title: Selenium-dependent glutathione peroxidases from ovine and bovine erythrocytes occur as longer chain forms than previously recognized.
abstract: From the cDNA sequence of bovine glutathione peroxidase [G. T. Mullenbach, A. Tabrizi, B. D. Irvine, G. I. Bell, J. A. Tainer, and R. A. Hallewell (1988) Protein Eng. 2, 239-246], it is known that the full transcript represents a 205-residue protein with the N-terminal sequence of MCAAQRSAAALAAAAPRTV. However, a protein primary structure determination on what is believed to be the mature protein showed an N-terminal sequence of AAALAAAAPRTV [W. A. Günzler, G. J. Steffens, A. Grossmann, S. A. Kim, F. Otting, A. Wendel, and L. Flohe (1984) Hoppe-Seyler's Z. Physiol. Chem. 365, 195-212], suggesting processing of the N-terminal 7 residues to give the mature protein. It is shown here that the normal form of bovine erythrocyte glutathione peroxidase is most likely the full transcript lacking the N-terminal methionine, and that subsequent proteolysis during isolation results in formation first of the form characterized by Günzler et al. and then of two slightly smaller species, the smaller of which appears to be similar to that examined crystallographically [O. Epp, R. Ladenstein, and A. Wendel (1983) Eur. J. Biochem. 133, 51-69]. For ovine erythrocyte glutathione peroxidase the same behavior is seen, with initial isolation of a high-molecular-weight form that is subsequently proteolyzed to two intermediate forms and a final form that migrates at the same position on acrylamide gels as the lowest-molecular-weight form of the bovine enzyme. The contaminating protease can be inhibited by the addition of 10 mM EDTA, suggesting that it is a metalloprotease. Activity measurements on the intact and processed forms of the ovine enzyme show no significant differences with both hydrogen peroxide and t-butyl hydroperoxide as substrates. These results suggest that both bovine and ovine erythrocyte glutathione peroxidases consist of a globular core that is relatively resistant to proteolysis and an N-terminal tail of approximately 17 residues that seems to be exposed and is very sensitive to proteolysis. This tail, which contains many hydrophobic residues, is predicted to be largely alpha-helical and may be involved in anchoring the enzyme at or close to the membrane surface.
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
authors.forename: P
authors.initials: P
authors.lastname: Gettins
authors.forename: D
authors.initials: D
authors.lastname: Dyal
authors.forename: B
authors.initials: B
authors.lastname: Crews
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology*; D005979:Glutathione Peroxidase / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010196:Pancreatic Elastase / Q000378:metabolism; D012643:Selenium / Q000494:pharmacology*; D012756:Sheep; D014357:Trypsin / Q000378:metabolism
chemical_list: D005979:Glutathione Peroxidase; D010196:Pancreatic Elastase; D014357:Trypsin; D012643:Selenium