Curtis F. Brewer

Determination of the concentrations of oligosaccharides, complex type carbohydrates, and glycoproteins using the phenol-sulfuric acid method☆

The concentrations of methyl glycosides, oligosaccharides, glycopeptides, and glycoproteins can be accurately determined by using calibration curves composed of the appropriate monosaccharide(s) obtained with a modified version of the colorimetric phenol-sulfuric acid method. Calibration curves of micrograms sugar vs. 490 nm for Man, Glc, or Gal are shown to provide reliable determinations (typically +/- 3-4%) of corresponding methyl glycosides and linear and branched-chain oligosaccharides containing the corresponding reactive hexose residue. For complex oligosaccharides containing a known mixture of reactive hexose units, the appropriate mixture of monosaccharides are shown to provide equally accurate calibration curves for concentration determinations. In the case of the soybean agglutinin, which is a tetramer possessing one Man9 oligomannose-type chain per subunit, the protein concentration was determined from the Man calibration curve which agreed with that obtained from the molar extinction coefficient of the protein.

X-ray crystallographic studies of unique cross-linked lattices between four isomeric biantennary oligosaccharides and soybean agglutinin

Soybean agglutinin (SBA) (Glycine max) is a tetrameric GalNAc/Gal-specific lectin which forms unique cross-linked complexes with a series of naturally occurring and synthetic multiantennary carbohydrates with terminal GalNAc or Gal residues [Gupta et al. (1994) Biochemistry 33, 7495-7504]. We recently reported the X-ray crystal structure of SBA cross-linked with a biantennary analog of the blood group I carbohydrate antigen [Dessen et al. (1995) Biochemistry 34, 4933-4942]. In order to determine the molecular basis of different carbohydrate-lectin cross-linked lattices, a comparison has been made of the X-ray crystallographic structures of SBA cross-linked with four isomeric analogs of the biantennary blood group I carbohydrate antigen. The four pentasaccharides possess the common structure of (beta-LacNAc)2Gal-beta-R, where R is -O(CH2)5COOCH3. The beta-LacNAc moieties in the four carbohydrates are linked to the 2,3-, 2,4-, 3,6-, and 2,6-positions of the core Gal residue(s), respectively. The structures of all four complexes have been refined to approximately 2.4-2.8 A. Noncovalent lattice formation in all four complexes is promoted uniquely by the bridging action of the two arms of each bivalent carbohydrate. Association between SBA tetramers involves binding of the terminal Gal residues of the pentasaccharides at identical sites in each monomer, with the sugar(s) cross-linking to a symmetry-related neighbor molecule. While the 2,4-, 3,6-, and 2,6-pentasaccharide complexes possess a common P6422 space group, their unit cell dimensions differ. The 2, 3-pentasaccharide cross-linked complex, on the other hand, possesses the space group I4122. Thus, all four complexes are crystallographically distinct. The four cross-linking carbohydrates are in similar conformations, possessing a pseudo-2-fold axis of symmetry which lies on a crystallographic 2-fold axis of symmetry in each lattice. In the case of the 3,6- and 2,6-pentasaccharides, the symmetry of their cross-linked lattices requires different rotamer orientations about their beta(1,6) glycosidic bonds. The results demonstrate that crystal packing interactions are the molecular basis for the formation of distinct cross-linked lattices between SBA and four isomeric pentasaccharides. The present findings are discussed in terms of lectins forming unique cross-linked complexes with glycoconjugate receptors in biological systems.

Stereochemical course of hydrolysis and hydration reactions catalysed by cellobiohydrolases I and II from Trichoderma reesei

Cellobiohydrolase I from Trichoderma reesei catalyzes the hydrolysis of methyl β‐D‐cellotrioside (K m = 48μM, k cat = 0.7 min−1) with release of the β‐cellobiose (retention of configuration). The same enzyme catalyzes the (trans‐hydration of cellobial (K m = 116 μM, K cat = 1.16 min−1) and lactal (K m = 135 μM, k cat = 1.35 min−1), presumably with glycosyi oxo‐carbonium ion mediation. Protonation of the double bond is from the direction opposite that assumed for methyl β‐cellotrioside, but products formed from these prochiral substrates are again of β configuration. Cellobiohydrolase II from the same microrganism hydrolyzes methyl β‐D‐cellotetraoside (K m = 4 μM, k cat = 112 min−1) with inversion of configuration to produce α‐cellobiose. The other reaction product, methyl β‐cellobioside, is in turn partly hydrolysed by Cellobiohydrolase II to form methyl β‐D‐glucoside and D‐glucose, presumably the α‐anomer. Reaction with cellobial is too slow to permit unequivocal determination of product configuration, but clear evidence is obtained that protonation occurs from the si‐direction, again opposite that assumed for protonating glycosidic substrates. These results add substantially to the growing evidence that individual glycosidases create the anomeric configuration of their reaction products by means that are independent of substrate configuration.

