John H. Pazur

Glycoenzymes: A note on the role for the carbohydrate moieties☆

Abstract The carbohydrate moieties of the glycoenzyme, glucoamylase I from Aspergillus niger are linked by O -glycosidic bonds to approximately 45 serine and threonine residues, presumably on the surface of the enzyme molecule. The glucoamylase is remarkably stable on storage at low temperatures. Extensive oxidation of the carbohydrate residues in the enzyme by periodate markedly affects the stability of the enzyme. It is suggested that the carbohydrate moieties function as stabilizers of the tridimensional structure of the glycoenzyme, and, in turn, of the catalytic property of the molecule.

Glycoenzymes: Structure and properties of the two forms of glucoamylase from Aspergillus niger

Abstract Aspergillus niger produces two forms (isoenzymes) of glucoamylase that are separable by electrophoresis or by chromatography on DEAE-cellulose and that are designated glucoamylase I and II. The molecular weight of glucoamylase I is 99,000, and that of glucoamylase II is 112,000. Both forms of the glucoamylase contain covalently linked carbohydrate (containing d -mannose, d -glucose, and d -galactose residues) and are therefore glycoenzymes. The carbohydrate-protein linkage in the glycoenzyme is primarily glycosidic to the hydroxyl group of l -serine and l -threonine residues, but glycosylamine linkages to l -asparagine and l -glutamine may also be present. Glucoamylase I and II possess identical amino acid compositions and, presumably, identical amino acid sequences. However, the two glycoenzymes differ in carbohydrate content, glucoamylase II containing nearly twice as many carbohydrate residues per molecule as glucoamylase I. Accordingly, it is suggested that the two forms of glucoamylase are isoglycoenzymes. The difference in electrophoretic and chromatographic properties of the isoglycoenzymes is probably due to a difference in the number of amide groups or glycosylaminically linked carbohydrate units in the polypeptide chains.

Affinity chromatography of macromolecular substances on adsorbents bearing carbohydrate ligands.

Publisher Summary This chapter provides information on the methods for preparing affinity adsorbents having carbohydrate ligands and illustrates the use of these adsorbents for the purification of representative, macromolecular substances. Affinity chromatography is possible because of the stereospecific interactions that can occur between the functional groups of one compound and the complementary groups of another. Affinity adsorbents prepared from cellulose may exhibit nonspecific adsorption, and the purification of the desired substance could be difficult to achieve. The p -aminophenyl glycosides have been most widely used for preparing affinity adsorbents having carbohydrate ligands. Many types of affinity adsorbent have been prepared by coupling ligands containing primary amino groups to agarose activated with cyanogen bromide. Antibodies that are induced by carbohydrate antigens and that combine with carbohydrate residues of these antigens have been designated antiglycosyl antibodies. Additional studies are required to establish whether the anti-isomaltose, myeloma protein is synthesized as a homogeneous protein or in multimolecular form.

Properties of the glucoamylase from Rhizopus delemar

Abstract The glucoamylase from Rhizopus delemar was purified by filtration and chromatography on Sephadex gel and ion-exchange resins. The purified enzyme is shown to be a glycoprotein containing residues of D -mannose and 2-amino-2-deoxy- D -glucose in its structure. The action pattern of the glucoamylase from R. delemar on

Glycoenzymes: Enzymes of Glycoprotein Structure

Publisher Summary Glycoenzymes contain carbohydrate residues covalently linked in their molecular structure. These enzymes are found in wide variety of biological materials, including plant tissues, animal organs, and microbial extracts. Glycoenzymes often occur in multimolecular forms and such forms are isoglycoenzymes, differing only in the carbohydrate portion of the molecules. It is noted that the hydrolase group of enzymes contains the largest number of glycoenzymes. The most common monosaccharide constituents of the carbohydrate moieties of glycoenzymes are D-mannose and 2-acetamido-2-deoxy-D-glucose. Other monosaccharides encountered are D-glucose, D-galactose, D-xylose, L-arabinose, L-fucose, and sialic acid. The monosaccharides can occur either as oligomeric or polymeric chains, attached to specific amino acid residues of the polypeptide chains of the enzymes. However, some glycoenzymes possess a single carbohydrate chain attached to a specific amino acid residue of the protein, others contain many such chains attached to many amino acid residues. The monosaccharide residues are glycosidically joined as in typical glycans.

Glycoenzymes: an unusual type of glycoprotein structure for a glucoamylase.

Glucoamylase, (1 leads to 4)(1 leads to 6)-alpha-D-glucan glucohydrolase (EC 3.2.1.3), hydrolyzes starch and glycogen completely to D-glucose and is used industrially in the manufacture of D-glucose from starch. The enzyme is elaborated by many types of fungi and occurs in two isoenzymic forms (glucoamylase I and glucoamylase II) in extracts from certain fungi. The isoenzymes from Aspergillus niger are glycoenzymes containing D-mannose, D-glucose, and D-galactose as integral structural components. New data from experiments on reductive alkaline beta-elimination and from methylation analyses show that the carbohydrate chains of glucoamylase I are linked O-glycosidically from D-mannose residues to L-serine or L-threonine residues of the protein moiety. In this enzyme, the carbohydrate residues are present as 20 single D-mannose residues, 11 disaccharides components having the structure 2-O-D-mannopyranosyl-D-mannose, 8 trisaccharides, and 5 tetrasaccharides composed of various combinations of D-mannose, D-glucose, and D-galactose residues joined by (1 leads to 3) and (1 leads to 6) glycosidic linkages. Such an array of carbohydrate chains in a glycoprotein is unusual, and may account for some of the unique properties exhibited by glucoamylase.

