Isolation, characterization, and properties of a trypsin-chymotrypsin inhibitor from amaranth seeds.
A trypsin-chymotrypsin inhibitor was isolated from the seeds of amaranth--a highly nutritious protein source. The purification of the inhibitor (AmI) was carried out by affinity chromatography on trypsin-Sepharose and by HPLC. AmI is a single-chain protein of 8 kD, as determined by electrophoresis on SDS-polyacrylamide gels and by gel exclusion on Sephadex G-50 column. It is stable at neutral and alkaline pH and is relatively thermostable. AmI inhibits trypsin and chymotrypsin from the digestive system of insects such as Tribolium castaneum and Locusta migratoria, supporting the hypothesis that inhibitors may have evolved as defense mechanisms of seeds against insects. AmI lost its inhibitory activities when submitted to limited proteolysis with trypsin, while limited proteolysis with chymotrypsin had almost no effect. The partial amino acid sequence of 45 amino acids from the amino terminus of AmI differs significantly from the known sequences of legume-seed and cereal-grain protease inhibitor families. Differences in the chemistry at the inhibitory site(s) and in the amino acid sequence of AmI in comparison to that of other cereal and legume inhibitors suggest that AmI is a member of a new family of serine protease inhibitors. AmI was found to inhibit the anchorage-independent growth of MCF-7 breast cancer cells, suggesting that AmI may have anticarcinogenic activity.
Tamir
S
S
Department of Biochemistry, Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot, Israel.
Bell
J
J
Finlay
T H
TH
Sakal
E
E
Smirnoff
P
P
Gaur
S
S
Birk
Y
Y
D000595:Amino Acid Sequence; D016588:Anticarcinogenic Agents / Q000494:pharmacology; D001943:Breast Neoplasms; D019684:Magnoliopsida / Q000737:chemistry*; D008969:Molecular Sequence Data; D012639:Seeds / Q000737:chemistry; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects
D016588:Anticarcinogenic Agents; D014361:Trypsin Inhibitors