Adenosine triphosphate hydrolysis in rat dental tissues

Summary The effect of EDTA-decalcification, reactivating and activating procedures on the hydrolysis of ATP was studied histochemically in developing dental tissues in the rat. The incubation media contained lead citrate at alkaline pH and lead nitrate at neutral pH, and the results with ATP as substrate were compared with those obtained with -glycerophosphate.The ion dependency of ATP hydrolysis could only be ascertained in decalcified sections. As in earlier studies on the hydrolysis of -glycerophosphate in dental tissues, this hydrolysis could readily be reactivated through preincubation of the sections in a series of 0.1 M solutions of divalent cations; Zn²⁺ being the most efficient. This treatment was now found also to give rise to an ATP hydrolysis, which occurred without the need for activating ions in the incubation medium. This ATP hydrolysis should thus be described as nonspecific and, in terms of ion dependency, as due to a metalloenzyme, i.e. alkaline phosphatase. Activating ion dependent ATP hydrolysis in the dental tissues was found in the blood vessels and in the apical part of the secretory ameloblasts. The former was activated by Mg²⁺, Ca²⁺ and Mn²⁺, and the latter by Ca²⁺ and -almost specifically—by Sr²⁺. Preincubation with Zn²⁺ always inhibited the ion dependant ATP hydrolysis in the dental tissues.     

Adenosine triphosphate hydrolysis in rat dental tissues. A histochemical study to differentiate the enzymes involved.

The purpose of this study was to try to differentiate histochemically between the various enzymes which may catalyze the hydrolysis of ATP in developing rat dental tissues. Freeze cut and freeze dried sections of molar and incisor teeth were incubated in lead capture-based media at pH 5.0, 7.2 or 9.4 with one of the following substrates: beta-glycerophosphate, AMP, ADP, ATP, AMP-PNP and tetrasodium pyrophosphate. To establish the enzymatic nature of the hydrolysis parallel sections were incubated after prior fixation in either formaldehyde or glutaraldehyde. By comparing the enzymatic stainings obtained with the various substrates and at the different pH:s, it was concluded that ATP can be visibly hydrolyzed in rat dental tissues by alkaline phosphatase (stratum intermedium, apical part of maturation ameloblasts, basal part of all ameloblasts, odontoblasts and subodontoblastic layer), specific ATPase (apical and basal parts of secretory ameloblasts) and ATP pyrophosphatase and/or adenylate cyclase (stratum intermedium, odontoblasts). Acid phosphatase, specific ADPase, 5'-nucleotidase, inorganic pyrophosphatase, 3':5'-cyclic-AMP-phosphodiesterase and adenylate kinase on the other hand, seem not to be engaged in the ATP hydrolysis to such a degree as to complicate the interpretation of the histochemical staining. The alkaline phosphatase part of the ATP hydrolysis appeared to be rather insensitive to aldehyde fixation, while the hydrolysis effected by specific ATPase and ATP pyrophosphatase and/or adenylate cyclase was extinguished after fixation with formaldehyde for 4 h or glutaraldehyde for 10 min.     

Integumental phosphatase isoenzymes from white puparia of Ceratitis capitata: isolation and characterization.

Two specific alkaline phosphatase forms were identified in the integument of wild-type Ceratitis capitata during transition of larvae to pupae. The separation was achieved by DEAE-cellulose chromatography; alkaline phosphatase 1 and alkaline phosphatase 2 were eluted in 0.1 and 0.4 M KCl, respectively. Both isoenzymes have a molecular weight of approximately 180,000. The pH curve reveals two peaks for both alkaline phosphatases: one at 9.4 and the other at 11.0. The two isoenzymes at both pH optima catalyze the hydrolysis of phosphotyrosine and beta-glycerophosphate, but not phosphoserine, phosphothreonine, ATP, or AMP. However, at pH 9.4, alkaline phosphatase 1 is more effective than ALPase 2 and exhibits a preference for phosphotyrosine. The divalent cations Mn2+, Mg2+, and Ba2+ activate the enzymes, while Cu2+ and Zn2+ are inhibitors for both isoenzymes. Both isoenzymes are inactivated by EDTA. The effect of amino acids on enzyme activity was also tested. Alkaline phosphatase 1 is inhibited by L-tyrosine, while alkaline phosphatase 2 is unaffected. L-Phenylalanine has no effect on either isoenzyme. Both isoenzymes are inhibited by urea and 2-mercaptoethanol. Simultaneous addition of urea and 2-mercaptoethanol reveals that ALPase 1 is more sensitive to these inhibitors than ALPase 2.     

