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Cited by 153Cynara cardunculus studiesPineapple and bromelain studiesEnzyme Production and Characterization

Purification, Characterization and Partial Amino Acid Sequencing of Two New Aspartic Proteinases from Fresh Flowers of Cynara cardunculus L.

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Veríssimo, Paula, Faro, Carlos, Moir, Arthur J. G., Lin, Yingzhang, Tang, Jordan, Pires, Euclides·1996·European Journal of Biochemistry
DOI 10.1111/j.1432-1033.1996.00762.x
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Abstract

Two new aspartic proteinases have been isolated from stigmas of the cardoon Cynara cardunculus L. by a two‐step purification procedure including extraction at low pH, gel filtration on Superdex 200, and ion‐exchange chromatography on Mono Q. To follow the conventional nomenclature for aspartic proteinases, we have named these proteinases cardosin A and cardosin B. On SDS/PAGE, cardosin A migrated as two bands with apparent molecular masses of 31 000 Da and 15000 Da where as the chains of cardosin B migrated as bands of 34000 Da and 14000 Da. The partial amino acid sequences of the two cardosins revealed that they are similar but not identical, and that they differ horn the previously reported cardoon proteinases named cynarases, which were assumed to be derived from a common precursor. Although the cardosins show some degree of similarity to each other, we could detect no immunological cross‐reactivity between them. Both cardosins were active at low pH and were inhibited by pepstatin, with Ki values of 3 nM for cardosin A and 1 nM for cardosin B, indicating that they belong to the class of aspartic proteinases. Significant differences between the two enzymes were also found for the Kcat/Km values for the hydrolysis of two chromophoric synthetic peptides. The active‐site ionization constants, pK e1 and pK e2, for cardosin A are 2.5±0.2 and 5.3±20.2, whereas for cardosin R they are 3.73±10.09 and 6.7±50.1. The results herein described on the structural and kinetic properties of the cardosins indicate that they are the products of distinct genes which have probably arisen by gene duplication. A scheme for the proteolytic processing of the two enzymes is also proposed.

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  • Those two enzymes appear chiefly in the female part of the flowers of C. cardunculus, i.e. in the upper portion (where the ratio of concentrations of cardosin A to cardosin B is also higher); in the lower portion, only cardosin B is present.

    “More recent studies [21,63,64] have indicated that those two enzymes appear chiefly in the female part of the flowers of C. cardunculus, i.e. in the upper portion (where the ratio of concentrations of cardosin A to cardosin B is also higher); in the lower portion, only cardosin B is present [9].”

    From Advances in the role of a plant coagulant ( <i>Cynara cardunculus</i> ) in vitro and during ripening of cheeses from several milk species (2002) · cites this paper as [64]

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  • Aspartic proteases from C. cardunculus flowers share specificity and kinetic parameters with chymosin and pepsin.

    “These flowers contain aspartic proteases, previously described, which have been shown to share specificity and kinetic parameters with chymosin and pepsin (Verissimo et al. 1995(Verissimo et al. , 1996)).”

    From Plant proteases as milk-clotting enzymes in cheesemaking: a review (2014) · cites this paper as Verissimo et al. 1996

  • These proteinases share pH optima in the acid range, inhibition by pepstatin and preferential cleavage of peptide bonds between hydrophobic residues.

    “These proteinases share pH optima in the acid range, inhibition by pepstatin and preferential cleavage of peptide bonds between hydrophobic residues (as happens with other aspartic proteinases) [64].”

