A novel ribosome-inactivating protein designated Moschatin from the mature seeds of pumpkin (Cucurbita moschata) has been successively purified to homogeneity, using ammonium sulfate precipitation, CM-cellulose 52 col...A novel ribosome-inactivating protein designated Moschatin from the mature seeds of pumpkin (Cucurbita moschata) has been successively purified to homogeneity, using ammonium sulfate precipitation, CM-cellulose 52 column chromatography, Blue Sepharose CL-6B Affinity column chromatography and FPLC size-exclusion column chromatography. Moschatin is a type 1 RIP with a pI of 9.4 and molecular weight of~29 kD. It is a rRNA N-glycosidase and potently blocked the protein synthesis in the rabbit reticulocyte lysate with a IC_(50) of 0.26 nM. Using the anti-human melanoma McAb Ng76, a novel immunotoxin Moschatin-Ng76 was prepared successfully and it efficiently inhibited the growth of targeted melanoma cells M_(21) with a IC_(50) of 0.04 nM, 1500 times lower than that of free Moschatin. The results implied that Moschatin could be used as a new potential anticancer agent.展开更多
The complete amino acid sequence of β-momorcharin, a ribosome-inactivating protein from the seeds of Momordica charantia Linn (Cucurbitaceae) has been determined. This has been done by the sequence analysis of peptid...The complete amino acid sequence of β-momorcharin, a ribosome-inactivating protein from the seeds of Momordica charantia Linn (Cucurbitaceae) has been determined. This has been done by the sequence analysis of peptides obtained by enzymatic digestion with trypsin, chymotrypsin and S. aureus V8 protease, as well as by chemical cleavage with BNPS-skatole. The protein consists of 249 amino acid residues containing one asparagine - linked sugar group attached to the site of Asn 5 1 and has a calculated relative molecular mass of 28,452 Da without addition of the carbohydrate. Comparison of this sequence with those of trichosanthin and other ribosome-inactivating proteins from different species of plants shows a significant homology with each other. Regarding the similarity of their biological properties, an active domain of these proteins has been predicted here.展开更多
Organellar biogenesis is mainly regulated by nucleus-encoded factors, which act on various steps of gene expression including RNA editing, processing, splicing, stabilization, and translation initiation. Among these r...Organellar biogenesis is mainly regulated by nucleus-encoded factors, which act on various steps of gene expression including RNA editing, processing, splicing, stabilization, and translation initiation. Among these regulatory factors, pentatricopeptide repeat （PPR） proteins form the largest family of RNA binding proteins, with hundreds of members in flowering plants. In striking contrast, the genome of the unicellular green alga Chlamydomonas reinhardtii encodes only 14 such proteins. In this study, we analyzed PPR7, the smallest and most highly expressed PPR protein in C. reinhardtii. Green fluorescent protein-based localization and gel-filtration analysis revealed that PPR7 forms a part of a high-molecular-weight ribonu- cleoprotein complex in the chloroplast stroma. RIP-chip analysis of PPRT-bound RNAs demonstrated that the protein associates with a diverse set of chloroplast transcripts in vivo, i.e. rrnS, psbH, rpoC2, rbcL, atpA, cemA-atpH, tscA, and atpl-psaJ. Furthermore, the investigation of PPR7 RNAi strains revealed that deple- tion of PPR7 results in a light-sensitive phenotype, accompanied by altered levels of its target RNAs that are compatible with the defects in their maturation or stabilization. PPR7 is thus an unusual type of small multi- functional PPR protein, which interacts, probably in conjunction with other RNA binding proteins, with numerous target RNAs to promote a variety of post-transcriptional events.展开更多
文摘A novel ribosome-inactivating protein designated Moschatin from the mature seeds of pumpkin (Cucurbita moschata) has been successively purified to homogeneity, using ammonium sulfate precipitation, CM-cellulose 52 column chromatography, Blue Sepharose CL-6B Affinity column chromatography and FPLC size-exclusion column chromatography. Moschatin is a type 1 RIP with a pI of 9.4 and molecular weight of~29 kD. It is a rRNA N-glycosidase and potently blocked the protein synthesis in the rabbit reticulocyte lysate with a IC_(50) of 0.26 nM. Using the anti-human melanoma McAb Ng76, a novel immunotoxin Moschatin-Ng76 was prepared successfully and it efficiently inhibited the growth of targeted melanoma cells M_(21) with a IC_(50) of 0.04 nM, 1500 times lower than that of free Moschatin. The results implied that Moschatin could be used as a new potential anticancer agent.
基金Project supported by Grants from the High Technology Development Program of China from the National Natural Science Foundation of China(No. 29272074)
文摘The complete amino acid sequence of β-momorcharin, a ribosome-inactivating protein from the seeds of Momordica charantia Linn (Cucurbitaceae) has been determined. This has been done by the sequence analysis of peptides obtained by enzymatic digestion with trypsin, chymotrypsin and S. aureus V8 protease, as well as by chemical cleavage with BNPS-skatole. The protein consists of 249 amino acid residues containing one asparagine - linked sugar group attached to the site of Asn 5 1 and has a calculated relative molecular mass of 28,452 Da without addition of the carbohydrate. Comparison of this sequence with those of trichosanthin and other ribosome-inactivating proteins from different species of plants shows a significant homology with each other. Regarding the similarity of their biological properties, an active domain of these proteins has been predicted here.
文摘Organellar biogenesis is mainly regulated by nucleus-encoded factors, which act on various steps of gene expression including RNA editing, processing, splicing, stabilization, and translation initiation. Among these regulatory factors, pentatricopeptide repeat （PPR） proteins form the largest family of RNA binding proteins, with hundreds of members in flowering plants. In striking contrast, the genome of the unicellular green alga Chlamydomonas reinhardtii encodes only 14 such proteins. In this study, we analyzed PPR7, the smallest and most highly expressed PPR protein in C. reinhardtii. Green fluorescent protein-based localization and gel-filtration analysis revealed that PPR7 forms a part of a high-molecular-weight ribonu- cleoprotein complex in the chloroplast stroma. RIP-chip analysis of PPRT-bound RNAs demonstrated that the protein associates with a diverse set of chloroplast transcripts in vivo, i.e. rrnS, psbH, rpoC2, rbcL, atpA, cemA-atpH, tscA, and atpl-psaJ. Furthermore, the investigation of PPR7 RNAi strains revealed that deple- tion of PPR7 results in a light-sensitive phenotype, accompanied by altered levels of its target RNAs that are compatible with the defects in their maturation or stabilization. PPR7 is thus an unusual type of small multi- functional PPR protein, which interacts, probably in conjunction with other RNA binding proteins, with numerous target RNAs to promote a variety of post-transcriptional events.
