Complexes of muscle aldolase in equilibrium with fructose 1,6-bisphosphate

Minimum values for the content of covalent intermediates in the equilibria of muscle aldolase with its cleavable substrates have been determined by acid denaturation/precipitation. Ribulose 1,5-bisphosphate, a nonsubstrate that binds well to aldolase in the native state, does not form a covalent complex that is acid precipitable. The insoluble protein complexes with substrates fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate, representing approximately 50% and approximately 60% of total bound substrate, are much more stable in acid and alkali than that with substrate 5-deoxyfructose 1,6-bisphosphate, suggesting that they have the form of protein-bound N-glycosides. Whether such complexes exist on the enzyme in the native state in addition to being formed subsequent to denaturation is unresolved. Both the acid-precipitable and nonprecipitable forms of fructose 1,6-bisphosphate are converted to triose phosphate products at the same rate, providing no kinetic evidence for a pool that is not on the main reaction path. Total fructose 1,6-bisphosphate liganded to enzyme returns to the free solution about 9 times for each net cleavage reaction. It is still not clear whether this is limited by the cleavage step or by release of glyceraldehyde phosphate.     

Molecular cloning, expression, purification, and characterization of fructose 1,6-bisphosphate aldolase from Mycobacterium tuberculosis--a novel Class II A tetramer

The Class II fructose 1,6-bisphosphate aldolase (fda, Rv0363c) from the pathogen Mycobacterium tuberculosis H37RV was subcloned in the Escherichia coli vector pT7-7 and purified to near homogeneity. The specific activity (35 U/mg) is approximately 9 times higher than previously reported for the enzyme partially purified from the pathogen. Attempts to express the enzyme with an N-terminal fusion tag yielded inactive, mostly insoluble protein. The native recombinant enzyme is zinc-dependent and has a catalytic efficiency for fructose 1,6-bisphosphate cleavage higher than most Class II aldolases characterized to date. The aldolase has a Km of 20 microM, a kcat of 21 s(-1), and a pH optimum of 7.8. The molecular mass of the enzyme subunits as determined by mass spectrometry is in agreement with the mass calculated on the basis of its gene sequence minus the terminal methionine, 36,413 Da. The enzyme is a homotetramer and retains only two zinc ions per tetramer when transferred to a metal-free buffer, as determined by ICP-MS and by a colorimetric assay using 4-(2-pyridylazo)-resorcinol (PAR) as a chelator. The E. coli expression system reported in this study will facilitate the further characterization of this enzyme and the screening for potential inhibitors.     

Fructose 2,6-bisphosphate. A new activator of phosphofructokinase

A new activator of rat liver phosphofructokinase was partially purified from rat hepatocyte extracts by DEAE-Sephadex chromatography. The activator, which eluted in the sugar diphosphate region, was sensitive to acid treatment but resistant to heating in alkali. Mild acid hydrolysis resulted in the appearance of a sugar monophosphate which was identified as fructose 6-phosphate by gas chromatography/mass spectroscopy. These observations suggest that the activator is fructose 2,6-bisphosphate. This compound was synthesized by first reacting fructose 1,6-bisphosphate with dicyclohexylcarbodiimide and then treating the cyclic intermediate with alkali. The structure of the synthetic compound was definitively identified as fructose 2,6-bisphosphate by 13C NMR spectroscopy. Fructose 2,6-bisphosphate had properties identical with those of the activator purified from hepatocyte extracts. It activated both the rat liver and rabbit skeletal muscle enzyme in the 0.1 microM range and was several orders of magnitude more effective than fructose 1,6-bisphosphate. Fructose 2,6-bisphosphate was not a substrate for aldolase or fructose 1,6-bisphosphatase. It is likely that this new activator is an important physiologic factor of phosphofructokinase in vivo.     

Characterization of the fructose 1,6-bisphosphate-activated, L(+)-lactate dehydrogenase from Thermoanaerobacter ethanolicus.

