The mammalian peroxisomal membrane is permeable to both GSH and GSSG - Implications for intraperoxisomal redox homeostasis.

Despite the large amounts of H2O2 generated in mammalian peroxisomes, cysteine residues of intraperoxisomal proteins are maintained in a reduced state. The biochemistry behind this phenomenon remains unexplored, and simple questions such as "is the peroxisomal membrane permeable to glutathione?" or "is there a thiol-disulfide oxidoreductase in the organelle matrix?" still have no answer. We used a cell-free in vitro system to equip rat liver peroxisomes with a glutathione redox sensor. The organelles were then incubated with glutathione solutions of different redox potentials and the oxidation/reduction kinetics of the redox sensor was monitored. The data suggest that the mammalian peroxisomal membrane is promptly permeable to both reduced and oxidized glutathione. No evidence for the presence of a robust thiol-disulfide oxidoreductase in the peroxisomal matrix could be found. Also, prolonged incubation of organelle suspensions with glutaredoxin 1 did not result in the internalization of the enzyme. To explore a potential role of glutathione in intraperoxisomal redox homeostasis we performed kinetic simulations. The results suggest that even in the absence of a glutaredoxin, glutathione is more important in protecting cysteine residues of matrix proteins from oxidation by H2O2 than peroxisomal catalase itself.

The relationship between serum thiol levels and thiol/disulfide homeostasis in women with tubal ectopic pregnancy.

OBJECTIVE: The aim of this study was to investigate the thiol/disulfide homeostasis in tubal ectopic pregnancies in terms of early diagnosis of the disease. DESIGN: A prospective case-control study was carried out between June 2017-February 2018 in the Gynaecology Department of Umraniye Medical and Research Hospital. MATERIALS AND METHODS: A total of 42 women with ectopic pregnancy were compared with 44 healthy women who have intrauterine first trimester pregnancies. The thiol/disulfide homeostasis is evaluated with the spectrophotometric measurement method that was recently developed by Erel&Neselioglu. RESULTS: Disulfide/native thiol and disulfide/total thiol ratios were increased (p = 0.018 and p = 0.023 respectively), while native thiol/total thiol ratios and native thiol levels were decreased in tubal ectopic pregnancy group according to control group (p = 0.023). Between control and tubal ectopic pregnancy groups no differences were measured in disulfide levels (p = 0.350). The area under curve for native thiol and total thiol were 0.937 and 0.927, respectively. The optimum cut off value for native thiol was 379.95 µmol/l with a sensitivity of 90% and specificity of 81%. The optimum cut off value for total thiol was 432.5 µmol/l had 92% sensitivity and 79% specificity. LIMITATIONS: In the study, whether intrauterine pregnancies resulted in miscarriage or delivery can be examined. CONCLUSION: Increased disulfide/native thiol levels, disulfide/total-thiol ratio and decreased native/total thiol ratio were found to be significantly associated with the presence of tubal ectopic pregnancy which can be useful for the early diagnosis of the disease.

ERp19 contributes to tumorigenicity in human gastric cancer by promoting cell growth, migration and invasion.

ERp19, a mammalian thioredoxin-like protein, plays a key role in defense against endoplasmic reticulum stress. It belongs to the protein disulfide isomerize (PDI) family, whose members have been implicated in development of breast, ovarian and gastrointestinal cancers. However, the role of ERp19 in gastric cancer (GC) remains undefined. Therefore, we sought to investigate the expression and prognostic value of ERp19 in GC patients, and to explore the role of ERp19 in tumorigenicity. Expression of ERp19 in gastric tissues was assessed by immunohistochemical staining and real-time PCR in clinical samples of GC patients. Statistical analysis of clinical cases revealed that the expression levels of ERp19 were higher in tumor tissues than non-tumor tissues. And the level of ERp19 expression was correlated with tumor size, lymph node involvement and poor clinical prognosis. Furthermore, ERp19 knockdown dramatically suppressed gastric cancer cell growth, inhibited cellular migration/invasion and down regulated the phosphorylation of FAK and paxillin, whereas ERp19 over-expression reversed these changes. We conclude that ERp19 contributes to tumorigenicity and metastasis of GC by activating the FAK signaling pathway, and may function as an oncogene in GC. ERp19 may represent a new diagnostic and prognostic marker and a novel target for the treatment of GC.

Investigation of protein FTT1103 electroactivity using carbon and mercury electrodes. Surface-inhibition approach for disulfide oxidoreductases using silver amalgam powder.

Recently, it was shown that electrochemical methods can be used for analysis of poorly water-soluble proteins and for study of their structural changes and intermolecular (protein-ligand) interactions. In this study, we focused on complex electrochemical investigation of recombinant protein FTT1103, a disulfide oxidoreductase with structural similarity to well described DsbA proteins. This thioredoxin-like periplasmic lipoprotein plays an important role in virulence of bacteria Francisella tularensis. For electrochemical analyses, adsorptive transfer (ex situ) square-wave voltammetry with pyrolytic graphite electrode, and alternating-current voltammetry and constant-current chronopotentiometric stripping analysis with mercury electrodes, including silver solid amalgam electrode (AgSAE) were used. AgSAE was used in poorly water-soluble protein analysis for the first time. In addition to basic redox, electrocatalytic and adsorption/desorption characterization of FTT1103, electrochemical methods were also used for sensitive determination of the protein at nanomolar level and study of its interaction with surface of AgSA microparticles. Proposed electrochemical protocol and AgSA surface-inhibition approach presented here could be used in future for biochemical studies focused on proteins associated with membranes as well as on those with disulfide oxidoreductase activity.

