Self-assembling, pore-forming cytolysins are illustrative molecules for the study of the assembly and membrane insertion of transmembrane pores. Here we purified pleurotolysin, a novel sphingomyelin-specific two-component cytolysin from the basidiocarps of Pleurotus ostreatus and studied the pore-forming properties of the cytolysin. Pleurotolysin consisted of non-associated A (17 kDa) and B (59 kDa) components, which cooperatively caused leakage of potassium ions from human erythrocytes and swelling of the cells at nanomolar concentrations, leading to colloid-osmotic hemolysis. Hemolytic assays in the presence of poly(ethylene glycol)s with different hydrodynamic diameters suggested that pleurotolysin formed membrane pores with a functional diameter of 3.8 -5 nm. Pleurotolysin-induced lysis of human erythrocytes was specifically inhibited by the addition of sphingomyelin-cholesterol liposomes to the extracellular space. Pleurotolysin A specifically bound to sphingomyelin-cholesterol liposomes and caused leakage of the internal carboxyfluorescein in concert with pleurotolysin B. Experiments including solubilization of pleurotolysin-treated erythrocytes with 2% (w/v) SDS at 25°C and SDS-polyacrylamide gel electrophoresis/Western immunoblotting showed that pleurotolysin A and B bound to human erythrocytes in this sequence and assembled into an SDS-stable, 700-kDa complex. Ring-shaped structures with outer and inner diameters of 14 and 7 nm, respectively, were isolated from the solubilized erythrocyte membranes by a sucrose gradient centrifugation. Pleurotolysin A and B formed an SDS-stable, ring-shaped complex of the same dimensions on sphingomyelin-cholesterol liposomes as well.

Organ-specific variations in blood group H-like activity were observed in developing radish plants. A temporary increase in serological activity was found to occur in the roots at the earlier stages of development. Arabinogalactan-proteins (AGPs) were isolated from primary and mature roots, and investigated for changes in their physicochemical properties, structure, and serological activities. These root AGPs were composed mainly of L-arabinose and D-galactose but were distinguishable from each other in their contents of L-fucose as well as of protein and hydroxyproline. The structures of the carbohydrate moieties of the root AGPs were essentially similar to those of AGPs isolated from seeds and mature leaves in that they consisted of consecutive (1-_3)-}inked B-D-galactosyl backbone chains having side chains of (1-16)-linked ft-D-galactosyl residues, to which a-L-arabinofuranosyl residues were attached in the outer regions. One prominent feature of the prmary root AGPs was that they contained appreciable amounts of L-fucose, which was presumably responsible for expression of the serological activity. In their immunological reactions with rabbit anti-radish leaf AGP antibody, the root AGPs were shown to share common antigenic determinant(s) with those of seed and leaf AGPs.The wide distribution of AGs' and AGPs in various plants has drawn much attention to their structure, cellular localization, and physiological function (7, 9). L-Fucose-containing AGPs from aqueous extracts of mature radish leaves exhibit a potent blood group H-like activity, inhibiting the hemagglutination of human 0 erythrocytes with eel anti-H agglutinin (19,26). In contrast, an AG and an AGP isolated by us from radish seeds were found to contain D-xylose instead of L-fucose, thus lacking the serological activity (27). Further, it has been shown that leaf and seed AGPs differ from each other not only in the structures of their carbohydrate moieties, especially in the outer regions, but also in their amino acid compositions. These findings suggest that the composition and structure of AG and AGPs in radish plants are variable, depending on the physiological functions of the organs where the specific proteoglycans are produced.The observation of a transient increase in H-like activity in developing radish roots prompted us to examine the relationship between the appearance of the serological activity, and the com-' Abbreviations: AG, arabinogalactan; AGP, arabinogalactan-protein; PBS, 14.5 mM phosphate buffer, pH 7.2, containing 0.13 M NaCl; PBSN, PBS plus 0.02% (w/v) NaN3.positions and structures of the polysaccharides relevant to differentiation and growth of the roots. In this paper, we describe the characterization of AGPs isolated from primary and mature radish roots, and compare their properties and structures with those of AG and AGPs from other organs. MATERIALS AND METHODSPlant Materials. Seeds of the radish (Raphanus sativus L. var. hortensis cv Aokubi) were purchased from Tokita Seed and Plant Co., Ltd., Saitama, Japan. Th...

