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| [PMID]: | 26083455 |
| [Au] Autor: | Baeyens-Volant D; Matagne A; El Mahyaoui R; Wattiez R; Azarkan M |
| [Ad] Endereço: | Université Libre de Bruxelles, Faculty of Medicine, Protein Chemistry Unit, Campus Erasme (CP 609), 808 route de Lennik, 1070 Brussels, Belgium. |
| [Ti] Título: | A novel form of ficin from Ficus carica latex: Purification and characterization. |
| [So] Source: | Phytochemistry;117:154-67, 2015 Sep. | | [Is] ISSN: | 1873-3700 |
| [Cp] País de publicação: | England |
| [La] Idioma: | eng |
| [Ab] Resumo: | A novel ficin form, named ficin E, was purified from fig tree latex by a combination of cation-exchange chromatography on SP-Sepharose Fast Flow, Thiopropyl Sepharose 4B and fplc-gel filtration chromatography. The new ficin appeared not to be sensitive to thiol derivatization by a polyethylene glycol derivative, allowing its purification. The protease is homogeneous according to PAGE, SDS-PAGE, mass spectrometry, N-terminal micro-sequencing analyses and E-64 active site titration. N-terminal sequencing of the first ten residues has shown high identity with the other known ficin (iso)forms. The molecular weight was found to be (24,294±10)Da by mass spectrometry, a lower value than the apparent molecular weight observed on SDS-PAGE, around 27 kDa. Far-UV CD data revealed a secondary structure content of 22% α-helix and 26% ß-sheet. The protein is not glycosylated as shown by carbohydrate analysis. pH and temperature measurements indicated maxima activity at pH 6.0 and 50 °C, respectively. Preliminary pH stability analyses have shown that the protease conserved its compact structure in slightly acidic, neutral and alkaline media but at acidic pH (<3), the formation of some relaxed or molten state was evidenced by 8-anilino-1-naphtalenesulfonic acid binding characteristics. Comparison with the known ficins A, B, C, D1 and D2 (iso)forms revealed that ficin E showed activity profile that looked like ficin A against two chromogenic substrates while it resembled ficins D1 and D2 against three fluorogenic substrates. Enzymatic activity of ficin E was not affected by Mg(2+), Ca(2+) and Mn(2+) at a concentration up to 10mM. However, the activity was completely suppressed by Zn(2+) at a concentration of 1mM. Inhibitory activity measurements clearly identified the enzyme as a cysteine protease, being unaffected by synthetic (Pefabloc SC, benzamidine) and by natural proteinaceous (aprotinin) serine proteases inhibitors, by aspartic proteases inhibitors (pepstatin A) and by metallo-proteases inhibitors (EDTA, EGTA). Surprisingly, it was well affected by the metallo-protease inhibitor o-phenanthroline. The enzymatic activity was however completely blocked by cysteine proteases inhibitors (E-64, iodoacetamide), by thiol-blocking compounds (HgCl2) and by cysteine/serine proteases inhibitors (TLCK and TPCK). This is a novel ficin form according to peptide mass fingerprint analysis, specific amidase activity, SDS-PAGE and PAGE electrophoretic mobility, N-terminal sequencing and unproneness to thiol pegylation. |
| [Mh] Termos MeSH primário: |
Cisteína Proteases/metabolismo
Ficina/isolamento & purificação
Ficus/química
Látex/química
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| [Mh] Termos MeSH secundário: |
Cromatografia em Gel
Cisteína Proteases/efeitos dos fármacos
Inibidores de Cisteína Proteinase/farmacologia
Eletroforese em Gel de Poliacrilamida
Ficina/química
Ficina/metabolismo
Concentração de Íons de Hidrogênio
Látex/isolamento & purificação
Leucina/análogos & derivados
Leucina/farmacologia
Peso Molecular
Ressonância Magnética Nuclear Biomolecular
Fenantrolinas/farmacologia
Polietilenoglicóis
Estrutura Secundária de Proteína
Compostos de Sulfidrila/química
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| [Pt] Tipo de publicação: | JOURNAL ARTICLE |
[Nm] Nome de substância:
| 0 (Cysteine Proteinase Inhibitors); 0 (Latex); 0 (Phenanthrolines); 0 (Sulfhydryl Compounds); 30IQX730WE (Polyethylene Glycols); EC 3.4.- (Cysteine Proteases); EC 3.4.22.3 (Ficain); EC 3.4.22.3 (ficin E, Ficus); GMW67QNF9C (Leucine); R76F7856MV (E 64); W4X6ZO7939 (1,10-phenanthroline) |
| [Em] Mês de entrada: | 1604 |
| [Cu] Atualização por classe: | 150828 |
[Lr] Data última revisão:
| 150828 |
| [Sb] Subgrupo de revista: | IM |
| [Da] Data de entrada para processamento: | 150618 |
| [St] Status: | MEDLINE |
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