Regulation of sedoheptulose-1,7-bisphosphatase by sedoheptulose-7-phosphate and glycerate,and of fructose-1,6-bisphosphatase by glycerate in spinach chloroplasts

Using partially purified sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea L.) chloroplasts the effects of metabolites on the dithiothreitoland Mg²⁺-activated enzyme were investigated. A screening of most of the intermediates of the Calvin cycle and the photorespiratory pathway showed that physiological concentrations of sedoheptulose-7-phosphate and glycerate specifically inhibited the enzyme by decreasing its maximal velocity. An inhibition by ribulose-1,5-bisphosphate was also found. The inhibitory effect of sedoheptulose-7-phosphate on the enzyme is discussed in terms of allowing a control of sedoheptulose-1,7-bisphosphate hydrolysis by the demand of the product of this reaction. Subsequent studies with partially purified fructose-1,6-bisphosphatase from spinach chloroplasts showed that glycerate also inhibited this enzyme. With isolated chloroplasts, glycerate was found to inhibit CO₂ fixation by blocking the stromal fructose-1,6-bisphosphatase. It is therefore possible that the inhibition of the two phosphatases by glycerate is an important regulatory factor for adjusting the activity of the Calvin cycle to the ATP supply by the light reaction.Abbreviations DTT dithiothreitol - FBPase fructose-1,6-bisphosphatase - Fru-1,6-P₂ fructose-1,6-bisphosphate - Fru-6-P fructose-6-phosphate - 3-PGA 3-phosphoglycerate - Ru-1,5-P₂ ribulose-1,5-bisphosphate - Ru-5-P ribulose-5-phosphate - SBPase sedoheptulose-1,7-bisphosphatase - Sed-1,7-P₂ sedoheptulose-1,7-bisphosphate - Sed-7-P sedoheptulose-7-phosphate This work was supported by the Deutsche Forschungsgemein-schaft.     

Reactions of antibodies against ferredoxin, ferredoxin-NADP+ reductase and plastocyanin with spinach chloroplasts

Purified antisera against ferredoxin, ferredoxin-NADP+ reductase and plastocyanin agglutinated osmotically shocked and washed spinach chloroplasts, prepared according to standard procedures. The monomeric antibody (immunoglobulin G fraction) of the reductase antiserum agglutinated chloroplasts specifically and directly, indicating that protruding structures (for example, the coupling factor) do not act as steric hindrances as has been suggested. With ferredoxin antiserum, the presence of a pentameric antibody (immunoglobulin M fraction) was obligatory to observe a positive agglutination reaction. Immunoglobulin G only inhibited ferredoxin-dependent reactions, like NADP+-photoreduction, but did not cause agglutination. Ferredoxin seems to be located in depressions of the membrane, possibly caused by a partial release of this protein in shocked chloroplasts. Similar results were obtained with purified immunoglobulins from a plastocyanin antiserum. Again the immunoglobulin G fraction inhibited electron transport reactions catalyzed by plastocyanin, whereas immunoglobulin M showed a positive agglutination, but had no influence on electron transport. It is concluded that ferredoxin, ferredoxin-NADP+ reductase and plastocyanin are peripheral electron transport components, located at the outer thylakoid membrane.     

Light induced activation of fructose-1, 6-bisphosphatase in isolated intact chloroplasts.

Thermolysin, a commercial bacterial proteinase, greatly activated the arginine transport system of isolated yeast vacuoles. Pronase had the same effect at low concentrations, but rapidly inactivated the transport system at higher concentrations. Arginine specifically protected the transport system form the inactivation by pronase. The protective effect of other amino acids correlated well with their affinity for the transport system. It is concluded that both thermolysin and pronase attack a membrane protein which restrains the transport of arginine, whereas the protein which carries the specific binding site of this transport system can be destroyed only by pronase.     

