GSH/GSSG

The possible role of cellular glutathione status in the controlling sarcolemmal protein activity has been addressed by studying the effect of GSH, GSSG and several other thiol and disulphide compounds on Na/K ATPase activity using (1) an isolated bovine ventricular Na/K ATPase preparation (2) crude sarcolemmal preparations (biochemical studies) (c) Langendorff-perfiised isolated hearts (cytochemical studies) and (4) isolated ventricular myocytes (electrophysiologjcal studies). 

Glutathione reductase (GR) catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) using NADPH provided from the hexose monophosphate pathway. GR, a ubiquitous flavoenzyme, maintains a high value of two for the GSH/GSSG ratio in the red blood cells. l,3-Bis(2-chloroethyl)-nitrosourea (BCNU) selectively inhibits cellular GR. GR is composed of two identical subunits, each of molecular mass 50 kDa (S8). The three-dimensional structure and mechanism of catalysis have been established for human GR (K17). 

III. Glutathione reductase (EC 1.6.4.2) It is a flavoprotein that catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG) to glutathione (GSH). This enzyme is essential for the GSH redox cycle which maintains adequate levels of reduced cellular GSH. A high GSH/GSSG ratio is essential for protection against oxidative stress. 

GSH, GSSG quantitation Cc, Dp, Pc Liver (Cc), digestive gland (Dp, Pc) Oxidative stress, mercury poisoning... 

Conversely, when 6 was extended N-terminally by a small portion of the prosequence, that is by the Lys-Arg dipeptide to give KR-ET-1 (7), and subjected to oxidation in 0.1 M NH4OAC buffer (pH 9.5) at 25 °C for 24 hours the ratio of native to nonnative-type disulfide isomer increases remarkably (88 12 vs 75 25 for 6), whilst isomer 3 is not detectable. In the presence of GSH/GSSG an additional increase to almost quantitative formation of the native isomer was observed (Table 2). 58 This improvement was completely abolished by substituting Asn for Asp at position 8 (D8N-KR-ET-1), whereas most of the increase was maintained with similar carboxamide analogues in positions 10 and 18 (Table 2). 

The reduced form of Na+, K+-ATPase inhibitor-I (10) was obtained by treatment of the protected peptide synthesized by the soln procedure with HF, followed by reaction with Hg(OAc)2. After purification of the crude product on Sephadex G-25, the reduced peptide (110 mg) was dissolved in 0.1 M NH4OAc buffer (1L, pH 7.8) at a peptide concentration of 0.018 mM and then stirred at rt. After 24 h, the major peak in the HPLC, which coeluted with the natural product, corresponded to 55% of the product distribution. The mixture was acidified to pH 3 with AcOH and 10 was purified by RP-HPLC. When the oxidation was carried out in the presence redox reagents at a peptide/GSH/GSSG ratio of 1 100 10, after 24 h the major oxidation product increased to 69%. The mixture was acidified with AcOH and the product (10) isolated by preparative HPLC yield 20%. The product was characterized by MALDI-TOF-MS and amino acid analysis a combination of enzymatic peptide mapping and synthetic approaches were applied to assign the cystine connectivities. 

As exemplarily shown in the case of charybdotoxin, a 37-residue peptide with three intramolecular disulfide bonds,[70] operating in redox buffer was crucial for efficient formation of the correct disulfide bonds.[71] When the reduced peptide was oxidized in 0.1 M NHtOAc buffer (pH 8.0) at 0.11 mM concentration in the presence of redox reagents (peptide/GSH/GSSG ratio of 1 60 6), the main product was the native peptide contaminated... 

Figure 3 HPLC Profile of the Effects of Reaction Temperature and NH4OAc Concentration on the Folding of Reduced co-Conotoxin MVIIC in the Presence of Redox Reagents Buffer NH4OAc pH 7.7 Peptide/GSH/ GSSG 1 100 10 Time 24hl la b...

Paolisso G, Di MG, Pizza G, D Amore A, Sgambato S, et al. 1992. Plasma GSH/GSSG affects glucose homeostasis in healthy subjects and non-insulin-dependent diabetics. Am... 

Very dynamic glutathione redox cycle activity to maintain GSH/GSSG ratio of about 50-100... 

Active glutathione redox cycle that maintains a GSH/GSSG ratio of about 25-50... 

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