Biological Activities of Leupeptins

Aoyagi, Takaaki; Miyata, Setsuko; Nanbo, Masako; Kojima, Fukiko; Matsuzaki, M; Ishizuka, Masaaki; Takeuchi, Tomio; Umezawa, Hamao

doi:10.7164/antibiotics.22.558

Cited by 217 publications

(65 citation statements)

References 14 publications

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“…1.0. The enzyme protein migrated as a single band which was stained for both protein (Coomassie blue) and cathepsin B activity [3] when run in a 7.5 % polyacrylamide gel electrophoresis system at pH 4.0.…”

Section: Resultsmentioning

confidence: 99%

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The Slow, Tight‐Binding Inhibition of Cathepsin B by Leupeptin

Baici¹,

1982

European Journal of Biochemistry

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Leupeptin was found to be a slow, tight-binding inhibitor of cathepsin B from human spleen and rabbit liver. During the enzyme-catalyzed reaction in the presence of inhibitor a concentration-dependent transient state, lasting several minutes, preceded the attainment of the steady state and was characterized by a concave upward or a concave downward lag phase depending on whether the enzyme had been preincubated with the inhibitor or not, respectively. From the pre-steady-state phase of the curves both k,, and k o f f for the formation of the enzyme-inhibitor complex could be calculated. Ki, as the ratio koff/kon, was in good agreement with the inhibition constant obtained using a steady-state treatment. k,, was 1.8 x lo5 M-' s p l and 2 . 0~ lo5 M-' s-' for the human and rabbit enzyme, respectively and the slowness of the binding process fitted into the general concept of enzyme hysteresis. The activation of the essential cysteine residue of cathepsin B by dithiothreitol was also a very slow process characterized by a second-order rate constant of 4.1 M -' S -' . The kinetic features of leupeptin binding allow the prediction of the possible efficiency of this inhibitor on cathepsin B in vivo. It is shown that in order for leupeptin to be a physiologically significant inhibitor of cathepsin B, its concentration at the target site must exceed 20 pM, at least. This contrasts with the predictions drawn from the value of K , ( z 5 nM), which would suggest an effective inhibition of the enzyme already at a concentration of 0.05 pM.Leupeptins are peptide derivatives isolated from culture filtrates of some Strqitomjxvs species [I, 21 and identified as mixtures of N-u-acyl-tripeptides containing the unusual amino aldehyde arginal at the C-terminal position. Leupeptins are potent competitive inhibitors of cysteine proteinases such as cathepsin B and papain [1,3] and serine proteinases such as trypsin, plasmin, kallikrein and acrosin [4,5].Cathepsin B is a lysosomal proteinase containing an essential cysteine residue in the active center and is found in the tissues of many animal species 161. While the normal physiological function of cathepsin B may be the intracellular breakdown of proteins [7,8], an implication of this enzyme has been proposed also in some pathological situations as cartilage destruction 191, tumor invasion [lo, 1 I] and muscular dystrophy [I 21.Recent work in this laboratory on the role of extracellular proteinases in the processes of tissue destruction in rheumatoid arthritis 1131 and tumor invasion [11,14] led us to the study of the inhibition mechanisms of these enzymes by some agents of established therapeutic significance [I5 -171. We have undertaken the present investigation on the inhibition of cathepsin B by leupeptin in virro in order to gain more information on the properties of the system before attempting experiments in vivo.The present paper reports on: (a) the purification of human spleen cathepsin B ; (b) the theory of tight-binding inhibition as a necessary complement to that which is k...

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Section: Resultsmentioning

confidence: 99%

“…Leupeptins are potent competitive inhibitors of cysteine proteinases such as cathepsin B and papain [1,3] and serine proteinases such as trypsin, plasmin, kallikrein and acrosin [4,5].…”

mentioning

confidence: 99%

The Slow, Tight‐Binding Inhibition of Cathepsin B by Leupeptin

Baici¹,

1982

European Journal of Biochemistry

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“…The residual proteolytic activity was determined with casein as a substrate. 13 ) Anti-(Xl-antitrypsin activity; (Xl-Antitrypsin and API-13782 were preincubated in O.4ml of VN buffer at 37 c C for 5 min, then 0.1 ml of trypsin (1.5/lg) was added and incubated at 3TC for 10 min. The residual trypsin activity was determined with protamine sulfate as a substrate as described above.…”