In vitro phosphorylation of fructose-1,6-bisphosphatase from rabbit and pig liver with cyclic AMP-dependent protein kinase

Homogeneous preparations of fructose-1,6-bisphosphatase from mouse, man, rabbit, pig, and rat were tested as substrates for cyclic AMP-dependent protein kinase. Up to 1 mol of [32P]phosphate per mole enzyme subunit was incorporated into fructose-1,6-bisphosphatase from pig and rabbit liver, which should be compared with 2.6 mol of phosphate per mole enzyme subunit in the case of the rat liver enzyme. The phosphorylation of fructose-1,6-bisphosphatase from the livers of man and mouse was negligible. Phosphorylation of pig and rabbit fructose-1,6-bisphosphatase decreased the apparent Km for fructose-1,6-bisphosphate, but in contrast to the case of the rat liver enzyme it did not change the inhibition constants for AMP and fructose-2,6-bisphosphate. The phosphorylation sites in rabbit and pig liver fructose-1,6-bisphosphatase were located close to the carboxyterminal of the polypeptide chains, since trypsin treatment of the phosphorylated enzyme quantitatively removed all of the protein-bound radioactivity without significantly altering the subunit molecular weight and with a maintained neutral pH optimum.     

Co-operative activation of chloroplast fructose-1,6-bisphosphatase by reductant,pH and substrate

Intact chloroplasts capable of high rates of photosynthesis fail to reduce CO₂ when illuminated in the absence of oxygen. While anaerobiosis limits proton gradient formation leading to ATP deficiency (Ziem-Hanck, U. and Heber, U. (1980) Biochim. Biophys. Acta 591, 266–274), light activation of fructose-1,6-bisphosphatase was also inhibited by anaerobiosis, whereas light activation of NADP-malate dehydrogenase was stimulated by anaerobiosis, indicating that reductant was still available for light activation. The chloroplast pool of NADP was largely reduced during illumination under anaerobiosis and electron transport to oxaloacetate was not inhibited by anaerobic conditions. Significant light activation of fructose-bisphosphatase was observed in anaerobic chloroplasts with 3-phosphoglycerate as substrate, but not with dihydroxyacetone phosphate (3-phosphoglycerate supports electron transport and hence proton gradient formation). In the absence of added substrates, illumination of anaerobic chloroplasts resulted in some light activation of fructose-bisphosphatase when the pH of the medium was increased. Under these conditions, light activation was stimulated by dihydroxyacetone phosphate. Dihydroxyacetone phosphate added together with oxaloacetate allowed light activation of fructose-bisphosphatase in anaerobic chloroplasts, while neither substrate added alone was effective. Formation of a transthylakoid proton gradient can therefore substitute for an alkaline suspension medium by causing an alkaline shift of the stromal pH on illumination. The data are interpreted as indicating that fructose-bisphosphatase, but not NADP-malate dehydrogenase, requires an alkaline pH and the presence of substrate for rapid reductive light activation and they bear on the interpretation of the lag observed in photosynthesis in chloroplasts and leaves on illumination after a prolonged dark period.     

In vitro synthesis and post-translational insertion into microsomes of the integral membrane protein, NADH-cytochrome b5 oxidoreductase

RNA extracted from a free polysome fraction from rat liver was used to direct translation in nuclease-treated rabbit reticulocyte lysates, and the [35S]methionine-labelled, in vitro-synthesized, cytochrome b5 reductase was isolated with specific antibodies. Analysis by SDS-polyacrylamide gel electrophoresis, non-equilibrium pH gradient electrophoresis and one-dimensional peptide mapping failed to reveal any difference between the in vitro-synthesized reductase and the enzyme endogenous to rat liver microsomes. To study the integration of the in vitro-synthesized reductase into membranes, carboxypeptidase Y was used as a proteolytic probe. The reductase endogenous to rat liver microsomes was resistant to attack by carboxypeptidase Y, but was degraded to a smaller form when the microsomes were solubilized by detergent. Likewise, the enzyme synthesized in vitro was attacked by carboxypeptidase Y, but became largely resistant after post-translational incubation with dog pancreatic microsomes, indicating that an integration into membranes similar to the physiological one had occurred. It is concluded that cytochrome b5 reductase is probably not synthesized as a precursor and inserts post-translationally into the membrane. The results are discussed in relation to the particular subcellular distribution of the reductase and to the possible topology in the lipid bilayer of its C-terminal non-polar membrane-binding segment.     

