Kinetic analysis of the mechanism and specificity of protein-disulfide isomerase using fluorescence-quenched peptides
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Kinetic analysis of the mechanism and specificity of protein-disulfide isomerase using fluorescence-quenched peptides. / Westphal, V; Spetzler, J C; Meldal, M; Christensen, U; Winther, Jakob R.
I: Journal of Biological Chemistry, Bind 273, Nr. 39, 1998, s. 24992-9.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - Kinetic analysis of the mechanism and specificity of protein-disulfide isomerase using fluorescence-quenched peptides
AU - Westphal, V
AU - Spetzler, J C
AU - Meldal, M
AU - Christensen, U
AU - Winther, Jakob R.
PY - 1998
Y1 - 1998
N2 - Protein-disulfide isomerase (PDI) is an abundant folding catalyst in the endoplasmic reticulum of eukaryotic cells. PDI introduces disulfide bonds into newly synthesized proteins and catalyzes disulfide bond isomerizations. We have synthesized a library of disulfide-linked fluorescence-quenched peptides, individually linked to resin beads, for two purposes: 1) to probe PDI specificity, and 2) to identify simple, sensitive peptide substrates of PDI. Using this library, beads that became rapidly fluorescent by reduction by human PDI were selected. Amino acid sequencing of the bead-linked peptides revealed substantial similarities. Several of the peptides were synthesized in solution, and a quantitative characterization of pre-steady state kinetics was carried out. Interestingly, a greater than 10-fold difference in affinity toward PDI was seen for various substrates of identical length. As opposed to conventional PDI assays involving larger polypeptides, the starting material for this assay is homogenous. It is furthermore simple and highly sensitive (requires less than 0.5 microgram of PDI/assay) and thus opens the possibility for quantitative determination of PDI activity and specificity.
AB - Protein-disulfide isomerase (PDI) is an abundant folding catalyst in the endoplasmic reticulum of eukaryotic cells. PDI introduces disulfide bonds into newly synthesized proteins and catalyzes disulfide bond isomerizations. We have synthesized a library of disulfide-linked fluorescence-quenched peptides, individually linked to resin beads, for two purposes: 1) to probe PDI specificity, and 2) to identify simple, sensitive peptide substrates of PDI. Using this library, beads that became rapidly fluorescent by reduction by human PDI were selected. Amino acid sequencing of the bead-linked peptides revealed substantial similarities. Several of the peptides were synthesized in solution, and a quantitative characterization of pre-steady state kinetics was carried out. Interestingly, a greater than 10-fold difference in affinity toward PDI was seen for various substrates of identical length. As opposed to conventional PDI assays involving larger polypeptides, the starting material for this assay is homogenous. It is furthermore simple and highly sensitive (requires less than 0.5 microgram of PDI/assay) and thus opens the possibility for quantitative determination of PDI activity and specificity.
KW - Amino Acid Sequence
KW - Catalysis
KW - Disulfides
KW - Humans
KW - Kinetics
KW - Microscopy, Fluorescence
KW - Oligopeptides
KW - Oxidation-Reduction
KW - Protein Disulfide-Isomerases
KW - Substrate Specificity
M3 - Journal article
C2 - 9737954
VL - 273
SP - 24992
EP - 24999
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 39
ER -
ID: 152444