Author/Editor		Rogelj, Boris
Title		Inhibitorji cisteinskih proteniaz kot potencialne obrambne molekule za zaščito rastlin
Translated title		Cysteine proteinase inhibitors as potential plant defense molecules
Type		monografija
Place		Ljubljana
Publisher		Medicinska fakulteta
Publication year		1999
Volume		str. 157
Language		slo
Abstract		One of the mechanisms of plant defense against pests involves proteinase inhibitors (PI), which interfere with pest's digestive proteolytic activity. With transgenic technology, PI genes from other species can be introduced into plants to improve their defensive capabilities. This procedure involves screening for and isolation of new PIs, in vitro and in vivo bioassays, introduction of the chosen gene into the plant and optimisation of expression of the recombinant PI in the plant. Our screening of various plant and fungal material for cysteine prateirmse inhibitors showed that protein extracts from the greater celandine, Chelidonium majus, and a basidiomycete mushroom, Clitocybe nebularis, strongly inhibit papain. Two novel inhibitors of cysteine proteinases were isolated, chelidocystatin (from C majus) and clitocypin (from C. nebularis). Chelidocystatin showed a mass of 10 kDa and a pI of 9.3. It is a strong inhibitor of cathepsin L (Ki = 0.056 nM), papain (Ki = 0.11 nM) and cathepsin H (Ki = 7.5 nM). Comparison of the complete amino acid sequence showed that it is a member of phytocistatin family within the superfamily of cystatins. Clitocypin has a larger mass of 34 kDa which, under strong denaturating conditions only, yielded a single band at 17 kDa, suggesting a homodimer composition with no disulphide bonds. The inhibitor has an isoelectric point of 4.4. It was found to be a tight-binding inhibitor of papain (Ki = 0.58 nM), cathepsin L (Ki = 0.83 nM ) and cathepsin B (Ki = 480 nM), but a very poor inhibitor of cathepsin H, and has no activity towards trypsin or cathepsin D. The complete amino acid sequence of clitocypin bears no similarity to any other cysteine proteinase inhibitor (CPI) nor to any other protein, suggesting a completely novel type of CPI. (Abstract truncated at 2000 characters.)
Descriptors		PLANTS, TRANSGENIC CYSTEINE PROTEINASE INHIBITORS POTATOES BEETLES LARVA PAPAVER TRANSFORMATION, GENETIC RECOMBINANT PROTEINS RECOMBINATION, GENETIC AMINO ACID SEQUENCE YEASTS