Protein disulfide-isomerase systems

Reoxidation of pro-a chains of types I and II procollagen into triple helical procollagen was studied using various concentrations of reduced (GSH) and oxidized (GSSG) glutathione as an oxidizing system in the reaction mixture. Pure protein disulfide-isomerase (PDI 5ng) was used in the reaction. 

A pathway analogous to the DsbA-DsbB system was recently shown to operate in the endoplasmic reticulum of eukaryotes (Frand and Kaiser, 1998 1999 Pollard et al., 1998). This pathway includes the membrane-bound protein EROl, which keeps protein disulfide isomerase (PDI) in an oxidized state in vivo. Consequently, PDI plays a role somewhat similar to that of DsbAin catalyzing the formation of disulfide bonds during the folding of secreted proteins. The mechanisms driving oxidative protein folding in eukaryotes are described in more detail in another chapter in this volume. 

There are at least two types of enzyme systems involved in the formation and breakage of disulfide bonds of cystine residues in proteins. A thiol-disulfide interchange enzyme (protein disulfide-isomerase EC 5.3.4.1 other name, S-S-rearrangase) was first described in 1963 ( 47) and was subsequently purified from beef liver (48,49). The molecular weight of the enzyme is 42,000. The enzyme contains three half-cystine residues, one of which must be cysteine in order for the enzyme to be active. The enzyme catalyzed the rearrangement of random incorrect pairs of half-cystine residues to the native disulfide bonds in several protein substrates. Low levels of mercaptoethanol were required for activity unless the enzyme was reduced prior to use. The efficiency of the enzyme in catalyzing the interconversion of disulfide bonds was found to be a function of the number of disulfide bonds in the substrate. Purification of a thiol-disulfide interchange enzyme from Candida claussenii has been described recently (50). 

By analogy with animal systems, the function of signal sequences in plant storage proteins is to facilitate the translocation of the storage protein into the lumen of the endoplasmic reticulum (ER) as the first step in intracellular transport. Protein folding and disulfide bond formation are considered to occur within the lumen of the endoplasmic reticulum, and may be assisted by molecular chaperones and by the enzyme protein disulfide isomerase respectively [83]. The precise mechanism of intracellular transport of storage proteins from their site of synthesis to their site of deposition are still largely unknown but a two-way hypothesis has been proposed by Shewry [45]. 

It is possible to reduce enzymatically both AFR and DHA, regenerating ascorbate and thus decreasing nutritional requirements in animals unable to carry out the synthesis de novo. Enzymatic systems that reduce AFR usually utilize NADH as an electron donor (one-electron reduction pathway or NADH-ascorbate free radical reductase). Reduction of DHA to ascorbate involves two electrons and has been attributed to thioltransferase (glutaredoxin) and protein disulfide isomerase (Wells et aL, 1990). However, taking into consideration the reported values for DHA (millimolar range) it is unlikely that the latter enzymes are involved in the maintenance of ascorbate in its reduced state in vivo and that function is carried out by NADH-AFR reductase (Minetti et aL, 1992). 

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