A novel type-1 ribosome-inactivating protein isolated from the supernatant of transformed suspension cultures of Trichosanthes kirilowii (original) (raw)

A type-1 ribosome-inactivating protein (RIP) designated TK-35 has been purified from the supernatant of suspension cultures of Agrobacterium rhizogenes-transformed stem sections of Trichosanthes kirilowii. The protein was purified from the supernatant by PerSeptive SH/M cation exchange and Sephadex G-75 S gel permeation chromatography. The protein occurs as a monomer, with a molecular weight of 35,117, and is glycosylated. A protein translation inhibition assay indicates that TK-35 has an IC 50 value of 2.45 nM and is able to release the rRNA N-glycosidase diagnostic fragment from rabbit reticulocyte lysate. TK-35 is quite thermally stable. Analysis of its N-terminal sequence and two lys-C-protease-digested polypeptides (internal) amino acid sequence indicates that this protein is not homologous to trichosanthin and other type-1 RIPs in Cucurbitaceae family. of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254 Casellas P, Dussossoy S, Falasca AI, Barbieri L, Guillemot JC, Ferrara P, Bolognesi A, Cenini P, Stirpe F (1988) Trichokirin a ribosome-inactivating protein from the seeds of Trichosanthis kirilowii Maximowicz. Am J Biochem 176:581-588 Collins EJ, Robertus JD, Lopresti M, Stone KL, Williams KR, Wu P, Hwang K, Piatak M (1990) Primary amino acid sequence of alpha-trichosanthin and molecular models for abrin α-chain and alpha-trichosanthin. J Biol Chem 265:8665-8669 De Benito FM, Citores L, Iglesias R, Ferreras JM, Soriano F, Arias J, Mendez E, Girbés T (1995) Ebulitins -a new family of type-1 ribosome-inactivating proteins (Rrna N-glycosidases) from leaves of Sambucus ebulus L that coexist with the type 2 ribosome-inactivating protein ebulin 1. FEBS Lett 360:299-302 Diouf D, Gherbi H, Prin Y, Franche C, Duhoux E, Bogusz D (1995) Hairy root nodulation of Casuarina glauca -a system for the study of symbiotic gene expression in an actinorhizal tree. Mol Plant Microbe Interact 4:532-537 Dong TX, Ng TB, Yeung HW, Wong RNS (1994) Isolation and characterization of a novel ribosome-inactivating protein beta-kirilowin from the seeds of Trichosanthes kirilowii. Biochem Biophys Res Commun 199:387-393 Ermayanti TM, McComb JA, O'Brien PA (1994) Stimulation of synthesis and release of swainosnine from transformed roots of