Glutathione-insulin Transhydrogenase Research Articles

Proteomic Evaluation to Identify Biomarkers for Carpal Tunnel Syndrome: A Comparative Serum Analysis

Carpal tunnel syndrome (CTS) is the most common peripheral nerve entrapment, causing pain, impairment, and disability. To identify proteins of CTS comprehensively, a comparative serum analysis of CTS patients and normal control subjects was performed. The two-dimensional electrophoresis patterns of serum obtained from six CTS patients and six normal control subjects were compared. We found 10 proteins that were significantly altered in the serum of CTS patients, among which four were upregulated and six were downregulated. The upregulated spots were identified as Chain A, heat shock 70-kDa protein, 42-kDa ATPase N-terminal domain; glutathione-insulin transhydrogenase (216AA); cAMP-dependent protein kinase inhibitor alpha; and mutant β-globin. The downregulated spots were identified as vitamin D-binding protein (VDBP), fibrinogen gamma chain, apolipoprotein A-IV (ApoA-IV), clusterin, heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1), and one unidentified protein. The information obtained from this proteomic analysis will be very useful in understanding the pathophysiology of CTS and in finding suitable proteins that can serve as new diagnostic biomarkers of CTS.

The Expression of and Insulin Binding to Cellular Thyroid Hormone Binding Protein, but Not Insulin Degrading Enzyme, Is Increased during 3T3-L1 Adipocytes Differentiation

Specific insulin binding to several proteins (cytosolic insulin binding proteins; CIBPs) in the isolated cytoplasm of numerous cell types has been demonstrated. CIBPs include insulin degrading enzyme (IDE), CIBP p55 (identified as cellular thyroid hormone binding protein (CTHBP), which is also known as protein disulfide isomerase, or glutathione insulin transhydrogenase). To assess the possible role of CIBP p55\\CTHBP in insulin action, we compared125I-insulin binding to CIBP in cytosol isolated from 3T3-L1 cells at various time points during differentiation of the adipocytes. Insulin did not bind to CTHBP in fibroblasts, but the labeling was markedly increased during adipocyte differentiation. In contrast, insulin binding to IDE did not change during differentiation. Protein expression level of CTHBP in the cytosol fraction increased gradually during the differentiation of adipocytes, whereas that of IDE did not change throughout the period. These data indicate that CTHBP, but not IDE, was up-regulated during differentiation of the adipocytes, suggesting that CIBP p55\\CTHBP may play a role in regulating some insulin action, especially the counter regulation between insulin and other hormones during adipocyte differentiation.

Glutathione-related detoxication functions in streptozotocin-induced diabetic rats.

In order to analyze the detoxication functions in rats with diabetes induced by streptozotocin, the authors administered to the diabetic animals two drugs, ethionine and benzo(a)pyrene, which affect mainly the liver and are metabolized through a glutathione conjugation process, and examined the changes in the content of glutathione and activities of related enzymes in the liver. In the liver of the rats with streptozotocin-induced diabetes, the total glutathione content, glutathione S-transferase activity and glutathione-insulin transhydrogenase activity were lower than those of normal rat livers, while the glutathione peroxidase activity showed high values. Although specific changes in the glutathione-related detoxication functions were observed in the rats to which ethionine or benzo(a)pyrene had been administered, these changes were not revealed under diabetic conditions. It is suggested that in diabetic rats responses to toxic stimuli are suppressed.

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.

Characterization of a cDNA for human glutathione-insulin transhydrogenase (protein-disulfide isomerase / oxidoreductase)

