Lagenin, a novel ribosome-inactivating protein with ribonucleolytic activity from bottle gourd (Lagenaria siceraria) seeds.
The seeds of Lagenaria siceraria (Family Cucurbitaceae) were extracted with water and the extract was lyophilized. The lyophilized extract was chromatographed on a DEAE-cellulose column in 10 mM Tris-HCl buffer (pH 7.2). The unadsorbed fraction was applied to an Affi-gel Blue gel column previously equilibrated with the same buffer. After removal of unadsorbed materials, the adsorbed proteins were eluted with 1.5 M NaCl in the Tris-HCl buffer. After dialysis the adsorbed fraction was loaded on a CM-Sepharose CL-6B column which had been equilibrated with and was eluted with the same buffer. After elution of unadsorbed proteins, the column was eluted with a gradient of 0-1 M NaCl in 10 mM Tris-HCl buffer (pH 7.2). The fraction eluting at about 0.55 M NaCl, which represented pure ribosome inactivating protein (RIP), inhibited cell-free translation in a rabbit reticulocyte system with an IC50 of 0.21 nM and exerted ribonuclease activity on yeast tRNA with an activity of 45 U/mg. The RIP was designated lagenin. It possessed a molecular weight of 20 kDa, smaller than the range of 26-32 kDa reported for other RIPs. The N-terminal sequence of lagenin exhibited a lesser extent of similarity to those of other Cucurbitaceae RIPs, characterized by a deletion of the first three amino acid residues and a replacement of the 4th (Phe), 17th (Phe), 18th (Ile) and 22nd (Arg) residues which are invariant in other RIPs.
Wang
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HX
Department of Microbiology, China Agricultural University, Beijing, Peoples Republic of China.
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D000595:Amino Acid Sequence; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D019662:Cucurbitaceae / Q000737:chemistry*; D004365:Drugs, Chinese Herbal / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010936:Plant Extracts / Q000302:isolation & purification / Q000494:pharmacology*; D010940:Plant Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D011817:Rabbits; D012260:Ribonucleases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000494:pharmacology*; D012270:Ribosomes / Q000187:drug effects*; D012639:Seeds / Q000737:chemistry*
D004365:Drugs, Chinese Herbal; D010936:Plant Extracts; D010940:Plant Proteins; D012260:Ribonucleases; C417219:lagenin