AN EXTRACELLULAR INSOLUBLE INHIBITOR OF CYSTEINE PROTEINASES IN CELL-CULTURES AND SEEDS OF CARROT

An 18 kDa extracellular insoluble protein (EIP18) was found previously in amorphous particles suspended in the culture medium and in the int erspaces of cell clusters of carrot (Daucus car-ota L.) callus, as wel l as in the extracellular spaces of carrot seeds, being located both i n the embryo and at the inner edge of the endosperm. We purified EIP18 by washing the amorphous particles with the mixture of Triton X-100, NaCl and ethylenediaminetetraacetic acid (EDTA). We determined several partial amino acid sequences, and then we cloned and sequenced a cDNA for EIP18. EIP18 was found to consist of 133 amino acid residues that included a signal sequence, but it did not contain cysteine, sites fo r N-linked glycosylation or hydrophobic regions. Since its sequence wa s found to be homologous to that of inhibitors of cysteine proteinases , namely cystatins, EIP18 was renamed EICC (extracellular insoluble cy statin of carrot). EICC expressed in yeast was also found in an insolu ble form in yeast cell walls. EICC prepared from the culture medium of carrot cells inhibited commercial cysteine proteinases and a proteina se extracted from germinating carrot seeds. The expression of the gene for EICC was detected in developing seeds, and the level of its trans cript was markedly enhanced upon treatment of somatic embryos with abs cisic acid.