An adenosine 3':5'-monophosphate-dependent protein kinase from the human erythrocyte membrane. Purification and characterization.

An adenosine 3':5'-monophosphate-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) has been isolated from the human erythrocyte memebrane and the phosphotransferase activity exhibited by this enzyme has been purified 800-fold. In concentrated solutions, the membrane-derived protein kinase undergoes aggregation with a concomitant loss in observed phosphotransferase activity. This loss of activity can be restored by means of inducing deaggregation. The phosphotransferase activity of the protein kinase is virtually obliterated in the presence of high (300 mM) concentrations of sodium chloride. This effect is also reversible. The pH optimum for the phosphotransferase reaction that is catalyzed by the membrane-derived protein kinase is approximately 8. Micromolar concentrations of cAMP are optimal with respect to promoting the phosphotransferase reaction. Initial velocity and product inhibition studies were conducted on the cAMP-independent protein kinase derived from the cAMP-dependent enzyme. These studies indicate that the phosphotransferase reaction proceeds by a sequential kinetic mechanism.     

A Limulus glucose-6-phosphatase with phosphotransferase activity characteristic of vertebrate liver microsomes. Its possible evolutionary significance.

1. Limulus hepatopancreas, coxal glands and intestine contain a particulate enzyme which can synthesize glucose 6-phosphate from glucose and inorganic pyrophosphate or carbamyl phosphate as well as hydrolyze glucose 6-phosphate. This has been clearly differentiated from hydrolysis by lysosomal or soluble phosphatases. 2. The enzyme resembles vertebrate glucose-6-phosphatase in its specific anatomical distribution, pH optimum, kinetic properties, donor specificity and phospholipid dependence, as indicated by its satency and lability to detergent treatment. 3. A variety of other invertebrates tested exhibited little or no PPi-glucose phosphotransferase activity with these properties. A similar phosphotransferase activity of lobster hepatopancreas had somewhat different kinetic properties and pH optimum. 4. The hypothesis that a specific glucose-6-phosphatase is to be found only in those animals which utilize free glucose as an important circulating form of energy is presented and discussed. It appears that a variety of transport compounds, such as trehalose and glucose, was tried at the evolutionary level of the Arthropods.     

Aminoglycoside-3'-phosphotransferase from Actinomyces fradiae. Its isolation, purification and properties]

Aminoglycoside phosphotransferase was isolated from the mycelium of Act. fradiae, the neomycin-producing organism, with paromomycin, neomycin and to a less extent ribostamycin being substrates of aminoglycoside-phosphotransferase. It was purified to homogenous state. The maximum activity of the enzyme preparations was observed at pH 7.7--7.8;KM for neomycin and paromomycin was about 20 micron and KM for ATP was 150 micron. Mg2+ ions were necessary for the enzyme activity. None of the divalent cations tested could replace the magnesium ions in the reaction of phosphorylation catalyzed by the enzyme. High sensitivity to the ionic strength of the buffer was characteristic of the enzyme. It lost about 80 per cent of the initial activity at a concentration of KC1 equal to 1.0 M. The molecular mass of the enzyme from the mycelium of Act. fradiae was determined by the method of gel-filtration through sefadex G-100. It was about 22,000. High stability was characteristic of the enzyme. The fingings indicate that aminoglycoside phosphotransferase from Act. fradiae differs from the described aminoglycoside-3'-phosphotransferases isolated from antibiotic resistant bacteria.     

Allosteric regulation of glycerol kinase by enzyme IIIglc of the phosphotransferase system in Escherichia coli and Salmonella typhimurium.

