Arabidopsis UDP-sugar pyrophosphorylase: Evidence for two isoforms
| Autor: | Carroll P. Vance, Susan S. Miller, John W. Gronwald |
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| Rok vydání: | 2008 |
| Předmět: |
Gene isoform
Physiology Immunoprecipitation Arabidopsis Plant Science Tandem mass spectrometry Mass Spectrometry law.invention law Genetics Differential centrifugation chemistry.chemical_classification biology Arabidopsis Proteins Hydrolysis biology.organism_classification Nucleotidyltransferases Isoenzymes Enzyme chemistry Biochemistry Polyclonal antibodies biology.protein Recombinant DNA Electrophoresis Polyacrylamide Gel |
| Zdroj: | Plant Physiology and Biochemistry. 46:1101-1105 |
| ISSN: | 0981-9428 |
| DOI: | 10.1016/j.plaphy.2008.07.001 |
| Popis: | Arabidopsis UDP-sugar pyrophosphorylase (AtUSP, EC 2.7.7.64) is a broad substrate pyrophosphorylase that exhibits activity with GlcA-1-P, Gal-1-P and Glc-1-P. Immunoblots using polyclonal antibodies raised to recombinant AtUSP demonstrated the presence of two USP isoforms of approximately 70 kDa (USP1) and 66 kDa (USP2) in crude extracts of Arabidopsis tissues. The 66 kDa isoform was not the result of proteolytic cleavage of USP1 during extraction. Trypsin digestion of bands on SDS gels corresponding to the location of the two isoforms followed by tandem mass spectrometry confirmed that USP peptides were present in both bands. Both USP isoforms were detected in the cytosol as determined by immunoblots of cellular fractions obtained by differential centrifugation. However, some USP1 was also detected in the microsomal fraction. Immunoprecipitation assays demonstrated that AtUSP antibodies removed USP activity (UDP-GlcA→GlcA-1-P) measured in floret extracts. These results indicate that USP is the only pyrophosphorylase that utilizes UDP-GlcA as a substrate and suggest that it serves as the terminal enzyme of the myo-inositol oxidation pathway. |
| Databáze: | OpenAIRE |
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