The L(+)-lactate dehydrogenase from Thermoanaerobacter ethanolicus wt was purified to a final specific activity of 598 mumol pyruvate reduced per min per mg of protein. The specific activity of the pure enzyme with L(+)-lactate was 0.79 units per mg of protein. The M(r) of the native enzyme was 134,000 containing a single subunit type of M(r) 33,500 indicating an apparent tetrameric structure. The L(+)-lactate dehydrogenase was activated by fructose 1,6-bisphosphate in a cooperative manner affecting Vmax and Km values. The activity of the enzyme was also effected by pH, pyruvate and NADH. The Km for NADH at pH 6.0 was 0.05 mM and the Vmax for pyruvate reduction at pH 6.0 was 1082 units per mg in the presence of 1 mM fructose 1,6-bisphosphate. The enzyme was inhibited by NADPH, displaying an uncompetitive pattern. This pattern indicated that NADPH was a negative modifier of the enzyme. The role of L(+)-lactate dehydrogenase in controlling the end products of fermentation is discussed.     

Binding of rabbit muscle aldolase to band 3, the predominant polypeptide of the human erythrocyte membrane.

Aldolase is a trace protein in isolated human red cell membrane preparations. Following total elution of the endogenous enzyme by a saline wash, the interaction of this membrane with rabbit muscle aldolase was studied. At saturation, exogenous aldolase constituted over 40% of the repleted membrane protein. Scatchard analysis revealed two classes of sites, each numbering approximately 7 X 10(5) per ghost. Specificity was suggested by the exclusive binding of the enzyme to the membrane's inner (cytoplasmic) surface. Furthermore, milimolar levels of fructose 1,6-bisphosphate eluted the enzyme from ghosts, while fructose 6-phosphate and NADH (a metabolite which elutes human erythrocyte glyceraldehyde-3-phosphate dehydrogenase (G3PD) from its binding site) were ineffectuve. Removing peripheral membrane proteins with EDTA and lithium 3,5-diiodosalicylate did not diminish the binding capacity of the membranes. An aldolase-band 3 complex, dissociable by high ionic strength or fructose 1,6-bisphosphate treatment, was demonstrated in Triton X-100 extracts of repleted membranes by rate zonal sedimentation analysis on sucrose gradients. We conclude that the association of rabbit muscle aldolase with isolated human erythrocyte membranes reflects its specific binding to band 3 at the cytoplasmic surface, as is also true of G3PD.     

Relationship between thiol group modification and the binding site for fructose 2,6-bisphosphate on rabbit liver fructose-1,6-bisphosphatase

A thiol group present in rabbit liver fructose-1,6-bisphosphatase is capable of reacting rapidly with N-ethylmaleimide (NEM) with a stoichiometry of one per monomer. Either fructose 1,6-bisphosphate or fructose 2,6-bisphosphate at 500 microM protected against the loss of fructose 2,6-bisphosphate inhibition potential when fructose-1,6-bisphosphatase was treated with NEM in the presence of AMP for up to 20 min. Fructose 2,6-bisphosphate proved more effective than fructose 1,6-bisphosphate when fructose-1,6-bisphosphatase was treated with NEM for 90-120 min. The NEM-modified enzyme exhibited a significant loss of catalytic activity. Fructose 2,6-bisphosphate was more effective than the substrate in protecting against the thiol group modification when the ligands are present with the enzyme and NEM. 100 microM fructose 2,6-bisphosphate, a level that should almost saturate the inhibitory binding site of the enzyme under our experimental conditions, affords only partial protection against the loss of activity of the enzyme caused by the NEM modification. In addition, the inhibition pattern for fructose 2,6-bisphosphate of the NEM-derivatized enzyme was found to be linear competitive, identical to the type of inhibition observed with the native enzyme. The KD for the modified enzyme was significantly greater than that of untreated fructose-1,6-bisphosphatase. Examination of space-filling models of the two bisphosphates suggest that they are very similar in conformation. On the basis of these observations, we suggest that fructose 1,6-bisphosphate and fructose 2,6-bisphosphate occupy overlapping sites within the active site domain of fructose-1,6-bisphosphatase. Fructose 2,6-bisphosphate affords better shielding against thiol-NEM modification than fructose 1,6-bisphosphate; however, the difference between the two ligands is quantitative rather than qualitative.     