Crystal structures of a peptidyl‐Lys metalloendopeptidase (MEP) from the edible mushroom Grifola frondosa (GfMEP) were solved in four crystal forms. This represents the first structure of the new family `aspzincins' with a novel active‐site architecture. The active site is composed of two helices and a loop region and includes the HExxH and GTxDxxYG motifs conserved among aspzincins. His117, His121 and Asp130 coordinate to the catalytic zinc ligands. An electrostatically negative region composed of Asp154 and Glu157 attracts a positively charged Lys side chain of a substrate in a specific manner. A Tyr133 side chain located on the S1′ pocket had different configurations in two crystal forms and was not observed in the other crystal forms. The flexible Tyr133 plays two roles in the enzymatic function of GfMEP. The first is to provide a hydrophobic environment with Phe83 in order to accommodate the alkyl part of the Lys side chain of a substrate and the second is as a `proton donor' to the oxyanion of the tetrahedral transition state to stabilize the reaction transition state.

A basic,8-galactosidase (,8-Galase) has been purified 281-fold from imbibed radish (Raphanus sativus L.) seeds by conventional purification procedures. The purified enzyme is an electrophoretically homogeneous protein consisting of a single polypeptide with an apparent molecular mass of 45 kilodaltons and pi values of 8.6 to 8.8. The enzyme was maximally active at pH 4.0 on pnitrophenyl 8-D-galactoside and 8l-1,3-linked galactobiose. The enzyme activity was inhibited strongly by Hg2 and 4-chloromercuribenzoate. D-Galactono-(1_-4)-lactone and D-galactal acted as potent competitive inhibitors. Using galactooligosacchandes differing in the types of linkage as the substrates, it was demonstrated that radish seed ,B-Galase specifically split off j0-1,3-and ,8-1,6-linked D-galactosyl residues from the nonreducing ends, and their rates of hydrolysis increased with increasing chain lengths. Radish seed and leaf arabino-3,6-galactan-proteins were resistant to the 0-galase alone but could be partially degraded by the enzyme after the treatment with a fungal a-L-arabinofuranosidase leaving some oligosaccharides consisting of D-galactose, uronic acid, L-arabinose, and other minor sugar components besides D-galactose as the main product.,3-Galactosidases (EC 3.2.1.23) are widely distributed in various plant tissues (8). The enzymic properties, multiple forms, and specificities relevant to structural studies on glycoproteins have been investigated (2,8,19). Recently, interest in this enzyme has been focused on its in vivo functions concerning the degradation of such galactose-containing cell wall polysaccharides as galactan-pectin polymers and xyloglucan in relation to cell growth (16), fruit ripening (25), and seed germination (9). Further, characterization of a thylakoid bound f,-Galase' in wheat leaf chloroplasts implicated the role for the intermediary degradation of mono-and digalactosylglycerol abundant in thylakoid membranes during senescence of chloroplasts (4).Working with organs that develop after germination of radish seeds, we have recently reported the formation of organ-specific AGPs in primary and mature roots and leaves, which were clearly distinguishable from the seed proteogly-' Abbreviations: ,8-Galase, ,B-galactosidase; PNP-P-DGal, p-nitrophenyl l-D-galactoside; ONP-,#--Gal, o-nitrophenyl ,-D-galactoside; 4-MU-fl-n-Gal, 4-methylumbelliferyl fl-Dgalactoside; X-Gal, 5-bromo4-chloro-3-indolyl t-n-galactoside; AG, arabinogalactan; AGP, arabinogalactan-protein; a-L-Arafase, a-L-arabinofuranosidase; 2-ME, 2-mercaptoethanol. 567 cans in chemical composition, structure, and serological properties (21,30,31).In the present paper, we report the purification and characterization of a radish seed ,B-Galase whose specificity is highly restricted to f3-1,3-and :3-1,6-linked D-galactosyl residues, thereby participating in the degradation ofthe backbone structure of radish AGPs. MATERIALS AND METHODS Plant MaterialSeeds of the radish (Raphanus sativus L. var hortensis cv Aokubi) were purchased from Tokita Seed ...

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