B.L. Horecker

The purification of properties of rat liver fructose 1,6-bisphosphatase.

Abstract Fructose 1,6-bisphosphatase has been isolated in homogeneous form from rat liver by a simple and convenient procedure, including adsorption on carboxymethyl cellulose and substrate elution. It is a tetrameric protein, with molecular weight of approximately 140,000. The amino acid composition shows some similarity to that of the rabbit liver enzyme, with a blocked NH 2 -terminus and leucine, rather than alanine, as the COOH-terminal residue. The purified enzyme contains no tryptophan. It requires Mg 2+ or Mn 2+ and is activated by NH 4 + , which also decreases the affinity for both substrate and divalent cations. In the absence of chelating agents, the pH optimum is 8.0, but this is shifted to 7.0, 7.2, and 7.5 on addition of EDTA, histidine, and citrate, respectively. Ca 2+ is a potent inhibitor and appears to compete for the Mg 2+ -binding site. The rat liver enzyme is inhibited by AMP in a cooperative manner, with a Hill coefficient of about 2. Saturation is reached when approximately 2 mol of AMP are bound per mole of enzyme, but no AMP binding is observed in the absence of the substrate. Antibody to the purified enzyme, produced in the rabbit, reacts with fructose 1,6-bisphosphatase in rat liver and rat kidney with equal affinity. It also reacts with rat muscle fructose 1,6-bisphosphatase, with 1/100th the affinity.

On the mechanism of inhibition of fructose 1,6-bisphosphatase by fructose 2,6-bisphosphate

Abstract The inhibition of rabbit liver fructose 1,6-bisphosphatase (EC 3.1.3.11) by fructose 2,6-bisphosphate (Fru-2,6-P2) is shown to be competitive with the substrate, fructose 1,6-bisphosphate (Fru-1,6-P2), with Ki for Fru-2,6-P2 of approximately 0.5 μ m . Binding of Fru-2,6-P2 to the catalytic site is confirmed by the fact that it protects this site against modification by pyridoxal phosphate. Inhibition by Fru-2,6-P2 is enhanced in the presence of a noninhibitory concentration (5 μ m ) of the allosteric inhibitor AMP and decreased by modification of the enzyme by limited proteolysis with subtilisin. Fru-2,6-P2, unlike the substrate Fru-1,6-P2, protects the enzyme against proteolysis by subtilisin or lysosomal proteinases.

The activation of rabbit muscle, liver, and kidney fructose bisphosphatases by histidine and citrate

Abstract Purified fructose 1,6-bisphosphatases from rabbit muscle, liver, and kidney require a metal chelator for optimal activity at neutral pH. This requirement is satisfied by physiological concentrations of histidine and citrate, and at pH 7 their effects are additive. In the presence of both histidine and citrate the optimum activity is shifted from about pH 8 to pH 7.2, and the activity is greater than that obtained with optimal concentrations of EDTA. Carnosine, anserine, and 1-methyl histidine are also effective, but only at much higher concentrations, while 3-methyl histidine is effective in the same concentration range as is histidine. Isocitrate can replace citrate. The results suggest that fructose bisphosphatases possess distinct binding sites for divalent cations (Mg 2+ or Mn 2+ ) and also for histidine and citrate complexes of these cations.

Transformation of neutral to alkaline fructose 1,6-bisphosphatase: Converting enzyme activity in the large-particle fraction from rabbit liver☆☆☆

Abstract A liver particle fraction containing lysosomes catalyzes the conversion of native rabbit liver fructose 1,6-bisphosphatase (EC 3.1.3.11), having a neutral pH optimum, to a modified form with an alkaline pH optimum. The “converting enzyme” activity is partially recovered with the membranes from disrupted particles, and is also detected in “intact” particles isolated and maintained in isotonic buffered sucrose. The converting enzyme activity associated with the membrane fraction is expressed at pH 6.5, but not at pH 4.5, although activity at the lower pH appears when the enzyme is released from the membranes with Triton X-100. In contrast, proteolytic activity as measured with peptide and protein substrates is maximal at pH 5.0 or below, and is the same for the membrane-bound or solubilized proteases. The results suggest that a specific converting enzyme, at least partially associated with a particle (possibly lysosomal) membrane, is responsible for the modification of fructose bisphosphatase and the change in its catalytic properties.

