Protease a from germinating vetch seeds: Purification by affinity chromatography and substrate specificity studies

Shutov, Andrei D.; Bulmaga, V. P.; Vaintraub, I. A.

doi:10.1016/s0015-3796(84)80077-3

Cited by 15 publications

(7 citation statements)

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“…For purification of the enzyme the isolation procedure described by Shutov et al (1984) was used as a guide. The first three steps effectively removed other proteinases from the proteinase A extract.…”

Section: Discussionmentioning

confidence: 99%

“…These steps efficiently removed contaminating proteins. The final purification step was gel filtration on Sephadex G-50, as used earlier (Shutov et al, 1984). Similar to the enzyme prepared previously, proteinase A prepared here was retarded on the column material and eluted by 37 mM sodium phosphate, pH 6.5.…”

Section: Discussionmentioning

confidence: 99%

“…Gel filtration was used as the final purification step. The enzyme was retarded on Sephadex G-SO (1.2 cmX60 cm) as shown previously (Shutov et al, 1984). It was eluted by 37 mM sodium phosphate, pH 6.5.…”

Section: Methodsmentioning

confidence: 99%

“…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 increases in proteolytic activity, which results from de novo synthesis of proteinases.According to Shutov and Vaintraub (1987) a cysteine endopeptidase, termed proteinase A (Shutov et al, 1984), is responsible for the triggering of limited proteolytic cleavages during stage 1 of globulin degradation in cotyledons of germinating vetch seeds. As a consequence, conformational changes are expected to occur that open the proteins to the combined attack by proteinase A with a second cysteine endopeptidase, called proteinase B (Shutov et al, 1982), and stored carboxy-peptidases leading to rapid complete breakdown during stage 2.…”

mentioning

confidence: 99%

“…According to Shutov and Vaintraub (1987) a cysteine endopeptidase, termed proteinase A (Shutov et al, 1984), is responsible for the triggering of limited proteolytic cleavages during stage 1 of globulin degradation in cotyledons of germinating vetch seeds. As a consequence, conformational changes are expected to occur that open the proteins to the combined attack by proteinase A with a second cysteine endopeptidase, called proteinase B (Shutov et al, 1982), and stored carboxy-peptidases leading to rapid complete breakdown during stage 2.…”

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confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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