A human liver cDNA expression library in λ-phage gt11 was screened with monoclonal antibodies to rat liver protein-disulfide isomerase / oxidoreductase (EC 5.3.4.1 / 1.8.4.2), also known as glutathione-insulin transhydrogenase (GIT). The nucleotide sequence of the largest cDNA insert (hgit-1) was determined. It contained approx. 1500 basepairs, representing an estimated 65% of the glutathione-insulin transhydrogenase message. The amino-acid sequence deduced from this cDNA insert contains a 7-amino-acid long polypeptide determined by sequencing the active-site fragment isolated from the rat GIT protein. A comparison of the nucleotide sequence of hgit-1 and a previously reported nucleotide sequence of rat glutathione-insulin transhydrogenase cDNA shows that the human hgit-1 clone corresponds to the middle of the transhydrogenase message at amino-acid residue number 275 of the rat protein, and codes for 206 amino-acid residues, including one of the two active-site regions of glutathione-insulin transhydrogenase, a stop codon (TAA), a long 3′-noncoding region of over 800 bases, a polyadenylation signal (AATAA), and a 29 base poly(A) tail. There exists high homology between the human and rat enzymes (94% in the overall amino-acid sequence, with 100% in the active site region and 81% in the nucleotide sequence within the coding portion of hgit-1). As with the rat enzyme, the human enzyme shows some identity with another dithiol-disulfide-exchange protein, Escherichia coli thioredoxin. Like rat cDNA, the human hgit-1 cDNA hybridized to rat mRNA of 2500 bases on a Northern blot. The relative quantitative abundance of GIT mRNA in nine rat tissues studied using hgit-1 as a hybridization probe was found to be in the same order as previously found with the rat cDNA. Thus, the above studies indicate that glutathione-insulin transhydrogenase is a highly conserved protein and that the human hgit-1 cDNA is suitable for use as a probe for further studies on gene regulation of this enzyme.

The occurrence of glutathione—insulin transhydrogenase (protein—disulfide interchange enzyme) in the lens

Glutathione-insulin transhydrogenase (GIT, thiol:protein-disulfide isomerase/oxidoreductase, E.C. 5.3.4.1/1.8.4.2) catalyzes via sulfhydryldisulfide interchange, the scission as well as formation of disulfide bonds in many diverse proteins. Using insulin as a substrate, the lens epithelial layer of cows, rats and rabbits was found to contain GIT activity. The enzyme's activity is activated by GSH and inhibited by N-ethylmaleimide. Subcellular distribution of bovine lens epithelial homogenates showed that the majority of GIT activity is located in the insoluble fraction (10,000 g pellet) and in the high molecular weight fraction (60,000 g pellet). Lens epithelial extracts were subjected to SDS-PAGE followed by Western blot, and probed with a polyclonal antibody to rat liver GIT, or with either of two monoclonal antibodies directed against different epitopes of the enzyme. Lens epithelium was found to contain two forms of GIT, one with the same molecular weight as the purified enzyme (Mr 56Kd), and a second having an Mr of 67Kd. Immunoblots using polyclonal antibodies revealed an additional major immunoreactive band of 32Kd in the cow lens epithelial layer as well as in the isolated cortical and nuclear portions. Rat lenses showed no immunoreactive 32Kd band. Using a bovine cortical/nuclear fraction the 32Kd reactivity was found to be associated with the beta H-crystallin fraction, but the extract failed to show GIT activity with the insulin substrate. This suggests that beta H-crystallin may share a common epitope with GIT.

Insulin-degrading neutral cysteine proteinase activity of adipose tissue and liver of nondiabetic, streptozotocin-diabetic, and insulin-treated diabetic rats

The activity of the insulin-degrading enzyme neutral cysteine proteinase (EC 3.4.22.11, insulinase) was studied in adipose tissue and in liver of nondiabetic, streptozotocindiabetic, and insulin-treated diabetic rats. Proteinase activity was found to be significantly decreased during diabetes and was restored to near normal levels in both tissues following insulin treatment. The insulin-mediated increase of proteinase activity in both tissues was partially or completely blocked by actinomycin D (an inhibitor of RNA synthesis) and by cyclohexamide (an inhibitor of protein synthesis). Kinetic analysis showed that the changes in proteinase activity of both liver and adipose tissues were accompanied by a change in V max (i.e., maximal enzyme activity) without a change in K m (i.e., substrate affinity). These data indicate that insulin functions as an inducer for neutral cysteine proteinase in both tissues. These alterations in the proteinase activity paralleled the alterations in the activity of a second insulin-degrading enzyme, glutathione-insulin transhydrogenase in adipose tissue (this paper) and in liver (previously published papers) under the same physiological conditions.

