Abstract
This chapter describes the purification and properties of the enzyme fructose-bisphosphatase (Fru-P2ase) from Rhodopseudomonas palustris . The Fru-P 2 ase of the photosynthetic bacterium Rhodopseudomonas palustris is allosterically regulated by substrate (fructose 1,6-bisphosphate), metal cofactor (Mn 2+ ), and inhibitor (GTP). Fructose-bisphosphatase catalyzes the hydrolysis of D-fructose 1,6-bisphosphate to D-fructose 6-phosphate and P i . The Fru-P 2 ase activity may be measured spectrophotometrically by following the reduction of NADP + in the presence of excess phosphohexoisomerase and glucose-6-phosphate dehydrogenase. The purification procedure involves preparation of crude extract, ammonium sulfate fractionation, protamine sulfate fractionation, ammonium sulfate fractionation, alumina C γ adsorption elution, diethylaminoethyl (DEAE)-column chromatography, ammonium sulfate fractionation, and Sephadex G-200 column chromatography. The maximum enzymic activity for Fru-P 2 ase occurs at pH 8.5. Fru-P 2 ase exhibits a requirement for divalent cations that can be satisfied by Mn 2+ or Mg 2+ . The physiological significance of the regulation of R. palustris Fru-P 2 ase may be that of controlling the Calvin–Basshan carbon-reduction cycle.
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