Nucleotide sequence of cDNA for sulfhydryl-endopeptidase (SH-EP) from cotyledons of germinating<i>Vigna mungo</i>seeds

Akasofu, Harue; Yamauchi, Daisuke; Mitsuhashi, Wataru; Minamikawa, Takao

doi:10.1093/nar/17.16.6733

Cited by 73 publications

(61 citation statements)

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“…6). In fact, our recent results indicated 96% homology in the amino acid sequence of the putative mature enzyme regions between EP-CI and SH-EP (Akasofu et al, 1989;Tanaka et al, 1991), although there was a small difference in molecular mass between the two (Fig. 6).…”

Section: Discussionmentioning

confidence: 78%

“…The enzyme protein and its activity were also detected in cotyledons and other organs of seedlings. EP-Cl was found to be immunologically identical to SH-EP, the major Cys endopeptidase expressed in cotyledons of germinating Vigna mungo seeds (Mitsuhashi et al, 1986;Akasofu et al, 1989;Mitsuhashi and Minamikawa, 1989), although there was a small difference in molecular mass between them. In fact, the amino acid sequence deduced from EP-Cl cDNA, which we reported separately (Tanaka et al, 1991), had a high degree of homology (94%) with that from SH-EP cDNA (Akasofu et al, 1989).…”

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confidence: 95%

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Expression of an Endopeptidase (EP-C1) in Phaseolus vulgaris Plants

et al. 1993

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38: 1988-1995). In the present studies, one of the major endopeptidase forms, designated EP-C1, was purified as a 34-kD polypeptide from pods of maturing French bean fruits. EP-C1 was found to be immunologically distinguished from other forms in extracts from pods, but homologous to SH-EP, the major cysteine endopeptidase expressed in cotyledons of germinating Vigna mungo seeds (W. Mitsuhashi, T. Minamikawa 119891 Plant Physiol89: 274-279). The leve1 of endopeptidase that reacted with the antiserum to EP-C1 increased in pods as the fruit maturation proceeded. EP-C1 was also immunologically detected in stems of French bean plants bearing fruits of later maturation stages. Protein immunoblotting showed that a 34-kD polypeptide corresponding to EP-C1 in molecular mass occurred in extracts from 7-to 9-d cotyledons of germinating French bean seeds. In addition, two other polypeptides with slightly higher molecular masses were observed in extracts from 3-to 5-d cotyledons. We suggest that these two polypeptides are intermediates involved in posttranslational processing of EP-C1. RNA blot hybridization with EP-C1 cDNA as a probe showed that EP-C1 mRNA occurred in pods of fruits at later maturing stages and also in cotyledons of 3-to 7-d germinating seeds.

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Section: Discussionmentioning

confidence: 78%

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confidence: 95%

Expression of an Endopeptidase (EP-C1) in Phaseolus vulgaris Plants

et al. 1993

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“…The EMBL Data Library (release 49) and Swiss-Prot Database (release 34) revealed 78.2% sequence identity of proteinase A to cysteine endopeptidase SH-EP, which is one of the major proteinases formed in cotyledons of Kgna mungo seedlings (Akasofu et al, 1989). The cDNA-derived amino acid sequence of SH-EP contains the tetrapeptide Lys-Asp-Glu-Leu (KDEL) at its C terminus (Okamoto et al, 1994).…”

Section: Preparation and Characterization Of Proteinase Amentioning

confidence: 99%

Proteinase A, A Storage‐Globulin‐Degrading Endopeptidase of Vetch (Vicia Sativa L.) Seeds, is not Involved in Early Steps of Storage‐Protein Mobilization

