Purification and characterization of serine proteinase inhibitors form gourd (Lagenaria leucantha Rusby var. Gourda Makino) seeds.
Gourd seed inhibitors were purified in the following manner: gourd seeds were ground and extracted with 10 mM ammonium carbonate, pH 7.8. The extract was precipitated with 65-90% acetone and the acetone precipitates were gel filtered in a Cellulofine GCL-90-m column. Fractions of 3000 Da showing trypsin inhibitory activity were combined and purified further by ion exchange and reversed phase chromatographies. Three inhibitors, LLTI-I, II, and III were thus purified to homogeneity and the amino acid sequences of these inhibitors were: [sequence: see text] The exact sequences are unique but very similar to proteinase inhibitors belonging to the squash family. Based on the sequence, it is assumed that the peptide bond (Arg-Ile) found in the three inhibitors is the reactive site for trypsin. The Ki values estimated for complexes of LLTI-I, II, and III with bovine trypsin were 3.6 x 10(-10) M, 6.5 x 10(-11) M, and 3.0 x 10(-11) M, respectively.
Department of Chemistry and Materials Technology, Faculty of Engineering and Design, Kyoto Institute of Technology, Japan.
N
N
Hamato
R
R
Takano
K
K
Kamei-Hayashi
S
S
Hara
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002850:Chromatography, Gel; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D012639:Seeds / Q000737:chemistry*; D015842:Serine Proteinase Inhibitors / Q000032:analysis / Q000302:isolation & purification*; D013045:Species Specificity
D000596:Amino Acids; D015842:Serine Proteinase Inhibitors