Specificity and pH dependence of ficin-catalyzed hydrolyses. Comparisons with bromelain specificity

Kortt, Alexander A.; Hinds, John A.; Zerner, Burt

doi:10.1021/bi00707a005

Cited by 18 publications

(5 citation statements)

References 34 publications

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“…Several plant cysteine proteases from latex have been reported. Latex of Carica papaya (Glazer and Smith, 1971;Robinson, 1975), Ficus carica (Kramer and Whitaker, 1964;Sgarbieri et al, 1964;Sugiura and Sasaki, 1974), Ficus glabrata (Englund et al, 1968;Jones and Glazer, 1970;Kortt et al, 1974aKortt et al, , 1974bWilliams and Whitaker, 1969), Ficus pumila (Perelló et al, 2000), Carica candamarcensis (Gravina de Moraes et al, 1994), Calotropis procera (Dubey and Jagannadham, 2003), and of Ervatamia coronaria (Kundu et al, 2000) contains distinct cysteine proteases.…”

Section: Introductionmentioning

confidence: 99%

Funastrain c II: A Cysteine Endopeptidase Purified from the Latex of Funastrum clausum

Morcelle

Trejo²,

Canals³

et al. 2004

Protein J

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A cysteine endopeptidase, named funastrain c II, was isolated and characterized from the latex of Funastrum clausum (Asclepiadaceae). The molecular mass (mass spectrometry) of the protease was 23.636 kDa. The analysis of funastrain c II by SDS-PAGE revealed a single polypeptide chain. The enzyme showed a remarkable stability of its caseinolytic activity after incubation at temperatures as high as 70 degrees C. Inhibition and activation assays indicated the cysteinic nature of the funastrain c II catalytic site. The optimum pH of funastrain c II enzymatic activity varied according to the substrate used (9.0-10.0 for casein and 6.2-6.8 for PFLNA). Kinetic parameters were determined for N-alpha-CBZ-Ala p-nitrophenyl ester (Km = 0.0243 mM, kcat = 1.5 s(-1)) and L-pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA; KM = 0.1011 mM, kcat = 0.9 s(-1)). The N-terminal sequence of funastrain c II showed considerable similarity to other proteases isolated from latex of different Asclepiadaceae species as well as to other cysteine proteinases belonging to the papain family.

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Section: Introductionmentioning

confidence: 99%

Funastrain c II: A Cysteine Endopeptidase Purified from the Latex of Funastrum clausum

Morcelle

Trejo²,

Canals³

et al. 2004

Protein J

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“…Although the Arg-X and Gln-X bonds may not be the most preferred sites of cleavage by bromelain (10), this cleavage is not inconsistent with the specificity of stem bromelain. Indeed, the enzyme is known to hydrolyze the Arg-Ala bond in the sequence, -Ser-Arg-Arg-Ala-Gln-, of glucagon (11), and the Gln-CyS0 3 H and Gin-Leu bonds in the oxidized insulin A chain (12), and Boc-Leu-Arg-Arg-MCA was shown to be a good substrate for the enzyme in the present study.…”

Section: Isolation Of Bromelain Isoinhibitors and Separation Of The mentioning

confidence: 52%

The Amino Acid Sequences of Isoforms of the Bromelain Inhibitor from Pineapple Stem

Hatano

Tanokura

Takahashi

1998

Journal of Biochemistry

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Eight bromelain inhibitor (BI) isoform fractions were isolated from pineapple stem, and then submitted to conventional amino acid sequencing after performic acid oxidation and subsequent separation of the resulting light and heavy chains. The results revealed that all fractions exhibited microheterogeneity, containing at least two major components, but that all the isoinhibitors have a common double-chain structure (Mr = ca. 5,700-5,900) with five disulfide bonds and similar amino acid sequences. Notably, Fraction BI-VIII exhibited less than 40% of the specific inhibitory activity toward stem bromelain as compared with the other inhibitor fractions. This fraction was a mixture of two isoforms, BI-VIII(1) and BI-VIII(2), the latter lacking the arginine or glutamine residue at the COOH-terminus of the light chain. Furthermore, the oxidized light chain of BI-III, used as a representative normal isoinhibitor, was found to exhibit significant inhibitory activity, whereas the oxidized light chain of BI-VIII(2) lacking the COOH-terminal Arg or Gln showed only very low inhibitory activity. Therefore, the major bromelain-inhibitory site was indicated to be the COOH-terminal residue, Arg or Gln, of the light chain. This is consistent with the three-dimensional structure model constructed by computer modeling for the hypothetical complex between BI-VI and papain, a close homolog of bromelain.

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“…They also showed the ratio of specific activity of bromelain to papain as being 1.1 using casein as substrate, the same as shown in our data. Kortt et al [10] described studies of peptides (bradykinin, insulin, etc.) as substrates for ficin and bromelain and indicated that these enzymes had several differences in their specificities.…”

Section: Resultsmentioning

confidence: 99%

Differences in Substrate Specificities for Cysteine Proteinases Used in Blood Group Serology, and the Use of Bromelain in a Two-Phase Inhibitor Technique

Ogasawara

Mazda²

1989

Vox Sanguinis

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Because substrate specificities differ between proteolytic enzymes and because knowledge of the optimal enzyme activity levels is necessary in order to standardize procedures used in antibody screening, a study was made of the best common assay method for the routinely employed enzymes bromelain, papain and ficin. Casein degradation was found better suited to this purpose than azoalbumin. With standardization achieved, a useful two-phase bromelain inhibitor technique was devised using bromelain at 20 casein units of activity. This method improved upon the one-stage bromelain technique in terms of sensitivity, freedom from false positive reactions and it compared well with the two-phase papain inhibitor technique.

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Specificity and pH dependence of ficin-catalyzed hydrolyses. Comparisons with bromelain specificity

Cited by 18 publications

References 34 publications

Funastrain c II: A Cysteine Endopeptidase Purified from the Latex of Funastrum clausum

Funastrain c II: A Cysteine Endopeptidase Purified from the Latex of Funastrum clausum

The Amino Acid Sequences of Isoforms of the Bromelain Inhibitor from Pineapple Stem

Differences in Substrate Specificities for Cysteine Proteinases Used in Blood Group Serology, and the Use of Bromelain in a Two-Phase Inhibitor Technique

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