Potato tuber pyrophosphate-dependent phosphofructokinase: effect of thiols and polyalcohols on its intrinsic fluorescence, oligomeric structure, and activity in dilute solutions.
The effect of dilution of homogeneous potato tuber pyrophosphate:fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90; PFP) on the enzyme's intrinsic fluorescence, activity, and oligomeric structure has been examined. A rapid decrease in PFP's intrinsic fluorescence occurred in response to dilution. The decay follows double-exponential kinetics and was accompanied by a reduction in catalytic activity (measured in the glycolytic direction). Gel filtration-HPLC indicated a concomitant deaggregation of the native alpha 4 beta 4 heterooctamer into the inactive free alpha- and beta-subunits, followed by random aggregation of the subunits into an inactive, high M(r) conglomerate. The addition of 2 mM dithiothreitol, 2 mM 2-mercaptoethanol, or 5% (w/v) polyethylene glycol, but not any of the substrates, Mg2+, or fructose 2,6-bisphosphate, prevented this process. When purified PFP was stored for 1 week at -20 degrees C in the presence of 50% (v/v) glycerol partial degradation of its alpha-subunit occurred. This resulted in a labile enzyme that was more susceptible to subunit dissociation. The intrinsic fluorescence of the degraded PFP could be stabilized by 5% (w/v) polyethylene glycol, but not by 2 mM dithiothreitol or 2-mercaptoethanol. It is proposed that the current assay procedures for PFP, which normally involve considerable dilution in the absence of added sulfhydryl reducing agents or polyhydroxy compounds, may underestimate the actual activity of the enzyme. This has important implications for the assessment of the functions and regulation of PFP in vivo.
Podestá
F E
FE
Department of Biology, Queen's University, Kingston, Ontario, Canada.
Moorhead
G B
GB
Plaxton
W C
WC
D000438:Alcohols / Q000494:pharmacology; D011756:Diphosphates / Q000378:metabolism; D005453:Fluorescence; D007700:Kinetics; D046911:Macromolecular Substances; D010732:Phosphofructokinase-1 / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011198:Solanum tuberosum / Q000201:enzymology*; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D014364:Tryptophan / Q000737:chemistry
D000438:Alcohols; D011756:Diphosphates; D046911:Macromolecular Substances; D012996:Solutions; D013438:Sulfhydryl Compounds; D014364:Tryptophan; D010732:Phosphofructokinase-1