with a halftime of 10-15 h in culture in defined medium. Most of these glycoproteins appear to be shed intact from the surface of the schistosomula rather than endocytosed and degraded, because greater than 50% of each of the lost proteins can be recovered by trichloroacetic acid precipitation of the culture medium and because there is no internalization of the radiolabels into cultured schistosomula examined by LMARG. In addition to glycoproteins, periodate labels at least seven glycolipids on the surface of mechanical schistosomula. After culture for 15 h, more than half of each of these periodate-labeled proteins and lipids are lost from the schistosomula, and their abundance relative to each other remains similar to that of freshly labeled organisms. Since both proteins and lipids are lost from the surface of the schistosomula at the same rate, we believe that we are observing a general loss of the parasite surface membrane.     

Stereospecific oxidation of aliphatic alcohols catalyzed by galactose oxidase

The stereospecificity of galactose oxidase (EC 1.1.3.9) from Dactylium dendroides in the oxidation of simple three-carbon alcohols has been examined. The enzyme oxidizes glycerol to optically pure S(?)glyceraldehyde. In addition to this prochiral stereospecificity, galactose oxidase also exhibits enantiomeric stereospecificity in the oxidation of 3-halo-1,2-propanediols: the R isomer appears to be a better substrate than its S counterpart. The above stereochemistry of galactose oxidase-catalyzed oxidation of “unnatural” substrates, non-sugar alcohols, can be predicted on the basis of the conformation of the natural substrate of the enzyme, D-galactose.     

Mechanism of "uncoupled" tetrahydropterin oxidation by phenylalanine hydroxylase

Phenylalanine hydroxylase can catalyze the oxidation of its tetrahydropterin cofactor without concomitant substrate hydroxylation. We now report that this "uncoupled" tetrahydropterin oxidation is mechanistically distinct from normal enzyme turnover. Tetrahydropterins are oxygenated to 4a-carbinolamines only during catalytic events involving substrate hydroxylation. In the absence of hydroxylation tetrahydropterins are oxidized directly to quinonoid dihydropterins. Stoichiometry studies define a ratio of two tetrahydropterins oxidized per O2 consumed in uncoupled enzyme turnover, thus indicating the complete reduction of O2 to H2O. Complementary results establish the lack of H2O2 production by the enzyme when uncoupled and define a tetrahydropterin oxidase activity for the enzyme. Thus, the hydroxylating intermediate of phenylalanine hydroxylase may be discharged in two ways, by substrate hydroxylation or by electron abstraction. A mechanism is proposed for the uncoupled oxidation of tetrahydropterins by phenylalanine hydroxylase, and the significance of these findings is discussed.