Synthesis of arbitrary frequency domain transmitting pulses applicable to pulsed NMR instruments

Abstract The synthesis of specialized excitation waveforms for pulsed NMR spin systems is an integral part of many present-day NMR experiments. Methods in use include various types of constant amplitude rectangular rf pulse trains with controlled interpulse delay times, modulating amplitudes of the envelopes of a chain of rectangular pulses, special long pulse rectangular pulses, and recently, single sideband spectral selections for NMR imaging. This paper describes, in quantitative fashion with experimental results, analysis and implementation of a method to generate pulsed NMR excitation waveforms using AM and FM techniques which yield individual pulses with controllable frequency spectral distributions. Examples given are of simplified cases, but the extension to complex frequency distribution requirements is clear.

Substrate-induced activation of maltose phosphorylase: Interaction with the anomeric hydroxyl group of α-maltose and α-d-glucose controls the enzyme's glucosyltransferase activity

Maltose phosphorylase, long considered strictly specific for beta-D-glucopyranosyl phosphate (beta-D-glucose 1-P), was found to catalyze the reaction beta-D-glucosyl fluoride + alpha-D-glucose----alpha-maltose + HF, at a rapid rate, V = 11.2 +/- 1.2 mumol/(min.mg), and K = 13.1 +/- 4.4 mM with alpha-D-glucose saturating, at 0 degrees C. This reaction is analogous to the synthesis of maltose from beta-D-glucose 1-P + D-glucose (the reverse of maltose phosphorolysis). In acting upon beta-D-glucosyl fluoride, maltose phosphorylase was found to use alpha-D-glucose as a cosubstrate but not beta-D-glucose or other close analogs (e.g., alpha-D-glucosyl fluoride) lacking an axial 1-OH group. Similarly, the enzyme was shown to use alpha-maltose as a substrate but not beta-maltose or close analogs (e.g., alpha-maltosyl fluoride) lacking an axial 1-OH group. These results indicate that interaction of the axial 1-OH group of the disaccharide donor or sugar acceptor with a particular protein group near the reaction center is required for effective catalysis. This interaction appears to be the means that leads maltose phosphorylase to promote a narrowly defined set of glucosyl transfer reactions with little hydrolysis, in contrast to other glycosylases that catalyze both hydrolytic and nonhydrolytic reactions.

Catalytic versatility of trehalase: Synthesis of α-d-glucopyranosyl α-d-xylopyranoside from β-d-glucosyl fluoride and α-d-xylose

Abstract Trehalase was previously shown (see ref. 5) to hydrolyze α- d -glucosyl fluoride, forming β- d -glucose, and to synthesize α,α-trehalose from β- d -glucosyl fluoride plus α- d -glucose. Present observations further define the enzymes separate cosubstrate requirements in utilizing these nonglycosidic substrates. α- d -Glucopyranose and α- d -xylopyranose were found to be uniquely effective in enabling Trichoderma reesei trehalase to catalyze reactions with β- d -glucosyl fluoride. As little as 0.2m m added α- d -glucose (0.4m m α- d -xylose) substantially increased the rate of enzymically catalyzed release of fluoride from 25m m β- d -glucosyl fluoride at 0°. Digest of β- d -glucosyl fluoride plus α- d -xylose yielded the α,α-trehalose analog, α- d -glucopyranosyl α- d -xylopyranoside, as a transient (i.e., subsequently hydrolyzed) transfer-product. The need for an aldopyranose acceptor having an axial 1-OH group when β- d -glucosyl fluoride is the donor, and for water when α- d -glucosyl fluoride is the substrate, indicates that the catalytic groups of trehalose have the flexibility to catalyze different stereochemical reactions.

Single-molecule pair studies of the interactions of the α-GalNAc (Tn-antigen) form of porcine submaxillary mucin with soybean agglutinin

Mucins form a group of heavily O‐glycosylated biologically important glycoproteins that are involved in a variety of biological functions, including modulating immune response, inflammation, and adhesion. Mucins are also involved in cancer and metastasis and often express diagnostic cancer antigens. Recently, a modified porcine submaxillary mucin (Tn‐PSM) containing GalNAcα1‐O‐Ser/Thr residues was shown to bind to soybean agglutinin (SBA) with ∼106‐fold enhanced affinity relative to GalNAcα1‐O‐Ser, the pancarcinoma carbohydrate antigen. In this study, dynamic force spectroscopy is used to investigate molecular pairs of SBA and Tn‐PSM. A number of force jumps that demonstrate unbinding or rebinding events were observed up to a distance equal to 2.0 μm, consistent with the length of the mucin chain. The unbinding force increased from 103 to 402 pN with increasing force loading rate. The position of the activation barrier in the energy landscape of the interaction was 0.1 nm. The lifetime of the SBA–TnPSM complex in the absence of applied force was determined to be in the range 1.3–1.9 s. Kinetic parameters describing the rate of dissociation of other sugar lectin interactions are in the range 3.3 × 10−3–2.5 × 10−3 s. The long lifetime of the SBA‐TnPSM complex is compatible with a binding model in which lectin molecules “bind and jump” from α‐GalNAc residue to α‐GalNAc residue along the polypeptide chain of Tn‐PSM before dissociating. These findings have important implications for the molecular recognition properties of mucins.