Regulation by glucagon of hepatic pyruvate kinase, 6-phosphofructo 1-kinase, and fructose-1,6-bisphosphatase

Glucagon stimulates gluconeogenesis in part by decreasing the rate of phosphoenolpyruvate disposal by pyruvate kinase. Glucagon, via cyclic AMP (cAMP) and the cAMP-dependent protein kinase, enhances phosphorylation of pyruvate kinase, phosphofructokinase, and fructose-1,6-bisphosphatase. Phosphorylation of pyruvate kinase results in enzyme inhibition and decreased recycling of phosphoenolpyruvate to pyruvate and enhanced glucose synthesis. Although phosphorylation of 6-phosphofructo 1-kinase and fructose-1,6-bisphosphatase is catalyzed in vitro by the cAMP-dependent protein kinase, the role of phosphorylation in regulating the activity of and flux through these enzymes in intact cells is uncertain. Glucagon regulation of these two enzyme activities is brought about primarily by changes in the level of a novel sugar diphosphate, fructose 2,6-bisphosphate. This compound is an activator of phosphofructokinase and an inhibitor of fructose-1,6-bisphosphatase; it also potentiates the effect of AMP on both enzymes. Glucagon addition to isolated liver systems results in a greater than 90% decrease in the level of this compound. This effect explains in large part the effect of glucagon to enhance flux through fructose-1,6-bisphosphatase and to suppress flux through phosphofructokinase. The discovery of fructose 2,6-bisphosphate has greatly furthered our understanding of regulation at the fructose 6-phosphate/fructose 1,6-bisphosphate substrate cycle.     

In vitro synthesis and assembly of photosystem II proteins of spinach chloroplasts

The synthesis and assembly of photosystem II (PS II) proteins of spinach chloroplasts were investigated in three different in vitro systems, i.e., protein synthesis in isolated chloroplasts (in organello translation), read-out translation of thylakoid-bound ribosomes, and transport of translation products from spinach leaf polyadenylated RNA into isolated chloroplasts. Polyacrylamide gel electrophoresis of labeled thylakoid polypeptides in the presence of sodium dodecyl sulfate revealed that the first two systems were capable of synthesizing the reaction center proteins of PS II (47 and 43 kDa), the herbicide-binding protein, and cytochrome b559. The reaction center proteins synthesized in organello were shown to bind chlorophyll and to assemble properly into the PS II core complex. One of the reaction center proteins translated by the thylakoid-bound ribosomes (47 kDa) was also found to be integrated in situ into the complex but was lacking bound chlorophyll. Incorporation of radioactivity into the three extrinsic proteins of the oxygen-evolution system (33, 24, and 18 kDa) was detected only when intact chloroplasts were incubated with the translation products from polyadenylated RNA, showing that these proteins are coded for by nuclear DNA. The occurrence of a precursor polypeptide 6 kDa larger than the 33-kDa protein was immunochemically detected in the translation products.     

Control of CO2 fixation regulation of stromal fructose-1,6-bisphosphatase in spinach by pH and Mg2+ concentration

The effect of pH and of Mg²⁺ concentration on the light activated form of stromal fructose-1,6-bisphosphatase (FBPase) was studied using the enzyme rapidly extracted from illuminated spinach chloroplasts. The (fructose-1,6-bisphosphate^4-)(Mg²⁺) complex has been identified as the substrate of the enzyme. Therefore, changes of pH and Mg²⁺ concentrations have an immediate effect on the activity of FBPase by shifting the pH and Mg²⁺ dependent equilibrium concentration of the substrate. In addition, changes of pH and Mg²⁺ concentration in the assay medium have a delayed effect on FBPase activity. A correlation of the activities observed using different pH and Mg²⁺ concentrations indicates, that the effect is not a consequence of the pH and Mg²⁺ concentration as such, but is caused by a shift in the equilibrium concentration of a hypothetical inhibitor fructose-1,6-bisphosphate^3- (uncomplexed), resulting in a change of the activation state of the enzyme. The interplay between a rapid effect on the concentration of the substrate and a delayed effect on the activation state enables a rigid control of stromal FBPase by stromal Mg²⁺ concentrations and pH. Fructose-1,6-bisphosphatase is allosterically inhibited by fructose-6-phosphate in a sigmoidal fashion, allowing a fine control of the enzyme by its product.Abbreviations Fru1,6 bis P fructose-1,6-bisphosphate - Fru6P fructose-6-phosphate - FBPase fructose-1,6-bisphosphatase Some of these results have been included in a preliminary report (Heldt et al. 1984)     

Specific, reversible inactivation of phosphofructokinase by fructose-1,6-bisphosphatase. Involvement of adenosine 5'-triphosphate, oleate, and 3-phosphoglycerate.