Section: Methodsmentioning

confidence: 99%

An α₂-Macroglobulin Inhibitor, API-13782, Produced byStreptomyces canusOFR-1267

Nakayama

Matsui

Takahashi

1985

Agricultural and Biological Chemistry

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“…In this connection it is of interest to note (199) that a novel class of protease inhibitors, the leupeptins, were shown to be peptide aldehydes (7). A possible transition state analog for chymotrypsin, the inhibitor 2-phenylethane boronic acid, was studied by KOEHLER and LIENHARD (96) who found a binding constant of 4 x 10-SM at pH 8.…”

Section: E-co-r + H20 )E-h + R-co-oh (Deacylation)mentioning

confidence: 99%

“…The finding that the peptides 21-29 and 22-29, obtained from cyanogen bromide cleavage of the Sprotein, were isolated in a ratio of 1:1 (38) or 1:1.5 (58) were taken to indicate that the initial attack of subtilisin BPN' occured at the Ser2~-Ser22 bond. The further degradation of the isolated S-peptide (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) by subtilisin BPN' (149) involved hydrolysis of the Ala,9-Alaz0 bond, indicating that the enzyme possesses carboxypeptidase actitivy. However, it should be pointed out that the free amino acids liberated in the experiments mentioned above have not been isolated, and that contamination of the subtilisin BPN' preparations by carboxypeptidase has not been ruled out.…”

Section: Natural Peptides and Proteinsmentioning

confidence: 99%

Chemical modifications of the subtilisins with special reference to the binding of large substrates. A review

Svendsen

1976

Carlsberg Res. Commun.

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Subtilisin type Carlsberg and subtilisin BPN' are two well characterized extracellular proteases from Bacillus subtilis and Bacillus amyloquefaciens, respectively. The present review summarizes the various chemical modification studies which have been performed with these enzymes. Of special interest has been those modifications which lead to changes in the enzymatic properties with regard to the hydrolysis of polypeptide substrates but not small synthetic ester substrates. In this way it is possible to obtain information about how large an area of the surface of the enzyme is involved in the binding of natural substrates (e.g. clupein, gelatine, casein). Nitration, iodination, glutarylation or succinylation of the subtilisins leads to increases in the hydrolyses of clupein and gelatine, while modification of carboxyl groups leads to a decrease. In all these cases the hydrolysis of small ester substrates is almost unaffected. A section of the review deals with the comparison between the results obtained by chemical modification studies and the two other methods available for the study of "secondary binding sites": X-ray diffraction analyses and kinetic studies with synthetic polypeptides. The productive binding mode for polypeptides proposed by KRAUT and coworkers is in agreement with the results obtained by nitration of the subtilisins. However, results from our laboratory and the literature point towards more than one mode of productive binding. These results are discussed. Finally a comparison is made between the secondary binding sites in subtilisin and other proteolytic enzymes, especially the serine proteases. The importance of secondary interactions between enzyme and substrate is discussed.

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Biological Activities of Leupeptins

Cited by 217 publications

References 14 publications

The Slow, Tight‐Binding Inhibition of Cathepsin B by Leupeptin

The Slow, Tight‐Binding Inhibition of Cathepsin B by Leupeptin

An α₂-Macroglobulin Inhibitor, API-13782, Produced byStreptomyces canusOFR-1267

Chemical modifications of the subtilisins with special reference to the binding of large substrates. A review

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Biological Activities of Leupeptins

Cited by 217 publications

References 14 publications

The Slow, Tight‐Binding Inhibition of Cathepsin B by Leupeptin

The Slow, Tight‐Binding Inhibition of Cathepsin B by Leupeptin

An α2-Macroglobulin Inhibitor, API-13782, Produced byStreptomyces canusOFR-1267

Chemical modifications of the subtilisins with special reference to the binding of large substrates. A review

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An α₂-Macroglobulin Inhibitor, API-13782, Produced byStreptomyces canusOFR-1267