Degradation of phosphoenolpyruvate carboxykinase (guanosine triphosphate) in vivo and in vitro

1. Phosphoenolpyruvate carboxykinase (GTP) in the cytosol fraction of liver was labelled in young rats by the injection of [(3)H]leucine and then isolated with specific antibody. Antibody-antigen precipitates from ;pulse'-labelled animals and from animals in which the content of radioactive enzyme had been decreased by a period of degradation were separated by electrophoresis on sodium dodecyl sulphate-polyacrylamide gels. No radioactive breakdown products were found. 2. (3)H-labelled phosphoenolpyruvate carboxykinase (GTP) was purified from rat liver and used to measure degradation in vitro. There was first a loss of catalytic activity, then a disappearance of immunological activity and finally a loss of solubility before any evidence of proteolytic cleavage. Proteolytic-cleavage fragments, when found, were also insoluble. 3. An analysis of the subcellular location of enzyme inactivation showed that phosphoenolpyruvate carboxykinase (GTP) was stable when incubated with liver cytosol fraction and was inactivated most rapidly by the microsomal fraction. 4. We propose that denaturation of the enzyme is the rate-limiting step in degradation in vivo, and precedes proteolytic cleavage when the enzyme is incubated with liver preparations in vitro.     

Inactivation of phosphoenolpyruvate carboxykinase by the guanosine nucleotide analogue, 5'-p-fluorosulfonylbenzoyl guanosine

Rat liver cytosolic phosphoenolpyruvate carboxykinase is inactivated by incubation with 0.84 mM 5′-p-fluorosulfonylbenzoyl guanosine, but is not appreciably affected by the adenosine analogue, 5′-p-fluorosulfonylbenzoyl adenosine, in correspondance with the known nucleotide specificity of this enzyme. Marked protection against inactivation by 5′-p-fluorosulfonylbenzoyl guanosine is provided (either in the presence or absence of divalent metal cation) by GTP or GDP but not by ATP or phosphoenolpyruvate. The inactivation appears to be due to covalent reaction since radioactive reagent remains associated with the enzyme after extensive dialysis and gel filtration on Sephadex G-25. These results are consistent with affinity labeling of the nucleotide binding site of phosphoenolpyruvate carboxykinase by the guanosine nucleotide analogue 5′-p-fluorosulfonylbenzoyl guanosine.     

Purification and molecular characteristics of mitochondrial phosphoenolpyruvate carboxykinase from bullfrog (Rana catesbeiana) liver

Phosphoenolpyruvate carboxykinase from bullfrog liver mitochondria has been purified to electrophoretical and immunological homogeneity by an improved method using hydrophobic chromatography on Sepharose-hexane-GMP and affinity chromatography on phosphocellulose. The molecular weight was determined to be 70,000 by SDS-gel electrophoresis, 65,000 by Sephadex G-100 gel filtration and 72,000 by glycerol gradient centrifugation. The isoelectric point was determined to be 6.2, differing from that of the cytosol enzyme. The rabbit IgG fraction against the mitochondrial PEP carboxykinase precipitated not only the mitochondrial but also the cytosol enzyme. The dissociation constant of the nucleotide-enzyme complex was determined to be 3 microM for GTP, 8.5 microM for GDP, and 171 microM for GMP. The affinity of GTP for the enzyme was reduced in the presence of phosphoenolpyruvate or Mn2+, whereas that of GDP was not changed. GMP inhibited the enzyme competitively with GDP for the phosphoenolpyruvate carboxylation and competitively with GTP for the exchange reaction between [14C]HCO3- and oxaloacetate. The purified enzyme was found to have a cysteine residue which reacted with iodoacetamide to form inactive enzyme. Guanine nucleotides or IDP and Mn2+ at a lower concentration prevented the inactivation by iodoacetamide of the enzyme in a competitive manner. Binding of guanine nucleotide to the enzyme and the relation of the sulfhydryl group to the nucleotide binding are discussed.     

Regulation of phosphoenolpyruvate carboxykinase (GTP) in adipose tissue in vivo by glucocorticoids and insulin.

