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Titolo:
Non-covalent binding of endogenous ligands to recombinant cellular retinol-binding proteins studied by mass spectrometric techniques
Autore:
Elviri, L; Zagnoni, I; Careri, M; Cavazzini, D; Rossi, GL;
Indirizzi:
Univ Parma, Dipartimento Biochem & Biol Mol, I-43100 Parma, Italy Univ Parma Parma Italy I-43100 Biochem & Biol Mol, I-43100 Parma, Italy Univ Parma, Dipartimento Chim Gen & Inorgan, I-43100 Parma, Italy Univ Parma Parma Italy I-43100 Chim Gen & Inorgan, I-43100 Parma, Italy
Titolo Testata:
RAPID COMMUNICATIONS IN MASS SPECTROMETRY
fascicolo: 22, volume: 15, anno: 2001,
pagine: 2186 - 2192
SICI:
0951-4198(2001)15:22<2186:NBOELT>2.0.ZU;2-P
Fonte:
ISI
Lingua:
ENG
Soggetto:
ESCHERICHIA-COLI; FATTY-ACID; VITAMIN-A; NONCOVALENT INTERACTIONS; TRANSPORT PROTEINS; RAT; PURIFICATION; COMPLEXES; SPECIFICITY; FAMILY;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Physical, Chemical & Earth Sciences
Citazioni:
29
Recensione:
Indirizzi per estratti:
Indirizzo: Elviri, L Univ Parma, Dipartimento Biochem & Biol Mol, Parco Area Sci 23-A, I-43100 Parma, Italy Univ Parma Parco Area Sci 23-A Parma Italy I-43100 Parma, Italy
Citazione:
L. Elviri et al., "Non-covalent binding of endogenous ligands to recombinant cellular retinol-binding proteins studied by mass spectrometric techniques", RAP C MASS, 15(22), 2001, pp. 2186-2192

Abstract

Recent developments in mass spectrometry have demonstrated the capability of this technique to transfer non-covalent protein complexes, involving lowand high molecular weight ligands, from a condensed state to the gas phase. In this work, electrospray mass spectrometry with a quadrupole analyzer (ES-MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) were used to analyze the non-covalent association between recombinant rat cellular retinol-binding protein type-I (CRBP) with its specific ligand, all-trans retinol (vitamin A), and with fatty acids. Under denaturing conditions, MALDI-TOFMS and ES-MS techniques allowed determination of the molecular weight of apo-CRBP with good accuracy (<0.01%) and to identify a protein fraction (similar to 20%) retaining the initial methionine. By adding saturating amounts of vitamin A, ES-MS studies on the protein in the holo-form under native conditions allowed detection of retinol bound within the cavity together with water molecules, as expected from its crystal structure. ES mass spectra of CRBP in the native state were also recorded under non-denaturing conditions, with the aim to study non-covalent interactions between CRBP and non-specific ligands such as fatty acids,bound to the protein as a result of expression in various strains of E. coli grown in different media. Since ES mass spectra do not elucidate which species interact with the protein, in order to investigate the ligands possibly retained in the active site of recombinant CRBP, liquid chromatography/ES-tandem mass spectrometry was used. In particular, this technique was applied to identify and quantify fatty acids bound to CRBP. Quantitative data indicated the presence of a few fatty acids at a total concentration lower than 2% of that of the protein. Similar findings were observed for the homolog rat cellular retinol-binding protein type-II, demonstrating the high degree of purity and homogeneity of apo-CRBP preparations derived from gene expression. Copyright (C) 2001 John Wiley & Sons, Ltd.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 05/04/20 alle ore 03:46:04