Saccharomyces cerevisiae aldolase mutants.

Six mutants lacking the glycolytic enzyme fructose 1,6-bisphosphate aldolase have been isolated in the yeast Saccharomyces cerevisiae by inositol starvation. The mutants grown on gluconeogenic substrates, such as glycerol or alcohol, and show growth inhibition by glucose and related sugars. The mutations are recessive, segregate as one gene in crosses, and fall in a single complementation group. All of the mutants synthesize an antigen cross-reacting to the antibody raised against yeast aldolase. The aldolase activity in various mutant alleles measured as fructose 1,6-bisphosphate cleavage is between 1 to 2% and as condensation of triose phosphates to fructose 1,6-bisphosphate is 2 to 5% that of the wild-type. The mutants accumulate fructose 1,6-bisphosphate from glucose during glycolysis and dihydroxyacetone phosphate during gluconeogenesis. This suggests that the aldolase activity is absent in vivo.     

Evidence for an interaction between fructose 1,6-bisphosphatase and fructose 1,6-bisphosphate aldolase.

Three distinct lines of evidence suggest interaction and possible complex formation between fructose 1,6-biphosphate aldolase (EC 4.1.2.13) and fructose 1,6-biphosphatase (EC 3.1.3.11) from rabbit liver. (1) Fructose 1,6-biphosphatase, which does not contain tryptophan, causes changes in the fluorescence emission spectrum of tryptophan in rabbit liver aldolase. (2) Aldolase reduces the affinity of binding of Zn²⁺ to the two high-affinity sites of fructose 1,6-biphosphatase. (3) Gel penetration coefficients are decreased for both enzymes when they are tested together, as compared to the coefficients observed when each is tested separately. These interactions were not observed when either liver enzyme was replaced by the corresponding enzyme purified from rabbit muscle; this specificity for enzymes purified from the same tissue excludes effects attributable to the catalytic activities of the enzyme. Maximum interaction was observed in the pH range between 8.0 and 8.5 and appeared to require the presence of two fructose 1,6-biphosphatase tetramers per tetramer of aldolase. The change in fluorescence emission spectrum was also observed, to a smaller extent, when muscle fructose 1,6-biphosphatase was added to a solution of muscle aldolase.     

Identification of a molecular target for the calcium-modulated protein S100. Fructose-1,6-bisphosphate aldolase

A rat brain S100-binding protein, R40,000, has been isolated, characterized, and identified as fructose-1,6-bisphosphate aldolase. R40,000 was purified by ammonium sulfate precipitation, hydroxylapatite chromatography, dye-binding chromatography, and electroelution from sodium dodecyl sulfate-polyacrylamide gels. Microsequence analysis of a fragment of R40,000 revealed a 15-residue amino acid sequence which shows a high degree of homology to the amino acid sequence of fructose-1,6-bisphosphate aldolase from rabbit muscle and rat liver. Further characterization demonstrated that R40,000 has an amino acid composition, subunit molecular weight, and cyanogen bromide map similar to aldolase. In addition, purified aldolase interacts with S100 alpha and S100 beta by gel overlay, and aldolase enzyme activity is stimulated 2-fold in vitro by S100 alpha and S100 beta. S100 interacts predominantly with the C or brain-specific form of the enzyme in gels and stimulates the activity of the C-enriched form of the enzyme in a calcium-dependent manner. Altogether, these data suggest that fructose-1,6-bisphosphate aldolase may be an intracellular target of S100 action in brain.     

Intracellular localization of fructose 1,6-bisphosphate aldolase.

Submission of a rat liver homogenate made in 250 mM sucrose-1 mM EDTA to centrifugation between 9,500 times g for 10 min and 105,000 times g for 60 min results in the sedimentation of 60 to 70% of the total cellular fructose 1,6-bisphosphate aldolase (EC 4.1.2.13). Under these conditions only about one-quarter of the total triose phosphate dehydrogenase and phosphoglycerate kinase appears in the microsomal fraction. Ultrastructural immunologic localization techniques have demonstrated that the aldolase is associated with the endoplasmic reticulum, in situ. The binding of this enzyme to the membrane is sensitive to changes in pH with an optimum at 6.0, and to increasing concentrations of NaCl and fructose 1,6-bisphosphate, being about 100-fold more sensitive to the ester than to the inorganic salt.     

