A Novel Glutathione Peroxidase in Bovine Eye

Abstract

Bovine ciliary body contains a selenium-independent glutathione peroxidase (GPX) with a molecular mass of about 100 kDa that is composed of four identical subunits and exhibits no glutathione S-transferase activity. In this study, we isolated cDNA clones and determined the nucleotide sequence to deduce the primary structure of the enzyme. The cDNA contained 672 base pairs encoding a polypeptide with an estimated molecular mass of 25,064 Da. Translation of bovine ciliary mRNA produced a protein which was immunologically indistinguishable from GPX and showed high enzyme activity. The encoded amino acid sequence of the protein was 95% identical with that of a human keratinocyte gene product expressed in response to keratinocyte growth factor. It also showed sequence identity to bacterial alkyl hydroperoxide reductases and thiol specific antioxidant enzymes. GPX mRNA level was highest in the ciliary body, followed by the retina and iris. In various rat organs, the level of GPX mRNA was highest in the lung, followed by the muscle, liver, eye, heart, testis, thymus, kidney, and spleen. A very low level of mRNA was detected in the brain. Enzyme-linked immunosorbent assay with an antibody raised against the NH 2 -terminal sequence of GPX detected GPX protein in all rat tissues examined.

1-Cys peroxiredoxin overexpression protects cells against phospholipid peroxidation-mediated membrane damage

“…Initial studies indicated that GSH could function as a reductant with 1-cysPrx isolated from bovine eye (20) or rat olfactory epithelium (21), although the latter result subsequently could not be reproduced (22). Studies with recombinant protein also have been inconclusive because GSH has been effective in some studies (7,23) but not in others (6). All reports agree that thioredoxin, a physiological reductant for other members of the peroxiredoxin family, does not reduce 1-cysPrx (7,24).…”

1-Cys Peroxiredoxin, a Bifunctional Enzyme with Glutathione Peroxidase and Phospholipase A2 Activities

“…Furthermore, based upon the cDNA sequence, this protein belongs to the thioredoxin peroxidase, or peroxiredoxin, family although with only one instead of the usual two conserved cysteine residues and thus has been called 1-Cys peroxiredoxin (5). The nomenclature is further confusing since the enzyme does not utilize thioredoxin (4,5), but rather functions as a GSH peroxidase (3,4). Although the cDNA sequence of aiPLA 2 (1) is identical to that of NSGPx (4), the presence of both activities up to the present has not been confirmed in the same peptide.…”

“…Evidence has emerged that a lysosomal-type Ca 2ϩ -independent phospholipase A 2 with acidic pH optimum (aiPLA 2 ) 1 (1,2) and a non-selenium glutathione peroxidase without glutathione S-tranferase activity (NSGPx) (3,4) are the same enzyme, based upon their identical cDNA sequence. Furthermore, based upon the cDNA sequence, this protein belongs to the thioredoxin peroxidase, or peroxiredoxin, family although with only one instead of the usual two conserved cysteine residues and thus has been called 1-Cys peroxiredoxin (5).…”

Activation of the antioxidant enzyme 1-CYS peroxiredoxin requires glutathionylation mediated by heterodimerization with πGST

“…By contrast, 1-cys peroxiredoxin (1-cysPrx or Prx VI) † has a single conserved cysteine (5) and does not use thioredoxin as reductant (5, 6). This peroxiredoxin is expressed in all tissues but at particularly high levels in brain, eye, testes, and lung (2,7,8). Expression of 1-cysPrx protein or mRNA is decreased in a mouse that is susceptible to experimental atherosclerosis (9) and is elevated in brains of patients with Parkinsonian dementia (10), sporadic Creutzfeldt-Jacob disease (11), and Pick disease (12), in lungs from newborns (13), in malignant mesothelioma (14), in the healing edge of skin wounds (15), and in experimental cellular premature senescence (16).…”