Purification and some properties of a neutral muscle pyrophosphatase.

In the water-soluble fraction of rabbit skeletal muscle, at least two types of inorganic pyro phosphatase (PPase) are distinguishable on ion exchange column chromatography. One of them, pyrophosphatase-A (PPase-A), was isolated in an electrophoretically homogeneous form. This enzyme catalyzed the hydrolysis of PPi but not that of other phosphate esters. Only Mg2+ was required for activity and stability. Other cations such as Ca2+, Co2+, Mn2+, and Zn2+ had no activating effect. The activity of this PPase was optimum at pH 7.4. ATP, ADP, sodium imidodiphosphate (PNP), p-chloromercuribenzoate, and Ca2+ inhibited its enzymic activity. The enzyme was protected by dithiothreitol (DTT) against heat denaturation. The molecular weight was estimated to be 67,000 by gel filtration and the molecular size of the subunit was found to be 35,000 by gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). The enzyme probably consists of two identical subunits of 35,000 daltons.     

ATP hydrolysis and synthesis by the membrane-bound ATP synthetase complex of Methanobacterium thermoautotrophicum.

The membrane-bound ATP synthetase complex of Methanobacterium thermoautotrophicum showed maximum activity for ATP hydrolysis at pH 8, at temperatures between 65 and 70 degrees C, and at an ATP-Mg2+ ratio of 0.5. Anaerobic conditions were not prerequisite for enzyme activity. The enzyme showed a Km value for ATP of 2 mM, and activity was Mg2+ dependent; Mn2+, Co2+, Ca2+, and Zn2+ could replace Mg2+ to some extent. Other nucleoside triphosphates could be hydrolyzed. N,N'-dicyclohexylcarbodiimide inhibited ATP hydrolysis. A proton-motive force, artificially imposed by a pH shift or valinomycin, resulted in ATP synthesis in whole cells. The ATP synthetase complex of the thermophilic methanogenic bacterium is similar to those described in aerobic and anaerobic microorganisms.     

The binding of a second divalent metal ion is necessary for the activation of ATP hydrolysis and its inhibition by tightly bound ADP in the ATPase from Halobacterium saccharovorum.

A binding site for divalent metal ions on the ATPase from Halobacterium saccharovorum is proposed. This site is different from the catalytic site which binds ATP and a complexed divalent metal ion. Occupation of the second site greatly stimulates the rate of ATP hydrolysis and the affinity of the catalytic site for the metal ion-ATP complex. The time-dependent inhibition of the ATPase, which occurs during catalysis and which is known to be caused by the retention of ADP, is also dependent on the occupation of this metal ion binding site. The binding of the metal ion apparently induces extremely tight binding of ADP after the departure of Pi. Mg2+, Mn2+, Zn2+, Co2+, and Ca2+ were tested and showed both the activating and the inhibitory effects, although their binding constants for ATP and the second metal ion binding site were quite different. The characteristic shapes of the nonlinear ATP hydrolysis curves obtained with different metal ions, and different ratios of metal ion and ATP, could be explained with the established dissociation constants. On this basis, a model for the ATPase was developed with two catalytic cycles: one in which the second metal ion binding site is occupied, and another in which it is empty. These pathways are connected by metal ion-dependent equilibria.     

Kinetics of Ca 2+ or Mg 2+ activated ATPase from lymphocyte plasma membranes]

The kinetic study of the C2+ ATPase activity of lymphocyte plasma memebranes allowed some properties of this enzyme to be evidenced. The Ca2+-activated hydrolysis of ATP is independent of a non-specific alkaline phosphatase. The substrate of the ATPase activity is the chelate Ca2+- ATP. Mg2+ may substitute for Ca2+ both as chelating ion and as activating ion. Several results suggest that we have only one ATPase, activated either by Ca2+-, or by Mg2+ with less efficiency; both chelates hve the same Km; pH values for maximum activity and transition temperatures are identical; the effects of free ions are also the same, activation at low concentration and inhibition at high concentration.     