1. The regulation of the synthesis of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) in epididymal adipose tissue, liver and kidney in vivo was studied immunochemically. 2. Phosphoenolpyruvate carboxykinase (GTP) synthesis in adipose tissue is increased by starvation, diabetes and noradrenaline, and decreased by re-feeding and insulin. These changes were also seen in adrenalectomized rats and are qualitatively similar to those observed for the liver enzyme. This indicates the involvement of cyclic AMP as an inducer and insulin as a de-inducer in the regulation of phosphoenolpyruvate carboxykinase (GTP) in both tissues. (Induction and de-induction are defined as selective increase and decrease respectively in the rate of enzyme synthesis, regardless of the mechanism involved.)3. Adrenalectomy had little effect on phosphoenolpyruvate carboxykinase (GTP) synthesis in liver and kidney, but increased the synthesis rate of the adipose-tissue enzyme. Starvation and adrenalectomy had additive effects in increasing the synthesis rate of adipose-tissue phosphoenolpyruvate carboxykinase (GTP). In adrenalectomized diabetic rats glucocorticoids increased phosphoenolpyruvate carboxykinase (GTP) synthesis in liver and kidney while decreasing enzyme synthesis in adipose tissue. De-induction of adipose tissue phosphoenolpyruvate carboxykinase (GTP) is therefore regulated independently by glucocorticoids and insulin. 4. Although liver, kidney and adipose-tissue phosphoenolpyruvate carboxykinases (GTP) are seemingly identical, there is an apparent tissue-specific differentiation in regulatory systems for the enzyme.     

Activation of liver cytosol phosphoenolpyruvate carboxykinase by Ca2+ through intracellular redistribution of Mn2+

Calcium has no known direct effect on phosphoenolpyruvate carboxykinase from rat liver cytosol. However, addition of calcium salts to liver postnuclear supernatant led to an increase in assayable enzyme activity in cytosols. This indicates that mitochondria and microsomes present in postnuclear supernatant can participate in observed enzyme activation. The stimulation of phosphoenolpyruvate carboxykinase was prevented by the manganese complexion 1-(2-pyridylazo)-2-naphthol, was not additive with activation by MnCl2 and was inhibited by La3+, Sr2+ and ruthenium red. These data indicate that manganese and mitochondrial or microsomal calcium carriers participate in the mechanism of indirect calcium effect. Measuring of manganese content in cytosols directly, by atomic absorption spectrometry, has provided evidence that there is a pool of manganese associated with mitochondrial and microsomal fraction of rat liver that can be mobilized to the cytosol by calcium ions. The direct addition of this pool of manganese to the cytosol caused the stimulation of phosphoenolpyruvate carboxykinase activity to the same levels as did calcium ions in the postnuclear supernatant. It is postulated that calcium can effect enzyme activity indirectly by releasing manganese from specific cellular compartments into the cytosol.     

An active-site lysine in avian liver phosphoenolpyruvate carboxykinase

The participation of lysine in the catalysis by avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification and by a characterization of the modified enzyme. The rate of inactivation by 2,4-pentanedione is pseudo-first-order and linearly dependent on reagent concentration with a second-order rate constant of 0.36 +/- 0.025 M-1 min-1. Inactivation by pyridoxal 5'-phosphate of the reversible reaction catalyzed by phosphoenolpyruvate carboxykinase follows bimolecular kinetics with a second-order rate constant of 7700 +/- 860 M-1 min-1. A second-order rate constant of inactivation for the irreversible reaction catalyzed by the enzyme is 1434 +/- 110 M-1 min-1. Treatment of the enzyme with pyridoxal 5'-phosphate gives incorporation of 1 mol of pyridoxal 5'-phosphate per mole of enzyme or one lysine residue modified concomitant with 100% loss in activity. A stoichiometry of 1:1 is observed when either the reversible or the irreversible reactions catalyzed by the enzyme are monitored. A study of kobs vs pH suggests this active-site lysine has a pKa of 8.1 and a pH-independent rate constant of inactivation of 47,700 M-1 min-1. The phosphate-containing substrates IDP, ITP, and phosphoenolpyruvate offer almost complete protection against inactivation by pyridoxal 5'-phosphate. Modified, inactive enzyme exhibits little change in Mn2+ binding as shown by EPR. Proton relaxation rate measurements suggest that pyridoxal 5'-phosphate modification alters binding of the phosphate-containing substrates. 31P NMR relaxation rate measurements show altered binding of the substrates in the ternary enzyme.Mn2+.substrate complex. Circular dichroism studies show little change in secondary structure of pyridoxal 5'-phosphate modified phosphoenolpyruvate carboxykinase. These results indicate that avian liver phosphoenolpyruvate carboxykinase has one reactive lysine at the active site and it is involved in the binding and activation of the phosphate-containing substrates.     

