Protein disulfide bond synthesis: a possible intracellular mechanism

Ziegler, Daniel M.; Poulsen, Lawrence L.

doi:10.1016/0968-0004(77)90042-1

Cited by 98 publications

(36 citation statements)

References 15 publications

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“…Egg white proteins are synthesized directly in the oviduct and it will be interesting to see whether these secretory cells themselves contain high levels of sulfhydryl oxidase activity. It should be noted that several other pathways for the net synthesis of disulfide bridges in proteins have been suggested including a microsomal hydroxylase (47) and a route involving the preferential transport of oxidized glutathione into the endoplasmic reticulum (48). Since egg white is a self-contained package of secreted proteins, it may prove useful in the study of the synthesis and isomerization of protein disulfides.…”

Section: Discussionmentioning

confidence: 99%

A Sulfhydryl Oxidase from Chicken Egg White

Hoober

Joneja

White

et al. 1996

Journal of Biological Chemistry

154

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A dimeric glycoprotein containing one FAD per ϳ80,000 M r subunit has been isolated from chicken egg white and found to have sulfhydryl oxidase activity with a range of small molecular weight thiols. Dithiothreitol was the best substrate of those tested, with a turnover number of 1030/min, a K m of 150 M, and a pH optimum of about 7.5. Oxidation of thiol substrates generates hydrogen peroxide in aerobic solution. Anaerobically, the ferricenium ion is a facile alternative electron acceptor. Reduction of the oxidase with dithionite or dithiothreitol under anaerobic conditions yields a two-electron intermediate (EH 2 ) showing a charge transfer band ( max 560 nm; ⑀ obs 2.5 mM ؊1 cm ؊1 ). Complete bleaching of the flavin and discharge of the charge transfer complex require a total of four electrons. Borohydride and catalytic photoreduction give the same spectral changes. EH 2 , but not the oxidized enzyme, is inactivated by iodoacetamide with alkylation of 2.7 cysteine residues/ subunit. These data indicate that the oxidase contains a redox-active disulfide bridge generating a thiolate to oxidized flavin charge transfer complex at the EH 2 level. Sulfite treatment does not form the expected flavin adduct with the native enzyme but cleaves the active site disulfide, yielding an air-stable EH 2 -like species. The close functional resemblance of the oxidase to the pyridine nucleotide-dependent disulfide oxidoreductase family is discussed.Sulfhydryl oxidases catalyze the oxidation of sulfhydryl groups to disulfides according to the following general reaction.They have been purified from a number of microbial and mammalian sources, but many questions concerning their mechanism and functional roles remain unresolved. Iron-dependent sulfhydryl oxidases have been found in bovine milk (1-3), in kidney (4), and in pancreatic zymogen granules (5). Coppercontaining sulfhydryl oxidases have been described from kidney and small intestine (6) and skin (7,8).A second distinct class of sulfhydryl oxidase is provided by FAD-dependent enzymes isolated from both rat seminal vesicles and fungal sources (9 -11). These careful studies show a range of diverse subunit composition and substrate specificity but clearly implicate the flavin cofactor in catalysis (9, 10). The present work reports the discovery and characterization of a flavoprotein sulfhydryl oxidase in chicken egg white.Our study began with the observation that egg white contains not only riboflavin (in the form of riboflavin-binding protein; Ref. 12) but also measurable levels of FAD (13). While the function of riboflavin-binding protein in the transport and storage of vitamin B 2 is well known (14), the reason for the presence of FAD in egg white was obscure. We first purified the FAD-containing protein by following its yellow color and subsequently identified it as a sulfhydryl oxidase active with a wide range of thiol substrates. This paper presents strong evidence for the involvement of a catalytically important disulfide as a second redox-active moiety in these flavin de...

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Section: Discussionmentioning

confidence: 99%

A Sulfhydryl Oxidase from Chicken Egg White

Hoober

Joneja

White

et al. 1996

Journal of Biological Chemistry

154

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“…la). The reducing potential of the cytoplasm, if not incompatible with the formation and maintenance of stable disulfide bonds in this compartment, is certain to antagonize the process (10,27). Disulfide bond formation would be expected to occur then only with passage of the protein into the oxidizing environment of the periplasm.…”

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confidence: 99%

Escherichia coli alkaline phosphatase fails to acquire disulfide bonds when retained in the cytoplasm