Becker

Senyuk

Shutov

et al. 1997

European Journal of Biochemistry

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Proteinase A is a papain-like cysteine endopeptidase of vetch (Vicia sativu L.) which was assumed to initiate storage-globulin breakdown just after the onset of seed germination. This enzyme was purified from cotyledons of vetch seedlings. On gelatin-containg SDS gels, active proteinase A migrated with an apparent molecular mass of 21 kDa, whereas after heat denaturation its molecular size on SDSPAGE was 29 kDa. Although proteinase A is capable of hydrolyzing storage globulins in vitro it could not be localized in the protein-body fraction of cotyledons from germinating seeds. cDNA clones encoding proteinase A precursor have been obtained by PCR. The precursor is composed of an N-terminal signal sequence followed by a propeptide, the region encoding mature proteinase A, and a C-terminal KDEL sequence. Mature proteinase A with a derived molecular mass of 25244 Da does not have the KDEL sequence. The derived amino acid sequence of the proteinase A precursor is 78.2% identical to sulfhydrylendopeptidase (SH-EP), a cysteine endopeptidase from germinating Vigna tnungo seedlings. Northern blot analysis indicated that proteinase A mRNA appears de novo in cotyledons of I-day-germinated vetch seeds, where its amount increases up to day 6. No proteinase A mRNA was detected in other vetch organs, not even in the embryo axis, which contains stored globulins. By means of antibodies raised against the purified and against recombinantly produced proteinase A, the 29-kDa bands of mature proteinase A were detected in cotyledon extracts of 6-day-germinated seeds when globulin degradation has already far proceeded. The reported data do not agree with the proposed triggering role of proteinase A in storage-globulin breakdown during germination.Keywords: triggering cysteine endopeptidase ; seed germination; Vicia sativa L. ; endoplasmic reticulum retention signal.The beginning of storage-protein degradation is indicated by slight mobility changes of globulins from cotyledon extracts of germinating dicotyledonous seeds on electrophoresis (Shutov and Vaintraub, 1973;Lichtenfeld et al., 1979Lichtenfeld et al., , 1981Boylan and Sussex, 1987; Dunaevsky and Belozersky, 1989). During this first stage of breakdown the holoproteins remain assembled, which is reflected by nearly unchanged positions in the sucrosedensity gradient after centrifugation and in gels after electrophoresis under non-denaturing conditions. Only small trichloroacetic-acid-soluble peptides are lost as the result of limited proteolysis (Shutov and Vaintraub, 1987). These first modifications are thought to be catalyzed either by stored endopeptidases that have to be activated just after imbibition, as in buckwheat (Elpidino et al., 1991), or by newly formed endopeptidases, as assumed for the majority of legumes (Chrispeels et al., 1976; Lichtenfeld et al., 1979;Muntz et al., 1985; Shutov and Vaintraub, 1987). Only after 3-6 days depending on the species and germination conditions, a second stage with rapid storageglobulin degradation begins, accompanied by strong increa...

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“…Characterization of GCP-1 at the enzyme level (33,34), and includes the presence of a KDEL sequence at the C-terminus of its precursor. These results clearly indicate DRCP26 is not a proteinase A-related enzyme.…”

Section: Discussionmentioning

confidence: 99%

Enzymatic characterization of germination-specific cysteine protease-1 expressed transiently in cotyledons during the early phase of germination

Tsuji¹,

Tsukamoto²,

Iwamoto³

et al. 2012

Journal of Biochemistry

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Papain-like cysteine protease activity that shows a unique transient expression profile in cotyledons of daikon radish during germination was detected. The enzyme showed a distinct elution pattern on DEAE-cellulose compared with cathepsin B-like and Responsive to dessication-21 cysteine protease. Although this activity was not detected in seed prior to imbibition, the activity increased markedly and reached a maximum at 2 days after imbibition and then decreased rapidly and completely disappeared after 5 days. Using cystatin-Sepharose, the 26 kDa cysteine protease (DRCP26) was isolated from cotyledons at 2 days after imbibition. The deduced amino acid sequence from the cDNA nucleotide sequence indicated that DRCP26 is an orthologue of Arabidopsis unidentified protein, germination-specific cysteine protease-1, belonging to the C1 family of cysteine protease predicted from genetic information. In an effort to characterize the enzymatic properties of DRCP26, the enzyme was purified to homogeneity from cotyledons at 48 h after imbibition. The best synthetic substrate for the enzyme was carbobenzoxy-Phe-Arg-4-methylcoumaryl-7-amide. All model peptides were digested to small peptides by the enzyme, suggesting that DRCP26 possesses broad cleavage specificity. These results indicated that DRCP26 plays a role in the mobilization of storage proteins in the early phase of seed germination.

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Nucleotide sequence of cDNA for sulfhydryl-endopeptidase (SH-EP) from cotyledons of germinatingVigna mungoseeds

Cited by 73 publications

References 1 publication

Expression of an Endopeptidase (EP-C1) in Phaseolus vulgaris Plants

Expression of an Endopeptidase (EP-C1) in Phaseolus vulgaris Plants

Proteinase A, A Storage‐Globulin‐Degrading Endopeptidase of Vetch (Vicia Sativa L.) Seeds, is not Involved in Early Steps of Storage‐Protein Mobilization

Enzymatic characterization of germination-specific cysteine protease-1 expressed transiently in cotyledons during the early phase of germination

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