Isolation and characterization of glutathione reductase from Physarum polycephalum and stage-specific expression of the enzyme in life-cycle stages with different oxidation-reduction levels.
Physarum polycephalum has a life cycle with several distinct phases that have different oxidation-reduction requirements. To investigate the relationship between the life cycle and the oxidation-reduction state, we isolated glutathione reductase (GR; EC 1.6.4.2) from Physarum microplasmodia. The enzyme was found to be a homodimer with a subunit M(r) of 49,000, and K(m) values for oxidized glutathione and NADPH of 40 and 28.6 microM, respectively. We then constructed a cDNA library from microplasmodium mRNA and cloned GR cDNA from the library. The isolated cDNA consisted of 1,475 bp encoding a polypeptide of 452 amino acids. The amino acid sequence similarity was about 50% with GRs of other organisms, and several conserved sequence motifs thought to be necessary for activity are evident in the Physarum enzyme. Escherichia coli transformed with an expression vector containing the cDNA synthesized the active GR. Genomic Southern blot analysis indicated that the GR gene is present as a single copy in the Physarum genome. Immunoblot analysis and RT-PCR analysis detected GR mRNA expression in the microplasmodium, plasmodium, and sclerotium, but not in the spore or flagellate. GR activity was low in the spore and flagellate. These results suggest that the glutathione oxidation-reduction system relates to the Physarum life cycle.
Minami
Yoshiko
Y
Department of Biochemistry, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005, Japan. minami@dbc.ous.ac.jp
Kohama
Takeshi
T
Sekimoto
Yu-Ji
YJ
Akasaka
Kenichi
K
Matsubara
Hiroshi
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D004926:Escherichia coli / Q000235:genetics; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D005980:Glutathione Reductase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008018:Life Cycle Stages / Q000502:physiology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D016839:Physarum polycephalum / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D016053:RNA, Protozoan / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment
D016053:RNA, Protozoan; D011994:Recombinant Proteins; D005980:Glutathione Reductase