Factors affecting oligomerization status of UDP-glucose pyrophosphorylase.
UDP-glucose pyrophosphorylase (UGPase) is involved in the production of UDP-glucose, a key precursor to polysaccharide synthesis in all organisms. UGPase activity has recently been proposed to be regulated by oligomerization, with monomer as the active species. In the present study, we investigated factors affecting oligomerization status of the enzyme, using purified recombinant barley UGPase. Incubation of wild-type (wt) UGPase with phosphate or Tris buffers promoted oligomerization, whereas Mops and Hepes completely dissociated the oligomers to monomers (the active form). Similar buffer effects were observed for KK127-128LL and C99S mutants of UGPase; however, the buffers had a relatively small effect on the oligomerization status of the LIV135-137NIN mutant, impaired in deoligomerization ability and showing only 6-9% activity of the wt. Buffer composition had no effect on UGPase activity at UGPase protein concentrations below ca. 20 ng/ml. However, at higher protein concentration the activity in Tris, but not Mops nor Hepes, underestimated the amount of the enzyme. The data suggest that oligomerization status of UGPase can be controlled by subtle changes in an immediate environment (buffers) and by protein dilution. The evidence is discussed in relation to our recent model of UGPase structure/function, and with respect to earlier reports on the oligomeric integrity/activity of UGPases from eukaryotic tissues.
Kleczkowski
Leszek A
LA
Umeå Plant Science Center, Department of Plant Physiology, Umeå University, 901 87 Umeå, Sweden. leszek.kleczkowski@plantphys.umu.se
Martz
Françoise
F
Wilczynska
Malgorzata
M
D002021:Buffers; D006531:HEPES / Q000737:chemistry; D001467:Hordeum / Q000201:enzymology*; D009154:Mutation; D014325:Tromethamine / Q000737:chemistry; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D002021:Buffers; D014325:Tromethamine; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; D006531:HEPES