    From Advances in the role of a plant coagulant ( <i>Cynara cardunculus</i> ) in vitro and during ripening of cheeses from several milk species (2002) · cites this paper as [64]

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  1. [2]2. Dunn, B. M. (1992) Structure undfirnrtion qf tbe aspartic. protein- a . m (Dunn, B., ed.) Plenum Press, New York.
  2. [3]3. Tang, J. (1977) Acid pmteases. structure, ,func!ion arid biolcigy (Tang, J., ed.) Plenum Press, New York.
  3. [4]4. Kostka, V. (1985) Aspartic proteina.res trnd their inhihilors (Kostka, V., ed.) Walter de Gruyter, Berlin.
  4. [8]8. Polanowski, A., Wilusz, 1:. Kolaczkowska, M. K., Wieczorck, M. & Wilimowska-Pelc, A. (1985) Purification and charactcrization of aspartic protcinases from Cucumis sativus and Curcuhita maxima seeds, in Aspartic prutsinases and rhrir inhihitmi (Kostka, V., ed.) pp. 49-52, Walter de Gruyter, New York.
  5. [13]13. Juniper, B. E., Robins, K. J . & Joel, D. M. (1989) Tbe Carriivorous phZt.9 (Juniper, B. E., Robins. R. J. & Joel, D. M., eds) pp. 189- 207, Academic Press Inc., London.
  6. [29]29. Estevcc, C'. L. (199s) Bstudo comparativo das carracteristicas bio- quirriicas dos coalhos de Cywum r~arr1unculu.r L., Cynaru . r c d y - rriu.~ L. e Cynaru humilis L., MSc Thesis, University of Coirnhra, 30.
  7. [35]35. Barret, A. J , ( I 977) ProtPinases in rnurnninlirxn cdls und tissues, pp. 209-248, Elsevier-North Holland, New York.
  8. [38]38. FETkS I , P / ~. 3.52, 131 -136. 14321033, 1996, 3, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1996.00762.x, Wiley Online Library on [14/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

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Authors
Veríssimo, P., Faro, C., Moir, A. J. G., Lin, Y., Tang, J., & Pires, E.
Affiliations
  • University of Coimbra (PT)
  • University of Sheffield (GB)
  • Oklahoma Medical Research Foundation (US)
Corresponding author
Faro, Carlos
ORCID
Venue
European Journal of Biochemistry
Publisher
Wiley
Published
1996-02
Volume
235
Issue
3
Pages
762-768
ISSN
  • 0014-2956 (ISSN-L)
  • 1432-1033
DOI
10.1111/j.1432-1033.1996.00762.x
OpenAlex
W2086022519
Semantic Scholar
7a83370164b5faf18b9386004bed408419f09581
Cited by
153
References
39
Citations by year
2026: 1 · 2025: 2 · 2024: 1 · 2023: 4 · 2022: 6 · 2021: 2 · 2020: 11 · 2019: 2 · 2018: 6 · 2017: 5 · 2016: 3 · 2015: 4 · 2014: 2 · 2013: 5 · 2012: 4
FWCI
12.12
Type
journal article
Language
en
Open access
Licence
onlinelibrary.wiley.com/termsAndConditions#vor (vor, 3134 d)
Locations
  • European Journal of Biochemistry (publishedVersion)
  • PubMed (publishedVersion)
  • Portuguese National Funding Agency for Science, Research and Technology (RCAAP Project by FCT) (submittedVersion, other-oa)
  • Portuguese National Funding Agency for Science, Research and Technology (RCAAP Project by FCT) (submittedVersion)
Topics
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Cynara cardunculus studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pineapple and bromelain studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Enzyme Production and Characterization
Keywords
aspartic protease, Cynara cardunculus, stigma (botany), enzyme purification, partial amino acid sequence, pepstatin inhibition, enzyme kinetics, gene duplication, proteolytic processing, immunological cross-reactivity
MeSH terms
Amino Acid Sequence, Chromatography, Gel, Chromatography, High Pressure Liquid, Electrophoresis, Polyacrylamide Gel, Hydrogen-Ion Concentration, Kinetics, Molecular Sequence Data, Plant Proteins, Plants, Protein Processing, Post-Translational, Blotting, Western, Aspartic Acid Endopeptidases, Sequence Alignment

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