Sites of cleavage of rabbit muscle aldolase by purified cathepsin M from rabbit liver

Rabbit liver cathepsin M, a sulfhydryl proteinase similar in catalytic properties to cathepsin B, causes a decrease in the activity of rabbit muscle aldolase assayed with fructose 1,6-bisphosphate but not with fructose 1-phosphate. Proteolytic modification of aldolase by cathepsin M is limited to the removal of small peptides from the COOH-terminus, including the COOH-terminal hexapeptide NH2-Ile-Ser-Asn-His-Ala-TyrOH. Correlation of loss of aldolase activity with COOH-terminal modification indicates that only three of the four subunits of muscle aldolase contribute to the catalytic activity of the tetrameric enzyme.     

Isolation of buffalo muscle aldolase and comparison of its properties with those of rabbit muscle aldolase .

Fructose-1,6-bisphosphate aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phyosphate-lyase, EC 4.1.2.13) was isolated from buffalo muscle by fractionation with ammonium sulphate and subsequent purification by phosphocellulose column chromatography using a linear salt gradient. As judged by gel filtration and electrophoresis in polyacrylamide gel, the enzyme was homogeneous with respect to size and charge. The molecular weight and Stokes radius of the enzyme were determined from its elution profile on a calibrated Sephadex column and the respective values were 162000 and 4.55 nm. The diffusion coefficient and frictional ratio were computed to be 4.8-10(7) cm2-s-1 and 1.27, respectively. The molecular weight of the polypeptide chain as measured by aodium dodecyl sulphate polyacrylamide gel electrophoresis was 40750. This taken together with the native molecular weight suggested a four-subunit model for the protein. The N- AND C-terminal residues of polypeptide chains were identified to be proline and tyrosine, respectively. At pH 8.0 the Michaelis-Menten constant and maximum attainable velocity were found to be 8.1 muM and 27 muM Fru-1,6-P2 split/min per mg, respectively. The buffalo muscle aldolase was found to be similar to rabbit muscle aldolase in physico-chemical properties. However, the two enzymes differ significantly in pH optimum; the p optima of the buffalo and rabbit enzymes were determined under identical conditions to be 8.0 and 8.6, respectively.     

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.     

Recombinant anaerobic maize aldolase: overexpression, characterization, and metabolic implications

Complementary DNA sequence of anaerobically induced cytoplasmic maize aldolase was expressed under control of the tac promoter sequence in Escherichia coli using the pKK223-3 plasmid as a vehicle. Levels of recombinant protein expressed exceeded 20 mg of soluble aldolase per liter of culture. The purified recombinant enzyme displayed the expected molecular weight and tetrameric subunit assembly on the basis of mobilities on denaturing electrophoretic gels and gel filtration, respectively. Sequencing of the NH2 terminus and amino acid composition analysis of the recombinant protein including COOH-terminal peptides agreed with the cDNA sequence. Partial kinetic characterization based on product inhibition studies was consistent with the ordered uni-bi reaction mechanism expected of aldolases. Turnover with respect to substrates Fru-1,6-P2 and Fru-1-P by the recombinant enzyme is the highest reported to date for class I aldolases. Fru-1,6-P2 cleavage rate by recombinant cytoplasmic maize enzyme is three times greater than that of the chloroplast enzyme. Fru-1-P cleavage is 8-fold greater than that of the rabbit liver isozyme and 20-fold greater than that of the rabbit muscle isozyme to which maize aldolase exhibits the greatest homology. The implications of such a high Fru-1-P turnover on carbohydrate utilization under anaerobiosis is discussed.     

