Inhibition of Growth of L5178Y Leukemia Cells by a Fungal Folate-deaminating Enzyme |
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Authors: | Hitoshi Kusakabe Kenjiro Kodama Yuichiro Midorikawa Haruhiko Machida Akira Kuninaka Hiroshi Yoshino |
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Institution: | Research Laboratory, Yamasa Shoyu Co., Ltd., Choshi |
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Abstract: | Reactive sites of adzuki bean proteinase inhibitor II were determined by limited hydrolyses with catalytic amounts of trypsin EC 3.4.21.4] and chymotrypsin EC 3.4.21.1] at pH 3.0. Treatment of the trypsin-modified inhibitor with carboxypeptidase B EC 3.4.12.3] released lysine from the inhibitor and led to complete loss of the activity for trypsin, virtually, without affecting the chymotrypsin-inhibitory activity. Limited hydrolysis with chymotrypsin resulted in a selective cleavage of a single tyrosyi peptide bond in the inhibitor, and treatment of this modified inhibitor with carboxypeptidase A EC 3.4.12.2] abolished the chymotrypsininhibitory activity, having no effect on the trypsin-inhibitory activity. After reduction and S-carboxymethylation, the trypsin- and the chymotrypsin-modified inhibitors both could be separated into two components by gel-filtration on Sephadex G–50 and DEAE-cellulose chromatography. Amino acid and end group analyses of these components indicated that the reactive sites of inhibitor II are the Lys27-Ser28 bond against trypsin and the Tyr54-Ser55 against chymotrypsin.Chemical modification of inhibitor II with cyanogen bromide had a fatal effect on the inhibitory activity against trypsin but no effect against chymotrypsin. |
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Keywords: | cytochrome P450 Streptomyces indolocarbazole staurosporine biosynthesis |
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