Calixarene-dependent hydrolysis of ATP. II. The catalytic properties of reaction stimulated by calixarene C-107

In this paper we have investigated nonenzimatic hydrolysis of ATP stimulated by calixarene C-107. It has been shown the dependences of the kinetic characteristics from reagent concentration: the maximal value released Pi did not depend on ATP concentration and linearly increased with the growth of calixarene concentration. Besides the growth concentration of ATP or calixarene increased the maximum instantaneous velocity of the reaction and decreased characteristic time. It was identified that univalent cation of Na+, K+, Li+, choline+ and bivalent cation of Ca2+ or Mg2+ did not influence the reaction of ATP hydrolysis, in the presence of other bivalent cation the inhibition of the reaction occurred in line with the sequence: Cu2+ > Ba2+ > Pb2+ > Sr2+ > Ni2+ = Zn2+ > Mn2+ > > Co2+. The alkalization in the range of pH 6.0-8.0 stimulated the ATP hydrolysis. The magnitude of activation energy of the reaction was 50.7 +/- 8.9 kilojoules per mole. The specificity for nucleoside tri- and di-phosphates was not observed. Obtained data can be useful for designing the synthetic ATP-hydrolyzing catalysts and also for subsequent investigation of kinetics, energetics and mechanism of both enzymatic and nonenzymatic ATP hydrolysis reaction.     

Characterization of the pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate hydrolase activity in rat liver. Identity with alkaline phosphatase.

The tissue content of pyridoxal 5'-phosphate is controlled principally by the protein binding of this coenzyme and its hydrolysis by a cellular phosphatase. The present study identifies this enzyme and its intracellular location in rat liver. Pyridoxal-P is not hydrolyzed by the acid phosphatase of intact lysosomes. At pH 7.4 and 9.0, the subcellular distribution of pyridoxal-P phosphatase activity is similar to the for p-nitrophenyl-P, and the major portion of both activities is found in the plasma membrane fraction. The ratio of specific activities for pyridoxal-P and p-nitrophenyl-P hydrolysis remains relatively constant during the isolation of plasma membranes. These activities also behave concordantly with respect to pH rate profile, pH-Km profile, and response to chelating agents, Zn2+, Mg2+, and inhibitors. Kinetic studies indicate that pyridoxal-P binds to same enzyme sites as beta-glycerophosphate and phosphorylcholine. The data strongly favor alkaline phosphatase as the enzyme which functions in the control of pyridoxal-P and pyridoxamine-P metabolism in rat liver. Alkaline phosphatase was solubilized from isolated plasma membranes. The kinetic properties of the enzyme are not markedly altered by its dissociation from the membrane matrix. However, there are significant differences in its behavior toward Mg2+ which suggest a structural role for Mg2+ in liver alkaline phosphatase.     

Cytochemical localization and quantification of plasma membrane Ca2+-ATPase activity in mollusc digestive gland cells

A cytochemical method allowing the localization and quantification of plasma membrane Ca2+-ATPase (PMCA) in frozen sections obtained from digestive gland cells of Mytilus galloprovincialis, Tapes tapes and Chamelea gallina, is presented. The method utilizes lead as a trapping agent of PO4(2-) ions released by Ca2+-ATPase activity. The amount of lead sulphide precipitate proportionally related to PMCA activity was quantified by a light microscopy digital imaging analysis system. The optimal assay conditions of Ca2+-ATPase activity evaluated at pH 7.4 were: 200 microM free Ca2+, 200 mM KCl, 2 mM ATP, and under such analysis conditions the enzyme showed a linear trend up to 60 min (at 20 degrees C). The PMCA activity was substrate specific: ADP was utilized only at a low rate (24% with respect to an equimolar ATP concentration), while glucose-6-phosphate and beta-glycerophosphate were poorly hydrolyzed. The enzyme activity was strongly inhibited by sodium ortho-vanadate. Our detection of a Ca2-ATPase activity at nanomolar concentrations of free Ca2+ suggests that we have identified a plasma membrane Ca2-ATPase involved in Ca2+ homeostasis. The Ca2+-ATPase was found to be localized in the basal part of the plasma membrane in the digestive gland cells of Mytilus galloprovincialis and Tapes tapes, but in the apical plasma membrane of Chamelea gallina. The possible implications of the different cellular distributions of PMCA activity is discussed.     

Fe2+ and other divalent metal ions uncouple Ca2+ transport from (Ca2+-Mg2+)-ATPase in rat liver plasma membranes

