Ancordin, the major rhizome protein of madeira-vine, with trypsin inhibitory and stimulatory activities in nitric oxide productions.
Anredera cordifolia (Ten.) Steenis, or the synonymous name of Boussingaultia baselloides or Boussingaultia gracilis var. pseudobaselloides, is a South American species of ornamental succulent vine, commonly known as the madeira-vine. The fresh leaves of madeira-vine are frequently used as vegetables. A. cordifolia is an evergreen climber that grows from fleshy rhizomes. The rhizome contained one major (23kDa) protein band under non-reducing condition in the SDS-PAGE. The first 15 amino acids in the N-terminal region of the major protein band (23kDa), named tentatively ancordin, were KDDLLVLDIGGNPVV which were highly homologous to sequences of winged bean seed protein ws-1, Medicago truncatula proteinase inhibitor, soybean trypsin inhibitor, and sporamin. By using activity stains, the ancordin showed trypsin inhibitory activity in the SDS-PAGE gel which was found not only in rhizomes but also in aerial tubers, but few in fresh leaves. The crude extracts from rhizomes of madeira-vine were directly loaded onto trypsin-Sepharose 4B affinity column. After washing with 100mM Tris-HCl buffer (pH 7.9) containing 100mM NaCl, the ancordin was eluted directly by 0.2M KC1-HC1 buffer (pH 2.0). In calculation, the purified protein exhibited 0.0428mug trypsin inhibition/mug ancordin (corresponding to 0.53 unit of TPCK-treated trypsin inhibited/mug ancordin). The purified ancordin was used to evaluate the nitric oxide productions in RAW264.7 cells in the presence of polymyxin B (poly B, 50microg/ml) to eliminate the lipopolysaccharide (LPS) contaminations. It was found that ancordin (1.25-5microg/ml) could dose-dependently (R=0.954) stimulate the nitric oxide (NO) productions (expressed as nitrite concentrations) in RAW264.7 cells without significant cytotoxicity, and kept the similar effects in NO production in 6.25microg/ml ancordin.
Chuang
Mao-Te
MT
St. Martin De Porres Hospital, Chiayi 600, Taiwan.
Lin
Yin-Shiou
YS
Hou
Wen-Chi
WC
D000818:Animals; D029748:Caryophyllaceae / Q000737:chemistry; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D008264:Macrophages / Q000187:drug effects; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis*; D009573:Nitrites / Q000032:analysis / Q000378:metabolism; D010940:Plant Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D027343:Rhizome / Q000737:chemistry*; D014357:Trypsin / Q000494:pharmacology*; D014361:Trypsin Inhibitors / Q000494:pharmacology*
D009573:Nitrites; D010940:Plant Proteins; D014361:Trypsin Inhibitors; D009569:Nitric Oxide; D014357:Trypsin