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L W Ruddock

Publications and source records attributed to L W Ruddock.

22 records · Page 2Linked to original sources
PubMed1996

pH-dependence of the dithiol-oxidizing activity of DsbA (a periplasmic protein thiol:disulphide oxidoreductase) and protein disulphide-isomerase: studies with a novel simple peptide substrate.

A decapeptide containing two cysteine residues at positions 3 and 8 has been designed for use in monitoring the disulphide bond-forming activity of thiol:disulphide oxidoreductases. The peptide contains a tryptophan residue adjacent to one of the cysteine residues and an arginine residue adjacent to the other. Oxidation of this dithiol peptide to the disulphide state is accompanied by a significant change in tryptophan fluorescence emission intensity. This fluorescence quenching was used as the basis for monitoring the disulphide bond-forming activity of the enzymes protein disulphide-isomerase (PDI) and DsbA (a periplasmic protein thiol:disulphide oxidoreductase) in the pH range 4.0-7.5, where the rates of spontaneous or chemical oxidation are low. Reaction rates were found to be directly proportional to enzyme concentration, and more detailed analysis indicated that the rate-determining step in the overall process was the reoxidation of the reduced form of the enzyme by GSSG. The pH-dependence of the enzyme-catalysed reaction reflected primarily the pKa of the reactive cysteine residue at the active site of each enzyme. The data indicate a pKapp of 5.6 for bovine PDI and of 5.1 for Vibrio cholerae DsbA.

Amino Acid Sequence↗
PubMed1995

Kinetics of acid-mediated disassembly of the B subunit pentamer of Escherichia coli heat-labile enterotoxin. Molecular basis of pH stability.

The B-subunit pentamer of Escherichia coli heat-labile enterotoxin (EtxB) is highly stable, maintaining its quaternary structure in a range of conditions that would normally be expected to cause protein denaturation. In this paper the structural stability of EtxB has been studied as a function of pH by electrophoretic, immunochemical, and spectroscopic techniques. Disassembly of the cyclic pentameric structure of human EtxB occurs only below pH 2. As determined by changes in intrinsic fluorescence this process follows first-order kinetics, with the rate constant for disassembly being proportional to the square of the H+ ion concentration, and with an activation energy of 155 kJ mol-1. A C-terminal deletion mutant, hEtxB214, similarly shows first-order kinetics for disassembly but with a higher pH threshold, resulting in disassembly being seen at pH 3.4 and below. These findings are consistent with the rate-limiting step for disassembly of human EtxB being the simultaneous disruption of two interfaces by protonation of two C-terminal carboxylates. By comparison, disassembly of the B-subunit of cholera toxin (CtxB), a protein which shows 80% sequence identity with EtxB, exhibits a much lower stability to acid conditions; with disassembly of CtxB occurring below pH 3.9, with an activation energy of 81 kJ mol-1. Reasons for the observed differences in acid stability are discussed, and the implications of these findings to the development of oral vaccines using EtxB and CtxB are considered.

Bacterial Toxins↗
PubMedDate not supplied

pH dependence of the peptide thiol-disulfide oxidase activity of six members of the human protein disulfide isomerase family.

Protein folding in the endoplasmic reticulum is often associated with the formation of native disulfide bonds, a process which in vivo is one of the rate limiting steps of protein folding and which is facilitated by the enzyme protein disulfide isomerase (PDI). Higher eukaryotes have multiple members of the PDI family, for example, seventeen human PDIs have been reported to date. With multiple members of the same family being present, even within the same cell, the question arises as to what differential functions are they performing? To date there has been no systematic evaluation of the enzymological properties of the different members of the PDI-family. To address the question of whether different PDI family members have differing thioldisulfide chemistry, we have recombinantly expressed and purified six members of the family, PDI, PDIp, ERp57, ERp72, P5, and PDIr from a single organism, human. An examination of the pH-dependence and nature of the rate limiting step for the peptide thiol-disulfide oxidase activity of these enzymes reveals that, with the exception of PDIr, they are all remarkably similar. In the light of this data potential differential functions for these enzymes are discussed.

Catalytic Domain↗