Isolation and characterization of lectins and lectin-alliinase complexes from bulbs of garlic (Allium sativum) and ramsons (Allium ursinum)

A procedure developed to separate the homodimeric and heterodimeric mannose-binding lectins from bulbs of garlic (Allium sativum L.) and ramsons (Allium ursinum L.) also enabled the isolation of stable lectin-alliinase complexes. Characterization of the individual lectins indicated that, in spite of their different molecular structure, the homomeric and heteromeric lectins resemble each other reasonably well with respect to their agglutination properties and carbohydrate-binding specificity. However, a detailed analysis of the lectin-alliinase complexes from garlic and ramsons bulbs demonstrated that only the heterodimeric lectins are capable of binding to the glycan chains of the alliinase molecules (EC 4.4.1.4). Moreover, it appears that only a subpopulation of the alliinase molecules is involved in the formation of lectin-alliinase complexes and that the complexed alliinase contains more glycan chains than the free enzyme. Finally, some arguments are given that the lectin-alliinase complexes do not occur in vivo but are formed in vitro after homogenization of the tissue. This revised version was published online in November 2006 with corrections to the Cover Date.     

New mannose-specific lectins from garlic (Allium sativum) and ramsons (Allium ursinum) bulbs.

Two new mannose-binding lectins were isolated from garlic (Allium sativum, ASA) and ramsons (Allium ursinum, AUA) bulbs, of the family Alliaceae, by affinity chromatography on immobilized mannose. The carbohydrate-binding specificity of these two lectins was studied by quantitative precipitation and hapten-inhibition assay. ASA reacted strongly with a synthetic linear (1----3)-alpha-D-mannan and S. cerevisiae mannan, weakly with a synthetic (1----6)-alpha-D-mannan, and failed to precipitate with galactomannans from T. gropengiesseri and T. lactis-condensi, a linear mannopentaose, and murine IgM. On the other hand, AUA gave a strong reaction of precipitation with murine IgM, and good reactions with S. cerevisiae mannan and both synthetic linear mannans, suggesting that the two lectins have somewhat different binding specificities for alpha-D-mannosyl units. Of the saccharides tested as inhibitors of precipitation, those with alpha-(1----3)-linked mannosyl units were the best inhibitors of ASA, the alpha-(1----2)-, alpha-(1----4)-, and alpha-(1----6)-linked mannobioses and biosides having less than one eighth the affinity of the alpha-(1----3)-linked compounds. The N-terminal amino acid sequence of ASA exhibits 79% homology with that of AUA, and moderately high homology (53%) with that of snowdrop bulb lectin, also an alpha-D-mannosyl-binding lectin.     

Antifungal saponins from bulbs of garlic,Allium sativum L. var. Voghiera

A bioassay-guided phytochemical analysis of the polar extract from the bulbs of garlic, Allium sativum L., var. Voghiera, typical of Voghiera, Ferrara (Italy), allowed the isolation of ten furostanol saponins; voghieroside A1/A2 and voghieroside B1/B2, based on the rare agapanthagenin aglycone; voghieroside C1/C2, based on agigenin aglycone; and voghieroside D1/D2 and E1/E2, based on gitogenin aglycone. In addition, we found two known spirostanol saponins, agigenin 3-O-trisaccharide and gitogenin 3-O-tetrasaccharide. The chemical structures of the isolated compounds were established through a combination of extensive nuclear magnetic resonance, mass spectrometry and chemical analyses. High concentrations of two eugenol diglycosides were also found for the first time in Allium spp. The isolated compounds were evaluated for their antimicrobial activity towards two fungal species, the air-borne pathogen Botrytis cinerea and the antagonistic fungus Trichoderma harzianum.     

Phospholipase D from Allium sativum bulbs: A highly active and thermal stable enzyme

This is the first report on the identification and partial characterization of phospholipase D (EC 3.1.4.4) from Allium sativum (garlic) bulbs (PLD_GB). The enzyme shares the phenomenon of interfacial activation with other lipolytic enzymes, i.e. the hydrolytic rate increases when the substrate changes to a more aggregated state. The enzyme activity is highly temperature tolerant and the temperature optimum was measured to be 70 °C. PLD_GB unlike many plant PLDs exhibited high thermal stability. It was activated further after exposure to high temperatures, i.e. 80 °C, indicating that the enzyme refolds better upon cooling back to room temperature after short exposure to thermal stress. The activity of PLD_GB is optimum in 70 mM calcium ion concentration and the enzyme is activated further in the presence of phosphatidyl-4,5-bisphosphate (PIP₂). PLD_GB exhibited both hydrolytic and transphosphatidylation activities, both of which appear to be higher than those of PLD from cabbage leaves (PLD_CL).     

