Action of cathepsin L on the oxidized B‐chain of bovine insulin

Kärgel, H.-J.; Dettmer, R; Etzold, G.; Kirschke, Heidrun; Bohley, Peter; Langner, Jürgen

doi:10.1016/0014-5793(80)81128-8

Cited by 62 publications

(23 citation statements)

References 12 publications

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“…The analysis of the cleavage sites showed that the presence of hydrophobic amino acids at P2 and P3 positions was essential for cleavage. The specificity partially resembles those of cathepsin L [30] and papain [31], although in papain more unspecific cleavages have been shown t o occur. The specificity of the egg proteinase was rather different from those of cathepsin D [32], pepsin, trypsin, and chymotrypsin [33].…”

Section: Discussionmentioning

confidence: 89%

Purification and characterization of a cysteine proteinase from silkworm eggs

Kageyama

Takahashi

1990

European Journal of Biochemistry

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Eggs of the silkworm, Bombyx mori, contain a high level of a proteinase which is most active in acidic pH region. The proteinase was purified from an extract of eggs by a six-step procedure which included conventional chromatographic fractionations. The molecular mass of the proteinase was estimated to be 350 kDa by gel filtration and 47 kDa by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels, suggesting an octameric structure. The amino acid composition was found to resemble that of mammalian lysosomal cysteine proteinases, in particular cathepsin L. The NH2-terminal 10-residue sequence is Val-Gln-Phe-Phe-Asp-Leu-Val-Lys-Glu-Glu-.The enzyme appears to be a member of the class of cysteine proteinases since it was strongly inhibited by sulfkydrylreactive compounds and N-[N-(1,3-trans-carboxyoxiran-2-carbonyl)-~-leucyl]-agmatine (E-64). The enzyme hydrolyzed various protein substrates, such as hemoglobin, vitellogenin, vitellin, and lipophorin, with maximal activity around pH 3 -3.5. The specificity of the cleavage sites in the oxidized B chain of insulin was rather well defined and there was high affinity for hydrophobic residues at the P2 and P3 positions. The cysteine proteinase is thought to be involved in protein degradation during embryonic development of silkworm eggs.Silkworm eggs contain high levels of yolk proteins such as vitellin and egg-specific protein [l]. Since these proteins are known to be utilized during embryogenesis, proteinases are thought to be involved in the catabolism of such proteins [2]. Proteinases from insect cells and tissues have been studied by various authors [3, 41. However, at least in part because of the difficulties in obtaining sufficient starting material, the information about these enzymes is much less extensive than that which has been published about mammalian proteinases. lntracellular proteinases, such as cathepsins B, H, L, and D, are present in mammalian lysosomes and play a significant role in the intracellular degradation of proteins [5]. The occurrence of these proteinases in insects is still unproven and remains to be clarified.We reported previously that silkworm eggs contain high levels of activity associated with cysteine proteinases [6]. In our efforts to learn more about the role of proteinases in the developing embryo, we have purified and characterized a cysteine proteinase from silkworm eggs. It is unusual in its high molecular mass and its maximum activity around pH 3.0. The amino acid composition and proteolytic specificity, however, are rather similar to those of other cysteine proteinases such as cathepsin L. EXPERIMENTAL PROCEDURES MaterialsEggs of the silkworm (Bombyx mori) were obtained by extraction from the fully matured ovaries of female moths. They were stored frozen at -20°C until use. Bovine hemoglobin was purchased from Worthington, synthetic chromogenic peptides, synthetic oligopeptides, microbial inhibitors were obtained from the Protein Research Foundation, Osaka; DEAE-cellulose (DE-32) from Whatman, DEAE-Toyopearl from Toso...

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Section: Discussionmentioning

confidence: 89%

Purification and characterization of a cysteine proteinase from silkworm eggs

Kageyama

Takahashi

1990

European Journal of Biochemistry

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“…In contrast to serine proteases, the primary structural determinant for the substrate specificity of papain-like cysteine endopeptidases is the P2 amino acid residue. Cathepsins B, H, and L all prefer hydrophobic amino acids, including Phe in the P2 position (Kä rgel et al, 1980;Koga et al, 1992;Rothe and Dodt, 1992). Although all tested prodrugs contain phenol in the P2-like position that may mimic Phe residue, only cathepsins H and L were able to hydrolyze TFV amidates.…”

Section: Discussionmentioning

confidence: 97%

Activation of 9-[(R)-2-[[(S)-[[(S)-1-(Isopropoxycarbonyl)ethyl]amino] phenoxyphosphinyl]-methoxy]propyl]adenine (GS-7340) and Other Tenofovir Phosphonoamidate Prodrugs by Human Proteases