Characterization and application of monoclonal antibodies directed to separate epitopes of glutathione-insulin transhydrogenase

Five monoclonal antibodies specific for glutathione-insulin transhydrogenase were characterized. None of the monoclonal antibodies cross-reacted with another insulin-degrading enzyme, neutral thiopeptidase. The isotype of four antibodies was IgG1 and of the fifth IgG2b. Affinity studies, competitive binding studies and immunoblot analysis of CNBr and trypsin cleavage products of glutathione-insulin transhydrogenase demonstrated that the four IgG1 antibodies were directed to an epitope of the enzyme which was distinct from the epitope recognized by the IgG2b antibody. Inhibition studies indicated that each monoclonal antibody, when added singly to glutathione-insulin transhydrogenase, was unable to inhibit the insulin-degrading activity of the enzyme. However, when monoclonal antibodies directed against separate epitopes of glutathione-insulin transhydrogenase were presented together (i.e., the IgG2b with any one of the four IgG1 antibodies), a loss in enzymatic activity was noted. Immunoblot analysis of rat organ extracts with the IgG1 antibodies demonstrated one immunoreactive protein band of M r 56000 in all tissues examined (liver, fat, pancreas and kidney) except the spleen, which demonstrated two immunoreactive protein bands of M r 56 000 and 51 000. The same immunoblots, when probed with the IgG2b antibody, demonstrated the same immunoreactive protein banding pattern as above plus an additional immunoreactive protein band of M r 67 000 in all tissues. Studies with spleen extracts from steptozotocin-induced diabetic rats demonstrated that there was a loss of the 51 000 immunoreactive band in diabetes. This 51 000 protein was restored upon insulin treatment of the diabetic rats and nullified upon concomitant administration of cycloheximide or actinomycin D with insulin. Immunoblots of human liver, adipose and skeletal muscle extracts indicated that each monoclonal antibody cross-reacted with the human form of the enzyme which had a molecular weight of M r 63 000; a second minor immunoreactive band of 67 000 was detected with the IgG2b antibody. The physiological significance of additional molecular forms of the enzyme (i.e., 67 000 and 51 000) remains to be determined.

Degradation of porcine relaxin by glutathione-insulin transhydrogenase and a neutral peptidase

The susceptibility of porcine relaxin and 125I-polytyrosyl-porcine relaxin to degradation by 3 purified senzymes involved in the degradation of insulin and proinsulin was examined. Rat liver glutathione-insulin transhydrogenase (GIT), which cleaves disulfide bonds in insulin, catalyzed a time- and concentration-dependent increase in trichloroacetic acid (TCA)-soluble radioactivity of relaxin. The Sephadex G-50 profile of the reaction products revealed conversion to the A- and B-chains. Relaxin competitively inhibited the degradation of insulin by GIT; however, kinetic analysis revealed insulin to be preferred over relaxin as a substrate. Rat liver cytosol neutral thiol peptidase (NTP) catalyzed a time- and concentration-dependent increase in the TCA solubility of relaxin and a shift in the Sephadex G-50 radioactivity profile to low molecular weight products. Kinetic analysis revealed that insulin and B-chain are preferred over relaxin as substrates for NTP. A third enzyme, rat kidney neutral metalloendopeptidase, which degrades proinsulin and insulin C-peptide but not insulin, also did not degrade porcine relaxin.

Stabilization of insulin receptor subunit structure by glutathione-insulin transhydrogenase

A partially purified insulin receptor preparation from rat liver was incubated at 37°C with and without the protein-disulfide interchange enzyme, glutathione-insulin transhydrogenase (thiol: protein-disulfide oxidoreductase/isomerase, EC 1.8.4.2/5.3.4.1). Insulin-binding activity was then assessed by crosslinking receptor- 125 I-insulin complexes and subjecting them to electrophoresis on SDS-polyacrylamide gels in the absence and presence of reductant followed by autoradiography. Prior incubation of the receptor at 37°C in the absence of the enzyme markedly decreased the subsequent binding of 125I-insulin to the holoreceptor ( M r 350 000) and to its subunits ( M r 180 000 and 130 000), while addition of the enzyme to the preincubation medium served to substantially prevent this decrease. The loss in binding at 37°C was not restored by subsequent addition of the enzyme, nor was the loss prevented by any of the several known inhibitors of proteolysis. The apparent stabilization of receptor by transhydrogenase, as evidenced by the increase in binding above control levels, was proportional to both the enzyme concentration and the duration of incubation. These effects seem to be specific for transhydrogenase, since several other disulfide-containing proteins were found to be ineffective. These data suggest that the stabilization of the subunit structure of the insulin receptor at physiological temperatures may take place via a disulfide interchange reaction catalyzed by glutathione-insulin transhydrogenase.

