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KMID : 0857020060210010349
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Kosin Medical Journal
2006 Volume.21 No. 1 p.349 ~ p.357
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Purification and characterization of ubiquitin activating enzymes from porcine thymus
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Lee Song-Jae
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Abstract
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Backgrounds : The continuous synthesis and degradation of proteins in the cell are responsible for essential cellular functions such as the modulation of the levels of key enzymes and regulatory porteins and removal of abnormal proteins that arise by biosynthetic errors or postsynthetic damages. Intracellular protein degradation are largely occurred in lysosome and cytoplasm. Nonselective protein degradation appears to be largely occurred in lysosome. However, short-lived proteins or damaged and abnormal proteins are degraded in the cytoplasm. These kind of protein degradation in the cytoplasm (ubiquitin mediated protein degradation) require energy (ATP), ubiquitin and ubiquitin activating enzymes such as E1 (ubiquitin activating enzyme), E2 (ubiquitin carrier protein) and E3 (ubiquitin-protein ligase). To better understand the degradation mechanism in the cytoplasm, ubiquitin activating enzymes (such as E1 and E2 enzymes) was purified from cytosol fraction of porcine thymus and tested their conjugation activity with iodinated ubiquitin.
Materials and Methods : The cytosol fraction prepared from porcine thymus tissue were used as a enzyme source. The DEAE-cellulose column chromatography, ammonium sulfate precipitation and ubiquitin-sepharose affinity column and/or gel filtration column chromatography were adapted to purify the E1 and E2 enzymes from thymus tissue. The conjugation activity of purified E1 and E2 enzymes were tested in the presence of 125I-ubiquitin.
Results : The E1 and E2 enzymes of cytosol fraction from porcine thymus tissue was purified from 30-80% ammonium sulfate precipitant of DEAE-cellulose eluate fraction. Following ubiquitin-sepharose column and gel filtration column chromatography, the E1 enzyme showed homogeneous from (judged by SDS-polyacrylamide gel electrophoresis). The various kinds of well known E2 isoforms and putative new kind of E2 isoform (M.W 37Kda) were found in cytosol fraction. When purified E1 and E2 enzyme or both were incubated in the presence of 125I-ubiquitin, these enzymes were conjugated with ubiquitin.
Conclusion : Ubiquitin-activating enzymes were purified from cytosol fraction of porcine thymus tissue. The homogeneous form of E1 enzyme and E2 isoforms showed strong conjugation activity with 125I-ubiquitin. These results might be useful for elucidation of selective protein degradation in the cytoplasm.
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KEYWORD
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Cytoplasm, Ubiquitin activating enzyme, Protein degradation
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