Glycans from streptococcal cell walls: The molecular structure and immunological properties of an antigenic glycan from Streptococcus bovis

Abstract The molecular structure and immunological properties of an antigenic glycan from the cell wall of Streptococcus bovis , strain C3, a member of the Group D Streptococci , have been determined by methylation analysis, periodate oxidation, and hapten inhibition methods. The glycan is shown to be a tetraheteroglycan composed of 6-deoxy- l -talose, l -rhamnose, d -galactose, and d -glucuronic acid. The sugar sequence and the types of glycosidic linkages of the glycan are: a main chain of l -rhamnosyl-(1,3)- d -galactosyl- (1,2)- l -rhamnosyl-(1,3)-6-deoxy- l -talosyl-(1,3)- units with d -glucuronosyl residues attached to position 4 of the first rhamnose of each repeating unit of the main chain. The d -glucuronic acid moiety is the primary immunodeterminant group of the glycan. On the basis of hapten inhibition data, it has been concluded that the binding of the antigen to the antibody occurs at the hydroxyl groups at positions 2 and 3 and the carboxyl group at position 6 of the d -glucuronic acid moieties. The antigen has been used to prepare antiserum with anti-glucuronic acid antibodies.

The synthesis of 1,6-anhydro-β-d-glucopyranose and d-glucosyl oligosaccharides from maltose by a fungal glucosyltransferase

Abstract The formation of 1,6-anhydro-β- d -glucopyranose and several d -glucosyl oligosaccharides has been observed during the action of a purified, fungal glucosyltransferase (EC 2.4.1.24) on maltose. Such products are synthesized by a transglucosylation mechanism involving the formation of a d -glucosyl-enzyme complex and the displacement of the d -glucosyl group by appropriate acceptor-substrates. The formation of the 1,6-anhydro bond is a novel type of transfer reaction and occurs by displacement of the enzyme from the d -glucosyl-enzyme complex by the proton of the primary hydroxyl group of the same glucosyl group. This reaction is characterized by inversion of configuration at the position of glucosidic bond-cleavage of the substrate. Synthesis of the d -glucosyl oligosaccharides occurs by displacement of the d -glucosyl groups from the enzyme by suitable acceptor-substrates. In these cases, the reactions are characterized by retention of configuration of the d -glucosidic bonds of the substrate. The list of oligosaccharides produced from maltose includes nigerose, kojibiose, isomaltose, maltotriose, panose, isomaltotriose, and 6- O - d -glucosyl-panose. The identity of these compounds has been established by methylation analysis and enzymic hydrolysis. d -Glucose is also a product of the reaction and arises from both the reducing and the non-reducing groups of maltose.

Comparison of the action of glucoamylase and glucosyltransferase on D-glucose, maltose, and malto-oligosaccharides.

The action patterns of glucoamylase (amyloglucosidase) and glucosyltransferase (transglucosylase) on D-[1-14C]glucose, [1-14C]maltose, and [1-14C]malto-oligosaccharides (labeled at position 1 of the D-glucose group at the reducing end) have been investigated by paper-chromatographic and oligosaccharide-mapping techniques. Under the conditions of the experiments, the extent of conversion of D-glucose and of maltose into new oligosaccharides was 2.2 and 1.9% with glucoamylase, and 5.7 and 33% with glucosyltransferase. The major oligosaccharides produced by both enzymes were isomaltose (6-O-alpha-D-glucopyranosyl-alpha-D-glucose), panose (O-alpha-D-glucopyranosyl (1 leads to 6)-O-alpha-D-glucopyranosyl-(1 leads to 4)-alpha-D-glucose), and nigerose (3-O-alpha-D-glucopyranosyl-alpha-D-glucose). The glucosyltransferase also synthesized oligosaccharides from malto-oligosaccharides of higher molecular weight to yield compounds having alpha-(1 leads to 6)-linked D-glucosyl groups at the non-reducing ends. Glucoamylase exhibited little, if any, such activity on malto-oligosaccharides.

The preparation and characterization of antibodies with specificity for the carbohydrate units of gum arabic and gum mesquite

Abstract Antibodies directed against terminal carbohydrate units of gum arabic and gum mesquite were deteced in sera of rabbits immunized intramuscularly with solutions of the gums and Freunds complete adjuvant. The antibodies were isolated by affinity chromatography on adsorbents of AH-Sepharose 4B containing ligands of the appropriate gum. From the sera of animals immunized with gum arabic, two sets of anti-carbohydrate antibodies were isolated and these were shown to have specificity for different disaccharide units at the non-reducing ends of the gum molecule. From the sera of animals immunized with gum mesquite only one set of anti-carbohydrate antibodies with specificity for a terminal disaccharide unit was obtained. Isoelectric focusing coupled with agar diffusion of the purified antibodies showed that all of the sets of antibodies were composed of isomeric proteins with each isomer exhibiting antibody activity. The antibodies of a set are appropriately termed isoantibodies and it is likely that each isomer is synthesized by a different immunocyte of the host. Hapten inhibition studies with oligosaccharides isolated from the gums showed that gum arabic possesses two different immunodeterminants with the structure α- l -arabinofuranosyl-(1→4)- d -glucuronic acid and β- d -glucuronosyl-(1→6)- d -galactose while gum mesquite possessed only one determinant with the structure 4-methyl-β- d -glucuronosyl-(1→6)- d -galactose.