The mechanism by which enzyme IIIglc of the bacterial phosphotransferase system regulates the activity of crystalline glycerol kinase from Escherichia coli has been studied, and the inhibitory effects have been compared with those produced by fructose-1,6-diphosphate. It was shown that the free, but not the phosphorylated, form of enzyme IIIglc inhibits the kinase. Mutants of Salmonella typhimurium were isolated which were resistant to inhibition by either enzyme IIIglc (glpKr mutants) or fructose-1,6-diphosphate (glpKi mutants), and each mutant type was shown to retain full sensitivity to inhibition by the other regulatory agent. Other mutants were fully or partially resistant to regulation by both agents. The two regulatory sites on the kinase are evidently distinct but must overlap or interact functionally. Kinetic analyses have revealed several mechanistic features of the regulatory interactions. (i) Inhibition by both allosteric regulatory agents is strongly pH dependent, with maximal inhibition occurring at ca. pH 6.5 under the assay conditions employed. (ii) Binding of enzyme IIIglc to glycerol kinase is also pH dependent, the Ki being near 4 microM at pH 6.0 but near 10 microM at pH 7.0. (iii) Whereas fructose-1,6-diphosphate inhibition apparently requires that the enzyme exist in a tetrameric state, both the dimer and the tetramer appear to be fully sensitive to enzyme IIIglc inhibition. (iv) Inhibition by enzyme IIIglc (like that by fructose-1,6-diphosphate) is noncompetitive with respect to both substrates. (v) The inhibitory responses of glycerol kinase to fructose-1, 6-diphosphate and enzyme IIIglc show features characteristic of positive cooperativity at low inhibitor concentration. (vi) Neither agent inhibits completely at high inhibitor concentration. (vii) Apparent negative cooperativity with respect to ATP binding is observed with purified E. coli glycerol kinase, with glycerol kinase in crude extracts of wild-type S. typhimurium cells, and with glpKr and glpKi mutant forms of glycerol kinase from S. typhimurium. These results serve to characterize the regulatory interactions which control the activity of glycerol kinase by fructose-1,6-diphosphate and by enzyme IIIglc of the phosphotransferase system.     

Biosynthesis of phosphatidylcholine by enzyme preparations from spinach leaves

The enzymic incorporation of choline-1,2-(14)C from CDP-choline-1,2-(14)C into phosphatidylcholine by spinach leaf preparations was characterized. The enzyme catalyzing the incorporation, choline phosphotransferase, had a pH optimum of about 8.0 and required either Mn(2+) or Mg(2+) as cofactor. The saturation concentration of Mn(2+) was 0.3 mm and that for Mg(2+) was 13 mm. The K(m) for CDP-choline was 10 micro m. The choline phosphotransferase was inhibited by sulfhydryl reagents. The enzyme was inactivated at 30 degrees C, but this inactivation could be prevented by dithiothreitol and Mn(2+). Preincubation of the enzyme with Mn(2+) prevented inhibition by sulfhydryl reagents. The incorporation of diglyceride-U-(14)C into phosphatidylcholine was also studied. The enzyme did not show any diglyceride specificity when exogenous diglyceride was added, indicating that fatty acid distribution in phosphatidylcholine of spinach is not controlled by choline phosphotransferase.     

Purification and kinetic properties of pyruvate kinase from Brochothrix thermosphacta.

Pyravate kinase (ATP: pyruvate 2-0 phosphotransferase E.C.2.7.1.40) was purified from Brochothrix thermosphacta. The enzyme is a homotetramer of monomer Mr 58,000. Fructose-1,6-bisphosphate stimulates activity and promotes hyperbolic kinetics although it is not essential for enzyme activity. The positive effect of fructose-1,6-bisphosphate on activity is repressed by inorganic phosphate which enhances cooperative kinetics. Unlike pyruvate kinases from other sources, the Brochothrix enzyme is uncompetitively inhibited by glucose-6-phosphate, although at high concentration. ATP is a strong inhibitor of pyruvate kinase and shifts the residual activity/pH profile towards more alkaline values.     

A cyclic adenosine 3',5'-monophosphate-dependent histone kinase from pig brain. Purification and some properties of the enzyme.

A cyclic adenosine 3',5'-monophosphate-dependent histone kinase (ATP: protein phosphotransferase, EC 2.7.1.37) was isolated from pig brain. The enzyme has been purified 1140-fold; it is homogeneous on polyacrylamide gel electrophoresis and gel filtration. The estimated molecular weight of the enzyme is 120 000. Histone kinase dissociates into a catalytic subunit and a regulatory one (molecular weights 40 000 and 90 000, respectively). The catalytic subunit has been obtained in homogeneous state as evidenced by sodium dodecylsulphate-polyacrylamide gel electrophoresis. At all purification steps, enzymatic activity is stimulated 5-fold by cyclic AMP. An apparent Km value for cyclic AMP is about 3.3 - 10- minus 7 M. In the presence of cyclic AMP(5 - 10- minus 6 M), the Km value for ATP and F1 histone were 1.2 - 10- minus five and 3 - 10- minus 5 M, respectively. Optimum pH value for histone kinase is 6.5, its isoelectric point is situated at pH 4.6. The purified enzyme displays high specificity for the lysine-rich and moderately lysine-rich histones F1, F2a2 and F2b. Arginine-rich histones and other known protein substrates for cyclic AMP-dependent protein kinases (casein, Escherichia coli RNA polymerase, etc.) are extremely poor substrates for this enzyme.     

