A simple procedure for purifying the major chloroplast fructose-1,6-bisphosphatase from spinach (Spinacia oleracea) and characterization of its stimulation by sub-femtomolar mercuric ions.
A rapid procedure for the purification of the redox-regulated chloroplast fructose-1,6-bisphosphatase [EC 3.1.3.11] from spinach leaf extract to homogeneity is described. No thiol-reducing agents were present during the purification and the enzyme is > 99% in the oxidized form. A rapid procedure to reduce and activate the Fru-1,6-P2ase by dithiothreitol in the absence of thioredoxin is described. Reduction activates the enzyme up to several hundred-fold when assayed at pH 8.0 with 2 mM Mg2+. The activity of the purified oxidized enzyme is unusually sensitive to changes in Mg2+ and H+ concentration. Tenfold changes in Mg2+ or H+ concentration lead to > 100-fold increases in activity. The recoveries of fructose-1,6-bisphosphatase activity as determined by the activity of the oxidized enzyme at pH 8.0/20 mM Mg2+; pH 9.0/2 mM Mg2+; pH 8/2 mM Mg2+ plus 0.1 mM Hg(II) or of the reduced enzyme at pH 8.0/2 mM Mg2+ are similar (approximately 40%) indicating that the major proportion of these activities in a leaf extract is catalyzed by the same enzyme. Moreover, antibodies raised against the purified enzyme inhibit all of the above activities in crude leaf extracts. The kinetic properties of the purified enzyme suggest that the oxidized Mg(2+)-dependent enzyme can play no significant role in photosynthetic carbon assimilation. A survey of some kinetic properties of Fru-1,6-P2ase activity in extracts of various photosynthetic organisms reveals that all 11 species examined possess a redox- and pH/Mg(2+)-stimulated Fru-1,6-P2ase, whereas Fru-1,6-P2ase in extracts of Taxus baccata (a gymnosperm), Chlorella vulgaris (a green alga), and the cyanobacterium Nostoc muscorum were not activated by Hg(II). The heat stability that proved useful in the purification of the spinach enzyme was conserved in both angiosperms and gymnosperms. The oxidized enzyme (which normally has no thiol groups accessible to 5,5'-dithio-bis[2-nitrobenzoic acid]) but not the reduced enzyme can be stimulated many hundred-fold by addition of extraordinarily low concentrations of Hg(II) to a complete assay mixture. With the aid of EDTA as a Hg(II) buffer, half-maximal stimulation was achieved at 2 x 10(-16) M free Hg(II). Methylmercury also stimulates the enzyme many hundred-fold at very low concentrations. The concentration for half-maximal stimulation by methylmercury was determined with a cyanide buffer to be approximately 10(-16) M. This, together with the high affinity of the enzyme for Hg(II), suggests that Hg(II) stimulates the enzyme by binding to an enzyme thiol group that be comes exposed in the catalytically active enzyme, thereby stabilizing the oxidized enzyme in an active conformation. By contrast, in the absence of Fru-1,6-P2 and either Mg2+ or Ca2+, Hg(II) (even at 2 x 10(-16) M) rapidly inactivates the oxidized Fru-1,6-P2ase. This inactivation is similar to the inactivation of Fru-1,6-P2ase that occurred at high pH (> 9) and which is also prevented by Fru-1,6-P2 and either Mg2+ or Ca2+. Although the Hg(II)- and high pH-inactivated oxidized enzyme has no activity, both forms of the enzyme can be activated by reduction. The usefulness of buffers to maintain low, defined Hg(II) and organic mercurial concentrations is discussed.
Ashton
A R
AR
Lehrstuhl fur Pflanzenphysiologie, Universität Bayreuth, Germany. tony.ashton@pi.esiro.au
D000478:Alkylation; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002736:Chloroplasts / Q000737:chemistry* / Q000201:enzymology*; D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D006597:Fructose-Bisphosphatase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D017669:Mercury Compounds / Q000494:pharmacology; D010084:Oxidation-Reduction; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D018724:Spinacia oleracea; D013438:Sulfhydryl Compounds / Q000737:chemistry
D017669:Mercury Compounds; D013438:Sulfhydryl Compounds; D006597:Fructose-Bisphosphatase; D010744:Phosphoric Monoester Hydrolases; C022627:sedoheptulose-bisphosphatase; D008274:Magnesium