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 -