Optimal conditions necessary for the reversible inactivation of crystalline rabbit muscle phosphofructokinase by homogeneous rabbit liver fructose-1,6-bisphosphatase have been studied. At higher enzyme levels (to 530 mug/ml of phosphofructokinase) the two proteins were mixed and incubated in a pH 7.5 buffer composed of 50 mM Tris-HC1, 2 mM potassium phosphate, and 0.2 mM dithiothreitol. Aliquots were removed at various times and assayed for enzyme activity. A time dependent inactivation of phosphofructokinase caused by 1-2.3 times its weight of fructose-1,6-bisphosphatase was observed at 30, 23, and 0 degree C. This inactivation did not require the presence of adenosine 5'-triphosphate or Mg2+ in the incubation mixture, but an adenosine 5'-triphosphate concentration of 2.7 mM or greater was required in the assay to keep phosphofructokinase in an inactive form. A mixture of activators (inorganic phosphate, (NH4)2SO4, and adenosine 5'-monophosphate), when added to the assay cuvette, restored nearly all of the expected enzyme activity. Incubations with other proteins, including aldolase, at concentrations equal to or greater than the effective quantity of fructose-1,6-bisphosphatase had no inhibitory effect on phosphofructokinase activity. Removal of tightly bound fructose 1,6-bisphosphate from phosphofructokinase could not explain this inactivation, since several analyses of crystalline phosphofructokinase averaged less than 0.1 mol of fructose 1,6-bisphosphate/320 000 g of enzyme. Furthermore, the inactivation occurred in the absence of Mg2+ where the complete lack of fructose-1-6-bisphosphatase activity was confirmed directly. At lower phosphofructokinase concentrations (0.2-2 mug/ml) the inactivation was studied directly in the assay cuvette. Higher ratios of fructose-1,6-bisphosphatase to phosphofructokinase were necessary in these cases, but oleate and 3-phosphoglycerate acted synergistically with lower amounts of fructose-1,6-bisphosphatase to cause inactivation. The inactivation did not occur when high concentrations of fructose 6-phosphate were present in the assay, or when the level of adenosine 5'-triphosphate was decreased. However, the inactivation was found at pH 8, where the effects of allosteric regulators on phosphofructokinase are greatly reduced. Experiments with rat liver phosphofructokinase showed that this enzyme was also subject to inhibition by rabbit liver fructose 1,6-bisphosphatase under conditions similar to those used in the muscle enzyme studies. Attempts to demonstrate direct interaction between phosphofructokinase and fructose-1,6-bisphosphate by physical methods were unsuccessful. Nevertheless, our results suggest that, under conditions which approximate the physiological state, the presence of fructose-1,6bisphosphatase can cause phosphofructokinase to assume an inactive conformation. This interaction may have a significant role in vivo in controlling the interrelationship between glycolysis and gluconeogenesis.     

The control of glucose 1,6-bisphosphatase by Ca2+ and calmodulin.

1. The control of glucose 1,6-bisphosphatase activity, the enzyme that degrades glucose 1,6-bisphosphate, a metabolite that regulates hexose phosphate metabolism, has been examined in a rat muscle extract. 2. The enzyme has been found to be activated by physiological Ca2+ concentrations. Ca2+ can be replaced by Sr2+ and Mn2+, but the effects are not so pronounced. 3. The Ca2+ effect is inhibited by EGTA. Trifluoperazine also inhibits enzyme activity. This effect can be reversed by adding exogenous calmodulin.     

Concerted action of cosolvents, chaotropic anions and thioredoxin on chloroplast fructose-1,6-bisphosphatase. Reactivity to iodoacetamide