Isolation of alliumin, a novel protein with antimicrobial and antiproliferative activities from multiple-cloved garlic bulbs

A protein designated alliumin, with a molecular mass of 13 kDa and an N-terminal sequence similar to a partial sequence of glucanase, and demonstrating antifungal activity against Mycosphaerella arachidicola, but not against Fusarium oxysporum, was isolated from multiple-cloved garlic (Allium sativum) bulbs. The protein, designated as alliumin, was purified using ion exchange chromatography on DEAE-cellulose, CM-cellulose and Mono S, affinity chromatography on Affi-gel blue gel, and gel filtration on Superdex 75. Alliumin was unadsorbed on DEAE-cellulose, but was adsorbed on Affi-gel blue gel, CM-cellulose and Mono S. Its antifungal activity was retained after boiling for 1 h and also after treatment with trypsin or chymotrypsin (1:1, w/w) for 30 min at room temperature. Alliumin was inhibitory to the bacterium Pseudomonas fluorescens and exerted antiproliferative activity toward leukemia L1210 cells. However, it was devoid of ribonuclease activity, protease activity, mitogenic activity toward mouse splenocytes, and antiproliferative activity toward hepatoma Hep G2 cells.     

Isolation of allicepin, a novel antifungal peptide from onion (Allium cepa) bulbs.

From the bulbs of the onion Allium cepa, a novel antifungal peptide distinct from the antimicrobial peptide previously reported from onion seeds was isolated. The antifungal peptide, designated allicepin, was purified with a procedure that involved aqueous extraction, ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel and FPLC-gel filtration on Superdex 75. Allicepin was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel. The molecular weight of allicepin was estimated to be 10 K by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration on Superdex 75. Allicepin exerted an inhibitory activity on mycelial growth in several fungal species including Botrytis cinerea, Fusarium oxysporum, Mycosphaerella arachidicola and Physalospora piricola.     

Isolation and characterization of 5-lipoxygenase from tulip bulbs

An unique membrane bound lipoxygenase was isolated and purified from purple star tulip bulbs with a specific activity of 5.2 mu moles O2 consumed.min-1.mg-1 protein. The purified tulip enzyme exhibits regiospecificity for O2 insertion at C-5 of the arachidonic acid molecule. Identification of the reaction product was confirmed as 5-hydroperoxyeicosatetraenoic acid by analytical criteria which included: cochromatography with the authentic compound, as well as mass spectral and 1H-NMR analysis. Thus, the enzyme from tulip bulbs appears to be different from the cytosolic lipoxygenase from potato tubers, which exhibits non-regiospecificity in terms of O2 incorporation. However, the purified tulip lipoxygenase showed a strong immunological crossreactivity with antiserum raised against the purified potato lipoxygenase, indicating close immunological relationship with the other plant lipoxygenases.     

Isolation and characterization of a lectin from tulip bulbs, Tulipa gesneriana

A lectin, which agglutinated specifically the yeast cells of the Saccharomyces genus, was isolated from tulip bulbs (Tulipa gesneriana) using affinity chromatography on mannan-Sepharose 4B. Its relative molecular mass was determined by gel filtration to be approximately 67,000. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, a relative molecular mass of 17,000 was obtained, suggesting that the lectin is a tetramer. Binding studies performed with iodinated lectin indicated that Saccharomyces cerevisiae cells contained approximately 5.7 X 10(6) binding sites per cell, whereas little binding was observed with yeasts other than the Saccharomyces genus, bacteria and animal erythrocytes. D-Mannose, D-mannose 6-phosphate, L-fucose and L-fucosylamine were potent inhibitors of the lectin binding to S. cerevisiae cells, while, D-glucose, D-galactose and D-mannosamine were inactive, indicating that hydroxyl group at C-2 of D-mannose was essential for the lectin binding. Furthermore, inhibition experiments, using various manno-oligosaccharides, suggested that the lectin recognized (1----6)-linked manno-oligosaccharide units larger than mannobiose.     

