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Titolo:
SITE-DIRECTED MUTAGENESIS OF CONSERVED CYSTEINE RESIDUES IN PORCINE MEMBRANE DIPEPTIDASE - CYS-361 ALONE IS INVOLVED IN DISULFIDE-LINKED DIMERIZATION
Autore:
KEYNAN S; HABGOOD NT; 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:
Biochemistry
fascicolo: 38, volume: 35, anno: 1996,
pagine: 12511 - 12517
SICI:
0006-2960(1996)35:38<12511:SMOCCR>2.0.ZU;2-E
Fonte:
ISI
Lingua:
ENG
Soggetto:
KIDNEY MICROVILLAR MEMBRANE; HUMAN RENAL DIPEPTIDASE; PHOSPHOLIPASE-C; ECTO-ENZYMES; MICROSOMAL DIPEPTIDASE; DEHYDROPEPTIDASE-I; CATALYTIC ACTIVITY; MOLECULAR-CLONING; PROTEINS; EXPRESSION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
45
Recensione:
Indirizzi per estratti:
Citazione:
S. Keynan et al., "SITE-DIRECTED MUTAGENESIS OF CONSERVED CYSTEINE RESIDUES IN PORCINE MEMBRANE DIPEPTIDASE - CYS-361 ALONE IS INVOLVED IN DISULFIDE-LINKED DIMERIZATION", Biochemistry, 35(38), 1996, pp. 12511-12517

Abstract

Membrane dipeptidase (EC 3.4.1 3.19) is a glycosylphosphatidylinositol-anchored glycoprotein of the renal brush border which exists as a disulfide-linked homodimer. Porcine membrane dipeptidase has a subunit M(r) of 47 kDa, and the mature protein contains seven cysteine residuesper subunit, six of which are conserved in the human enzyme. Chemicalmodification established that cysteine residues are not involved in enzyme activity. In order to determine which of the cysteine residues are involved in the interchain disulfide bond, we have used a site-directed mutagenesis approach. Each of the conserved cysteine residues wasreplaced by glycine or alanine. The single mutants (C71G, C93A, C154G, C226A, C258G, and C361G) were expressed in COS-1 cells and their enzymatic activity and oligomeric structure determined. Only the C361G mutant migrated as a polypeptide of 47 kDa when subjected to denaturing polyacrylamide gel electrophoresis under nonreducing conditions. Thus,cysteine 361 is the only residue involved in disulfide linkage between the subunits. This places the disulfide bond close to the site of GPI anchor addition (Ser 368 in the porcine enzyme and to the membrane surface. Titration of the human and porcine proteins with 2-nitro-5-thiosulfabenzoate indicates that membrane dipeptidase additionally possesses two intrachain disulfide bonds. On native polyacrylamide gel electrophoresis, the C361G mutant migrates in a manner identical to that Sfthe wild type, indicating that the protein remains associated as a noncovalent homodimer. The expressed C361G mutant, unlike the wild type,is released from COS-1 cell membranes by trypsin and by an endogenousserine protease.

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Documento generato il 03/12/20 alle ore 21:02:02