Kunio Oishi

Sugar specificity of anti-B hemagglutinin produced by Streptomyces sp

Abstract A hemagglutinin specific for blood group B antigen has been purified to 190-fold from the culture fluid of a strain of Streptomyces sp. by conventional procedure involving ammonium sulfate fractionation and column chromatography. The molecular weight of the partially purified preparation was estimated to be approximately 5000±1000; this value is extremely small as compared with those of hemagglutinins which have been so far isolated from various sources. Hemagglutination-inhibition tests revealed that the Streptomyces agglutinin has a specificity to combine with D-galactose and several saccharides having D-galactose residues at the non-reducing terminal, and that the special configuration of the hydroxyl groups at C-2 and C-4, particularly the hydroxyl group at C-2, is essential for binding of the sugars to the hemagglutinin.

Formation and Regeneration of Protoplasts from Conidiobolus lamprauges

Summary: Protoplasts were formed from aged germlings and mycelia of Conidiobolus lamprauges, using a combination of chitinase and β-glucanase. Methanol, ethanol and propanol stimulated protoplast formation, apparently by a mechanism different from that of mercaptoethanol. Regeneration of protoplasts in soft agar was detectable by light microscopy 2 h after incubation and colonies were observable by the naked eye within 40 h; the regeneration rate was 65%.

Studies on carbohydrate binding to a lectin purified from Streptomyces sp.

The anti-B specific lectin produced by Streptomyces sp. was shown to have two carbohydrate-binding sites with binding constants of 8.3 . 10(3) M-1 (15 degrees C) and 2.2 . 10(3) M-1 (4 degrees C) for L-rhamnose and D-galactose, respectively, calculated according to Scatchard plots. The binding of specific sugars to the lectin not only induced a peculiar ultraviolet difference spectrum showing a blue shift of tryptophan absorption, but also caused crystallization of the lectin at a concentration of 1 mg per ml or more. The solvent-perturbation studies on the lectin showed that the number of solvent-exposed tryptophan (or average extent of exposure) was two in the absence of L-rhamnose, and three in the presence of the sugar. This suggests that one tryptophan residue appears outside as the result of sugar-binding to the lectin, which is reflected by the difference spectra. Oxidation of two tryptophan residues with N-bromosuccinimide led to complete loss of carbohydrate-binding activity of the lectin, indicating that these residues are important for retaining the activity.

Stereochemical structure recognized by the L-fucose-specific hemagglutinin produced by Streptomyces sp

A hemagglutinin has been purified 4000-fold from the culture filtrate of a strain of Streptomyces by affinity chromatography. The purified preparation was judged to be homogeneous by gel electrophoresis and its molecular weight was estimated to be about 70 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It may exhibit its full hemagglutinating activity in the monomer form. This hemagglutinin strongly agglutinated human blood group O erythrocytes and was inhibited by L-fucose. It was, however, distinct from the known L-fucose-specific hemagglutinins; first, the hemagglutinating activity of the purified preparation was more than 100-times stronger than that of others; second, D-mannose was a potent inhibitor of this hemagglutinin besides L-fucose but not or scarcely inhibitory to others; and third, p-nitrophenyl-beta-L-fucoside was more inhibitory to this hemagglutinin than p-nitrophenyl-alpha-L-fucoside as opposed to the case of others.

Specific inhibition of viral neuraminidases by an inhibitor, neuraminin, produced by Streptomyces sp.

Abstract Neuraminin was characterized with respect to its inhibition of viral and bacterial neuraminidases. It inhibits the viral but not the bacterial enzymes. In addition, neuraminin distinguishes influenza from Newcastle disease virus. Neuraminin inhibits hydrolysis of fetuin, orosomucoid, and human red blood cell surface glycoproteins by influenza virus or its isolated neuraminidase. The inhibition does not occur when neuraminlactose or colominic acid (a homopolymer of N -acetylneuraminic acid) is used as the substrate. In contrast, neuraminin equally effectively inhibits the action of the Newcastle disease virus toward these substrates. The inhibition was noncompetitive and saturated at a very low concentration of the inhibitor irrespective of the kind and concentration of the substrates.