Purification and properties of mitochondrial phosphoenolpyruvate carboxykinase from liver of Squalus acanthias

Liver from Squalus acanthias (spiny dogfish), a representative elasmobranch, contains approximately 1.4 units (mumol/min) of phosphoenolpyruvate carboxykinase activity per gram and approximately 90% of the total units of activity are localized in the mitochondria. The mitochondrial phosphoenolpyruvate carboxykinase was isolated and characterized. The purified enzyme has properties generally similar to those found in mammalian and avian species. The enzyme has a molecular weight of approximately 70,000 and exists in a functional state as a monomer. The isolated enzyme displays a dual cation requirement (e.g., 6 mM Mg2+ and 10 microM Mn2+) for maximal activity; very little activity is observed when Mg2+ is present alone, and the maximal activity attained with Mn2+ alone (millimolar concentrations required) is significantly less than that observed under optimal conditions with both cations present. When assayed in the direction of oxalacetate formation there is a lag in product formation with time; the lag can be eliminated by the presence of 50 microM GTP (product). The Km for substrates is not affected by Mn2+ concentration, suggesting that the role of Mn2+ may not be related to substrate binding. The apparent Km for phosphoenolpyruvate (approximately 1 mM) is substantially higher than that reported for phosphoenolpyruvate carboxykinase from other species. The activity of phosphoenolpyruvate carboxykinase is increased 70% by physiological concentrations of urea. Maximal velocity of the reaction in the direction of oxalacetate formation is approximately half that of the reverse reaction.     

Glucocorticoids and the regulation of phosphoenolpyruvate carboxykinase (guanosine triphosphate) in the rat.

The effect glucocorticoids on the synthesis and degradation of phosphoenolpyruvate carboxykinase (GTP)(EC4.1.1.32) in rat liver and kidney in vivo was studied immunochemically. The glucocorticoid analogue triamcinolone (9alpha-fluoro-11beta, 21-dihydroxy-16alpha,17alpha-isopropylidenedioxypregna-1,4-diene-3,20-dione) increased the synthesis rate of the kidney enzyme in starved animals. Both triamcinolone and cortisol decreased the synthesis rate of hepatic phosphoenolpyruvate carboxykinase (GTP) in fed and starved rats, but were without effect on the degradation rate of the enzyme. This effect of triamcinolone in liver was reversed by injection of dibutyryl cyclic AMP. However, in diabetic animals glucocorticoids increased the synthesis rate of hepatic phosphoenolpyruvate carboxykinase (GTP). Triamcinolone administration to starved rats in vivo is shown to cause an increase in the portal blood concentrations of insulin and glucose. Since the physiological de-inducer of liver phosphoenolpyruvate carboxykinase (GTP) is insulin, this is the probable cause of the decrease in the synthesis rate of the hepatic enzyme noted when glucocorticoids are administered to non-diabetic animals.     

Effects of Mn2+ on the exchange reaction of phosphoenolpyruvate carboxykinase in the presence of high concentrations of Mg2+

The mitochondrial phosphoenolpyruvate carboxykinase (GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32), purified from chick embryo liver, was synergistically activated by a combination of Mn2+ and Mg2+ in the oxaloacetate ---- H14CO-3 exchange reaction. Increases in the Mg2+ concentration caused decreases in the K0.5 value of Mn2+ in line with the earlier finding that the enzyme was markedly activated by low Mn2+ (microM) plus high Mg2+ (mM). In the presence of 2.5 mM Mg2+, increases in the Mn2+ level first enhanced the activity of phosphoenolpyruvate carboxykinase, and then suppressed it to the maximal velocity shown in the presence of Mn2+ alone. Kinetic studies showed that high Mn2+ inhibited the activity of Mg2+ noncompetitively, and those of GTP and oxaloacetate uncompetitively. The inhibition constant for oxaloacetate (K'i = 550 microM) was lower than that of Mg2+ (Ki = K'i = 860 microM) or GTP (K'i = 1.6 mM), and was nearly equal to the apparent half-maximal inhibition concentration of Mn2+. These results suggested that Mn2+ can play two roles, of activating and suppressing phosphoenolpyruvate carboxykinase activity in the presence of high Mg2+.     

