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.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Mieyal JJ,Starke DW,Gravina SA,Dothey C,Chung JSdoi
10.1021/bi00239a002subject
Has Abstractpub_date
1991-06-25 00:00:00pages
6088-97issue
25eissn
0006-2960issn
1520-4995journal_volume
30pub_type
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