Regulation of glycerol uptake by the phosphoenolpyruvate-sugar phosphotransferase system in Bacillus subtilis.

Enteric bacteria have been previously shown to regulate the uptake of certain carbohydrates (lactose, maltose, and glycerol) by an allosteric mechanism involving the catalytic activities of the phosphoenolpyruvate-sugar phosphotransferase system. In the present studies, a ptsI mutant of Bacillus subtilis, possessing a thermosensitive enzyme I of the phosphotransferase system, was used to gain evidence for a similar regulatory mechanism in a gram-positive bacterium. Thermoinactivation of enzyme I resulted in the loss of methyl alpha-glucoside uptake activity and enhanced sensitivity of glycerol uptake to inhibition by sugar substrates of the phosphotransferase system. The concentration of the inhibiting sugar which half maximally blocked glycerol uptake was directly related to residual enzyme I activity. Each sugar substrate of the phosphotransferase system inhibited glycerol uptake provided that the enzyme II specific for that sugar was induced to a sufficiently high level. The results support the conclusion that the phosphotransferase system regulates glycerol uptake in B. subtilis and perhaps in other gram-positive bacteria.     

Phosphotransferase activity associated with rat osseous plate alkaline phosphatase: a possible role in biomineralization.

1. Alkaline phosphatase from rat osseous plate catalyzed the transfer of phosphate from p-nitrophenylphosphate to glycerol, ethanolamines, Tris, glucose and 1-amino-1-methyl-2-propanol, in a wide range of pH. Serine did not stimulate phosphotransferase activity of the enzyme. 2. The best phosphotransferase acceptors were diethanolamine and glycerol while glucose was the poorest phosphotransferase acceptor used. 3. Diethanolamine and glycerol affected both VM and KM of p-nitrophenylphosphate hydrolysis with activation constants (KA) of 0.25 and 0.85 M, respectively. 4. A kinetic model was proposed for the phosphotransferase reaction observed with alkaline phosphatase from rat osseous plates.     

Regulation of aconitate hydratase activity from rat kidney cortex by bicarbonate.

1. The increase in pH value and bicarbonate concentration stimulated citrate synthesis from pyruvate and malate, inhibiting simultaneously conversion of isocitrate to citrate. 2. Bicarbonate inhibited competitively the activity of aconitate hydratase, probably binding with the two active sites of the enzyme. The Ki values for the cytoplasmic and mitochondrial enzyme were, respectively, 27 and 38 mM. The pH optimum for both forms of the enzyme in Tris-HCl buffer was in the range 7.8-8.6, and in bicarbonate buffer varied from 7.2 to 8.0, depending on the form of the enzyme and the substrate used. 3. Only free, completely dissociated citrate anion acts as a substrate for aconitate hydratase. 4. The role of aconitate hydratase as a factor controlling the rate of citrate metabolism in kidney in metabolic alkalosis is discussed.     

Unmasking of an essential thiol during function of the membrane bound enzyme II of the phosphoenolpyruvate glucose phosphotransferase system of Escherichia coli.

