Influence of glutathione on the reactivation of enzymes containing cysteine or cystine.
Refolding of dimeric porcine cytosolic or mitochondrial malate dehydrogenases and of tetrameric pig heart and skeletal muscle lactate dehydrogenases (containing 5-7 cysteine residues), as well as reformation of the four cystine cross-bridges of bovine pancreatic ribonuclease, were studied in the presence of reduced and oxidized glutathione (GSH and GSSG). At the intracellular GSH level (5 mM) reduced ribonuclease can be reoxidized by 0.01-0.5 mM GSSG (pH 7.4) both at 20 degrees C and 37 degrees C. In this physiological range of GSSG concentrations and pH, the dehydrogenases show at least partial reactivation. With GSSG concentrations greater than 5 mM, reactivation is found to be completely inhibited for all the enzymes given. The results show that at the intracellular level of GSH and GSSG, thiol groups in reduced, unfolded ribonuclease are oxidized to form intramolecular cystine cross-bridges, while thiol groups of typical cysteine enzymes, such as lactate and malate dehydrogenase, remain in their reduced state during refolding. The rate of reactivation of lactate dehydrogenase (porcine muscle) is not affected by GSSG. In the case of ribonuclease, increasing concentrations of GSSG increase the rate of reactivation: At 20 degrees C, the halftime of the correct disulfide bond formation varies from approximately equal to 80 h in the presence of 0.01 mM GSSG to approximately equal to 10 h in the presence of 0.25 mM GSSG. A further increase in the rate of reactivation at higher GSSG concentrations is accompanied by a decrease in yield. Reactivation of ribonuclease is also observed at the low glutathione level found in blood plasma (5-25 microM GSH).
D000818:Animals; D003545:Cysteine / Q000378:metabolism*; D003553:Cystine / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects*; D005978:Glutathione / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D011489:Protein Denaturation; D012260:Ribonucleases / Q000378:metabolism; D013552:Swine
D003553:Cystine; D010088:Oxidoreductases; D007770:L-Lactate Dehydrogenase; D012260:Ribonucleases; D005978:Glutathione; D003545:Cysteine