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Sang-Wook Park

Publications and source records attributed to Sang-Wook Park.

12 recordsLinked to original sources
PubMed2006

Isolation and purification of ribosome-inactivating proteins.

Ribosome-inactivating proteins (RIPs) are cytotoxic N-glycosidases identified in plants, fungi, and bacteria. RIPs inhibit protein synthesis by virtue of their enzymatic activity, selectively cleaving a specific adenine residue from a highly conserved, surface-exposed, stem-loop (S/R loop) structure in the 28S rRNA of ribosomes. Some RIPs also exhibit a number of other enzymatic activities such as RNase, DNase, phospholipase, and superoxide dismutase (SOD). RIPs are considered to be plant defense-related proteins as they are able to inhibit the multiplication and growth of several pathogenic virus, fungi, and bacteria either alone or in conjugation with other defense-related proteins. The mechanism of inhibitory activity of RIPs against fungal pathogens seems to be by directly inhibiting fungal growth rather than depurinating host plant ribosomes and causing cell death as previously envisaged. This chapter describes the protocol used to isolate and purify RIPs from plant tissues.

Glycoside Hydrolases↗
PubMed2005

Bacterial expression and enzymatic activity analysis of ME1, a ribosome-inactivating protein from Mirabilis expansa.

Ribosome-inactivating proteins (RIPs) are toxic proteins synthesized by many plants and some bacteria, that specifically depurinate the 28S RNA and thus interrupt protein translation. RIPs hold broad interest because of their potential use as plant defense factors against pathogens. However, study of the activity of type I RIPs has been hampered since their expression in Escherichia coli has typically been toxic to the model system. Mirabilis expansa, an Andean root crop, produces a type I RIP called ME1 in large quantities in its storage roots. In this study, the cDNA sequence of ME1 was used to successfully express the recombinant ME1 protein in E. coli. The production of recombinant ME1 in E. coli was confirmed by Western blot analysis using anti-ME1 antibodies. The studies with fluorescence-labeled ME1 showed that ME1 can enter bacteria and be distributed in the cytoplasm uniformly, indicating its ability to access the protein synthesis machinery of the bacteria. The recombinant enzyme was active and depurinated yeast ribosomes. However, both native and recombinant ME1 proteins failed to depurinate the E. coli ribosomes, explaining the non-toxicity of recombinant ME1 to E. coli. Structural modeling of ME1 showed that it has folding patterns similar to other RIPs, indicating that ME1 and PAP, which share a similar folding pattern, can show different substrate specificity towards E. coli ribosomes. The results presented here are very significant, as few reports are available in the area of bacterial interaction with type I RIPs.

Base Sequence↗
PubMed2004

The N-glycosidase activity of the ribosome-inactivating protein ME1 targets single-stranded regions of nucleic acids independent of sequence or structural motifs.

ME(1), a type I ribosome-inactivating protein (RIP), belongs to a family of enzymes long believed to possess rRNA N-glycosidase activity directed solely at the universally conserved residue A4324 in the sarcin/ricin loop of large eukaryotic and prokaryotic rRNAs. We have investigated the effect of modifying the structure of nonribosomal RNA substrates on their interaction with ME(1) and other RIPs. ME(1) was shown to depurinate a variety of partially denatured nucleic acids, randomly removing adenine residues from single-stranded regions and, to a lesser extent, guanine residues from wobble base-pairs in hairpin stems. A defined sequence motif was not required for recognition of non-paired adenosines and cleavage of the N-glycosidic bond. Substrate recognition and ME(1) activity appeared to depend on the physical availability of nucleotides, and denaturation of nucleic acid substrates increased their interaction with ME(1). Pretreatment of mRNA at 75 degrees C rather than 60 degrees C, for example, lowered the apparent K(D) from 87.1 to 73.9 nm, making it more vulnerable to depurination by RIPs. Exposure to ME(1) in vitro completely abolished the infectivity of partially denatured RNA transcripts of the potato spindle tuber viroid, suggesting that RIPs may target invading nucleic acids before they reach host ribosomes in vivo. Our data suggest that the extensive folding of many potential substrates interferes with their ability to interact with RIPs, thereby blocking their inactivation by ME(1) (or other RIPs).

Adenine↗
PubMed2004

Biochemical and physiological mechanisms mediated by allelochemicals.

Allelochemistry, the production and release of toxic chemicals produced by one species that affect a receiving susceptible species, has been the subject of diverse degrees of scientific enquiry. Recent advances in plant biology have permitted the revamp of allelochemistry as a biologically and ecologically sound explanation for plant invasion and plant-plant communication in the rhizosphere. Recent progress has been made in understanding the biochemical and molecular changes that are induced by allelochemicals in susceptible plant species, and the complex mechanisms that are used by allelochemical-resistant plants to defend against this toxic insult.