The addition of N-ethylmaleimide (MalNEt), or of fluoro dinitrobenzene to a suspension of Escherichia coli during the phosphorylating uptake of methyl-alpha-D-glucopyranoside (Me-Glc), a glucose analog, stops uptake and phosphorylation and causes the loss of previously accumulated sugar and of its phosphate ester. After removal of the reagents, the phosphotransferase system remains irreversibly inactive. Pretreatment of the bacteria with the same reagents under the same conditions of concentration, pH, temperature and for the same length of time causes very little inactivation. Mercuric chloride, a reversible inactivator, prevents the phosphotransferase system from reacting simultaneously with MaINEt or with fluorodinitrobenzene. This protection strongly suggests that all three reagents react with the same site, presumably an -SH group. The change which makes this site available to the reagents depends on the phosphorylative uptake of Me-Glc. Preload of the cells and efflux of Me-Glc do not achieve the same change. The rate of inactivation is directly proportional to the rate of phosphorylative uptake. When the Km of phosphorylative uptake is modified by an uncoupling agent, the substrate concentration allowing half maximal rate of inactivation by MaINEt changes accordingly. The reactive sites of the phosphotransferase system can also be made accessible to the -SH group reagents by fluoride inhibition of phosphoenolpyruvate synthesis. This suggests that the inactivator resistent form is an "energized form" of the enzyme. The unmasking of the reactive site is not due to a change in transmembrane penetration of the reagents since incubation of toluene treated cells with MaINEt in the presence of phosphoenolpyruvate fails to inactivate the phosphotransferase activity, while incubation with MaINEt plus Me-Glc causes fast inactivation.     

Ionic control of immobilized enzymes. Kinetics of acid phosphatase bound to plant cell walls.

When an enzyme is bound to an insoluble polyelectrolyte it may acquire novel kinetic properties generated by Donnan effects. It the enzyme is homogeneously distributed within the matrix, a variation of the electrostatic partition coefficient, when substrate concentration is varied, mimics either positive or negative co-operativity. This type of non-hyperbolic behaviour may be distinguished from true co-operativity by an analysis of the Hill plots. If the enzyme is heterogeneously distributed within the polyelectrolyte matrix, an apparent negative co-operativity occurs, even if the electrostatic partition coefficient does not vary when substrate concentration is varied in the bulk phase. If the partition coefficient varies, mixed positive and negative co-operativities may occur. All these effects must be suppressed by raising the ionic strength in the bulk phase. Attraction of cations by fixed negative charges of the polyanionic matrix may be associated with a significant decrease of the local pH. The magnitude of this effect is controlled by the pK of the fixed charges groups of the Donnan phase. The local pH cannot be much lower than the value of this pK. This effect may be considered as a regulatory device of the local pH. Acid phosphatase of sycamore (Acer pseudoplatanus) cell walls is a monomeric enzyme that displays classical Michaelis-Menten kinetics in free solution. However, when bound to small cell-wall fragments or to intact cells, it has an apparent negative co-operativity at low ionic strength. Moreover a slight increase of ionic strength apparently activates the bound enzymes and tends to suppress the apparent co-operativity. At I0.1, or higher, the bound enzyme has a kinetic behavior indistinguishable from that of the purified enzyme in free solution. These results are interpreted in the light of the Donnan theory. Owing to the repulsion of the substrate by the negative charges of cell-wall polygalacturonates, the local substrate concentration in the vicinity of the bound enzyme is smaller than the corresponding concentration in bulk solution. The kinetic results obtained are consistent with the view that there exist at least three populations of bound enzyme with different ionic environments: a first population with enzyme molecules not submitted to electrostatic effects, and two other populations with molecules differently submitted to these effects. The theory allows one to estimate the proportions of enzyme belonging to these populations, as well as the local pH values and the partition coefficients within the cell walls.     

Regulation of glycolysis in Neurospora crassa. Kinetic properties of pyruvate kinase.

Pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40), extracted from the mycelium of Neurospora crassa has been purified 560-fold by precipitation with ammonium sulphate, chromatography with DEAE-Sephadex, and gel filtration with Sephadex G-200. Potassium and magnesium are required for enzyme activity. Fructose, 1,6-diphosphate is the only physiological activator found for the enzyme. In decreasing order of potency, citrate, oxalacetate, calcium, and ATP are inhibitors. Phosphoenolpyruvate is cooperatively bound by the enzyme and the cooperatively is reduced by ATP and completely eliminated by fructose-1,6-diphosphate. Lowering of pH from 7-5 to 5-5 changes the Hill coefficient from 2-7 to 1-0. Substitution of ADP by other nucleotides reduces enzyme activity. Manganese can substitute for the cofactor magnesium, but the reaction velocity is then reduced. MgADP- is cooperatively bound by the enzyme and inhibition of the enzyme occurs only when either magnesium or ADP is in excess of the other beyond the optimum concentration. These kinetics properties of pyruvate kinase are compatible with the role of a regulator of glycolysis in Neurospora crassa.     

Adenylate cyclase is required for chemotaxis to phosphotransferase system sugars by Escherichia coli.