大蒜产香内生真菌的分离及其挥发性成分分析

大蒜(Allium sativum L.)是百合科葱属常见的植物,富含丰富的生物活性成分和营养物质。本研究采用组织分离法从新鲜的大蒜中分离内生真菌并进行纯化,获得1株产香浓郁的菌株KLBMPAS015。采用固相微萃取固定吸附,结合气相色谱-质谱解吸附分析技术(SPME-GC-MS)对菌株KLBMPAS015的挥发成分进行分析。结果表明:该菌株代谢产物的香气成分主要为:n-丁酸2-乙基己基酯(n-butyric acid 2-ethylhexyl ester)(1.36%)、1-丁醇3-甲基-丙酸(1-butanol,3-methyl-,propanoate)(25.40%)和戊基惕各酸(iso-amyl tiglate)(65.51%)。产香真菌KLBMPAS015的戊基惕各酸含量相对较高,在天然香料生产中具有重要的潜在应用价值。     

Isolation and characterization of two enzymatic activities from chick embryos which degrade double-stranded RNA

Enzymatic activities capable of degrading double-stranded RNA have been solubilized from whole 9-day-old chick embryos and separated by ion exchange chromatography on DEAE-cellulose into two classes, designated nucleases DI and DII. Nuclease DI exhibits an absolute requirement for Mn2+ in the range of 5 to 10 mM. Monovalent cations, including K+, Na+, and NH4+, are inhibitory. The molecular weight of DI is 60,000 to 62,500 as estimated from sedimentation in sucrose density gradients. Following gradient fractionation, nuclease DI possesses the ability to degrade several substrates exhibiting a 250-fold preference for poly(rC) as compared to poly(rC)-poly(rG). The activity responsible for degrading double-stranded RNA functions as an endonuclease generating oligonucleotides with 5'-phosphate termini. Nuclease DII requires both monovalent and divalent cations. Optimal degradation of poly[r(A-U)] is seen at 75 to 100 mM salt and 0.5 to 1.0 mM MgCl2 or MnCl2. The molecular weight estimated from sucrose gradient sedimentation is in the range of 38,000 to 40,000. Nuclease DII acts endonucleolytically producing oligonucleotides terminating in 5'-phosphates. During the isolation and characterization of nucleases DI and DII, a third activity was detected which degrades single-stranded RNA substrates but which, in the presence of either DII or RNase H, significantly enhances the degradation of poly[r(A-U)] or poly(rA)-poly(dT) substrates.     

Isolation,characterization and molecular cloning of the mannose-binding lectins from leaves and roots of garlic (Allium sativum L.)

Two novel lectins were isolated from roots and leaves of garlic. Characterization of the purified proteins indicated that the leaf lectin ASAL is a dimer of two identical subunits of 12 kDa, which closely resembles the leaf lectins from onion, leek and shallot with respect to its molecular structure and agglutination activity. In contrast, the root lectin ASARI, which is a dimer of subunits of 15 kDa, strongly differs from the leaf lectin with respect to its agglutination activity. cDNA cloning of the leaf and root lectins revealed that the deduced amino acid sequences of ASAL and ASARI are virtually identical. Since both lectins have identical N-terminal sequences the larger Mr of the ASARI subunits implies that the root lectin has an extra sequence at its C-terminus. These results not only demonstrate that virtually identical precursor polypeptides are differently processed at their C-terminus in roots and leaves but also indicate that differential processing yields mature lectins with strongly different biological activities. Further screening of the cDNA library for garlic roots also yielded a cDNA clone encoding a protein composed of two tandemly arrayed lectin domains. Since the presumed two-domain root lectin has not been isolated yet, its possible relationship to the previously described two-domain bulb lectin could not be studied at the protein level.     

Isolation and characterization of a Japanese flounder clonal line, reversed, which exhibits reversal of metamorphic left-right asymmetry.

We have isolated a clonal line reversed (rev) of homozygous Japanese flounder through gynogenesis. The homozygous offspring gynogenetically produced from rev exhibited reversal of organization of the metamorphic L/R asymmetry such as the direction of eye-migration at a high frequency (20-30%). The molecular analysis using a left-specific marker pitx2 revealed that the embryonic L/R axis was ambiguously established: in more than half of rev embryos, pitx2 was expressed bilaterally in the lateral plate mesoderm (LPM). Previous studies in other animals demonstrated that ectopic pitx2 expression in the LPM could cause laterality defects of the visceral organs. Likewise, our results using rev imply that bilateral pitx2 expression could lead to randomization of the visceral organs. Coincidence of ectopic pitx2 expression and reversal of the direction of eye-migration in the population of rev offspring suggests that the rev locus is critical in specification of both the metamorphic and the visceral L/R asymmetries. However, reversal of the sidedness of the orientation of the visceral organs was not always accompanied by reversal of the direction of metamorphic eye-migration, suggesting that different mechanisms should be involved downstream of the rev locus in directing these two phases of asymmetric morphogenesis in the Japanese flounder.     

