Spinach cytosolic fructose‐1,6‐bisphosphatase

Abstract

Cytosolic fructose-l,6-bisphosphatase from spinach leaves was purified to homogeneity and characterized. The pure enzyme has a subunit mass of 38 kDa, its K,,, values for fructose 1,6-bisphosphate and Mg2+ are 1.5 pM and 260 mM, respectively, and its V,,,,, is 110-120 units/mg. It is inhibited by fructose 2,6-bisphosphate and AMP with Ki values of 0.07 pM and 120 pM, respectively. About 90% of the primary structure of the spinach cytosolic fructose-l,6-bisphosphatase has been determined by amino-acid sequencing. The sequence data demonstrate that the cytosolic enzyme lacks the sequence insert characteristic of chloroplast fructose-l,6-bisphosphatase. The data include also the sequences of peptides containing all seven cysteine residues. Only two of the seven cysteines are conserved between the two isozymes, none of which is believed to be involved with the light regulation of the chloroplast enzyme. Sequence comparisons between the spinach cytosolic enzyme and gluconeogenic fructose-l,6-bisphosphatases from other species reveal similarity ranging over 47 -%yo, which is higher than the 40 -45% similarity between the chloroplast enzyme and gluconeogenic fructose-l,6-bisphosphatases. However, similarity between these isozymes and Escherichia coli fructose-l,6-bisphosphatase are 44% and 47% for the cytosolic and chloroplast enzymes, respectively. Similarity between the cytosolic and chloroplast counterparts is 52%, indicating wide divergence between these two fructose-l,6-bisphosphatases.

Fructose‐1,6‐bisphosphatase of the yeast Kluyveromyces lactis

“…It can be speculated that the insert is responsible for the decreased AMP inhibition shown by the K. lactis enzyme, in comparison with other AMP-sensitive Fru( 1,6)P,ases, because a residue close to the insert sequence (Argl40) interacts with AMP (see Table 1 in [53]). The Ki for AMP of the K. lactis enzyme, about 540 pM, is significantly higher than the 20-220 pM reported for S. cerevisiae [12], E. coli [7], spinach cytosolic [56], mammalian liver and kidney [3, 8 -101 Fru( 1,6)P,ases. The results of the in vitro mutagenesis studies indeed suggest that the unique insert present in K. lactis Fru(l,6)P2ase is responsible for decreased AMP inhibition.…”

cDNA and gene sequences of wheat chloroplast sedoheptulose‐1,7‐bisphosphatase reveal homology with fructose‐1,6‐bisphosphatases

“…Identity and similarity between wheat Sed(l,7)Pzase and a variety of Fru(l,6)Pzase amino acid sequences. Sources of sequences: wheat Sed(1 ,7)P2ase, this work; wheat chloroplast Fru(l,6)P2ase, Raines et al (1988); spinach cytosolic Fru(l,6)P2ase, Ladror et al (1990); pig Fru(l,6)P2ase, Marcus et al (1982); Saccharomyces cerevisiae Fru(l,6)P2ase, Rogers et al (1988); E. coli Fru(l,6)P2ase, Hamilton et al (1988); Rhodobacrer sphoeroides Fru(l,6)P2ase, Gibson et al (1990). The alignments used to generate these values were made using the Wisconsin GAP programme.…”

Regulation by Ca2+ of a Cytosolic Fructose-1,6-Bisphosphatase from Spinach Leaves

“…This purification level was higher than those showed in other reports on this enzyme (10,11,29). On Mono Q column chromatography, FBPase was eluted as a single symmetric peak, without any associated enzyme activity, such as aldolase, phosphofructokinase, pyrophosphate-dependent phosphofructokinase, nonspecific phosphatases, and chloroplast FBPase.…”