Okitoshi Abe

Reexamination of the primary structure of an antitumor protein, neocarzinostatin.

The primary structure of an antitumor protein, neocarzinostatin, has been reinvestigated by conventional and gas-phase Edman degradation procedures. Sequence analyses of tryptic peptides of both S-carboxymethylated and S-aminoethylated derivatives as well as peptic peptides of the native protein revealed a revised primary structure of neocarzinostatin. The present sequence of 113 amino acid residues thus established agrees with results obtained by fast atom bombardment and gas chromatographic mass spectrometry which were published recently [B.N. Gibson, W.C. Herlihy, T.S.A. Samy, K.S. Hahm, H. Maeda, J. Meienhofer, and K. Biemann (1984) J. Biol. Chem. 259, 10801-10806]. The assignment of four intriguing asparagine/aspartic acid residues has been also achieved.

Location of the disulfide bonds in the antitumor protein neocarzinostatin

Two disulfide bonds in the antitumor antibiotic neocarzinostatin were determined chemically. The peptic and peptic/thermolytic peptides from the native protein were isolated by gel filtration and ion-exchange chromatography followed by reverse-phase HPLC. The cystine peptides obtained were oxidized separately by performic acid treatment and further separated by HPLC into cysteic acid peptides. Sequence analyses of the isolated peptides revealed the location of the disulfide bonds at Cys37-Cys47 and Cys88-Cys93.

New fluorogenic substrate for esterase activity of α-chymotrypsin and related enzymes

Abstract A new fluorogenic substrate, benzyloxycarbonyl- l -phenylalanine 4-methylcoumaryl-7-ester, has been developed for determination of the esterase activity of α-chymotrypsin and related enzymes. Synthesis of the substrate was achieved simply by the carbodiimide condensation of benzyloxycarbonyl- l -phenylalanine and 7-hydroxy-4-methylcoumarin in a 86% yield. The esterase activity was measured by increase of the fluorescence intensity at excitation and emission wave-lengths of 325 and 465 nm, respectively. An initial rate of hydrolysis was linear over a 100-fold range of the enzyme concentration. As little as 2 ng of α-chymotrypsin could be detected in the standard assay. A typical enzyme assay, stability of the substrate, kinetic parameters, and specific activity have been reported.

Highly sensitive method for determination of esterase activity of α-chymotrypsin and α-chymotrypsin-like enzymes using micro high-performance liquid chromatography

Abstract A new substrate, Dns- L -phenylalanine ethyl ester, with high UV absorption has been developed for the determination of the esterase activity of α-chymotrypsin and α-chymotrypsin-like enzymes. The product, generated by the enzyme action, Dns- L -phenylalanine, was clearly separated from the ester substrate by micro reversed-phase high-performance liquid chromatography. The substrate was highly stable under the enzyme assay conditions used. As little as 0.15 ng of α-chymotrypsin and 1.49 ng of subtilisin BPN′ could be detected when a long reaction time was employed. Hydrolyses of the substrate by α-chymotrypsin and α-chymotrypsin-like enzymes were blocked by specific inhibitors of the enzymes.

Isolation and activities of the trypsin-modified vicia angustifolia proteinase inhibitor lacking carboxyl-terminal hexapeptide

The Vicia angustifolia proteinase inhibitor was incubated with p-toluenesulfonyl-L-phenylalanine chloromethyl ketone-trypsin (EC 3.4.21.4) and a main product was isolated. The purified product was different to the first trypsin-modified V. angustifolia inhibitor. The C-terminal residues of the new derivative were arginine, which was also the C-terminal of the cleaved antitryptic site; lysine was a newly exposed C-terminal. These results suggest that the new derivative lacks the C-terminal portion of the native inhibitor, which has asparagine at its C-terminus. The liberated C-terminal peptide had the following amino acid sequence: H-Glu-Glu-Val-Ile-Lys-Asn-OH. The derivative lacking the C-terminal hexapeptide still possesses inhibitory activities against trypsin and alpha-chymotrypsin (EC 3.4.21.1), however, its antichymotryptic activity was inactivated by incubation with chymotrypsin at pH 8.0.