A novel angiotensin-I-converting enzyme (ACE) inhibitory peptide, designated acein-1, was isolated from the tryptic hydrolysate of human plasma. Gel filtration and cation exchange chromatography were performed to purify this peptide, followed by reversed-phase gradient and isocratic high-performance liquid chromatography. Acein-1 was found to be a heptapeptide, Tyr-Leu-Tyr-Glu-Ile-Ala-Arg, corresponding to f(138^144) of human serum albumin. The synthetic heptapeptide, hexapeptide (Tyr-Leu-Tyr-Glu-Ile-Ala, des-U R acein-1) and octapeptide (Tyr-Leu-Tyr-Glu-Ile-Ala-Arg-Arg, acein-1R) showed dose-dependent inhibitions of ACE, and their IC SH values were 16 W Wmol/l, 500 W Wmol/l and 86 W Wmol/l, respectively. Acein-1 might be a non-competitive inhibitor, while acein-1R may be an uncompetitive inhibitor, as shown by Lineweaver-Burk plots.z 1998 Federation of European Biochemical Societies.

Lysosomal cysteine proteases are known to play important roles in intracellular protein degradation 1) and bone resorption.2) They have also been implicated in various pathological processes such as tumor invasion and metastasis, 3) cartilage degradation in arthritis, 4) and muscular dystrophy. 5) Among these lysosomal enzymes, cathepsin B (EC 3.4.22.1) represents a major component that exhibits both endopeptidase and exopeptidase activities. Cathepsin B has also been described as a strong candidate for a prorenin-processing enzyme. 6,7)Recently, cathepsin B inhibitory peptides derived from bcasein such as PFPGPI and GPFPI have been reported by Lee and Lee. 8) Several enzyme inhibitory peptides have also been isolated from enzymatic hydrolysates of proteins. [9][10][11][12][13][14] We previously isolated Acein-1 10) and Acein-2, 11) which are novel peptides that inhibit angiotensin I-converting enzyme (ACE). Acein-1 was isolated from a tryptic hydrolysate of human plasma with the amino acid sequence of Tyr-LeuTyr-Glu-Ile-Ala-Arg, which corresponds to f(138-144) of human serum albumin. Acein-2 (Leu-Ile-Tyr) was isolated from the same hydrolysate, which corresponds to f(518-520) of human a2-macroglobulin. These findings strongly suggest the possibility of isolating some novel cathepsin B inhibitory peptides from enzymatic hydrolysates of proteins.Almost all organisms, including animals, plants, and microorganisms, as well as a large number of synthetic compounds have been examined in the search for bioactive substances as starting materials for medicines.15-18) However, we believe that the human body might be the best resource for such bioactive peptides because such substances would be expected to be less toxic to humans. We have therefore been attempting to discover novel bioactive peptide sequences in human proteins.In this paper, we report the isolation of four novel peptides that inhibit cathepsin B, designated as Cabin-1, -2, -3, and -4, from a thermolysin (EC 3.4.24.4) digest of human plasma.To our knowledge, this is the first report on the isolation of cathepsin B inhibitory peptides from human protein. We also discuss their inhibitory mechanisms based on Lineweaver-Burk plot analysis. MATERIALS AND METHODSInhibitory Assay for Cathepsin B Inhibitory activity of cathepsin B was measured spectrofluorometrically using ZArg-Arg-MCA as the substrate as described by Barret and Kirschke 19) with a slight modification. Cathepsin B (from human placenta, Sigma, St. Louis, MO, U.S.A.) was dissolved in 50 mM sodium acetate buffer (pH 5.0) containing 0.2 M NaCl, 0.5 mM HgCl 2 , and 1 mM EDTA to a final concentration of 0.2 units/ml. A sample (100 ml) was mixed with 400 ml of 0.4 M sodium acetate buffer (pH 5.5) containing 4 mM EDTA, 200 ml of the enzyme solution, and 50 ml of 0.2 M L-cysteine in sodium acetate buffer (pH 5.5) and preincubated for 3 min at 37°C. A 250 ml aliquot of 40 mM Z-ArgArg-MCA (Peptide Institute, Osaka, Japan) in water was added and mixed to start the reaction at 37°C. After precisely 6 min, 1....