The incubation of chloroplast fructose-1,6-bisphosphatase with both dithiothreitol and protein denaturants made sulfhydryl groups available for reaction with [1-14C]iodoacetamide (10-12 mol iodoacetamide incorporated/mol enzyme). Digestion of S-carboxyamidomethylated enzyme with trypsin and polyacrylamide gel electrophoresis, in the presence of sodium dodecylsulfate, yielded two 14C-labeled fragments whose apparent molecular mass were 10 kDa and 16 kDa. In the absence of either dithiothreitol or protein denaturants the incorporation of iodoacetamide to the enzyme was lower than 4 mol. When chloroplast fructose-1,6-bisphosphatase was initially incubated with dithiothreitol (2.5 mM) and (a) high concentrations of both fructose 1,6-bisphosphate (4 mM) and Ca2+ (0.3 mM) or (b) low concentrations of both fructose 1,6-bisphosphate (0.8 mM) and Ca2+ (0.05 mM) in the presence of either 2-propanol (15%, by vol.), trichloroacetate (0.15 M) or chloroplast thioredoxin-f (0.5 microM) and subsequently subjected to proteolysis and electrophoresis, S-carboxyamidomethylated tryptic fragments had similar molecular masses. Thus, conditions that stimulated the specific activity of chloroplast fructose-1,6-bisphosphatase caused conformational changes which favoured both the reduction of disulfide bridges and the exposure of sulfhydryl groups. In this aspect, thioredoxin exerted structural and kinetic effects similar to compounds not involved in redox reactions (organic solvents, chaotropic anions). These results indicated that the modification of hydrophobic (intramolecular) interactions in chloroplast fructose-1,6-bisphosphatase constituted the underlying mechanism in light-activation by the ferredoxin-thioredoxin system.     

Design,synthesis and biological evaluation of 7-nitro-1H-indole-2-carboxylic acid derivatives as allosteric inhibitors of fructose-1,6-bisphosphatase

A series of novel indole derivatives was synthesized as inhibitors of fructose-1,6-bisphosphatase (FBPase). Extensive structure–activity relationships were conducted and led to a potent FBPase inhibitor 3.9 with an IC₅₀ of 0.99 μM. The binding mode of this series of indoles was predicted using CDOCKER algorithm. The results of this research will shed light on the further design and optimization of novel small molecules as FBPase inhibitors.     

Regulation of rabbit liver fructose-1,6-bisphosphatase by metals, nucleotides, and fructose 2,6-bisphosphate as determined from fluorescence studies

The fluorescent nucleotide analogue formycin 5'-monophosphate (FMP) inhibits rabbit liver fructose-1,6-bisphosphatase (I50 = 17 microM, Hill coefficient = 1.2), as does the natural regulator AMP (I50 = 13 microM, Hill coefficient = 2.3), but exhibits little or no cooperativity of inhibition. Binding of FMP to fructose-1,6-bisphosphatase can be monitored by the increased fluorescence emission intensity (a 2.7-fold enhancement) or the increased fluorescence polarization of the probe. A single dissociation constant for FMP binding of 6.6 microM (4 sites per tetramer) was determined by monitoring fluorescence intensity. AMP displaces FMP from the enzyme as evidenced by a decrease in FMP fluorescence and polarization. The substrates, fructose 6-phosphate and fructose 1,6-bisphosphate, and inhibitors, methyl alpha-D-fructofuranoside 1,6-bisphosphate and fructose 2,6-bisphosphate, all increase the maximal fluorescence of enzyme-bound FMP but have little or no effect on FMP binding. Weak metal binding sites on rabbit liver fructose-1,6-bisphosphatase have been detected by the effect of Zn2+, Mn2+, and Mg2+ in displacing FMP from the enzyme. This is observed as a decrease in FMP fluorescence intensity and polarization in the presence of enzyme as a function of divalent cation concentration. The order of binding by divalent cations is Zn2+ = Mn2+ greater than Mg2+, and the Kd for Mn2+ displacement of FMP is 91 microM. Methyl alpha-D-fructofuranoside 1,6-bisphosphate, as well as fructose 6-phosphate and inorganic phosphate, enhances metal-mediated FMP displacement from rabbit liver fructose-1,6-bisphosphatase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the allosteric binding pocket of human liver fructose-1,6-bisphosphatase by protein crystallography and inhibitor activity studies.

The structures of three complexes of human fructose-1,6-bisphosphatase (FB) with the allosteric inhibitor AMP and two AMP analogues have been determined and all fully refined. The data used for structure determination were collected at cryogenic temperature (110 K), and with the use of synchrotron radiation. The structures reveal a common mode of binding for AMP and formycine monophosphate (FMP). 5-Amino-4-carboxamido-1 beta-D-5-phosphate-ribofuranosyl-1H-imidazole (AICAR-P) shows an unexpected mode of binding to FB, different from that of the other two ligands. The imidazole ring of AICAR-P is rotated 180 degrees compared to the AMP and FMP bases. This rotation results in a slightly different hydrogen bonding pattern and minor changes in the water structure in the binding pocket. Common features of binding are seen for the ribose and phosphate moieties of all three compounds. Although binding in a different mode, AICAR-P is still capable of making all the important interactions with the residues building the allosteric binding pocket. The IC50 values of AMP, FMP, and AICAR-P were determined to be 1.7, 1.4, and 20.9 microM, respectively. Thus, the approximately 10 times lower potency of AICAR-P is difficult to explain solely from the variations observed in the binding pocket. Only one water molecule in the allosteric binding pocket was found to be conserved in all four subunits in all three structures. This water molecule coordinates to a phosphate oxygen atom and the N7 atom of the AMP molecule, and to similarly situated atoms in the FMP and AICAR-P complexes. This implies an important role of the conserved water molecule in binding of the ligand.     