Novel processing and localization of catA, ccdA associated thiol-disulfide oxidoreductase, in protein hyper-producing bacterium Brevibacillus choshinensis.

Previously, we have cloned ccdA and its associated thiol-disulfide oxidoreductase gene, catA, in Brevibacillus choshinensis. CcdA is known to be an integral membrane protein and its associated oxidoreductase homologues are believed to be membrane anchoring proteins, both providing reducing equivalents across the membrane to control correct disulfide bond formation. Here, we found that CatA is first localized as a membrane bound form and then slowly released into the cellular periphery and culture medium with cleavage at a novel processing site.

Di-fluoresceinthiocarbamyl-insulin: a fluorescent substrate for the assay of protein disulfide oxidoreductase activity.

We have developed a novel method for the continuous assay of protein disulfide oxidoreductase activity using as substrate bovine pancreas insulin in which both N-terminal amino groups are chemically modified with fluorescein isothiocyanate. The reduction of intercatenary disulfide bonds of di-fluoresceinthiocarbamyl-insulin with dithiothreitol initially lowers but subsequently enhances the emission intensity. In this biphasic kinetics, the rate of increase is sensitive enough for the estimation of Escherichia coli thioredoxin concentrations from 5 nM (0.06 microgram/ml) to 500 nM (6 micrograms/ml). Neither changes of pH over a range of 6.2 to 8.4 nor neutral salts (K+, Mg2+, and Ca2+) at concentrations lower than 100 mM affect this simple reaction system. Moreover, the fluorometric method is functional for measuring the reductive capacity of Brassica napus protein disulfide isomerase. Hence, a highly reproducible and accurate one-state assay for protein disulfide oxidoreductase activity not only greatly improves the sensitivity compared to the commonly used turbidimetric assay but also represents a reliable alternative to assays based on accessory enzymes or radiolabeled substrates.

A novel HPLC method for glutathione-insulin transhydrogenase assay using fluorescence labeled insulin.

The activity of rat liver glutathione-insulin transhydrogenase (GIT) was measured by HPLC. The degradation of fluorescein isothiocyanate-I (FITC-I)-labeled insulin is separated into several peaks, which are bound different amount of FITC-I. We selected mono-fluorescein-thiocarbamylated insulin to estimate the decrease of insulin content and it became possible to assay GIT activity. This novel method was time-saving and simple, and this system could utilize instead of previous method.

Localization of glutathione-insulin transhydrogenase (protein-disulfide interchange enzyme) in pancreas using immunocytochemistry, immunodiffusion, and enzymatic activity assay techniques.

The localization of the protein-disulfide interchange enzyme, glutathione-insulin transhydrogenase (GIT), in rat and mouse pancreas was studied by protein A-gold immunocytochemistry, immunodiffusion, and assay of enzymatic activity. Immunocytochemistry on tissue sections using antibody to GIT and protein A-gold complex indicated the presence of GIT in alpha and beta cells in islets as well as acinar cells. The beta cells in obese (ob/ob) hyperinsulinemic mice showed increased GIT immunoreactivity. In both alpha and beta cells, GIT immunoreactive sites were associated predominantly with secretory granules. In pancreas from rats injected with glibenclamide, the degranulated beta cells contained GIT immunoreactive sites on the cisternal surface of the rough endoplasmic reticulum (RER). In acinar cells, the RER, Golgi elements, condensing vacuoles, and zymogen granules possessed GIT immunoreactive sites as did mitochondria. Immunocytochemistry on sections of isolated subcellular fractions showed that GIT was associated with different membranes. The enzymatic activity of GIT was found in the following order: Golgi elements greater than mitochondria greater than microsomes greater than zymogen granules greater than cytosol. In Ouchterlony immunodiffusion tests, each subcellular fraction showed a precipitin band which was continuous with that of purified GIT, a result indicating the presence of immunologically identical GIT in all fractions.

Insulin degradation: radioimmunoassay for glutathione-insulin transhydrogenase and its application.