Birkuš

Kutty²,

He³

et al. 2008

Mol Pharmacol

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9-[(R)-2-[[(S)-[[(S)-1-(Isopropoxycarbonyl)ethyl]amino] phenoxyphosphinyl]-methoxy]propyl]adenine (GS-7340) is an isopropylalaninyl phenyl ester prodrug of the nucleotide HIV reverse transcriptase inhibitor tenofovir (TFV; 9-[(2-phosphonomethoxy)propyl]adenine) exhibiting potent anti-HIV activity and enhanced ability to deliver parent TFV into peripheral blood mononuclear cells (PBMCs) and other lymphatic tissues in vivo. The present study focuses on the intracellular metabolism of GS-7340 and its activation by a variety of cellular hydrolytic enzymes. Incubation of human PBMCs in the presence of GS-7340 indicates that the prodrug is hydrolyzed slightly faster to an intermediate TFV-alanine conjugate (TFV-Ala) in quiescent PBMCs compared with activated cells (0.21 versus 0.16 pmol/min/10 6 cells). In contrast, the conversion of TFV-Ala to TFV and subsequent phosphorylation to TFV-diphosphate occur more rapidly in activated PBMCs. The activity of GS-7340 hydrolase producing TFV-Ala in PBMCs is primarily localized in lysosomes and is sensitive to inhibitors of serine hydrolases. Cathepsin A, a lysosomal serine protease has recently been identified as the primary enzyme activating GS-7340 in human PBMCs. Results from the present study indicate that in addition to cathepsin A, a variety of serine and cysteine proteases cleave GS-7340 and other phosphonoamidate prodrugs of TFV. The substrate preferences displayed by these enzymes toward TFV amidate prodrugs are nearly identical to their preferences displayed against oligopeptide substrates, indicating that GS-7340 and other phosphonoamidate derivatives of TFV should be considered peptidomimetic prodrugs of TFV. (De Clercq, 2003). TFV contains catabolically stable phosphonate moiety; therefore, unlike with nucleoside analogs, its intracellular activation requires only two phosphorylation steps to yield its active form, TFV diphosphate (TFVpp). The phosphorylation of TFV is catalyzed by AMP kinase and nucleoside diphosphate kinase (Robbins et al., 1995). TFVpp is a potent competitive inhibitor of HIV reverse transcriptase, acting as an obligatory DNA chain terminator (Suo and Johnson, 1998). However, the presence of two negative charges on the TFV molecule limits its cellular permeability and precludes oral administration. To overcome these limitations, various TFV prodrugs containing lipophilic groups masking the charged phosphonate moiety have been designed. Among these, tenofovir disoproxil fumarate (TDF; Viread) has been approved for the treatment of HIV infection. Because of its favorable resistance profile and long-term tolerability, TDF therapy is broadly used in treatment of both naive and previously drug-treated HIV-infected patients (for review, see Antoniou et al, 2003;Grim and Romanelli, 2003). Tenofovir (TFV) is an acyclic nucleotide analog active against a variety of retroviruses including human immunodeficiency virus (HIV) and hepatitis B virusGS-7340 (Fig. 1) is a prototype molecule representing a novel class of TFV mono-phosphonoamidate prodru...

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“…41 Cathepsins B and L are cysteine endopeptidases that prefer to hydrolyze proteins containing hydrophobic amino acid residues interacting with the S 2 , and S 3 , and SЈ 1 active site subsites of the enzymes. 40,41,44 The hydrophobic amino acid residues along the copolymeric substrates may not arrange in a way that all fit into the active sites of the pertinent enzymes properly; however, they may enhance the interaction of the enzymes with the substrates to some extent. Therefore, moderately increasing the contents of the hydrophobic amino acid residues in the copolymeric substrates undoubtedly increases their enzymatic binding capability and, consequently, their enzymatic degradability.…”

Section: Discussionmentioning

confidence: 97%

Lysosomal degradability of poly(?-amino acids)

Chiu

Kopečková

Deshmane

et al. 1997

J. Biomed. Mater. Res.

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The lysosomal degradability of poly(alpha-amino acids) based on poly(L-glutamic acid) and its derivatives/copolymers was evaluated to gain insight into the subcellular fate of the macromolecules as water soluble polymeric drug carriers. The results indicate that both the incorporation of hydrophobic comonomers and modification of the carboxylic groups of glutamic acid side chains with hydroxyalkylamine increase the lysosomal degradability of the copolymers. Decreased lysosomal degradability of L-glutamic acid copolymers containing tripeptides terminated in p-nitroanilide (drug model) in the side chains confirmed that drug conjugation alters the degradation pattern of the polymeric carriers. The percentages of the enzymatic release of p-nitroaniline from its polymeric complex with time is relatively independent of the contents of the tripeptidyl p-nitroanilides attached to the polymeric conjugates. Determination of the degradation products by electrospray mass spectroscopy showed that no fragments less than 10(3) D were generated by lysosomal enzymes, whereas the main degradation products by papain and chymotrypsin were tripeptides and tetrapeptides. The conclusions derived from these data strongly suggest that these macromolecules, if used as lysosomotropic drug carriers, may accumulate in the lysosomes and limit their usefulness in some applications.

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Action of cathepsin L on the oxidized B‐chain of bovine insulin

Cited by 62 publications

References 12 publications

Purification and characterization of a cysteine proteinase from silkworm eggs

Purification and characterization of a cysteine proteinase from silkworm eggs

Activation of 9-[(R)-2-[[(S)-[[(S)-1-(Isopropoxycarbonyl)ethyl]amino] phenoxyphosphinyl]-methoxy]propyl]adenine (GS-7340) and Other Tenofovir Phosphonoamidate Prodrugs by Human Proteases

Lysosomal degradability of poly(?-amino acids)

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