Conversion of Human Blood Group B and AB Red Blood Cells to Group O and A Cells by Streptomyces Enzyme

Human blood group B and AB cells were converted to O and A active cells, respectively, by treatment with an α-d-galactosidase from Streptomyces 9917S2. Accompanying these conversions a 2 to 8-fold increase was observed in the group H activity on these cells. The conversion reaction proceeded rapidly in phosphate buffered saline without any supplement, Hemolysis, discoloration, and other visible deterioration of the cell were not observed during the process. Conditions for the optimal conversion as well as the susceptibility to the inhibition by sugars were somewhat different from those for the reaction with soluble substrates.

Blood Group Substance-degrading Enzymes Obtained from Streptomyces sp.: Part II. Purification and Characteristics of α-Galactosidase from Streptomyces 9917S2

The blood group B substance-degrading activity of Streptomyces 9917S2 is induced by galactosides as α-galactosidase activity is. Purification of the α-galactosidase was attempted by chromatography on DEAE-Sephadex and Sephadex. The purified preparation was shown to be free from α- and β-glucosidases, β-galactosidase, α- and β-glucosaminidases, and α- and β-galactosaminidases activities. The blood group B substance-degrading activity was present only in this fraction. This enzyme preparation cleaves α-(1→3)- and α-(1→6)-galactosidic linkages. The activity is inhibited by d-galactose, melibiose, and raffinose and also by l-arabinose and d-xylose.

CHITIN-BINDING LECTINS FROM FUNGI

The interaction of chitin and related saccharides with a fungal lectin (CLA) and plant lectins (WGA and STA) was studied. All these lectins are equally known to be chitin-and chitosaccharide-binding lectins, but their detailed specificities for erythrocytes and chitosaccharides were quite different from each other. WGA and STA agglutinated rabbit and sheep erythrocytes as well as human erythrocytes, whereas CLA did not. Agglutination of human erythrocytes by STA was strongly inhibited by water-soluble chitin derivatives such as ethylene glycol chitin and carboxymethyl chitin. Hemagglutination by CLA was inhibited only by ethylene glycol chitin, and that by WGA was never inhibited by these chitin derivatives. The precipitation reaction, however, occurred independently of this hemagglutination inhibition. It was observed only between ethylene glycol chitin and WGA, and not between carboxymethyl chitin and WGA, and other combinations of chitin derivatives and lectins. Water-insoluble chitin and chitosan derivatives adsorbed WGA and STA efficiently, but adsorbed CLA relatively less. The mechanism of interaction between chitosaccharides and lectins and possibility of application of these lectins to isolation and structural analysis of chitosaccharides are discussed.

Inhibition of restriction endonucleases by commercial polysaccharides

Abstract Commercial polysaccharide preparations were investigated for their restriction enzyme-inhibitory activities. Dextran sulfate (S content 18.5%) and laminaran from Eisenia arborea (0.88%) had marked inhibitory activity and haparin (13.1%) and ξ-, κ-, and λ-carrageenans (3.0, 3.8, and 4.3%) showed moderate inhibition. The effects of sulfation level and the structure of the carbohydrate moiety on the inhibitory activity were discussed.

Flocculation of influenza virus by a neuraminidase inhibitor, neuraminin, produced by Streptomyces sp.

Abstract Flocculation of virions by a viral neuraminidase inhibitor, neuraminin, was examined by low speed centrifugation and electron microscopy. The inhibitor could flocculate virions of influenza viruses but not those of Newcastle disease viruses. Virion-free neuraminidase of influenza virus was also flocculated by this inhibitor. The flocculation was temperature dependent and proceeded rapidly over a wide pH range. The amount of the inhibitor required for complete flocculation of virions was similar to that for the maximum inhibition of neuraminidase. Viral neuraminin receptors were stable to various enzymological, physical, and chemical treatments which caused a loss or reduction of the activity of neuraminidase.