Phosphorylated human lectin galectin-3: analysis of ligand binding by histochemical monitoring of normal/malignant squamous epithelia and by isothermal titration calorimetry.

The human lectin galectin‐3 is a multifunctional effector with special functions in regulation of adhesion and apoptosis. Its unique trimodular organization includes the 12‐residue N‐terminal sequence, a substrate for protein kinase CK1‐dependent phosphorylation. As a step towards elucidating its significance, we prepared phosphorylated galectin‐3, labelled it and used it as a tool in histochemistry. We monitored normal and malignant squamous epithelia. Binding was suprabasal with obvious positive correlation to the degree of differentiation and negative correlation to proliferation. The staining pattern resembled that obtained with the unmodified lectin. Basal cell carcinomas were invariably negative. The epidermal positivity profile was akin to distribution of the desmosomal protein desmoglein, as also seen with keratinocytes in vitro. In all cases, binding was inhibitable by the presence of lactose, prompting further investigation of the activity of the lectin site by a sensitive biochemical method, i.e. isothermal titration calorimetry. The overall affinity and the individual enthalpic and entropic contributions were determined. No effect of phosphorylation was revealed. This strategic combination of histo‐ and biochemical techniques applied to an endogenous effector after its processing by a protein kinase thus enabled a detailed monitoring of the binding properties of the post‐translationally modified lectin. It underscores the value of using endogenous lectins as a histochemical tool. The documented approach has merit for applications beyond lectinology.

Preparation and use of α-maltosyl fluoride as a substrate by beta amylase

Abstract The preparation of pure (amorphous) α-maltosyl fluoride is described. A modification of the procedure of Brauns was used to obtain analytically pure, crystalline hepta- O -acetyl-α-maltosyl fluoride, the structure of which was assigned by 19 F-and 1 H-n.m.r. spectroscopy. α-Maltosyl fluoride was obtained by deacetylating the heptaacetate. It behaved as a single compound on thin-layer and paper chromatography, and was essentially completely hydrolyzed to maltose and hydrogen fluoride by 0.01 M sulfuric acid in 10 min at 100°. Crystalline beta amylase, likewise, catalyzed essentially complete hydrolysis of α-maltosyl fluoride to give maltose and hydrogen fluoride. The rates of hydrolysis catalyzed by beta amylase preparations from sweet potatoes and soybeans acting on a range of concentrations of the substrate produced linear curves for the relationship, 1/ v vs 1/ S ; reaction constants for crystalline, sweet-potato enzyme were K m 3.6 m M and V max ~ 2 μ mol/min/mg. The finding that α-maltosyl fluoride is hydrolyzed 30–60 times faster than maltotriose demonstrates for the first time that beta amylase is capable of effecting hydrolysis at an appreciable rate of a substrate having only two d -glucose residues.

Hydrolysis of β-d-glucopyranosyl fluoride to α-d-glucose catalyzed by Aspergillus niger α-d-glucosidase

Abstract Aspergillus niger α- d -glucosidase, crystallized and free of detectable activity for β- d -glucosides, catalyzes the slow hydrolysis of β- d -glucopyranosyl fluoride to form α- d -glucose. Maximal initial rates, V, for the hydrolysis of β- d -glucosyl fluoride, p-nitrophenyl α- d -glucopyranoside, and α- d -glucopyranosyl fluoride are 0.27, 0.75, and 78.5 μmol.min−1.mg−1, respectively, with corresponding V/K constants of 0.0068, 1.44, and 41.3. Independent lines of evidence make clear that the reaction stems from β- d -glucosyl fluoride and not from a contaminating trace of α- d -glucosyl fluoride, and is catalyzed by the α- d -glucosidase and not by an accompanying trace of β- d -glucosidase or glucoamylase. Maltotriose competitively inhibits the hydrolysis, and β- d -glucosyl fluoride in turn competitively inhibits the hydrolysis of p-nitrophenyl α- d -glucopyranoside, indicating that β- d -glucosyl fluoride is bound at the same site as known substrates for the α-glucosidase. Present findings provide new evidence that α-glucosidases are not restricted to α- d -glucosylic substrates or to reactions providing retention of configuration. They strongly support the concept that product configuration in glycosylase-catalyzed reactions is primarily determined by enzyme structures controlling the direction of approach of acceptor molecules to the reaction center rather than by the anomeric configuration of the substrate.