High level production of the Magnaporthe grisea fructose 1,6-bisphosphate aldolase enzyme in Escherichia coli using a small volume bench-top fermentor

The Class II fructose 1,6-bisphosphate aldolase from the Rice Blast causative agent Magnaporthe grisea was subcloned in the Escherichia coli vector pT7-7. The enzyme was overexpressed using fed-batch fermentation in a small bench-top reactor. A total of 275 g of cells and 1.3 g of highly purified enzyme with a specific activity of 70 U/mg were obtained from a 1.5L culture. The purified enzyme is a homodimer of 39.6 kDa subunits with a zinc ion at the active site. Kinetic characterization indicates that the enzyme has a K(m) of 51 microM, a k(cat) of 46 s(-1), and a pH optimum of 7.8 for fructose 1,6-bisphosphate cleavage. The fermentation system procedure reported exemplifies the potential of using a lab-scale bioreactor for the large scale production of recombinant enzymes.     

Inhibition of fructose-1,6-bisphosphate aldolase from rabbit muscle and Bacillus stearothermophilus

Phosphoglycollohydroxamic acid and phosphoglycollamide are inhibitors of rabbit muscle fructose-1,6-bisphosphate aldolase. The binding dissociation constants determined by enzyme inhibition and protein fluorescence quenching suggest that two distinct enzyme inhibitor complexes may be formed. The binding dissociation constants of the two inhibitors to Bacillus stearothermophilus cobalt (II) fructose-1,6-bisphosphate aldolase have also been determined. The hydroxamic acid is an exceptionally potent inhibitor (Ki = 1.2 nM) probably due to direct chelation with Co(II) at the active site. The inhibition, however, is time-dependant and the association and dissociation constants have been estimated. Ethyl phosphoglycollate irreversibly inhibits rabbit muscle fructose-1,6-bisphosphate aldolase in the presence of sodium borohydride, presumably by forming a stable secondary amine through the active-site lysine reside. A new condensation assay for fructose-1,6-bisphosphate aldolases has been developed which is more sensitive than currently used assay procedures.     

An exploration of the binding site of aldolase using alkanediol monoglycolate bisphosphoric esters

Alkanediol monoglycolate bisphosphoric esters (P-O-CH2-CO-O-(CH2)n-O-P), which are analogues of the aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) substrate fructose 1,6-bisphosphate, were synthesized and used for probing its active site. The Ki value was lowest when the maximum distance between the phosphorus atoms of the bisphosphate was brought close to that of fructose 1,6-bisphosphate. The binding constants estimated from difference spectra correlate well with Ki values for the substrate analogues. Propanediol monoglycolate bisphosphoric ester protected aldolase from inactivation by 1,2-cyclohexanedione, which preferentially attacks arginine-55. However, propanol phosphate had little protective effect. The synthesized phosphate compounds protected the enzyme against inactivation by trypsin, and also against spontaneous denaturation. These results suggest that the synthesized phosphate compounds bind to aldolase at the active site, which tends to keep the distance constant between the two phosphate-binding sites for the open-chain form of fructose 1,6-bisphosphate, and stabilize the natural conformation of the enzyme. Both arginine-55 and lysine-146 are shown to participate in the phosphate-binding site for the C-1-phosphate of fructose 1,6-bisphosphate.     

Fructose-6-phosphate aldolase is a novel class I aldolase from Escherichia coli and is related to a novel group of bacterial transaldolases

We have cloned an open reading frame from the Escherichia coli K-12 chromosome that had been assumed earlier to be a transaldolase or a transaldolase-related protein, termed MipB. Here we show that instead a novel enzyme activity, fructose-6-phosphate aldolase, is encoded by this open reading frame, which is the first report of an enzyme that catalyzes an aldol cleavage of fructose 6-phosphate from any organism. We propose the name FSA (for fructose-six phosphate aldolase; gene name fsa). The recombinant protein was purified to apparent homogeneity by anion exchange and gel permeation chromatography with a yield of 40 mg of protein from 1 liter of culture. By using electrospray tandem mass spectroscopy, a molecular weight of 22,998 per subunit was determined. From gel filtration a size of 257,000 (+/- 20,000) was calculated. The enzyme most likely forms either a decamer or dodecamer of identical subunits. The purified enzyme displayed a V(max) of 7 units mg(-)1 of protein for fructose 6-phosphate cleavage (at 30 degrees C, pH 8.5 in 50 mm glycylglycine buffer). For the aldolization reaction a V(max) of 45 units mg(-)1 of protein was found; K(m) values for the substrates were 9 mm for fructose 6-phosphate, 35 mm for dihydroxyacetone, and 0.8 mm for glyceraldehyde 3-phosphate. FSA did not utilize fructose, fructose 1-phosphate, fructose 1,6-bisphosphate, or dihydroxyacetone phosphate. FSA is not inhibited by EDTA which points to a metal-independent mode of action. The lysine 85 residue is essential for its action as its exchange to arginine (K85R) resulted in complete loss of activity in line with the assumption that the reaction mechanism involves a Schiff base formation through this lysine residue (class I aldolase). Another fsa-related gene, talC of Escherichia coli, was shown to also encode fructose-6-phosphate aldolase activity and not a transaldolase as proposed earlier.     