Ligand-induced conformational states of rabbit liver fructose 1,6-bisphosphatase as revealed by digestion with subtilisin

Abstract Fructose 1,6-bisphosphatase undergoes specific conformational changes in the presence of the substrate fructose 1,6-bisphosphate and of the allosteric modifier, AMP and also on activation by cystamine. These changes can be monitored by observing the changes in sensitivity to digestion by subtilisin. In the presence of AMP the enzyme is protected against the action of subtilisin. Some protection is also observed with high concentrations of fructose bisphosphate while low concentrations of this substrate, which are ineffective alone, enhance the protective effect of low concentrations of AMP. The results suggest that AMP induces a resistant conformation, and that fructose bisphosphate promotes the binding of AMP. Divalent cations, although essential for activity, do not protect the enzyme against digestion by subtilisin. The native enzyme is activated by disulfide exchange with cystamine, and the activated enzyme is also more resistant to subtilisin. Thus, the enzyme in both inhibited (AMP) and activated conformations (cystamine) is rendered resistant to modification by proteolysis.

Structural studies on fructose-1,6-bisphosphatases from rabbit liver, kidney, and muscle

Abstract Fructose-1,6-bisphosphatases (EC 3.1.3.11) isolated from rabbit liver and kidney appear to have identical primary structures, as deduced from their tryptic peptide maps and the peptide patterns obtained after cleavage with cyanogen bromide and chromatography on Sephadex G75. The enzyme isolated from rabbit skeletal muscle, on the other hand, yields distinctly different fingerprints and cyanogen bromide cleavage products. The results indicate that animal cells possess two genes that code for fructose-bisphosphatase. Native rabbit liver fructose bisphosphatase contains a single tryptophan located near the NH 2 -terminus, and the NH 2 terminal-BrCN peptide containing this residue has been identified in the Sephadex G75 filtrates.

Binding of monoclonal antibody to cathepsin M located on the external surface of rabbit lysosomes

A monoclonal antibody raised against rabbit liver cathepsin M binds to intact rabbit liver lysosomes. The binding is specific and is abolished by treating the lysosomes with trypsin, which has previously been shown to digest the membrane-bound cathepsin M [S. Pontremoli, E. Melloni, M. Michetti, F. Salamino, B. Sparatore, and B. L. Horecker (1982) Biochem, Biophys. Res. Commun. 106, 903-909]. Rabbit liver lysosomes are adsorbed onto Sepharose 4B coupled to anti-cathepsin M, but not to Sepharose 4B itself or to Sepharose coupled to a nonspecific antibody. The results confirm the location of membrane-bound cathepsin M on the outer surface of the lysosomal membrane.

Evidence for the modification of fructose 1,6-bisphosphatase by two distinct lysosomal proteases

Two lysosomal proteases have been detected, each capable of catalyzing a different modification of the NH2-terminal region of rabbit liver fructose bisphosphatase. Protease I has optimum activity near pH 5.0, in contrast to Protease II, which is most active only at lower pH. The peptide formed by the action of Protease I is acid-insoluble, with a molecular weight of approximately 7,000, whereas Protease II releases a small acid-soluble peptide, containing the tryptophan residue that is located near the NH2-terminus. In fasted rabbits, Protease II appears to be selectively released from the lysosomes.

Cooperative interactions between native and modified subunits of rabbit liver fructose 1,6-bisphosphatase.

Abstract Rabbit liver fructose 1,6-bisphosphatase is converted by subtilisin to a form with smaller subunits and modified catalytic and allosteric properties. Analysis of the changes in the catalytic properties of the enzyme during digestion with subtilisin indicates that these properties depend on the presence of strong functional interactions between all four subunits in the molecule. On the other hand, sensitivity to inhibition by AMP appears to depend only on intrachain interactions. Changes in subunit interaction relating to relaxation in protein conformation during digestion with subtilisin were also inferred from the changes in concentration dependency for the effects of urea on the fluorescent emission spectrum. Structural changes around the region containing the single tryptophan residue appear to be related to the changes in catalytic properties.

Specific Proteolytic Modification of Rabbit Liver Fructose Bisphosphatase under Gluconeogenic Conditions

Evidence has been obtained for the presence in rabbit liver lysosomes of two distinct proteinases (Pontremoli et al., 1974b, 1975a) capable of modifying Fru-P2ase. These activities are detected by analysis of the products formed at pH 4.0 and pH 5.0 (Fig. 1) during incubation of purified native Fru-P2ase with the lysosomal enzymes released by freezing and thawing.