We report that in Escherichia coli, chemotaxis to sugars transported by the phosphotransferase system is mediated by adenylate cyclase, the nucleotide cyclase linked to the phosphotransferase system. We conclude that adenylate cyclase is required in this chemotaxis pathway because mutations in the cyclase gene (cya) eliminate or impair the response to phosphotransferase system sugars, even though other components of the phosphotransferase system known to be required for the detection of these sugars are relatively unaffected by such mutations. Moreover, merely supplying the mutant bacteria with the products of this enzyme, cyclic AMP and cyclic GMP, does not restore the chemotactic response. Because a residual chemotactic response is observed in certain strains with residual cyclic GMP synthesis but no cyclic AMP synthesis, it appears that the guanylate cyclase activity rather than the adenylate cyclase activity of the enzyme may be required for chemotaxis to sugars transported by the phosphotransferase system. Mutations in the cyclic nucleotide phosphodiesterase gene, which increase the level of both cyclic AMP and cyclic GMP, also reduce chemotaxis to these sugars. Therefore, it appears that control of the level of a cyclic nucleotide is critical for the chemotactic response to phosphotransferase system sugars.     

The possible role of the closed-open transition in proton pumping by cytochrome c oxidase: the pH dependence of cyanide inhibition

The rate of oxidation of reduced cytochrome c catalyzed by cytochrome oxidase in the presence and absence of cyanide has been measured spectrophotometrically at pH 5.5, 6.4, 7.4 and 8.3. At the cytochrome c concentration used (272 microM), the uninhibited rate is maximal at pH 6.4 and drops to a value about one sixth of this maximum at pH 8.3. In the presence of cyanide, the rate initially drops rapidly, but with the cyanide concentration used (5.5 microM) there is still a measurable rate of oxidation when maximal inhibition has been reached. This inhibited rate decreases as the pH increases, whereas the apparent rate constant for cyanide binding is almost independent of pH. The results have been analyzed on the basis of a model in which two-electron reduction of the oxidized enzyme triggers a transition from a closed to an open conformation. It is assumed that cyanide can only bind to the open conformation and, furthermore, that rapid internal electron transfer to the dioxygen-reducing site occurs in this state alone. The analysis shows that the true constant for cyanide binding decreases with decreasing pH to a constant value at low pH. It also indicates that the increase in the catalytic constant with decreasing pH is associated with an increase in the rate of the closed-open conformational transition on protonation of the enzyme, and it is proposed that this transition is operative in electron gating in the proton-pump function of the enzyme.     

The β-glucoside system of Escherichia coli III. Properties of A P–HPr: β-Glucoside phosphotransferase extracted from membranes with detergent

A P-HPr:β-glucoside phosphotransferase (enzyme II^bgl)

1 The nomenclautre of the enzymes II is that suggested by Lin (1)

has been extracted from membranes of a β-glucoside fermenting strain of Escherichia coli K 12 using the nonionic detergent Triton X–100. The extracted enzyme was rendered virtually free of both lipid and detergent by chromatography on DEAE-cellulose. At this stage, the partially purified enzyme had negligible activity, but activity was restored effectively by the addition of (1) nonionic detergents of the Tween or Triton series and (2) crude E. coli phospholipids or an anionic lipid enriched fraction, but not phosphatidylethanolamine. Detergent activators were most effective at or near the critical micelle concentration, but were inhibitory when added at concentrations above the critical micelle concentration. In order to obtain maximal initial rates of phosphotransferase activity, it was necessary to incubate the extracted, partially purified enzyme with detergent activator and HPr prior to the addition of the other assay system components. High detergent concentration inhibited the initial rate of phosphorylation by interfering with an essential step (or steps) that occur during this preliminary incubation. The activation occuring during the preliminary incubation was also highly temperature dependent; a precipitous decrease in activation was detected below 16° when Tween 40 was employed as the detergent activator. Phosphorylation mediated by the membrane associated form of the phosphotransferase was not influenced by the physical state of the lipid components of the membrane. This is in marked contrast to the properties of the phosphorylation reaction mediated by the phosphotransferase in intact cells.     

The mechanism of the reaction catalysed by adenosine triphosphate-creatine phosphotransferase.

