Affinity Purification and Properties of Cathepsin‐E‐Like Acid Proteinase from Rat Spleen

Yamamoto, Kazuo; Katsuda, Nobuo; Kato, Keitaro

doi:10.1111/j.1432-1033.1978.tb12772.x

Cited by 104 publications

(29 citation statements)

References 29 publications

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“…After electrophoresis at 4OC, the gels were incubated at low pH and the proteolytic activity was visualized by Coomassie blue staining. The two bands obtained are typical for the two forms of cathepsin E (Yamamoto et al, 1978; Kageyama and Takahashi, 1980). This fraction 23 which contained the higher amount of cathepsin E was used for subsequent digestions with the peptide library GAX,,AG, the native protein gp96 or the substrate melittin.…”

Section: Methodsmentioning

confidence: 99%

Substrate Specificity of Cathepsins D and E Determined by N‐Terminal and C‐terminal Sequencing of Peptide Pools

Arnold

Keilholz

Schild

et al. 1997

European Journal of Biochemistry

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Degradation of protein antigens by cellular proteases is a crucial step in the initiation of a T-cellmediated immune response. But still little is known about the enzymes responsible for the proceysing of antigens, including their specificity. In this paper, we show that the combination of automated N-terminal sequencing with a newly developed method for C-terminal sequencing of peptide pools generated by the aspartic proteases cathepsins D and E is a fast and easy method to obtain detailed information of the substrate specificity of these endopeptidases. Using a 15-residue synthetic peptide library and a native protein as substrates, we confirm and extend the knowledge about the cleavage motif of cathepsin E where positions P1 and PI' of the substrate must be occupied exclusively by hydrophobic amino acids with aromatic or aliphatic side chains. However, Val and Ile residues are not allowed at position P1. Position P2' accepts a broad range of amino acids, including charged and polar ones. Additional requirements concerning the substrate positions P3' and P4' were also defined by pool sequencing. Furthermore, pool sequencing analysis of melittin digests with the aspartic proteases cathepsin D and E provided evidence that both enzymes share the same cleavage motif, identical to the one derived from the peptide library and the native protein. Therefore, pool sequencing analysis is a valuable and fast tool to determine the substrate specificity of any endopeptidase.

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Section: Methodsmentioning

confidence: 99%

Substrate Specificity of Cathepsins D and E Determined by N‐Terminal and C‐terminal Sequencing of Peptide Pools

Arnold

Keilholz

Schild

et al. 1997

European Journal of Biochemistry

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“…The final phases of purification are affinity chromatography on agarose bound to pepstatin or to an antibody against cathepsin E [2]. A homogenous preparation of mature cathepsin E has been obtained from human gastric mucus [4], human erythrocytes [33] and rat spleen [34], neutrophiles [35] and epidermis [36]. Cathepsin E preparations are kept at -20°C, in buffer with pH 7.0 containing 50% glycerol.…”

Section: Purificationmentioning

confidence: 99%

Cathepsin E (EC 3.4.23.34) — a review

Chlabicz¹,

Gacko²,

Worowska³

et al. 2012

Folia Histochem Cytobiol.

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Cathepsin E belongs to the third class of enzymes -hydrolases, a subclass of peptide bond hydrolases and a sub-subclass of endopeptidases with aspartic catalytic sites. Cathepsin E is an endopeptidase with substrate specificity similar to that of cathepsin D. In a human organism, cathepsin E occurs in: erythrocytes, thymus, dendritic cells, epithelial M cells, microglia cells, Langerhans cells, lymphocytes, epithelium of gastrointestinal tract, urinary bladder, lungs, osteoclasts, spleen and lymphatic nodes. In human cells, loci of the gene of pre-procathepsin E are located on chromosome 1 in the region 1231-32. The catalytic site of cathepsin E is two residues of aspartic acid -Asp96 and Asn281, occurring in amino acid triads with sequences DTG96-98 and DTG281-283. To date, no particular role of cathepsin E in the metabolism of proteins in normal tissues has been found. However, it is known that there are many documented pathological conditions in which overexpression of cathepsin E occurs.

