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Titolo:
IDENTIFICATION BY SITE-DIRECTED MUTAGENESIS OF 3 ESSENTIAL HISTIDINE-RESIDUES IN MEMBRANE DIPEPTIDASE, A NOVEL MAMMALIAN ZINC PEPTIDASE
Autore:
KEYNAN S; HOOPER NM; TURNER AJ;
Indirizzi:
UNIV LEEDS,DEPT BIOCHEM & MOL BIOL LEEDS LS2 9JT W YORKSHIRE ENGLAND UNIV LEEDS,DEPT BIOCHEM & MOL BIOL LEEDS LS2 9JT W YORKSHIRE ENGLAND
Titolo Testata:
Biochemical journal
, volume: 326, anno: 1997,
parte:, 1
pagine: 47 - 51
SICI:
0264-6021(1997)326:<47:IBSMO3>2.0.ZU;2-V
Fonte:
ISI
Lingua:
ENG
Soggetto:
HUMAN RENAL DIPEPTIDASE; KIDNEY MICROVILLAR MEMBRANE; MICROSOMAL DIPEPTIDASE; DEHYDROPEPTIDASE-I; CATALYTIC ACTIVITY; MOLECULAR-CLONING; PHOSPHOLIPASE-C; ECTO-ENZYMES; EXPRESSION; PROTEINS;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
33
Recensione:
Indirizzi per estratti:
Citazione:
S. Keynan et al., "IDENTIFICATION BY SITE-DIRECTED MUTAGENESIS OF 3 ESSENTIAL HISTIDINE-RESIDUES IN MEMBRANE DIPEPTIDASE, A NOVEL MAMMALIAN ZINC PEPTIDASE", Biochemical journal, 326, 1997, pp. 47-51

Abstract

Membrane dipeptidase (EC 3.4.13.19) is a plasma membrane zinc peptidase that is involved in the renal metabolism of glutathione and its conjugates, such as leukotriene D-4. The enzyme lacks the classical signatures of other zinc-dependent hydrolases and shows no homology with any other mammalian protein. We have used site-directed mutagenesis to explore the roles of five histidine residues in pig membrane dipeptidase that are conserved among mammalian species. When expressed in COS-1 cells, the mutants H49K and H128L exhibited a specific activity and K-m for the substrate Gly-D-Phe comparable with those of the wild-type enzyme. However, the mutants H20L, H152L and H198K were inactive, but were expressed at the cell surface at equivalent levels to the wild-type, as assessed by immunoblotting and immunofluorescence. These three mutants were compared with regard to their ability to bind to the competitive inhibitor cilastatin, which binds with equal efficacy to nativeand EDTA-treated pig kidney membrane dipeptidase. Expressed wild-typeenzyme and mutants H20L and H198K were efficiently bound by cilastatin-Sepharose, but H152L failed to bind. Thus His-152 appears to be involved in the binding of substrate or inhibitor, whereas His-20 and His-198 appear to be involved in catalysis. Membrane dipeptidase shares some similarity with a dipeptidase recently cloned from Acinetobacter calcoaceticus. In particular, His-20 and His-198 of membrane dipeptidaseare conserved in the bacterial enzyme, as are Glu-125 and His-219, previously shown to be required for catalytic activity.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 03/12/20 alle ore 21:07:51