Exploring CP12 binding proteins revealed aldolase as a new partner for the phosphoribulokinase/glyceraldehyde 3-phosphate dehydrogenase/CP12 complex--purification and kinetic characterization of this enzyme from Chlamydomonas reinhardtii

Possible binding proteins of CP12 in a green alga, Chlamydomonas reinhardtii, were investigated. We covalently immobilized CP12 on a resin and then used it to trap CP12 partners. Thus, we found an association between CP12 and phosphoribulokinase (EC 2.7.1.19), glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) and aldolase. Immunoprecipitation with purified CP12 antibodies supported these data. The dissociation constant between CP12 and fructose 1,6-bisphosphate (EC 4.1.2.13) aldolase was measured by surface plasmon resonance and is equal to 0.48 +/- 0.05 mum and thus corroborated an interaction between CP12 and aldolase. However, the association is even stronger between aldolase and the phosphoribulokinase/glyceraldehyde 3-phosphate dehydrogenase/CP12 complex and the dissociation constant between them is equal to 55+/-5 nm. Moreover, owing to the fact that aldolase has been poorly studied in C. reinhardtii, we purified it and analyzed its kinetic properties. The enzyme displayed Michaelis-Menten kinetics with fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate, with a catalytic constant equal to 35 +/- 1 s(-1) and 4 +/- 0.1 s(-1), respectively. The K(m) value for fructose 1,6-bisphosphate was equal to 0.16 +/- 0.02 mm and 0.046 +/- 0.005 mm for sedoheptulose 1,7-bisphosphate. The catalytic efficiency of aldolase was thus 219 +/- 31 s(-1).mm(-1) with fructose 1,6-bisphosphate and 87 +/- 9 s(-1).mm(-1) with sedoheptulose 1,7-bisphosphate. In the presence of the complex, this parameter for fructose 1,6-bisphosphate increased to 310 +/- 23 s(-1).mm(-1), whereas no change was observed with sedoheptulose 1,7-bisphosphate. The condensation reaction of aldolase to form fructose 1,6-bisphosphate was also investigated but no effect of CP12 or the complex on this reaction was observed.     

Inactivation of liver aldolase in fasting rabbits: Evidence for the accumulation of two different immunoreactive forms

During prolonged starvation the activity of aldolase in crude rabbit liver extracts decreases to less than one-half the value observed in extracts of livers from fed animals. The specific activity of the enzyme purified by adsorption on phosphocellulose and elution with substrate is also approximately one-half that of the purified native enzyme. Both the level of enzyme activity and the specific activity are restored to normal within 36 h of refeeding. After removal of active aldolase from the liver extracts by adsorption on phosphocellulose an additional immunoreactive protein can be isolated by adsorption on antialdolase-Sepharose and elution with 4 m MgCl₂. This protein is devoid of catalytic activity and in livers of fasted rabbits accounts for nearly 40% of the total immunoreactive material. It has also been detected in extracts prepared from livers of fed rabbits, where it accounts for 10–20% of the total protein adsorbed by antialdolase-Sepharose. The low-activity enzyme isolated from livers of fasted rabbits cannot be reactivated by sulfhydryl compounds; it shows similar sensitivity to heat and denaturing agents as the enzyme isolated from livers of fed rabbits. The activity ratios with fructose 1,6-bisphosphate, fructose 1-phosphate, and triose phosphate are similar to those observed for the native liver enzyme.