Inhibition of glutathione-insulin transhydrogenase by metal ions and activation by histidine and other chelating agents

The catalytic activity of purified glutathione-insulin transhydrogenase (thiol:protein-disulfide oxidoreductase/isomerase, EC 1.8.4.2) from bovine pancreas is markedly stimulated by histidine and other chelating agents. The activation produced was highest with EDTA, followed by EGTA, 8-hydroxyquinoline and 1,10-phenanthroline. Of the many amino acids tested, histidine was the only one that activated the enzyme; the structurally related compounds, 3-methylhistidine and imidazole also stimulated the enzyme, but 1-methylhistidine and histamine were without effect. The activation of EDTA was negated by metal ions, most effectively by Se 2+, Hg 2+, Cu 2+ and Zn 2+, and less effectively by Ca 2+ and Ni 2+. Likewise, activation by histidine was negated by Zn 2+ but not by Ca 2+ or Mg 2+. Thus, activation of glutathione-insulin transhydrogenase is apparently achieved in part by the chelation of inhibitory metal ion(s). These findings are consistent with a regulatory scheme for glutathione-insulin transhydrogenase in which (a) the enzyme is inhibited by selenium and heavy metal ions normally present in tissues and (b) this inhibition can be relieved by the addition of histidine or chelating agents.

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.

Characterization of insulin-degrading activity of intact and subcellular components of human fibroblasts.

We have studied insulin degrading activity (IDA) in cultured human fibroblasts and assessed the effect of various inhibitors of insulin processing on IDA. To evaluate the role of three enzymes of insulin degradation (neutral protease, microsomal glutathione insulin transhydrogenase, and lysosomal acid protease), we subfractionated homogenized fibroblasts into membrane (and nuclei) cytosol, mitochondria, microsomes, and lysosomes. Greater than 90% of IDA was found to be present in the cytosolar fraction containing neutral protease. IDA in intact fibroblasts was completely inhibited by 1 mM N-ethylmaleimide and partially by 0.5 mM dansylcadaverine (75%), 0.5 mM chloroquine (48%), 1 mg/ml bacitracin (32%) and Trasylol (30%). Lidocaine (5 mM) and glucagon (10(-6)M) exhibited about 15% inhibition with minimal inhibition (7%) by nonsuppressible insulin-like activity. Study of similar inhibitors on subfractionated components indicated inhibition of cytosolar enzyme by N-ethylmaleimide (100%), glucagon (30%), chloroquine (41%), nonsuppressible insulin-like activity (30%), Lidocaine (25%), dansylcadaverine (16%), and bacitracin (11%). Incubation of ammonium sulfate-fractionated cytosolar enzyme at 37 C with A14-125I-insulin resulted in generation of two intermediate peaks as early as 1 min. These peaks could be identified by HPLC but not by molecular sieve chromatography. These intermediates exhibited less immunoprecipitability with antiinsulin antibody and receptor binding with liver membrane preparations than intact insulin. Further incubation of A14-125I-insulin with the cytosolar enzyme(s) resulted in reduction of these peaks as well as insulin and formation of 125Iodotyrosine peak. We conclude that human fibroblast is capable of metabolizing cell-associated A14-125I-insulin in a time- and temperature-dependent manner. This process is inhibited by various inhibitors of insulin processing. The bulk of IDA consists of soluble neutral protease(s) with properties similar to other more purified neutral insulin protease preparations. This fraction, similar to the intact fibroblast degrades insulin to two intermediates with similar molecular weight to that of intact insulin but with more hydrophilicity and less binding affinity to antiinsulin antibody and liver membrane than intact insulin.

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: Igγ1 heavy chains and κ 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 NaDodSO 4/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.

Insulin-degrading activity in mononuclear and polymorphonuclear circulating leukocytes of nondiabetic and diabetic subjects.

Insulin-degrading activity in mononuclear (MN) and polymorphonuclear (PMN) fractions of circulating leukocytes obtained from 7 nondiabetic and 13 insulin-dependent diabetic subjects was studied. Insulin-degrading activity in both MN and PMN fractions was activated by reduced glutathione and was inhibited completely by N-ethylmaleimide. Both fractions had Michaelis-Menten constant (Km) (insulin) values within the range of values reported for purified glutathione-insulin transhydrogenase (GIT). In double immunodiffusion tests with antibody to human liver GIT, the MN fraction showed immunoprecipitin bands continuous with those of purified liver enzyme, but the PMN fraction showed little or no reaction with the antibody. These data indicate that both leukocyte fractions contain thiol-dependent insulin-degrading activity; however, only in the MN fraction was the degrading activity immunologically similar to that of liver GIT. Kinetic studies showed that the insulin-degrading activity of MN and PMN cells from diabetic patients had a 3.6- and 14.5-fold, respectively, higher maximal capacity (Vmax) than the insulin-degrading activity of these cells from nondiabetic subjects, without any change in the half-saturation constant for the substrate (Km for insulin). These results demonstrate that diabetes and/or insulin therapy result in increased leukocyte glutathione-dependent insulin-degrading activity.