In vitro synthesis of the cyanelle proteins of Cyanophora paradoxa by isolated cyanelles and cyanelle RNA

Cyanelles isolated from the alga Cyanophora paradoxa Korschikoff synthesized cyanelle proteins in vitro. This synthesis was stimulated by light and totally inhibited by chloramphenicol. Cycloheximide had only a small inhibitory effect. Electrophoretic separation of the labelled soluble cyanelle proteins yielded at least 20 discrete polypeptides. The RNA isolated from the cyanelles and the whole cells was successfully translated in a rabbit reticulocyte-lysate system.Abbreviations poly(A)^-RNA, poly(A)⁺RNA nonadenylated, polyadenylated RNA; - SDS sodium dodecyl sulfate     

Lysine and glutamate production by Corynebacterium glutamicum on glucose, fructose and sucrose: roles of malic enzyme and fructose-1,6-bisphosphatase

In the biotechnological production of L-lysine and L-glutamate by Corynebacterium glutamicum media based on glucose, fructose or sucrose are typically used. Glutamate production by C. glutamicum was very similar on glucose, fructose, glucose plus fructose and sucrose. In contrast, lysine production of genetically defined C. glutamicum strains was significantly higher on glucose than on the other carbon sources. To test whether malic enzyme or fructose-1,6-bisphosphatase might limit growth and lysine on fructose, glucose plus fructose or sucrose, strains overexpressing either malE which encodes the NADPH-dependent malic enzyme or the fructose-1,6-bisphosphatase gene fbp were generated. Overexpression of malE did not improve lysine production on any of the tested carbon sources. Upon overexpression of fbp lysine yields on glucose and/or fructose were unchanged, but the lysine yield on sucrose increased twofold. Thus, fructose-1,6-bisphosphatase was identified as a limiting factor for lysine production by C. glutamicum with sucrose as the carbon source.     

Design, synthesis, and biological evaluation of substituted 2,3-dihydro-1H-cyclopenta[b]quinolin-9-ylamine related compounds as fructose-1,6-bisphosphatase inhibitors

In a search for structurally new inhibitors of fructose-1,6-bisphosphatase (F16BPase), substituted 2,3-dihydro-1H-cyclopenta[b]quinoline derivatives were synthesized. It has been shown that the 2,3-dihydro-1H-cyclopenta[b]quinoline moiety may represent a suitable scaffold for the synthesis of potent F16BPase inhibitors endowed with significantly lower EGFR tyrosine kinase inhibitory activity.     

Characterization of the gene for fructose-1,6-bisphosphatase from Saccharomyces cerevisiae and Schizosaccharomyces pombe. Sequence, protein homology, and expression during growth on glucose

We have determined the nucleotide sequence of the gene for fructose-1,6-bisphosphatase from both Saccharomyces cerevisiae and Schizosaccharomyces pombe. The predicted protein sequence for fructose-1,6-bisphosphatase from S. cerevisiae contains 347 amino acids and has a molecular weight of 38,100; that from S. pombe, contains 346 amino acids and has a molecular weight of 38,380. Comparison of these amino acid sequences with each other and that of pig kidney fructose-1,6-bisphosphatase shows several regions of strong homology separated by regions of divergence. These homologous regions are likely candidates for functional domains. A gene cassette was constructed for fructose-1,6-bisphosphatase from S. cerevisiae and the gene cassette expressed from the regulated PHO5 and GAL1 promoters of yeast. Yeast cells expressing fructose-1,6-bisphosphatase, while growing on glucose, accumulated large amounts of enzyme intracellularly, suggesting that glucose-regulated proteolytic inactivation does not operate efficiently under these conditions. Growth on glucose was not inhibited by the expression of fructose 1,6-bisphosphatase.