Affinity labeling of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase with the 2',3'-dialdehyde derivative of ATP

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] is completely inactivated by the 2',3'-dialdehyde derivative of ATP (oATP) in the presence of Mn2+. The dependence of the pseudo-first-order rate constant on reagent concentration indicates the formation of a reversible complex with the enzyme (Kd = 60 +/- 17 microM) prior to covalent modification. The maximum inactivation rate constant at pH 7.5 and 30 degrees C is 0.200 +/- 0.045 min-1. ATP or ADP plus phosphoenolpyruvate effectively protect the enzyme against inactivation. oATP is a competitive inhibitor toward ADP, suggesting that oATP interacts with the enzyme at the substrate binding site. The partially inactivated enzyme shows an unaltered Km but a decreased V as compared with native phosphoenolpyruvate carboxykinase. Analysis of the inactivation rate at different H+ concentrations allowed estimation of a pKa of 8.1 for the reactive amino acid residue in the enzyme. Complete inactivation of the carboxykinase can be correlated with the incorporation of about one mole of [8-14C]oATP per mole of enzyme subunit. The results indicate that oATP can be used as an affinity label for yeast phosphoenolpyruvate carboxykinase.     

Purification and characterization of cytosol-specific phosphoenolpyruvate carboxykinase from chicken liver

Phosphoenolpyruvate carboxykinase of chicken liver cytosol was purified to homogeneity by procedures including affinity chromatography with GTP as a ligand. The purified enzyme showed a molecular weight of 68,000 on gel electrophoresis in the presence of dodecyl sulfate. Comparative studies on this enzyme and its isozyme purified from chicken liver mitochondria were performed. As regards amino acid composition, the cytosolic enzyme was quite different from the mitochondrial enzyme, but was rather similar to rat liver cytosolic phosphoenolpyruvate carboxykinase. Specific activities of the cytosolic enzyme were 30-100% higher than those of the mitochondrial enzyme for oxaloacetate-CO2 exchange, oxaloacetate decarboxylation, and phosphoenolpyruvate carboxylation reactions, though the relative rates of the activities were similar, decreasing in the order given. Apparent Michaelis constants for oxaloacetate in the oxaloacetate decarboxylation reaction were 11.6 and 17.9 microM for the cytosolic and the mitochondrial enzyme, respectively, but the values for GTP, GDP, phosphoenolpyruvate, and CO2 in the oxaloacetate decarboxylation and phosphoenolpyruvate carboxylation reactions were 1.3-2.2 times higher for the cytosolic enzyme than for the mitochondrial enzyme. Thus, the fundamental catalytic properties of the chicken liver phosphoenolpyruvate carboxykinase isozymes were rather similar, despite the marked difference in amino acid compositions.     

The relative stability of liver cytosol enzymes incubated in vitro

1. Relative rates of enzyme inactivation were measured in liver slices, homogenates and cytosol fractions as well as in the presence of trypsin and at acid pH. The enzymes chosen are all present in the cytosol fraction of rat liver, and have widely different degradation rate constants in vivo. 2. The inactivation rates of lactate dehydrogenase, fructose bisphosphate aldolase, glucose 6-phosphate dehydrogenase, glucokinase, phosphoenolpyruvate carboxykinase (GTP), l-serine dehydratase and thymidine kinase in liver preparations at neutral pH are in a similar order to the rate constants of degradation of these enzymes in the intact animal. 3. The two exceptions of this general correlation were tyrosine aminotransferase, which was stable in vitro but not in vivo, and glyceraldehyde phosphate dehydrogenase, which shows the reverse pattern. 4. These findings generally support the concept that the same factors are responsible for enzyme inactivation in vitro as occur in the intact tissue.     