Production and characterization of a monoclonal antibody to rat liver thiol: Protein-disulfide oxidoreductase/glutathione-insulin transhydrogenase

Rat liver thiol: protein-disulfide oxidoreductase/glutathione-insulin transhydrogenase (glutathione: protein disulfide oxidoreductase EC 1.8.4.2.) was purified and found to give two bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis. A monoclonal antibody was produced against this enzyme preparation and found to remove all the insulin degrading activity of purified preparations of the enzyme. This monoclonal antibody was also found to react with the two different forms of the enzyme observed on gel electrophoresis. These results suggest that glutathione-insulin transhydrogenase can exist in more than one state.

Nonlatent and latent hepatic glutathione-insulin transhydrogenase activity during perinatal development and liver regeneration in rats and in rat placenta

We have studied glutathione-insulin transhydrogenase (GIT) activity in differentiating rat liver during parturition and neonatal growth and during compensatory liver growth. Parturition is characterized by a rapid but transient increase in total (i.e., nonlatent plus latent) hepatic GIT activity resulting from changes in the quantity (Vm) of the enzyme while neonatal growth is characterized by an increase in the nonlatent (active) form which persists until just prior to weaning. During liver regeneration following partial hepatectomy, GIT activity/mg protein is lowest after 12 h of regeneration and then progressively increases exceeding the control levels after 72 h of regeneration. Placenta from near-term rats contain a significant concentration of GIT which is immunologically similar to hepatic GIT.

Immunological identity between bovine preparations of thiol: Protein-disulphide oxidoreductase, glutathione-insulin transhydrogenase and protein-disulphide isomerase

Five preparations of bovine thiol:protein-disulphide oxidoreductase/glutathione-insulin transhydrogenase (EC 1.8.4.2) and one preparation of bovine liver protein-disulphide isomerase (EC 5.3.4.1) from four different laboratories showed immunological identity in double immunodiffusion and rocket-line immunoelectrophoresis. Consequently, thiol:protein-disulphide oxidoreductase/glutathione-insulin transhydrogenase and protein-disulphide isomerase, formerly classified as two separate enzymes, should be considered as alternative activities of the same enzyme.

Topology of glutathione-insulin transhydrogenase in rat liver microsomes

Glutathione-insulin transhydrogenase (EC 1.8.4.2) catalyzes the inactivation of insulin through scission of the disulfide bonds to form insulin A and B chains. In the liver, the transhydrogenase occurs primarily in the microsomal fraction where most of the enzyme is present in a latent (‘inactive’) state. We have isolated rat hepatic microsomes with latent transhydrogenase activity being an integral part of the vesicles. We have used these vesicles to study the topological location of glutathione-insulin transhydrogenase by investigating the effects of detergents (Triton X-100 and sodium deoxycholate), phospholipase A 2 and proteinases (trypsin and thermolysin) on the latent enzyme activity. Treatment of intact vesicles with variable concentrations of detergents and phospholipase A 2 resulted in the unmasking of latent transhydrogenase activity. The extent of unmasking of transhydrogenase activity is dependent upon the concentration of detergent or phospholipase used and is accompanied by a parallel release of the enzyme into the soluble fraction. Activation of the transhydrogenase by phospholipase A 2 is partially inhibited by bovine serum albumin and the extent of inhibition is inversely proportional to the phospholipase concentration. In intact vesicles, latent transhydrogenase activity is resistant to proteolytic inactivation by both trypsin and thermolysin, while in semipermeable and permeable vesicles these proteases inactivate 60 and 25% of the total transhydrogenase activity, respectively. Together these results indicate that in microsomes transhydrogenase is probably weakly bound to membrane phospholipid components and that most of the enzyme is present on the cisternal surface (i.e., the luminal surface of endoplasmic reticulum) of microsomes. Each detergent and phospholipase apparently unmasks glutathione-insulin transhydrogenase activity through disruption of the phospholipid-enzyme interaction followed by translocation of the enzyme to the soluble (cytoplasmic) fraction and not through increases in substrate availability.

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.