Photoaffinity labelling of lactate dehydrogenase from pig heart with a bifunctional NAD(+)-analogue.
Biochim Biophys Acta
; 1293(2): 277-83, 1996 Apr 16.
Article
em En
| MEDLINE
| ID: mdl-8620041
ABSTRACT
P1-N6-(4-azidophenylethyl)adenosine-P2-4-(3-azidopyridinio)b utyl diphosphate was synthesized with an [8-14C]adenine label. This bifunctional photoaffinity labelling reagent inactivates lactate dehydrogenase from pig heart upon irradiation with light of wavelength 300-380 nm. Stoichiometry of binding and enzymatic parameters suggest that the analogue is bound to the coenzyme binding site and that adjacent residues are modified. Four radioactive peptides were isolated by reverse-phase HPLC after tryptic digestion of the labelled protein. Amino-acid sequence analysis identified the peptides and correlation with the three-dimensional structure of dogfish lactate dehydrogenase reveals that the peptides correspond to positions affecting the coenzyme binding site, consistent with proper affinity labelling. Two of the peptides, Ile-77 --> Lys-81 and Asp-82 --> Asn-88, are located close to the adenine binding site. Low recovery of Thr-86 in combination with the detection of additional products in the sequence analysis indicates that this residue is modified by the photoaffinity label. The two other peptides (positions 119-124 and 318-328) are located next to the substrate binding site; their label is lost upon treatment with pyrophosphatase, showing that they are linked to the pyridinio moiety of the coenzyme analogue.
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Base de dados:
MEDLINE
Assunto principal:
Azidas
/
Marcadores de Afinidade
/
L-Lactato Desidrogenase
/
Miocárdio
/
NAD
Limite:
Animals
Idioma:
En
Revista:
Biochim Biophys Acta
Ano de publicação:
1996
Tipo de documento:
Article
País de afiliação:
Alemanha