Induction by cAMP of the mRNA encoding the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the chicken. Identification and characterization of a cDNA clone for the enzyme

Previous work from our laboratory (Hod, Y., Utter, M. F., and Hanson, R. W. (1982) J. Biol. Chem. 257, 13787-13794) has demonstrated that chicken kidney contains both mitochondrial and cytosolic forms of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) and that the two forms are distinct proteins. Using poly(A+) RNA from chicken kidney, a double-stranded cDNA library was constructed. DNA clones containing sequences complementary to the mRNA for the cytosolic form of phosphoenolpyruvate carboxykinase were initially identified by colony hybridization with 32P-labeled cDNA transcribed from an RNA fraction enriched for the enzyme mRNA. The identity of plasmids containing phosphoenolpyruvate carboxykinase cDNA was confirmed by hybrid-selected translation. Mature mRNA for cytosolic phosphoenolpyruvate carboxykinase of the chicken is 2.8 kilobases in length, similar to that previously noted for mRNA coding for the same enzyme in the rat. The cDNA for the chicken enzyme hybridizes with several restriction fragments of the corresponding cDNA for the rat cytosolic phosphoenolpyruvate carboxykinase, indicating conservation of nucleotide sequences during evolution. Wide spread conservation of sequence homology is also demonstrated by the hybridization of the cDNA for the rat phosphoenolpyruvate carboxykinase with a 2.8-kilobase RNA from the livers of a variety of vertebrates including amphibian, avian, and primate species. Specific mRNA coding for the cytosolic form of phosphoenolpyruvate carboxykinase was present in chicken kidney but absent from the liver, even in animals starved for 48 h. However, the administration of cAMP to normal fed chickens caused a rapid induction of phosphoenolpyruvate carboxykinase mRNA. These findings suggest that the gene for the cytosolic enzyme in chicken liver can be expressed if the proper hormonal stimuli are present.     

Formation of an intramolecular cystine disulfide during the reaction of 8-azidoguanosine 5'-triphosphate with cytosolic phosphoenolpyruvate carboxykinase (GTP) causes inactivation without photolabeling

Phosphoenolpyruvate carboxykinase (GTP) (PEPCK) specifically utilizes a guanosine or inosine nucleotide as a substrate, yet it does not share extended sequence homology with other GTP-binding proteins, and the molecular basis for its nucleotide specificity is not understood. In an effort to locate the enzyme's nucleotide-binding site, we have studied the interaction of cytosolic PEPCK from rat liver with the photoprobe 8-azidoGTP, which fulfills the criteria of a specific photoaffinity label for PEPCK. The photoprobe binds reversibly to the enzyme prior to modification and at low concentrations causes greater than 60% inactivation (Ki = 1.2 microM). GTP provides nearly complete protection against inactivation by 8-azidoGTP, whereas phosphoenolpyruvate and metal ions provide partial protection. In addition, the photoprobe is a substrate for the enzyme and has a Km similar to that for GTP. However, the extent of covalent modification by [32P]8-azidoGTP as measured by three independent techniques is significantly lower than the extent of enzyme inactivation. Further investigation of this anomaly has revealed that the loss in enzymatic activity is caused by modification of a critical cysteine residue in a reaction that does not terminate with covalent attachment of the photolabel. Quantitation of the total free thiols of modified PEPCK shows that 2 mol of cysteine is lost per mole of inactivated enzyme. These results indicate that the photoinactivation of PEPCK by 8-azidoGTP is caused by the formation of an intramolecular cystine disulfide bridge, thus providing evidence for the existence of a pair of proximal cysteine residues within the GTP-binding site. The interaction of cysteine residues with the reactive photogenerated derivatives of 8-azidopurines is discussed.     

