Purification,characterization and chemical modification studies on a translation inhibitor protein from Luffa cylindrica.
A ribosome-inactivating protein (RIP), luffin has been isolated from the seeds of Luffa cylindrica of Cucurbitaceae family by ammonium sulfate fractionation followed by cation exchange and gel-filtration chromatography. Extensive physico-chemical, immunological and biological characterizations were carried out on luffin and compared with that of gelonin. The molecular mass of luffin was -28 kDa as determined by gel-filtration chromatography and SDS-PAGE. The epsilon-NH2 group(s) of luffin were sequentially modified by N-succinimidyl 6-[3-(2-pyridyldithio) propionamido] hexanoate (LC-SPDP), N-succinimidyl-3-(2-pyridylthio)propionate (SPDP) and 2-iminothiolane (2IT) and their effect on immunoreactivity and ribosome inactivating property was evaluated. Modification of single amino group resulted in about 80% inhibition of immunoreactivity and more than 90% loss of protein synthesis inhibition activity. Modification of 2-3 amino groups further hampered both immunoreactivity and protein-synthesis inhibition property LC-SPDP modification played more pronounced effects on immunoreactivity and RIP activity than that of SPDP. However, 2IT modification retained both the immunoreactivity and RIP activity of luffin-LC-SPDP substantially. SPDP showed more pronounced effect on immunoreactivity and RIP activity as compared to 2IT. Therefore, it seems that the positive charge on lysine residues plays an important role in immunological as well as protein synthesis inhibitory effect of luffin.
Institute of Self-Organising Systems and Biophysics, North-Eastern Hill University, Permanent Campus, Shillong 793 022, Meghalaya, India
Ranjit C
RC
Singh
Anis
A
Alam
Vinod
V
Singh
D000818:Animals; D002417:Cattle; D007096:Imidoesters / Q000737:chemistry; D029776:Luffa / Q000201:enzymology*; D014176:Protein Biosynthesis / Q000187:drug effects; D054788:Ribosome Inactivating Proteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013388:Succinimides / Q000737:chemistry
D007096:Imidoesters; D013388:Succinimides; C085779:N-succinimidyl 6-(3-(2-pyridyldithio)propionamido)hexanoate; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; C005905:methyl 4-mercaptobutyrimidate; D054788:Ribosome Inactivating Proteins