Enzymatic properties of the 20S proteasome in wheat endosperm and its biochemical characteristics after seed imbibition.
The 20S proteasome from wheat (Triticum aestivum L., Yangmai 158) endosperm was purified to apparent homogeneity by three sequential centrifugations and gradient PAGE (GPAGE). The purified 20S proteasome clearly cleaved peptidyl-arylamide bonds in the model synthetic substrates Z-GGL-AMC and Z-GGR-AMC, which are used to reflect chymotrypsin-like and trypsin-like activity, respectively. For both substrates, the optimum pH was 8.0, but the optimum temperatures for chymotrypsin-like and trypsin-like activity were 55 degrees C and 37 degrees C, respectively. Both enzyme activities were clearly inhibited by MG115 and PMSF. Polyubiquitinated proteins remained constant from 0 to 7 days after seed imbibition, but caseinolytic activity and the amount of the 20S proteasome associated with the aleurone layer decreased from 1 to 2 days after imbibition (DAI), then increased from 2 to 4 DAI, and reached a maximum at 4 DAI that was retained until 7 DAI. An increase was seen in the mRNA level of the beta5 subunit of the 20S proteasome from 2 DAI, and caseinolytic activity and the amount of the 20S proteasome increased from 3 DAI onwards. In addition, the main storage proteins of the wheat endosperm could not be hydrolyzed by the 20S proteasome. The evidence suggests that the main role of the 20S proteasome may not be to degrade massive proteins of the wheat endosperm after seed imbibition.
College of Life Sciences, Nanjing Agricultural University, Nanjing, China.
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D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000302:isolation & purification / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D012639:Seeds / Q000201:enzymology* / Q000254:growth & development; D013379:Substrate Specificity; D013696:Temperature; D014908:Triticum / Q000196:embryology / Q000201:enzymology*; D057149:Ubiquitinated Proteins / Q000378:metabolism
D010940:Plant Proteins; D011480:Protease Inhibitors; D012333:RNA, Messenger; D057149:Ubiquitinated Proteins; D046988:Proteasome Endopeptidase Complex