Purification of phosphoenolpyruvate carboxykinase (GTP) by affinity chromatography on agarose-hydrazide-GTP

The cytosolic form of phosphoenolpyruvate carboxykinase (GTP; EC 4.1.1.32) from rat liver was purified by a procedure involving affinity chromatography on agarose-hydrazide-GTP. Phosphoenolpyruvate carboxykinase is retained quantitatively by the affinity medium in the presence of manganese and can be specifically eluted by a pulse of GTP. On the contrary, no binding to agarose-hydrazide-GTP occurs in the absence of manganese. This suggests that the affinity of the enzyme for GTP is enhanced by prior interaction with manganese. A combination of several conventional purification steps followed by affinity chromatography provides pure phosphoenolpyruvate carboxykinase in good yields. The final specific activity is 19 U/mg protein. The enzyme migrates as a single polypeptide of molecular weight 70,600 during electrophoresis on sodium dodecyl sulfate polyacrylamide gels.     

Fluorescent labeling of the nucleotide site in cytosolic rat liver phosphoenolpyruvate carboxykinase

Reaction of rat liver phosphoenolpyruvate carboxykinase (GTP: oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32) with the alkylating fluorescent probe N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (1,5-I-AEDANS), results in complete loss of enzymatic activity. One mole of the fluorescent reagent is incorporated per mole of the inactivated enzyme. When the modification is carried out in the presence of GDPMn, the enzyme retains 97% of its activity with almost no incorporation of label. The specificity of the reaction is further supported by the detection of a unique fluorescent peptide from the trypsin-treated modified enzyme. Fluorescence emission of enzyme-bound AEDANS shows a broad band centered at 470 nm and presents a monoexponential decay with a lifetime of 19 ns. These data indicate that the probe-binding site is considerably less polar than water and similar in polarity to ethanol. Anisotropy determinations give evidence for restricted rotational freedom for AEDANS bound to the rat carboxykinase, while acrylamide quenching studies reveal limited accessibility to the probe site. The results are consistent with specific labeling of rat liver phosphoenolpyruvate carboxykinase at or near the GDP site. The characteristics of the nucleotide-binding sites of rat liver and yeast (ATP) phosphoenolpyruvate carboxykinase are compared.     

Phosphoenolpyruvate carboxykinase of kidney. Subcellular distribution and response to acid–base changes

1. A method for the assay of phosphoenolpyruvate carboxykinase is presented, based on the enzymic determination of the phosphoenolpyruvate produced by the enzyme reaction. 2. The subcellular distribution of phosphoenolpyruvate carboxykinase in the kidney of several animal species resembled the distribution in the liver. 3. The rise in enzyme activity in the kidney cortex of rats made acidotic by feeding with ammonium chloride was not prevented by administration of ethionine or actinomycin. 4. The possibility is suggested that in the kidney acidosis causes activation of an inactive form of the enzyme already present.     

Comparison of the rat microsomal Mg-ATPase of various tissues

The microsomal Mg-ATPase from various rat tissues was compared. After fractionating the microsomal vesicles by sucrose gradient centrifugation, the highest specific activity of the Mg-ATPase was found in the low-density vesicles which contained plasma membrane. A large fraction (25-90%) of the microsomal Ca-independent Mg-ATPase found in each tissue had the following properties: (1) the Km for ATP was 0.2 mM; (2) the rate of ATP hydrolysis by the Mg-ATPase was nonlinear due to an ATP-stimulated inactivation of the enzyme; (3) wheat germ agglutinin, concanavalin A, glutaraldehyde, and antiserum prevented inactivation induced by ATP or AdoPP[NH]P; (4) detergents at relatively low detergent:protein ratios increased the rate of inactivation with little change in the initial rate of ATP hydrolysis; (5) the Mg-ATPase was inactivated by irradiation in the presence of 8-azido ATP. (6) in addition to ATP, the Mg-ATPase was able to hydrolyze CTP, GTP, UTP, ITP, and GTP but was unable to hydrolyze any of the 10 nonnucleotide phosphocompounds which were tested; (7) the bivalent cation requirement of the Mg-ATPase could be provided by Mg2+, Ca2+, Mn2+, Zn2+, or Co2+ but the enzyme was inactive in the presence of Cu2+, Sr2+, Ba2+, or Be2+; (8) the Mg-ATPase activity was not altered by ionophores or inhibitors of the Na,K-ATPase, the Ca,Mg-ATPase or the mitochondrial F1ATPase. These data suggest that a major portion of the microsomal, basal Mg-ATPase activity is due to one unique enzyme found in most if not all tissues.