Hyperaccumulation of cadmium by roots, bulbs and shoots of garlic (Allium sativum L.)

The effects of cadmium chloride concentration on root, bulb and shoot growth of garlic (Allium sativum L.), and the uptake and accumulation of Cd2+ by garlic roots, bulbs and shoots were investigated. The range of cadmium chloride (CdCl2 x 2.5H2O) concentrations was 10(-6) - 10(-2) M. Cadmium stimulated root length at lower concentrations (10(-6) - 10(-5) M) significantly (P < 0.005) during the entire treatment period. The seedlings exposed to 10(-3) - 10(-2) M Cd exhibited substantial growth reduction (P < 0.005), but did not develop chlorosis. Garlic has considerable ability to remove Cd from solutions and accumulate it. The Cd content in roots of garlic increased with increasing solution concentration of Cd2+. The roots in plants exposed to 10(-2) M Cd accumulated a large amount of Cd. approximately 1,826 times the control. The Cd contents in roots of plants treated with 10(-3), 10(-4), 10(-5) and 10(-6) M Cd were approximately 114, 59, 24 and 4 times the control, respectively. However, the plants transported only a small amount of Cd to their bulbs and shoots and concentrations in these tissues were low.     

Isolation and characterization of a calmodulin-like protein from Halobacterium salinarium.

The first evidence for a calmodulin-like protein in an archaeon, Halobacterium salinarium, is reported here. The calmodulin-like protein, with a molecular mass of 24 kDa and an estimated pI of 4.8, stimulated cyclic nucleotide phosphodiesterase in a calcium-dependent manner. This stimulation could be suppressed by calmodulin inhibitors. The Ca(2+)-binding ability was verified by 45Ca autoradiography.     

Isolation and characterization of a novel calmodulin-binding protein from potato.

Tuberization in potato is controlled by hormonal and environmental signals. Ca(2+), an important intracellular messenger, and calmodulin (CaM), one of the primary Ca(2+) sensors, have been implicated in controlling diverse cellular processes in plants including tuberization. The regulation of cellular processes by CaM involves its interaction with other proteins. To understand the role of Ca(2+)/CaM in tuberization, we have screened an expression library prepared from developing tubers with biotinylated CaM. This screening resulted in isolation of a cDNA encoding a novel CaM-binding protein (potato calmodulin-binding protein (PCBP)). Ca(2+)-dependent binding of the cDNA-encoded protein to CaM is confirmed by (35)S-labeled CaM. The full-length cDNA is 5 kb long and encodes a protein of 1309 amino acids. The deduced amino acid sequence showed significant similarity with a hypothetical protein from another plant, Arabidopsis. However, no homologs of PCBP are found in nonplant systems, suggesting that it is likely to be specific to plants. Using truncated versions of the protein and a synthetic peptide in CaM binding assays we mapped the CaM-binding region to a 20-amino acid stretch (residues 1216-1237). The bacterially expressed protein containing the CaM-binding domain interacted with three CaM isoforms (CaM2, CaM4, and CaM6). PCBP is encoded by a single gene and is expressed differentially in the tissues tested. The expression of CaM, PCBP, and another CaM-binding protein is similar in different tissues and organs. The predicted protein contained seven putative nuclear localization signals and several strong PEST motifs. Fusion of the N-terminal region of the protein containing six of the seven nuclear localization signals to the reporter gene beta-glucuronidase targeted the reporter gene to the nucleus, suggesting a nuclear role for PCBP.     

Isolation and characterization of a proteolytic enzyme from the adult hookworm Ancylostoma caninum

The adult hookworm Ancylostoma caninum releases a proteolytic enzyme which is thought to be essential for its adaption to parasitism. The protease was purified from parasite extracts by ion-exchange chromatography followed by gel filtration and hydrophobic interaction chromatography. The purified enzyme exhibited a molecular weight of 37,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had an NH2-terminal sequence of Arg-His-His-Gln-Pro-Lys-Val-Ala-Leu-Leu-Gly-Ala-His-Gly-Gly-Ile. Using 125I-fibrin as substrate, the enzyme displayed optimal activity at pH 9-11 and was inactivated by dialysis against EDTA. The enzyme degraded [3H]elastin and both elastin and trypsin-labile glycoproteins in a rat vascular smooth muscle extracellular matrix. Antiserum raised to the protease in rabbits cross-reacted with extracts from the infective larval stage of A. caninum, suggesting that the production of the enzyme begins in an earlier developmental stage of the parasite life cycle. The role of the protease in the histolytic and anticlotting processes of the hookworm and its importance in immunity to ancylostomiasis is discussed.