Trypsin inhibitors from the garden four o'clock (Mirabilis jalapa) and spinach (Spinacia oleracea) seeds: isolation, characterization and chemical synthesis.
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.
Kowalska
Jolanta
J
Faculty of Biotechnology, University of Wrocław, Tamka 2, 50-137 Wrocław, Poland.
Pszczoła
Katarzyna
K
Wilimowska-Pelc
Anna
A
Lorenc-Kubis
Irena
I
Zuziak
Ewa
E
Ługowski
Mateusz
M
Łegowska
Anna
A
Kwiatkowska
Anna
A
Sleszyńska
Małgorzata
M
Lesner
Adam
A
Walewska
Aleksandra
A
Zabłotna
Ewa
E
Rolka
Krzysztof
K
Wilusz
Tadeusz
T
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
D010940:Plant Proteins; D014361:Trypsin Inhibitors