1991

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The cysteines of the Escherichia coli periplasmic enzyme alkaline phosphatase, which are involved in disulfide bonds in the native enzyme, were found to be fully reduced when the protein was retained in the cytoplasm. Under these circumstances the cysteines remained reduced for at least several minutes after the synthesis of the protein was completed. This contrasted with the normally exported protein, wherein disulfide bonds formed rapidly. Disulfide bond formation accompanied export and processing. The implications of these findings for the inactivity of the enzyme in the cytoplasm are discussed.The properties of the enzyme bacterial alkaline phosphatase (AP) have provided the basis for a convenient in vivo strategy for investigating protein localization and topology in Escherichia coli (for a review, see reference 18). This strategy has been developed around the observation that the normally periplasmic AP is enzymatically active only if exported from the cytoplasm (5). Retention in the cytoplasm by a variety of signal sequence mutations or deletions yields an inactive protein that is typically unstable (11,17,20). In cases where some fraction of wild-type export is preserved, the amount of activity correlates well with the amount of export (19). Thus the gene for AP, phoA, modified so as to no longer contain the information that codes for its signal sequence, can be fused to a gene of interest. Export of AP and enzymatic activity will thereby require that export information be present in the target protein.The inactivity of AP in the cytoplasm of E. coli has not been explained. One possibility, that rapid degradation in the cytoplasm preempts proper folding, has been discounted (5). Rather, the reverse appears to be true-that the failure of the protein to assume a native, enzymatically active conformation in the cytoplasm results in its rapid degradation. Indeed, cytoplasmic AP is susceptible to proteolytic digestion at concentrations where the native AP is fully resistant, indicating that cytoplasmic AP is either misfolded or unfolded (1).The native enzyme is well characterized both structurally and enzymologically (15). The crystal structure has been refined to a 2.0-A (0.2-nm) resolution (14

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“…On the other hand, several microsomal enzymes (cytochrome P-450s, NADPH cytochrome P-450 reductase, gulonolactone oxidase, microsomal iron protein, NADPH-dependent oxidase, sulfydryl oxidase, etc.) can produce reactive oxygen species (5)(6)(7)(8)(9)(10). The recent exploration of the ER oxidase protein (ERO1) and its role in the protein folding also support the latter mechanism (11,12).…”

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confidence: 99%

Preferential Transport of Glutathione versusGlutathione Disulfide in Rat Liver Microsomal Vesicles

Bánhegyi¹,

Lusini²,

Puskás³

et al. 1999

Journal of Biological Chemistry

114

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A bi-directional, saturable transport of glutathione (GSH) was found in rat liver microsomal vesicles. GSH transport could be inhibited by the anion transport blockers flufenamic acid and 4,4-diisothiocyanostilbene-2,2-disulfonic acid. A part of GSH taken up by the vesicles was metabolized to glutathione disulfide (GSSG) in the lumen. Microsomal membrane was virtually nonpermeable toward GSSG; accordingly, GSSG generated in the microsomal lumen could hardly exit. Therefore, GSH transport, contrary to previous assumptions, is preferred in the endoplasmic reticulum, and GSSG entrapped and accumulated in the lumen creates the oxidized state of its redox buffer. The endoplasmic reticulum (ER)1 of the cell is the site of the synthesis, posttranslational modification, and folding of proteins transported along the secretory pathway. The oxidizing environment in the lumen of the ER is necessary for the formation of disulfide bonds and for the proper folding of these proteins (1). The oxidative effects are reflected in and supported by the GSH redox buffer; the ratio of GSH and GSSG is around 2:1 within the lumen of ER and along the secretory pathway, whereas the cytosolic ratio ranges from 30:1 to 100:1 (2). However, the primary source(s) of the oxidative effect has not been demonstrated. Recent observations suggest two possible mechanisms. First, the preferential uptake of the oxidized member of a redox couple through the ER membrane and/or the efflux (or exocytosis) of its reduced form could ensure the oxidative environment. Alternatively, enzymes resident in the membrane or lumen of the ER could produce oxidizing compounds (e.g. reactive oxygen species) toward the lumen. Experimental evidence supports both mechanisms. Favoring the transport-based hypothesis, the preferential transport of dehydroascorbate (the oxidized form of ascorbate) has been described in rat liver microsomal vesicles (3). Similarly, the selective microsomal transport of GSSG was also reported (2, 4). On the other hand, several microsomal enzymes (cytochrome P-450s, NADPH cytochrome P-450 reductase, gulonolactone oxidase, microsomal iron protein, NADPH-dependent oxidase, sulfydryl oxidase, etc.) can produce reactive oxygen species (5-10). The recent exploration of the ER oxidase protein (ERO1) and its role in the protein folding also support the latter mechanism (11, 12). Because of the conflicting opinions, the microsomal transport of GSH and GSSG has not been unequivocally established. The presently available data are based on the detection of microsome-associated radioactivity by applying a rapid filtration method and radiolabeled compounds (2, 4); however, intraluminal GSH or GSSG contents upon transport have not been directly demonstrated. Therefore, experiments were undertaken to reinvestigate the transport of GSH and GSSG through the ER membrane.The main difficulties in the investigation of microsomal transport processes are deriving from the very small intraluminal space, the presence of (intraluminal) reactions affecting the transported c...

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Protein disulfide bond synthesis: a possible intracellular mechanism

Cited by 98 publications

References 15 publications

A Sulfhydryl Oxidase from Chicken Egg White

A Sulfhydryl Oxidase from Chicken Egg White

Escherichia coli alkaline phosphatase fails to acquire disulfide bonds when retained in the cytoplasm

Preferential Transport of Glutathione versusGlutathione Disulfide in Rat Liver Microsomal Vesicles

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