Models, Chemical↗
PubMed2004

How plants communicate using the underground information superhighway.

The rhizosphere is a densely populated area in which plant roots must compete with invading root systems of neighboring plants for space, water, and mineral nutrients, and with other soil-borne organisms, including bacteria and fungi. Root-root and root-microbe communications are continuous occurrences in this biologically active soil zone. How do roots manage to simultaneously communicate with neighboring plants, and with symbiotic and pathogenic organisms within this crowded rhizosphere? Increasing evidence suggests that root exudates might initiate and manipulate biological and physical interactions between roots and soil organisms, and thus play an active role in root-root and root-microbe communication.

Communication↗
PubMed2003

Isolation and characterization of an RIP (ribosome-inactivating protein)-like protein from tobacco with dual enzymatic activity.

Ribosome-inactivating proteins (RIPs) are N-glycosidases that remove a specific adenine from the sarcin/ricin loop of the large rRNA, thus arresting protein synthesis at the translocation step. In the present study, a protein termed tobacco RIP (TRIP) was isolated from tobacco (Nicotiana tabacum) leaves and purified using ion exchange and gel filtration chromatography in combination with yeast ribosome depurination assays. TRIP has a molecular mass of 26 kD as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed strong N-glycosidase activity as manifested by the depurination of yeast rRNA. Purified TRIP showed immunoreactivity with antibodies of RIPs from Mirabilis expansa. TRIP released fewer amounts of adenine residues from ribosomal (Artemia sp. and rat ribosomes) and non-ribosomal substrates (herring sperm DNA, rRNA, and tRNA) compared with other RIPs. TRIP inhibited translation in wheat (Triticum aestivum) germ more efficiently than in rabbit reticulocytes, showing an IC50 at 30 ng in the former system. Antimicrobial assays using highly purified TRIP (50 microg mL(-1)) conducted against various fungi and bacterial pathogens showed the strongest inhibitory activity against Trichoderma reesei and Pseudomonas solancearum. A 15-amino acid internal polypeptide sequence of TRIP was identical with the internal sequences of the iron-superoxide dismutase (Fe-SOD) from wild tobacco (Nicotiana plumbaginifolia), Arabidopsis, and potato (Solanum tuberosum). Purified TRIP showed SOD activity, and Escherichia coli Fe-SOD was observed to have RIP activity too. Thus, TRIP may be considered a dual activity enzyme showing RIP-like activity and Fe-SOD characteristics.

Adenine↗
PubMed2002

Enzymatic specificity of three ribosome-inactivating proteins against fungal ribosomes, and correlation with antifungal activity.

Ribosome-inactivating proteins (RIPs) are enzymes that cleave a specific adenine base from the highly conserved sarcin/ricin (S/R) loop of the large ribosomal RNA, thus arresting protein synthesis at the translocation step. In the present study, we employed three RIPs to dissect the antifungal activity of RIPs as plant defense proteins. We measured the catalytic activity of RAT (the catalytic A-chain of ricin from Ricinus communis L.), saporin-S6 (from Saponaria officinalis L.), and ME (RIP from Mirabilis expansa R&P) against intact ribosomal substrates isolated from various pathogenic fungi. We further determined the enzymatic specificity of these three RIPs against fungal ribosomes, from Rhizoctonia solani Kuhn, Alternaria solani Sorauer, Trichoderma reesei Simmons and Candida albicans Berkhout, and correlated the data with antifungal activity. RAT showed the strongest toxicity against all tested fungal ribosomes, except for the ribosomes isolated from C. albicans, which were most susceptible to saporin. RAT and saporin showed higher enzymatic activity than ME against ribosomes from all of the fungal species assayed, but did not show detectable antifungal activity. In contrast, ME showed substantial inhibitory activity against fungal growth. Using N-hydroxysuccinimide-fluorescein labeling of RIPs and fluorescence microscopy, we determined that ME was targeted to the surface of fungal cells and transferred into the cells. Thus, ME caused ribosome depurination and subsequent fungal mortality. In contrast, saporin did not interact with fungal cells, correlating with its lack of antifungal activity.

Catalysis↗
PubMed2002

Nuclear and microtubule reorganization in nuclear-transferred bovine embryos.

