title: Trypsin inhibitors from the garden four o'clock (Mirabilis jalapa) and spinach (Spinacia oleracea) seeds: isolation, characterization and chemical synthesis.
abstract: Five serine proteinase inhibitors (Mirabilis jalapa trypsin inhibitors, MJTI I and II and Spinacia oleracea trypsin inhibitors, SOTI I, II, and III) from the garden four-o'clock (M. jalapa) and spinach (S. oleracea) seeds were isolated. The purification procedures included affinity chromatography on immobilized methylchymotrypsin in the presence of 5M NaCl, ion exchange chromatography and/or preparative electrophoresis, and finally RP-HPLC on a C-18 column. The inhibitors, crosslinked by three disulfide bridges, are built of 35 to 37 amino-acid residues. Their primary structures differ from those of known trypsin inhibitors, but showed significant similarity to the antimicrobial peptides isolated from the seeds of M. jalapa (MJ-AMP1, MJ-AMP2), Mesembryanthemum crystallinum (AMP1), and Phytolacca americana (AMP-2 and PAFP-S) and from the hemolymph of Acrocinus longimanus (Alo-1, 2 and 3). The association equilibrium constants (K(a)) with bovine beta-trypsin for the inhibitors from M. jalapa (MJTI I and II) and S. oleracea (SOTI I-III) were found to be about 10(7)M(-1). Fully active MJTI I and SOTI I were obtained by solid-phase peptide synthesis. The disulfide bridge pattern in both inhibitors (Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6) was established after their digestion with thermolysin and proteinase K followed by the MALDI-TOF analysis.
authors.affiliation: Faculty of Biotechnology, University of Wrocław, Tamka 2, 50-137 Wrocław, Poland.
authors.forename: Jolanta
authors.initials: J
authors.lastname: Kowalska
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Pszczoła
authors.forename: Anna
authors.initials: A
authors.lastname: Wilimowska-Pelc
authors.forename: Irena
authors.initials: I
authors.lastname: Lorenc-Kubis
authors.forename: Ewa
authors.initials: E
authors.lastname: Zuziak
authors.forename: Mateusz
authors.initials: M
authors.lastname: Ługowski
authors.forename: Anna
authors.initials: A
authors.lastname: Łegowska
authors.forename: Anna
authors.initials: A
authors.lastname: Kwiatkowska
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Sleszyńska
authors.forename: Adam
authors.initials: A
authors.lastname: Lesner
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Walewska
authors.forename: Ewa
authors.initials: E
authors.lastname: Zabłotna
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Rolka
authors.forename: Tadeusz
authors.initials: T
authors.lastname: Wilusz
mesh_terms: D000595:Amino Acid Sequence; D039282:Mirabilis / Q000737:chemistry*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification; D012639:Seeds / Q000737:chemistry*; D016415:Sequence Alignment; D018724:Spinacia oleracea / Q000737:chemistry*; D014361:Trypsin Inhibitors / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D010940:Plant Proteins; D014361:Trypsin Inhibitors