A double-antibody radioimmunoassay for the insulin-degrading enzyme, glutathione-insulin transhydrogenase (GIT), has been developed with the use of rabbit antiserum against human liver GIT and [125I]-GIT. The method can determine as little as 32 fmol of GIT, thus allowing measurements in needle tissue biopsy samples and in plasma, which have not been possible with previous enzymatic procedures. Relative competition in the radioimmunoassay by unlabelled GITs purified from other sources are in agreement with homologies in GITs previously found using the enzymatic assay. No competition was observed with pork insulin, bovine ribonuclease, human albumin or human gamma-globulin, indicating that the radioimmunoassay is highly specific for GIT. Similar competition curves were observed for native GIT; active, reduced GIT; or for the inactive, S-(ethylsuccinimido) derivative of GIT. The radioimmunoassay thus measures total (active + inactive) GIT and permits determinations in the presence of materials which react with the active site and render the enzymatic methods unusable. Radioimmunoassay of plasma and extracts of liver, muscle and adipose tissues from diabetic and non-diabetic subjects showed parallel competition curves with standard purified human GIT indicating that GITs of non-diabetic and diabetic persons are immunologically very similar or identical. Concentrations of GIT in plasma determined by radioimmunoassay were significantly higher in diabetic than those in non-diabetic subjects (1620 +/- 80 versus 1070 +/- 30 fmol/l, p less than 0.001). Tissue GIT levels found by the radioimmunoassay as well as by the enzyme assay, both in non-diabetic and diabetic subjects, were highest in the liver, intermediate in the adipose tissue and lowest in the muscle.

Production of a monoclonal antibody directed against rat liver glutathione-insulin transhydrogenase.

A hybridoma cell line secreting monoclonal antibody specific for glutathione-insulin transhydrogenase has been produced by fusing mouse myeloma cells with spleen cells from mice immunized to purified rat liver glutathione-insulin transhydrogenase. The secreted antibody isotypes were found to be: Ig gamma 1 heavy chains and kappa light chains. This monoclonal antibody has been used to screen glutathione-insulin transhydrogenase in various rat tissue extracts (liver, fat, heart, testis, spleen, lung and kidney) following separation on NaDodSO4/urea polyacrylamide disc-gel electrophoresis and electrophoretic transfer to nitrocellulose. Screening with the monoclonal antibody showed the presence of one immunoreactive protein band equal in molecular weight to that of purified rat liver GIT (Mr 53,000) in extracts of all tissues studied and a second immunoreactive protein band of lower molecular weight (Mr 49,000) in spleen and lung tissue extracts. Separation of these two proteins by HPLC using a TSK-DEAE column demonstrated that both proteins exhibit insulin degrading activity. These data indicate that GIT may occur in multiple forms in some tissues.

Studies on the subcellular localization and role of glutathione-insulin transhydrogenase in rat liver.

125I-insulin was shown to be internalized in vivo to a discrete population of low-density membranes (ligandosomes), distinct from the +Golgi, endoplasmic reticulum, plasma membrane, and lysosomes. However, analytical subcellular fractionation shows that glutathione-insulin transhydrogenase is localized to the endoplasmic reticulum. Measurement of the specific enzyme activity of glutathione-insulin transhydrogenase showed no differences between normal, diabetic, and hyperinsulinaemic rats. These results suggest that glutathione-insulin transhydrogenase is not directly involved in the subcellular processing of receptor-bound internalized insulin.

Insulin degradation. XXVIII. Immunocytochemical localization of glutathione-insulin transhydrogenase in the pancreas, kidney and liver of normal and streptozotocin-diabetic rats and of lean and obese (ob/ob) mice.

Glutathione-insulin transhydrogenase catalyzed the inactivation of insulin by splitting the hormone into A and B chains. We have localized this enzyme immunocytochemically by light microscopy in the pancreas, kidney and liver of both lean and obese (ob/ob) mice and similarly in normal and streptozotocin-diabetic rats. Localization was achieved by an antibody to glutathione-insulin transhydrogenase using a peroxidase-antiperoxidase technique. In comparison with tissues from control animals, positive immunostaining for glutathione-insulin transhydrogenase was increased in the obese mouse but reduced in the diabetic rat. Different tissues showed considerable variation in the amount of glutathione-insulin transhydrogenase which could be detected. In the pancreatic islets there was little or no evidence for the presence of the enzyme in peripheral cells. In the kidney, immunocytochemical staining was found only in the proximal tubules. In the liver there was a generalised distribution of the enzyme, but the greatest concentration was in the periportal region. These observations parallel the biochemical data relating to glutathione-insulin transhydrogenase, indicating that different amount of insulin degrading activity exist in different regions of tissues.

The immunochemical identification of a thiol-protein disulfide oxidoreductase (glutathione-insulin transhydrogenase) in pancreatic islets.

Pancreatic islets contained insulin-degrading activity that was completely removed by antisera to purified microsomal thiol-protein disulfide oxidoreductase from rat liver. In Ouchterlony double-diffusion experiments with these antisera, extracts of islet homogenates showed a single precipitation band of identity with the purified liver enzyme. Two dimensional immunoelectrophoresis also gave a single precipitate peak like that of the liver enzyme. The concentration of the enzyme in rat islets as determined by quantitation of the precipitates obtained in the electroimmunodiffusion analysis was in the order of 1.0% of total islet protein. The results suggest that, in vitro, cleavage of insulin into its polypeptide chains is catalyzed by the thiol-protein disulfide oxidoreductase. This enzyme promoting thiol-protein disulfide inter-change may be important for regulating the content of pancreatic insulin.