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Philibertain g I, the Most Basic Cysteine Endopeptidase Purified from the Latex of Philibertia gilliesii Hook. et Arn. (Apocynaceae)

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Abstract

A new papain-like cysteine peptidase isolated from latex of Philibertia  gilliesii Hook. et Arn., Apocynaceae (formerly Asclepiadaceae) has been purified and characterized. The enzyme, named philibertain g I, is the most basic component present in latex extracts and was purified by acetone fractionation followed by cation exchange chromatography (SP-Sepharose HR) using FPLC system. Homogeneity was confirmed by SDS-PAGE and mass spectroscopy (MS). Molecular mass of the enzyme was 23,530 Da (MALDI-TOF MS), its isoelectric point was >10.25, and maximum proteolytic activity (casein) was achieved at pH 7–8. The new protease was inhibited by E-64 a cysteine peptidases inhibitor. K m was 0.15 mM, using PFLNA as substrate. The N-terminal sequence of philibertain g I (LPASVDWRKEGAVLPIRHQGQCG) was compared with those of twenty plant proteases. Philibertain g I showed the higher degree of identity (73%) with caricain, one of the Carica  papaya endopepetidases.

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Abbreviations

AMPSO:

3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxy-propanesulfonic acid

BLAST:

basic local alignment search tool

CAPS:

3-(ciclohexylamino)-1-propanesulfonic acid

DMSO:

dimethyl sulfoxide

DTT:

dithiothreitol

E-64:

trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane

EDTA:

ethylendiaminetetraacetic acid

IEF:

isoelectric focusing

MALDI-TOF MS:

matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

MES:

2-(N-Morpholino)ethanesulfonic acid

MOPS:

3-(N-morpholino) propanesulfonic acid

PFLNA:

L-pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide

PMSF:

phenylmethylsulfonyl fluoride

SP-Sepharose:

sulphopropyl-Sepharose

TAPS:

N-tris-(hydroxymethyl)-methyl-3-aminopropanesulfonic acid.

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Authors and Affiliations

  1. CENPAT-CONICET Centro Nacional Patagónico, Blvd. Brown s/n, 9120, Puerto Madryn, Chubut, Argentina

    C. Sequeiros & J. L. Esteves

  2. LIPROVE, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, CC 711, 1900, La Plata, Argentina

    M. J. Torres, S. A. Trejo, C. L. Natalucci & L. M. I. López

Authors
  1. C. Sequeiros
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  2. M. J. Torres
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  3. S. A. Trejo
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  4. J. L. Esteves
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  5. C. L. Natalucci
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  6. L. M. I. López
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Correspondence to L. M. I. López.

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Sequeiros, C., Torres, M.J., Trejo, S.A. et al. Philibertain g I, the Most Basic Cysteine Endopeptidase Purified from the Latex of Philibertia gilliesii Hook. et Arn. (Apocynaceae). Protein J 24, 445–453 (2005). https://doi.org/10.1007/s10930-005-7640-0

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