Submitted to: American Society of Plant Biologists Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: April 1, 2008
Publication Date: June 26, 2008
Citation: Gronwald, J.W., Miller, S.S., Vance, C.P. 2008. Arabidopsis UDP-Sugar Pyrophosphorylase: Evidence for Two Isoforms [abstract]. 2008 American Society of Plant Biologists Annual Meeting, Pan American Congress on Plants and BioEnergy, June 22-25, 2008, Merida, Mexico. Abstract No. P17009. Available: http://abstracts.aspb.org/pb2008/public/P17/P17009.html Technical Abstract: Arabidopsis UDP-sugar pyrophosphorylase (AtUSP, EC 220.127.116.11) is a broad substrate pyrophosphorylase that exhibits activity with GlcA-1-P, Gal-1-P, and Glc-1-P. AtUSP, a single gene in Arabidopsis, is widely expressed in tissues. Although USP exhibits activity with GlcA-1-P, it is not clear whether USP is the only pyrophosphorylase exhibiting this activity and hence the pyrophosphorylase that catalyzes the terminal step of the myo-inositol oxidation pathway. A polyclonal antibody was generated to Arabidopsis recombinant USP and used in immunoblots and immunoprecipitation assays to further characterize the enzyme. Immunoblots 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, USP1 was also detected in the microsomal fraction. Immunoprecipitation assays demonstrated that AtUSP antibodies removed USP activity (pyrophosphorolysis reaction) 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.