Purification and characterization of phospholipid hydroperoxide glutathione peroxidase from rat testis mitochondrial membranes.
The selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) is highly expressed in rat testis, where it is under gonadotropin control. In this organ a relevant PHGPx activity is strongly linked to mitochondria of cells undergoing differentiation to spermatozoa. This prompted a study on the possible difference between the soluble and the mitochondrial enzyme and the nature of the binding. The mitochondrial PHGPx activity could be solubilized by detergents or by the combined action of mild detergent treatment and ionic strength, thus suggesting an electrostatic binding of the protein to the inner surfaces of the organelle. The same chromatographic purification procedures were applied to cytosolic and membrane bound PHGPx, without revealing any significant difference between the two forms. Moreover, the electrophoretic mobility, the reactivity to antibodies and the fragmentation patterns also suggested the identity of the two forms of testis PHGPx. Eventually, testis cytosolic and membrane bound PHGPx showed the same substrate specificity for both peroxidic and thiol substrates. On the other hand, a complex behaviour on hydrophobic interaction chromatography, compatible with multiple forms of the enzyme, and with a different tertiary structure of the major peaks was observed for soluble and mitochondrial PHGPx. Accordingly, two-dimensional electrophoresis followed by immunostaining with monoclonal antibodies, showed the presence of multiple isoforms with a different pattern between the soluble and the mitochondrial enzyme. These differences are not accounted for by glycosylation or a different degree of phosphorylation of tyrosines. In both enzymes, indeed, no glycosylation was detected and no more than 10% of PHGPx molecules were shown to contain a phosphotyrosine residue.
Roveri
A
A
Department of Biological Chemistry, University of Padova, Italy.
Maiorino
M
M
Nisii
C
C
Ursini
F
F
D000818:Animals; D002846:Chromatography, Affinity; D003600:Cytosol / Q000201:enzymology; D005979:Glutathione Peroxidase / Q000737:chemistry / Q000302:isolation & purification*; D007425:Intracellular Membranes / Q000201:enzymology; D008297:Male; D008928:Mitochondria / Q000201:enzymology; D010449:Peptide Mapping; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D019000:Phosphotyrosine; D051381:Rats; D017208:Rats, Wistar; D012995:Solubility; D013379:Substrate Specificity; D013737:Testis / Q000201:enzymology*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D019000:Phosphotyrosine; D014443:Tyrosine; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D005979:Glutathione Peroxidase