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Titolo:
Parameters affecting in vitro oxidation/folding of maurotoxin, a four-disulphide-bridged scorpion toxin
Autore:
di Luccio, E; Azulay, DO; Regaya, I; Fajloun, Z; Sandoz, G; Mansuelle, P; Kharrat, R; Fathallah, M; Carrega, L; Esteve, E; Rochat, H; De Waard, M; Sabatier, JM;
Indirizzi:
CNRS, UMR 6560, F-13916 Marseille 20, France CNRS Marseille France 20CNRS, UMR 6560, F-13916 Marseille 20, France CEA, INSERM, EMI 99 31, F-38000 Grenoble, France CEA Grenoble France F-38000 INSERM, EMI 99 31, F-38000 Grenoble, France Inst Pasteur, Tunis, Tunisia Inst Pasteur Tunis TunisiaInst Pasteur, Tunis, Tunisia Hop St Marguerite, CIC9502, F-13274 Marseille, France Hop St Marguerite Marseille France F-13274 02, F-13274 Marseille, France
Titolo Testata:
BIOCHEMICAL JOURNAL
, volume: 358, anno: 2001,
parte:, 3
pagine: 681 - 692
SICI:
0264-6021(20010915)358:<681:PAIVOO>2.0.ZU;2-5
Fonte:
ISI
Lingua:
ENG
Soggetto:
PANCREATIC RIBONUCLEASE-A; PROTEIN-FOLDING DYNAMICS; POTASSIUM CHANNELS; K+ CHANNELS; HIGH-AFFINITY; PROLINE; MAURUS; ISOMERIZATION; MECHANISMS; CATALYSIS;
Keywords:
mass spectrometry; half-cystine pairing;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
34
Recensione:
Indirizzi per estratti:
Indirizzo: Sabatier, JM CNRS, UMR 6560, Bd Pierre Dramard, F-13916 Marseille 20, France CNRS Bd Pierre Dramard Marseille France 20 seille 20, France
Citazione:
E. di Luccio et al., "Parameters affecting in vitro oxidation/folding of maurotoxin, a four-disulphide-bridged scorpion toxin", BIOCHEM J, 358, 2001, pp. 681-692

Abstract

Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four disulphide bridges that acts on various K+ channel subtypes. MTX adopts a disulphidebridge organization of the type C1-C5, C2-C6, C3-C4 and C7-C8, and folds according to the common alpha/beta scaffold reported for other known scorpion toxins. Here we have investigated the process and kinetics of the in vitro oxidation/folding of reduced synthetic L-MTX (L-sMTX, where L-MTX contains only L-amino acid residues). During the oxidation/folding of reduced L-sMTX, the oxidation intermediates were blocked by iodoacetamide alkylation offree cysteine residues, and analysed by MS. The L-sMTX intermediates appeared sequentially over time from the least (intermediates with one disulphide bridge) to the most oxidized species (native-like, four-disulphide-bridged L-sMTX). The mathematical formulation of the diffusion-collision model being inadequate to accurately describe the kinetics of oxidation/folding of L-sMTX, we have formulated a derived mathematical description that better fits the experimental data. Using this mathematical description, we have compared for the first time the oxidation/folding of L-sMTX with that of D-sMTX, its stereoisomer that contains only D-amino acid residues. Several experimental parameters, likely to affect the oxidation/folding process, were studied further; these included temperature, pH, ionic strength, redox potential and concentration of reduced toxin. We also assessed the effects of some cellular enzymes, peptidylprolyl cis-trans isomerase (PPIase) and proteindisulphide isomerase (PDI), on the folding pathways of reduced L-sMTX and D-sMTX. All the parameters tested affect the oxidative folding of sMTX, andthe kinetics of this process were indistinguishable for L-sMTX and D-sMTX,except when stereospecific enzymes were used. The most efficient conditions were found to be: 50 mM Tris/HCl/1.4 mM EDTA, pH 7.5, supplemented by 0.5mM PPIase and 50 units/ml PDI for 0.1 mM reduced compound. These data represent the first report of potent stereoselective effects of cellular enzymes on the oxidation/folding of a scorpion toxin.

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Documento generato il 14/07/20 alle ore 13:21:43