Purification of human erythrocyte proteolytic enzyme responsible for degradation of oxidant-damaged hemoglobin. Evidence for identifying as a member of the multicatalytic proteinase family

Sacchetta, Paolo; Battista, Pasquale; Santarone, Stella; Cola, Domenico Di

doi:10.1016/0167-4838(90)90035-e

Cited by 33 publications

(6 citation statements)

References 33 publications

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“…Interestingly, ep24.15 is present in large amounts in human erythrocytes, where hemoglobin also occurs in large quantities (46). Short hemoglobin fragments have been shown to be generated directly by the proteolytic action of the proteasome (47,48). Whereas additional studies will be necessary to clarify the putative enzymes involved in the generation of hemopressin in vivo, it seems reasonable to suggest that ep24.15 and ep24.16 may function in the later steps of intracellular protein degradation.…”

Section: Resultsmentioning

confidence: 99%

Novel Natural Peptide Substrates for Endopeptidase 24.15, Neurolysin, and Angiotensin-converting Enzyme

Rioli

Gozzo²,

Heimann

et al. 2003

Journal of Biological Chemistry

159

165

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The hemoglobin ␣-chain fragment PVNFKFLSH, which we have named hemopressin, produced dose-dependent hypotension in anesthetized rats, starting at 0.001 g/kg. The hypotensive effect of the peptide was potentiated by enalapril only at the lowest peptide dose. These results suggest a role for hemopressin as a vasoactive substance in vivo. The identification of these putative intracellular substrates for ep24.15 and ep24.16 is an important step toward the elucidation of the role of these enzymes within cells. Endopeptidase EC 3.4.24.15 (ep24.15; also referred to as thimet oligopeptidase) and endopeptidase EC 3. 4.24.16 (ep24.16; also referred to as neurolysin) were initially detected in and purified from rat brain homogenates (1, 2). The cloned rat brain ep24.16 (3) showed 80% similarity and 63% identity with the previously cloned rat testis ep24.15 (4). Both peptidases share most of their natural substrates, including bradykinin, neurotensin, opioids, angiotensin I, and gonadotrophinreleasing hormone (5, 6

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Section: Resultsmentioning

confidence: 99%

Novel Natural Peptide Substrates for Endopeptidase 24.15, Neurolysin, and Angiotensin-converting Enzyme

Rioli

Gozzo²,

Heimann

et al. 2003

Journal of Biological Chemistry

159

165

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“…It had been shown previously that exposure of erythrocytes to 1 mM phenylhydrazine greatly enhances the susceptibility of erythrocyte proteins to degradation by proteasomes [7,9,15]. While untreated native hemoglobin is not noticeably degraded by proteasomes, Thermoplasma acidophilum proteasomes as well as the recombinant proteasomes are capable of degrading phenylhydrazine-treated human hemoglobin-Ao to an extent that only a broad band in the low molecular weight range remains (Fig.…”

Section: Degradation Of Phenylhydrazine-treated Hemoglobin-a Omentioning

confidence: 99%

“…The reaction was terminated after a 2 h incubation period at 37°C by adding cold perchloric acid to a final concentration of 0.5 M. Following 30 min in an ice-bath, the insoluble material was removed by centrifugation. The supernatant was neutralized with 0.05 ml of 10 M KOH and insoluble perchlorate was removed [9]. The proteolytic cleavage of the substrate protein was analyzed by gel electrophoresis and HPLC (see below).…”

Section: Degradation Of Phenylhydrazine-treated Hemoglobinmentioning

confidence: 99%

Thermoplasma acidophilum proteasomes degrade partially unfolded and ubiquitin‐associated proteins

Wenzel

Baumeister

1993

FEBS Letters

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It is shown that proteasomes from the archaebacterium Thermoplasma acidophilum selectively degrade substrate proteins partially unfolded by phenylhydrazine-or hydrogen peroxide-treatment. Surprisingly, the pre-incubation of the substrate proteins with ubiquitin is also sufficient to render them susceptible to proteolytic degradation by proteasomes. We propose that, upon spontaneously associating with the substrate protein, ubiquitin exerts a chaotropic effect on it; this may involve the exposure of hydrophobic segments of the polypeptide chain which are recognized by the binding sites of the proteasome.

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“…Erythrocyte membranes are known to be associated with several proteases, including calpain (14), cathepsin E (15,16), acid proteinase (17), and multicatalytic proteinase (18), while erythrocyte cytosoi contains proteases selective for oxidized hemoglobin or oxidized proteins (2,3). The erythrocyte cytosolic proteases include 670-kDa (on gel filtration) and 21.5-35.7-kDa proteins (on SDS-PAGE under reducing conditions) (2) or 700-kDa (on gel filtration) and 23-32-kDa proteins (on SDS-PAGE under reducing conditions) (3).…”

Section: Discussionmentioning

confidence: 99%

Purification and Characterization of a Serine Protease in Erythrocyte Cytosol That Is Adherent to Oxidized Membranes and Preferentially Degrades Proteins Modified by Oxidation and Glycation

Fujino

Tada

Beppu

et al. 1998

Journal of Biochemistry

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A serine protease that preferentially degrades oxidized and glycated proteins was shown to be present in erythrocyte cytosol. Human erythrocyte cytosol was labeled with [3H]diisopropyl fluorophosphate (DFP) and passed through a column of carboxymethyl-Sephadex to obtain radioactive fractions free of hemoglobin. The fractions contained a single radioactive 80-kDa protein, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE)/fluorography. The radioactive 80-kDa protein bound to unoxidized erythrocyte membranes, and more effectively to oxidized membranes. The radioactive protein was purified through a column of diethylaminoethyl-cellulose and by preparative native-PAGE in a purity of 80%. Antibody against the cytosolic 80-kDa protein bound to 80-kDa protein of erythrocyte membranes, indicating the presence of the same protein in the membrane. The antibody bound more effectively to oxidized membranes than to unoxidized membranes. The 80-kDa protein partially purified from unlabeled cytosol degraded more effectively oxidized bovine serum albumin (BSA), oxidized IgG, and glycated BSA more effectively than the corresponding unoxidized or unglycated proteins. Degradation of oxidized or glycated proteins was effectively inhibited by DFP. Hence, the protein is an 80-kDa serine protease that is adherent to oxidized membranes and is responsible for degradation of proteins modified by oxidation and glycation.

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Purification of human erythrocyte proteolytic enzyme responsible for degradation of oxidant-damaged hemoglobin. Evidence for identifying as a member of the multicatalytic proteinase family

Cited by 33 publications

References 33 publications

Novel Natural Peptide Substrates for Endopeptidase 24.15, Neurolysin, and Angiotensin-converting Enzyme

Novel Natural Peptide Substrates for Endopeptidase 24.15, Neurolysin, and Angiotensin-converting Enzyme

Thermoplasma acidophilum proteasomes degrade partially unfolded and ubiquitin‐associated proteins

Purification and Characterization of a Serine Protease in Erythrocyte Cytosol That Is Adherent to Oxidized Membranes and Preferentially Degrades Proteins Modified by Oxidation and Glycation

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