We studied the nuclear and microtubule dynamics in nonactivated and pre-activated chromatin-removed oocytes following transfer of nuclei from bovine fibroblast cells. Immediately after fusion between membranes of oocytes and fibroblasts, a microtubule aster containing a gamma-tubulin spot was seen near the transferred nucleus in most oocytes regardless of activation conditions. Most fibroblast nuclei transferred into nonactivated oocytes underwent premature chromosome condensation (PCC) and divided into two masses of chromosomes. In contrast, fibroblast nuclei in pre-activated oocytes rarely underwent PCC, but formed a swelled pronuclear-like structure. Under nonactivation condition, microtubular spindles surrounded condensed chromosomes during the division of two nuclear structures. Gamma-tubulins were detected in the vicinity of condensed chromosomes, suggesting transient spindle formation. Two pronuclear-like structures near the microtubular aster containing gamma-tubulin spot(s) later formed a syngamy-like nuclear structure. While 20% of reconstructed oocytes under nonactivated conditions developed to morulae and blastocysts, only 4% of reconstructed oocytes under pre-activated conditions developed to morulae and blastocysts. These results suggest introduction of a foreign centrosome during somatic cell nuclear transfer, which probably plays a role in nuclear remodeling and subsequent development.

Animals↗
PubMed2002

Exudation of fluorescent beta-carbolines from Oxalis tuberosa L roots.

Root fluorescence is a phenomenon in which roots of seedlings fluoresce when irradiated with ultraviolet (UV) light. Soybean (Glycine max) and rye grass (Elymus glaucus) are the only plant species that have been reported to exhibit this occurrence in germinating seedling roots. The trait has been useful as a marker in genetic, tissue culture and diversity studies, and has facilitated selection of plants for breeding purposes. However, the biological significance of this occurrence in plants and other organisms is unknown. Here we report that the Andean tuber crop species Oxalis tuberosa, known as oca in the highlands of South America, secretes a fluorescent compound as part of its root exudates. The main fluorescent compounds were characterized as harmine (7-methoxy-1-methyl-beta-carboline) and harmaline (3, 4-dihydroharmine). We also detected endogenous root fluorescence in other plant species, including Arabidopsis thaliana and Phytolacca americana, a possible indication that this phenomenon is widespread within the plant kingdom.

Arabidopsis↗
PubMed2002

Isolation and characterization of a novel ribosome-inactivating protein from root cultures of pokeweed and its mechanism of secretion from roots.

Ribosome-inactivating proteins are N-glycosidases that remove a specific adenine from the sarcin/ricin loop of the large rRNA, thus arresting protein synthesis at the translocation step. In the present study, a novel type I ribosome-inactivating protein, termed PAP-H, was purified from Agrobacterium rhizogenes-transformed hairy roots of pokeweed (Phytolacca americana). The protein was purified by anion- and cation-exchange chromatography. PAP-H has a molecular mass of 29.5 kD as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its isoelectric point was determined to be 7.8. Yeast (Saccharomyces cerevisiae) ribosomes incubated with PAP-H released the 360-nucleotide diagnostic fragment from the 26S rRNA upon aniline treatment, an indication of its ribosome-inactivating activity. Using immunofluorescence microscopy, PAP-H was found to be located in the cell walls of hairy roots and root border cells. PAP-H was determined to be constitutively secreted as part of the root exudates, with its secretion enhanced by a mechanism mediated by ethylene induction. Purified PAP-H did not show in vitro antifungal activity against soil-borne fungi. In contrast, root exudates containing PAP-H as well as additional chitinase, beta-1,3-glucanase, and protease activities did inhibit the growth of soil-borne fungi. We found that PAP-H depurinates fungal ribosomes in vitro and in vivo, suggesting an additive mechanism that enables PAP-H to penetrate fungal cells.

Amino Acid Sequence↗
PubMedDate not supplied

Purification of an isoform of patatin with antimicrobial activity against Phytophthora infestans.

Phytophthora infestans (Mont.) de Bary is infamous as the causal agent of the late blight epidemic contributing to the Irish potato famine of the mid 19th century and remains agriculture's most destructive disease as new mutations and migrations confound control measures. In efforts to develop resistant varieties, a somatic hybrid (the Wisconsin J series) between potato (Solanum tuberosum) and a wild relative (Solanum bulbocastanum) has been found to convey durable resistance against the pathogen. We screened the total protein (100 microg ml(-1)) of somatic hybrid varieties J138, J138A12, J101K12, J103K12, and J101K9 for in vitro spore germination inhibition of P. infestans. Since J138 exhibited maximum inhibition at 150 microg ml(-1) in comparison to other varieties, we purified a 40 kD protein from J138 tubers by assaying its ability to inhibit spore germination in P. infestans spores. The highly purified protein was able to inhibit P. infestans spore germination by 70% at the 2.5 microg ml(-1) concentration. The N-terminal sequence of this protein was found to have exact amino acid homology to patatin, the major storage protein of potato tubers. The inhibitory protein has the same molecular weight as patatin and cross-reacts with patatin antibodies. The infection of J138 plants with spores of P. infestans under greenhouse conditions showed that patatin is expressed in stem tissue 72 h after the plant is inoculated with field isolates of P. infestans (US8). In this communication, we report the purification, characterization and antifungal activity against spores of P. infestans of patatin-J from potato tubers.

Anti-Infective Agents↗