Characterization of Fe/Mn−Superoxide Dismutase from DiatomThallassiosira weissflogii:  Cloning, Expression, and Property

Abstract

A cDNA clone of 1114 bp encoding a putative Mn-superoxide dismutase (Mn-SOD) from diatom Thallassiosira weissflogii was cloned by the PCR technique. Nucleotide sequence analysis of this cDNA clone revealed that it was translated into 201 amino acid residues. When the sequence was compared with Mn-SODs from Vibrio mimicus and Escherichia coli, as well as two Fe-SODs from E. coli and Photobacterium leiognathi, this SOD showed higher homology to Mn-SOD. The amino acid residues required to coordinate the single manganese ion were conserved in all reported Mn-SOD sequences. This cDNA was introduced in an expression vector, pET-20b(+), and transformed into E. coli BL21(DE3)pLysS. The expressed SOD protein was then purified by a His-tag column. The recombinant enzyme was heated at 55 degrees C with a time-dependent assay; the time interval for 50% inactivation was 23 min, and its thermal inactivation rate constant K(d) was 3.03 x 10(-)(2) min(-)(1). The enzyme was inactivated either in acidic pH (below 4.0) or in the presence of imidazole (above 1.6 M) and had only a moderate effect under SDS (above 4%), whereas it was not affected under an alkaline pH (above 9.0). The atomic absorption spectrometric assay showed that 0.6 atom of iron/manganese (3:1) was present in each subunit of SOD. Reconstitution study was suggested that diatom SOD was cambialistic (Fe/Mn)-SOD. The finding of this SOD cDNA could be used for a reference in comparing the differences among marine phytoplankton species and as a probe to detect the transcription level of this enzyme, which can be applied in cosmetics for skin protection or defending unesthetic effects caused by oxygen-containing free radicals.

Functional Analysis of Insect Molting Fluid Proteins on the Protection and Regulation of Ecdysis

“…Approximately 30 g of cell lysates or 30 g of molting fluid were loaded on each lane for native gel separation. Methods to detect superoxide dismutase (14), catalase (15), protease (16), and esterase (17) activities in a native gel were performed as described previously.…”

Localization and Role of Manganese Superoxide Dismutase in a Marine Diatom

“…In contrast, MnSOD is found in the mitochondria of embryophytes, chlorophytes, and dinoflagellates (Kliebenstein et al, 1998;Kitayama et al, 1999;Wu et al, 1999;Okamoto et al, 2001;Okomoto et al, 2001;Fink and Scandalios, 2002). Although some information has been shown regarding SOD in diatoms Ken et al, 2005), no information on the subcellular localization of MnSOD and the associated kinetics in diatoms is available.…”

A Rigidoporus Vinctus Alcohol Dehydrogenase and Its Characterization

“…These cells were harvested and soluble proteins extracted in 1X PBS with glass beads as described before. 8 The …”