The uptake hydrogenase of chemolithotrophically grown Rhizobium japonicum was purified to apparent homogeneity with a final specific activity of 69 ,umol of H2 oxidized per min per mg of protein. The procedure included Triton extraction of broken membranes and DEAE-cellulose and Sephacryl S-200 chromatographies. The purified protein contained two polypeptides separable only by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They comigrated on native polyacrylamide gels and sucrose density gradients. The molecular weights were ca. 60,000 and 30,000. Densitometric scans of the sodium dodecyl sulfate gels indicated a molar ratio of 1.03 0.03. Antiserum was developed against the 60-kilodalton polypeptide for use in hydrogenase detection by an enzyme-linked immunosorbent assay. The antiserum did not cross-react with the 30-kilodalton polypeptide. Native gel electrophoresis of Triton-extracted cells grown in the presence of 63Ni showed comigration of the hydrogenase and radioactive Ni.
To know the mechanism of ammonia assimilation in non-heading Chinese cabbage (Brassica campestris L. ssp. chinensis (L.) Makino) leaves regulated by chitosan (CTS), a CTS-binding protein was isolated from non-heading Chinese cabbage leaves using the chitosan affinity chromatography approach and this CTS-binding protein was partially characterized. The profile of the 53.1 kDa purified protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was compared with the native molecular weight of 106.5 kDa, which indicated that the purified protein was a dimer with identical subunits. After isoelectric focusing, a band was obtained at pH 8.25. The agglutination test and periodic acid-Schiff staining further revealed that the protein was a glycoprotein with lectin activity. Moreover, the purified protein contained 17.4% (w/w) neutral carbohydrate and 82.56% (w/w) protein. The comparison of this protein and the 67 kDa CTS-binding protein isolated previously from Rubus culture tissue exhibited some differences in characterization. According to results of peptide mass fingerprinting analysis, the protein purified in the present study does not show any similarity with any protein in the protein data bank. Thus, it was deduced that the protein purified in the present study is a novel CTS-binding protein.Chemically, chitosan (CTS) is a polymeric Dglucosamine, a basic polysaccharide, and is a natural product derived from chitin found in the exoskeletons of most arthropods and in the cell walls of fungi. In addition, CTS has been found to act as a growth regulator and elicitor in plants. For example, CTS induced phytoalexin production in pea rods, inhibited fungal growth, and promoted protection from further infection (Hadwiger and Beckman, 1980). In addition, CTS has been demonstrated to improve nutrient qualities, to elevate the yield (Ou-Yang and Xu 2003), and to regulate the key enzymes of ammonia assimilation (Chen and Xu 2003) in non-heading Chinese cabbage.As a signal molecule, CTS plays an important role in regulating pathogenic host-fungal interactions and plant growth (Hadwiger and Loschke 1981; Liénart et al. 1991; Zhang et al. 1998). In 1991, Liénart et al.isolated a lectin specific for glucosamine oligomers from Rubus cell-suspension cultures. Further experiments showed that this lectin was a membrane-binding protein with a molecular weight of 67 kDa. However, little information is available concerning CTS-binding proteins in plants. In the present study, we describe the isolation and characterization of a CTS-binding protein from non-heading Chinese cabbage leaves, which provides important clues to the elucidation of the mechanism of CTS modulation in non-heading Chinese cabbage leaves. Materials and Methods Materials and plant cultureSeeds of non-heading Chinese cabbage (Brassica campestris L. ssp. chinensis (L.) Makino) were sterilized by immersion in 1% aqueous sodium hypochlorite
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