Thioltransferase in human red blood cells: purification and properties

Abstract

Thioltransferase activity was identified and the enzyme purified to apparent homogeneity from human red blood cells. Activity was measured as glutathione-dependent reduction of the prototype substrate hydroxyethyl disulfide; formation of oxidized glutathione (GSSG) was coupled to NADPH oxidation by GSSG reductase (1 unit of activity = 1 mumol/min of NADPH oxidized). The thioltransferase-GSH-GSSG reductase system was shown also to catalyze the regeneration of hemoglobin from the mixed disulfide hemoglobin-S-S-glutathione (HbSSG) and to reactivate the metabolic control enzyme phosphofructokinase (PFK) after oxidation of its sulfhydryl groups. On a relative concentration basis, thioltransferase was about 1200 times more efficient than dithiothreitol in reactivation of phosphofructokinase; e.g., 500 microM DTT was required to effect the same extent of reactivation as that of 0.4 microM TTase. The GSH plus GSSG reductase system without thioltransferase was ineffective for reduction of HbSSG or reactivation of PFK. The average amount of thioltransferase in intact erythrocytes was calculated to be 4.6 units/g of Hb at 25 degrees C. This level of activity is about the same as those of other enzymes that participate in sulfhydryl maintenance in red blood cells, such as GSSG reductase and glucose-6-phosphate dehydrogenase. These results suggest a physiological role for the thioltransferase in erythrocyte sulfhydryl homeostasis. Certain properties of the human erythrocyte thioltransferase resemble those of other mammalian thioltransferase and glutaredoxin enzymes. Thus, the human erythrocyte enzyme, purified about 28,000-fold to apparent homogeneity, is a single polypeptide with a molecular weight of 11,300. Its N-terminus is blocked, it is heat stable, and it contains four cysteine residues per protein molecule. However, the human erythrocyte thioltransferase is a distinct protein based on its amino acid composition. For example, it contains no methionine residues; whereas the related mammalian enzymes described to date have at least one internal methionine residue in their largely homologous sequences.

The Malaria Parasite Supplies Glutathione to its Host Cell — Investigation of Glutathione Transport and Metabolism in Human Erythrocytes Infected with Plasmodium Falciparum

“…This finding may be very relevant for the metabolism of the host cell as high [GSSG] are known to inhibit two major enzymes of glycolysis (phosphofructokinase and pyruvate kinase) and to activate glucose-&phosphate dehydrogenase, the rate determining enzyme of HMS (Ziegler, 1985). The low GSH/GSSG ratio could have resulted from a very intensive oxidative stress in addition to S-thiolatioddethiolation of protein sulfhydryls (Ziegler, 1985: Mieyal et al, 1991Thomas et al, 1995), or to a large efflux of GSSG from the parasite into the host-cell compartment or any combination of these disparate events. In contrast, the GSH/ GSSG ratio in the parasite compartment is similar to that found in normal eukaryotic cells, indicating that the parasite has ample reducing power and/or adequate capacity for de novo synthesis to maintain the physiological concentration of GSH.…”

The YeastSaccharomyces cerevisiaeContains Two Glutaredoxin Genes That Are Required for Protection against Reactive Oxygen Species

“…In addition, in vitro experiments indicate that glutaredoxins can reactivate a number of oxidized enzymes by reducing the mixed disulfides formed as a result of thiol oxidation (Terada et al, 1992;Terada, 1994;Yoshitake et al, 1994). This repair activity is not restricted to enzymes, because glutaredoxin from human erythrocytes is able to reactivate membrane proteins and to regenerate hemoglobin from the mixed disulfide hemoglobin-S-S-glutathione (Mieyal et al, 1991;Terada et al, 1992). More recently, glutaredoxin has been isolated from the human ocular lens and was found to dethiolate mixed disulfides formed during oxidative stress, preventing loss of lens transparency and cataract formation (Raghavachari and Lou, 1996).…”

Evolution of enzymes in a series is driven by dissimilar functional demands

“…† † Thus, under moderate GSR deficiency, GSSG consumption by hemoglobin glutathionylation may outcompete GSR-catalyzed reduction, which has a pseudo-first-order rate constant of 0.75 s Ϫ1 at normal GSR activity. Furthermore, glutathionyl-hemoglobin has a long half-life: Ϸ15 min, as estimated from the concentration of glutaredoxin (Ϸ1 M) (34) and from kinetic data about this enzyme (32). In healthy individuals, 1.2-16% of total hemoglobin (35-37) is glutathionylated (although these high estimates have been challenged by ref.…”