ABSTRACT
A novel peptide from the seeds of Trichosanthes kirilowii, trichokirin-S1, was purified by extraction of protein body, ammonia sulfate precipitation, Blue-gel affinity chromatography, FPLC Mono S ion exchange chromatography and Superose12 gel filtration chromatography. Its molecular weight was determined to be 11,426 by MALDI-TOF MS analysis. Its reaction mechanism to inactive ribosome was the same as that of the ribosome-inactivating protein trichosanthin, a rRNA N-glycosidase. The purified trichokirin-S1 showed a strong inhibitory activity on protein synthesis in cell-free rabbit reticulocyte lysate system, with IC(50) of 0.7 nmol/L. Therefore, trichokirin-S1 may be a promising and efficient toxin moiety of immunotoxins.
Subject(s)
Plant Proteins/isolation & purification , Seeds/chemistry , Trichosanthes/chemistry , Amino Acid Sequence , Animals , Cell-Free System , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , Molecular Weight , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Plant Extracts/pharmacology , Plant Proteins/chemistry , Plant Proteins/pharmacology , Protein Biosynthesis/drug effects , Rabbits , Rats , Ribosomes/drug effects , Ribosomes/metabolism , Sequence Analysis, Protein , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
A peptide, luffin P1, from seeds of Luffa cylindrica, was purified by ammonia sulfate precipitation, CM-52 ion exchange chromatography, Blue-gel affinity chromatography and FPLC Mono S ion exchange chromatography. Its molecular weight was 5226.5 as determined by MALDI-TOF-MS analysis. The sequence of N-terminal 11 amino acids of luffin P1 was identical with the partial N-terminal sequence (from G3 to R13) of 6.5K Arg/Glu rich peptide, which was also isolated from the seeds of Luffa cylindrica. Besides, luffin P1 had a very high homology with a trypsin inhibitor, named C2 peptide, from pumpkin seeds. Interestingly, the purified luffin P1 not only showed a strong inhibitory activity on protein synthesis in rabbit reticulocyte lysate cell-free translation system with IC(50) of 0.6 nmol/L, but also had trypsin inhibitory activity with IC(50) of 22 micromol/L.
Subject(s)
Luffa/chemistry , Plant Proteins/isolation & purification , Seeds/chemistry , Animals , Base Sequence , Chromatography, Ion Exchange/methods , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Molecular Weight , Plant Proteins/chemistry , Plant Proteins/pharmacology , Protein Biosynthesis/drug effects , Rabbits , Rats , Sequence Analysis, Protein , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Trypsin Inhibitors/pharmacologyABSTRACT
A peptide designated Luffin P1 was purified from the seeds of Luffa cylindrica. Its molecular mass was determined to be 5226.1 Da by MALDI-TOF MS analysis. The purified Luffin P1 shows a strong inhibitory activity on protein synthesis in the cell-free rabbit reticulocyte lysate with IC(50) of 0.88 nM. Its reaction mechanism is the same as that of the ribosome-inactivating protein trichosanthin, which is an rRNA N-glycosidase. Besides, the results of gel filtration chromatography suggested the existence of polymers of Luffin P1 and polymerization of Luffin P1 enhanced its rRNA N-glycosidase activity. Luffin P1 was the smallest peptide yet reported that has translational inhibitory activity. The cDNA and deduced amino acid sequence of Luffin P1 has also been determined.
