A serine protease that preferentially degrades oxidized and glycated proteins was shown to be present in erythrocyte cytosol. Human erythrocyte cytosol was labeled with [3H]diisopropyl fluorophosphate (DFP) and passed through a column of carboxymethyl-Sephadex to obtain radioactive fractions free of hemoglobin. The fractions contained a single radioactive 80-kDa protein, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE)/fluorography. The radioactive 80-kDa protein bound to unoxidized erythrocyte membranes, and more effectively to oxidized membranes. The radioactive protein was purified through a column of diethylaminoethyl-cellulose and by preparative native-PAGE in a purity of 80%. Antibody against the cytosolic 80-kDa protein bound to 80-kDa protein of erythrocyte membranes, indicating the presence of the same protein in the membrane. The antibody bound more effectively to oxidized membranes than to unoxidized membranes. The 80-kDa protein partially purified from unlabeled cytosol degraded more effectively oxidized bovine serum albumin (BSA), oxidized IgG, and glycated BSA more effectively than the corresponding unoxidized or unglycated proteins. Degradation of oxidized or glycated proteins was effectively inhibited by DFP. Hence, the protein is an 80-kDa serine protease that is adherent to oxidized membranes and is responsible for degradation of proteins modified by oxidation and glycation.
Oxidized protein hydrolase (OPH), an 80 kDa serine protease whose activity is inhibited by diisopropyl fluorophosphate (DFP), has been isolated from human erythrocytes [Fujino, T. et al. (1998) J. Biochem. 124, 1077-1085]. The presence of OPH in various biological samples was examined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting using an anti-OPH antibody raised against OPH purified from human erythrocytes, and by [(3)H]DFP-labeling and successive SDS-PAGE/fluorography. Solubilized samples of human cell lines including K-562 cells, THP-1 cells and Jurkat cells, and rat tissues including brain, heart, liver, kidney, and testis, inhibited the anti-OPH antibody binding to OPH in ELISA. Immunoblotting of lysates of K-562 cells, THP-1 cells and Jurkat cells showed four immunoreactive protein bands including an 80 kDa protein. Immunoprecipitation of the [(3)H]DFP-labeled K-562 cell lysate and successive SDS-PAGE/fluorography showed the presence of only the 80 kDa DFP-reactive protein with OPH antigenic activity. The level of the 80 kDa immunoreactive protein in K-562 cells rose as the cells differentiated toward erythrocytes. Immunoblotting of human and rat plasma showed two immunoreactive protein bands, including the 80 kDa protein, and SDS-PAGE/fluorography of [(3)H]DFP-labeled rat and human plasma showed the presence of only the 80 kDa DFP-reactive protein. The results indicate that OPH is present in a wide variety of biological samples.
cDNAs containing the open reading frames of a putative auxin influx carrier and/or facilitator, PsAUX1 (Accession No. AB488680), and putative auxin efflux carriers and/or facilitators, PsPIN1, PsPIN2 and PsPIN3 (Accession No. AB488679, AB488681 and AB488678, respectively) from etiolated epicotyls of an agravitropic pea mutant, ageotropum, have been isolated. In PsPIN1 and PsPIN2, a few deduced amino acids were different between ageotropum and Alaska showing a normal gravitropic response. No insertion and deletion were observed in PsPIN1 and PsPIN2 of ageotropum compared with those of Alaska. However, PsAUX1 of ageotropum had additional amino acids at the C-terminus compared to that of Alaska. Phylogenetic analyses based on deduced amino acid sequences revealed that PsPIN1 of ageotropum and Alaska belonged to the same clade as MtPIN4, while PsPIN2 of ageotropum and Alaska belonged to a subclade including MtPIN3, BjPIN2, BjPIN3, SlPIN3 and SlPIN4. PsAUX1 of ageotropum with additional amino acids at C-terminus belonged to the same subclade as MiAUX1 and PtAUX1. PsPIN1, PsPIN2, PsPIN3 mRNAs in 3.5-day-old etiolated ageotropum pea seedlings were significantly expressed in the proximal and the distal sides of the first internode of epicotyls to cotyledon. Judging from the results of the structure and the expression of genes related to auxin polar transport, an abnormal gravitropic response of etiolated ageotropum pea seedlings might be related to post transcriptional regulation and translocation of PsAUX1, PsPIN1, PsPIN2 and PsPIN3 as well as the perturbation of auxin transport due to additional amino acids at C-terminus of PsAUX1. ©2012 Jpn. Soc. Biol. Sci.
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