Isolation and characterization of E-64, a new thiol protease inhibitor.

Hanada, Kazunori; Tamai, Masamitsu; Yamagishi, Masaaki; Ohmura, Sadafumi; Sawada, Jiro; Tanaka, Ichiro

doi:10.1271/bbb1961.42.523

Cited by 329 publications

(194 citation statements)

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“…Possibly because of this, leupeptin administered to animals actually increases cathepsin L in various organs probably by inhibiting protease degradation. This has been ascribed to a role of the lysosomal cysteine proteases in their turnover [24] due to a similar effect of Ep-475, an epoxide inactivator of cysteine proteases [25]. On the other hand, a more specific inhibitor, Cbz-PheAlaCHN2 does not produce this elevated proteolytic activity in animals [23].…”

Section: Resultsmentioning

confidence: 99%

Active center differences between cathepsins L and B: The S₁ binding region

1988

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The substrate peptide bond cleaved by cathepsins B and L is determined not by the amino acid contributing the carboxyl group to this bond as in the case of serine proteases but rather by the presence of a neighboring amino acid with a large hydrophobic side chain. From a study of the inhibitory potency in a series, Cbz-Phe-X-CHN,, in which Phe promotes binding at S, (terminology of [(1968) Biochem. Biophys. Res. Commun. 32, 898-9021) while the amino acid X probes S,, it is shown that this region of cathepsin L also has the ability to accommodate large hydrophobic side chains. In this respect cathepsin L differs from cathepsin B. Thus Cbz-Phe-Tyr(O-l-Bu)CHN, inactivates cathepsin L with a rate 2.5 x IO4 greater than that for cathepsin B.

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Section: Resultsmentioning

confidence: 99%

Active center differences between cathepsins L and B: The S₁ binding region

1988

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“…The purified enzyme (9.1 mg/ml) in a solution containing 10% dimethyl sulfoxide, 0.1 M KCl, 50 mM cysteine and 10 mM EDTA (pH adjusted to 7.0) was treated with a 5 molar excess of E-64-c (dissolved in a minimal amount of dimethyl sulfoxide). This treatment led to complete inhibition of the enzyme, as confirmed by assaying caseinolytic activity according to standard methods [6,10]. The inhibited enzyme was thoroughly dialyzed against water.…”

Section: Methodsmentioning

confidence: 99%

“…Loxistatin was designed as a clinically usable drug for the treatment of muscular dystrophy [5], based on the prototype E-64 (3), a natural product isolated from cultures of Aspergillus juponicus [6,7]. The chemical structures of l-3 are shown in fig.1.…”

Section: E-64-c [( +)-(2and3s)-3-(l-[n-(3_methylbutyl)amino]leucylcarbomentioning

confidence: 99%

Mode of binding of E‐64‐c, a potent thiol protease inhibitor, to papain as determined by X‐ray crystal analysis of the complex

et al. 1989

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The three‐dimensional structure of the E‐64‐c‐papain complex has been determined by X‐ray crystal analysis at 2.5 Å resolution (conventional R = 26.9%). The structure determined indicates that: (i) the C2 atom of the oxirane ring of E‐64‐c is covalently bound by the Sγ atom of Cys‐25 of papain; (ii) this covalent bond formation results in a configurational conversion of the oxirane C2 atom from the S‐ to the R‐form; and (iii) extensive hydrogen bonding and hydrophobic interactions are responsible for the specific interaction of the E‐64‐c molecule with papain.

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“…Pepstatin A (PEP) and E-64 are known to be largely specific towards aspartyl (Barrett, 1977b) and cysteine proteinases (Hanada et al, 1978;Barrett et al, 1982), respectively. The inhibition pattern obtained with these inhibitors without added thiol activator is presented in Fig.…”

Section: Class-specific Inhibition Of Proteolytic Activitymentioning

confidence: 99%

Inhibition of cysteine and aspartyl proteinases in the alfalfa weevil midgut with biochemical and plant-derived proteinase inhibitors

Wilhite

Elden

Brzin

et al. 2000

Insect Biochemistry and Molecular Biology

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"Inhibition of cysteine and aspartyl proteinases in the alfalfa weevil midgut with biochemical and plant-derived proteinase inhibitors" (2000). AbstractProteolytic activities in alfalfa weevil (Hypera postica) larval midguts have been characterized. Effects of pH, thiol activators, low-molecular weight inhibitors, and proteinase inhibitors (PIs) on general substrate hydrolysis by midgut extracts were determined. Hemoglobinolytic activity was highest in the acidic to mildly acidic pH range, but was maximal at pH 3.5. Addition of thiolactivators dithiothreitol (DTT), 2-mercaptoethanol (2-ME), or l-cysteine had little effect on hemoglobin hydrolysis at pH 3.5, but enhanced azocaseinolytic activity two to three-fold at pH 5.0. The broad cysteine PI E-64 reduced azocaseinolytic activity by 64% or 42% at pH 5 in the presence or absence of 5 mM l-cysteine, respectively. Inhibition by diazomethyl ketones, Z-Phe-Phe-CHN 2 and Z-Phe-Ala-CHN 2 , suggest that cathepsins L and B are present and comprise approximately 70% and 30% of the cysteine proteolytic activity, respectively. An aspartyl proteinase component was identified using pepstatin A, which inhibited 32% (pH 3.5, hemoglobin) and 50% (pH 5, azocasein) of total proteolytic activity. This activity was completely inhibited by an aspartyl proteinase inhibitor from potato (API), and is consistent with the action of a cathepsin D-like enzyme. Hence, genes encoding PIs with specificity toward cathepsins L, B and D could potentially be effective for control of alfalfa weevil using transgenic plants.

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Isolation and characterization of E-64, a new thiol protease inhibitor.

Cited by 329 publications

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Active center differences between cathepsins L and B: The S₁ binding region

Active center differences between cathepsins L and B: The S₁ binding region

Mode of binding of E‐64‐c, a potent thiol protease inhibitor, to papain as determined by X‐ray crystal analysis of the complex

Inhibition of cysteine and aspartyl proteinases in the alfalfa weevil midgut with biochemical and plant-derived proteinase inhibitors

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Isolation and characterization of E-64, a new thiol protease inhibitor.

Cited by 329 publications

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Active center differences between cathepsins L and B: The S1 binding region

Active center differences between cathepsins L and B: The S1 binding region

Mode of binding of E‐64‐c, a potent thiol protease inhibitor, to papain as determined by X‐ray crystal analysis of the complex

Inhibition of cysteine and aspartyl proteinases in the alfalfa weevil midgut with biochemical and plant-derived proteinase inhibitors

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Active center differences between cathepsins L and B: The S₁ binding region

Active center differences between cathepsins L and B: The S₁ binding region