1. The forward and reverse reactions catalysed by ATP-creatine phosphotransferase have been studied kinetically at pH8.0 in the presence and absence of products, under conditions in which the free Mg(2+) concentration was maintained constant at 1mm. Thus at fixed pH the reaction may be considered as being bireactant and expressed as:MgATP(2-)+creatine(0)right harpoon over left harpoonMgADP(-)+phosphocreatine(2-)2. The initial-velocity pattern in the absence of products and the product-inhibition pattern have been determined. These are consistent with a random mechanism in which all steps are in rapid equilibrium except that concerned with the interconversion of the central ternary complexes, and in which two dead-end complexes (enzyme-MgADP-creatine and enzyme-MgATP-phosphocreatine) are formed. The results are in accord with previous suggestions that the enzyme possesses distinct sites for the combination of the nucleotide and guanidino substrates. 3. Values have been determined for the Michaelis and dissociation constants involved in the combination of each substrate with various enzyme forms. Although these values cannot be regarded as absolute, they appear to indicate that the presence of one substrate on the enzyme enhances the combination of the second substrate. In addition, it would seem that in the formation of the enzyme-MgADP-creatine complex the concentration of one reactant does not affect the combination of the other. This contrasts with the formation of the enzyme-MgATP-phosphocreatine complex, where each reactant hinders the combination of the other.     

Steady-state and pre-steady-state kinetics of propionaldehyde oxidation by sheep liver cytosolic aldehyde dehydrogenase at pH 5.2. Evidence that the release of NADH remains rate-limiting in the enzyme mechanism at acid pH values

The kcat value for the oxidation of propionaldehyde by sheep liver cytosolic aldehyde dehydrogenase increased 3-fold, from 0.16 s-1 at pH 7.6 to 0.49 s-1 at pH 5.2, in parallel with the increase in the rate of displacement of NADH from binary enzyme.NADH complexes. A burst in nucleotide fluorescence was observed at all pH values consistent with the rate of isomerization of binary enzyme.NADH complexes constituting the rate-limiting step in the steady state. No substrate activation by propionaldehyde was observed at pH 5.2, but the enzyme exhibited dissociation/association behavior. The inactive dissociated form of the enzyme was favored by low enzyme concentration, low pH, and low ionic strength. Propionaldehyde protected the enzyme against dissociation.     

Structure and function of D-amino acid oxidase. IX. Changes in the fluorescence polarization of FAD upon complex formation.

1. The fluorescence polarization, P, of FAD increased on complex formation with the apoenzyme of D-amino acid oxidase [D-amino acid: O2 ocidoreductase (deaminating), EC 1.4.3.3]. The time course of the increase was monophasic. The values of P were extimated to be 0.04, 0.4, and 0.4 for FAD, the enzyme and the enzyme-benzoate complex, respectively. 2. The value of P of the enzyme is dependent on its concentration, indicating that the degrees of dissociation of FAD in the monomer and dimer are different. The dissociation constant was calculated to be 7 times 10-minus 7 M for the monomeric form of the enzyme. This value is far larger than the value for the dimeric form of the enzyme, 1 times 10-minus 8 M, calculated from equilibrium dialysis data. 3. Changes in fluorescence polarization of the enzyme due to changes in solution pH or temperature can be explained in terms of the monomer-dimer equilibrium.     

Purification and properties of fructose 1,6-bisphosphatase from turkey liver.

Fructose 1,6-bisphosphatase (EC 3.1.3.11) has been purified 360-fold from turkey liver. The purified enzyme appears to be homogeneous by disc gel electrophoresis and has a pH profile indistinguishable from that of the enzyme in crude extracts. Mn²⁺ is significantly more effective than Mg²⁺ as the essential metal cofactor of this enzyme. The maximal effect of histidine is equivalent to that of EDTA except that EDTA is more efficient at lower concentrations. The histidine effect is decreased with an increase in pH or if substrate is first bound to the enzyme. The enzyme activity is activated equally by d- and l-forms of histidine. Enzyme affinity for the substrate decreases with an increase in pH. The inhibition by high substrate concentrations observed at pH 7.5 is markedly reduced in the absence of chelating activator or when Mg² is replaced by Mn²⁺ as the metal cofactor. Turkeys liver fructose 1,6-bisphosphatase resembles the enzyme from mammalian sources in that the sensitivity to AMP inhibition is decreased with the increase in pH, temperature, and Mg² concentration.