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“…Recently several workers have isolated cathepsin D by affinity chromatography using suitable ligands such as substrate [8] and inhibitors [12,21,22]. In the previous work we have shown that rat spleen possesses two types of acid proteinases strongly inhibited by pepstatin [23,24]. One of these was isolated in a pure form by the use of affinity chromatography on pepstatinSepharose 4B and concanavalin-A -Sepharose 4B, and identified as a cathepsin-E-like enzyme that differs from cathepsin D [24].…”

mentioning

confidence: 99%

Cathepsin D of Rat Spleen Affinity Purification and Properties of Two Types of Cathepsin D

Yamamoto

Katsuda

Himeno

et al. 1979

European Journal of Biochemistry

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101

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Two types of cathepsin D were purified from rat spleen by a rapid procedure involving an acid precipitation of tissue extract, affinity chromatography with pepstatin-Sepharose 4B and concanava h -A -Sepharose 4B, and chromatography on Sephadex G-100 and DEAE-Sephacel. The purified major enzyme (85 % of the cathepsin D activity after DEAE-Sephacel chromatography), termed cathepsin D-I, represented about a 1000-fold purification over the homogenate and about a 20% recovery. The purified minor enzyme (15 %), termed cathepsin D-11, represented about a 900-fold purification and about a 3% recovery. Both enzymes showed four (pl: 4.2, 4.9, 6.1 and 6.5) and three (pl: 4.6, 5.6 and 5.8) multiple forms after isoelectric focusing, respectively. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of about 44000. In sodium dodecylsulfate/polyacrylamide gel electrophoresis both enzymes showed a single protein band corrcsponding to a molecular weight of 44000. The enzymes had similar amino acid compositions except for serine, proline and methionine. Cathepsin D-I contained 6.6 % carbohydrate, consisting of mannose, glucose, galactose, fucose and glucosamine in a ratio of 8 : 2 : 1 : 1 : 5 with a trace of sialic acid. The properties of purified enzymes were also compared.Cathepsin D is a major proteolytic enzyme of lysosomes [l, 21 and widely distributed in animal tissues. It is presumed that cathepsin D plays an important role in physiological protein degradation and pathological processes [2 -51. However, the precise role of cathepsin D is still unclear, largely because the mechanism of protein degradation is presumably a complex and multistep process. Consequently it is clear that significant progress in understanding cathepsin D function can be made if a reasonably large quantity of pure enzyme can be obtained. For this purpose we used a rapid and convenient method for the purification of cathepsin D, which involved affinity chromatography with pepstatin -Sepharose 4B and concanavalin-A -Sepharose 4B. Although a number of lysosoma1 cathepsin D have been isolated from various tissues, including spleen [6-91, liver [lo-121, uterus [13,14], thyroid [15,16], small intestine [17], lung [18,19] and erythrocytes [20], we have undertaken a study of cathepsin D from rat spleen because cathepsin D from rat spleen has not been extensively studied and a specific activity of the enzyme in this organ is about 5 times higher than that of the liver. Recently several workers have isolated cathepsin D by affinity chromatography using suitable ligands such as substrate [8] and inhibitors [12,21,22]. In the previous work we have shown that rat spleen possesses two types of acid proteinases strongly inhibited by pepstatin [23,24]. One of these was isolated in a pure form by the use of affinity chromatography on pepstatinSepharose 4B and concanavalin-A -Sepharose 4B, and identified as a cathepsin-E-like enzyme that differs from cathepsin D [24]. In the study described here on the isolati...

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Affinity Purification and Properties of Cathepsin‐E‐Like Acid Proteinase from Rat Spleen

Abstract: A unique acid proteinase different from cathepsin D was purified from rat spleen by a method involving precipitation at pH 3.5, affinity chromatography on pepstatin -Sepharose 4B and concanavalin A -Sepharose 4B, chromatography on Sephadex G-100 and DEAE-Sephacel, and isoelectric focusing.

Cited by 104 publications

References 29 publications

Substrate Specificity of Cathepsins D and E Determined by N‐Terminal and C‐terminal Sequencing of Peptide Pools

Substrate Specificity of Cathepsins D and E Determined by N‐Terminal and C‐terminal Sequencing of Peptide Pools

Cathepsin E (EC 3.4.23.34) — a review

Cathepsin D of Rat Spleen Affinity Purification and Properties of Two Types of Cathepsin D

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