The addition of nanomolar concentrations of free Fe2+, Mn2+, or Co2+ to rat liver plasma membranes resulted in an activation of ATP hydrolysis by these membranes which was not additive with the Ca2+-stimulated ATPase activity coupled to the Ca2+ pump. Detailed analysis showed that, if fact, (i) as for the stimulation of (Ca2+-Mg2+)-ATPase by Ca2+, activation of ATP hydrolysis by Fe2+, Mn3+, or Co2+ followed a cooperative mechanism involving two ions; (ii) two interacting sites for ATP were involved in the activation of both Fe2+- and Ca2+-stimulated ATPase activities; (iii) micromolar concentrations of magnesium caused the same dramatic inhibition of both activities; and (iv) the subcellular distribution of Fe2+-activated ATP hydrolysis activity corresponded to that of plasma membrane markers. This suggests that the (Ca2+-Mg2+)-ATPase might be stimulated not only by Ca2+, but also by Fe2+, Mn2+, or Co2+. However, interaction of (Ca2+-Mg2+)-ATPase with Fe2+, Mn2+, or Co2+ inhibited the Ca2+ pump activity. Furthermore, neither the formation of the phosphorylated intermediate of (Ca2+-Mg2+)-ATPase, nor ATP-dependent (59Fe) uptake could be detected in the presence of Fe2+ concentrations which stimulated ATP hydrolysis. We conclude that: (i) under the influence of certain metal ions, the Ca2+ pump in the liver plasma membrane may be switched to an uncoupled state which displays ATP hydrolysis activity, but does not insure ion transport; (ii) therefore the Ca2+ pump in liver plasma membranes specifically insures Ca2+ transport.     

Acid ATPase from chicken liver lysosomes. II. Presence of metal ion-activated enzyme

Metal ion-activated acid ATPase was present in chicken liver lysosomes. The enzyme catalyzed the hydrolysis of nucleoside tri-, di-, and monophosphates and cleaved the phosphodiester linkage. Among the substrates studied, ATP was hydrolyzed at the highest rate at pH 5.4. The enzyme activity was stimulated 3.5 approximately 7.5-fold by divalent cations such as Ca2+, Mg2+, and Zn2+, but inhibited by EDTA or Hg2+.     

Transient state kinetic studies of phosphorylation by ATP and Pi of the calcium-dependent ATPase from sarcoplasmic reticulum.

The ATPase of the sarcoplasmic reticulum is phosphorylated by ATP in the presence of Ca2+. A rapid phosphorylation was observed when the enzyme was preincubated with Ca2+ prior to the addition of 0.1 or 1 mM ATP. The rate of phosphorylation was decreased when Ca2+ was omitted from the preincubation medium and added with ATP when the reaction was started. The rate of phosphorylation by ATP was further decreased when Pi was included in the preincubation medium without Ca2+. In this case, the enzyme was phosphorylated by Pi during the preincubation. When Ca2+ and ATP were added, a burst of phosphorylation by ATP was observed in the initial 16 ms. In the subsequent incubation intervals, the phosphorylation by ATP was synchronous with the hydrolysis of the phosphoenzyme formed by Pi. The rate of hydrolysis of the phosphoenzyme formed by Pi was measured when either the Pi concentration was decreased 10 fold, or when Ca2+, ATP or ATP plus Ca2+ was added to the medium. Upon the single addition of Ca2+, the time for half-maximal decay was in the range 500--1000 ms. In all other conditions it was in the range 70--90 ms.     

The substitution of calcium for magnesium in H+,K+-ATPase catalytic cycle. Evidence for two actions of divalent cations

In order to determine the role of divalent cations in the reaction mechanism of the H+,K+-ATPase, we have substituted calcium for magnesium, which is required by the H+,K+-ATPase for phosphorylation from ATP and from PO4. Calcium was chosen over other divalent cations assayed (barium and manganese) because in the absence of magnesium, calcium activated ATP hydrolysis, generated sufficiently high levels of phosphoenzyme (573 +/- 51 pmol.mg-1) from [gamma-32P]ATP to study dephosphorylation, and inhibited K+-stimulated ATP hydrolysis. The Ca2+-ATPase activity of the H+,K+-ATPase was 40% of the basal Mg2+-ATPase activity. However, the Ca2+,K+-ATPase activity (minus the Ca2+ basal activity) was only 0.7% of the Mg2+,K+-ATPase, indicating that calcium could partially substitute for Mg2+ in activating ATP hydrolysis but not in K+ stimulation of ATP hydrolysis. Approximately 0.1 mM calcium inhibited 50% of the Mg2+-ATPase or Mg2+,K+-ATPase activities. Inhibition of Mg2+,K+-ATPase activity was not competitive with respect to K+. Inhibition by calcium of Mg2+,K+ activity p-nitrophenyl phosphatase activity was competitive with respect to Mg2+ with an apparent Ki of 0.27 mM. Proton transport measured by acridine orange uptake was not detected in the presence of Ca2+ and K+. In the presence of Mg2+ and K+, Ca2+ inhibited proton transport with an apparent affinity similar to the inhibition of the Mg2+, K+-ATPase activity. The site of calcium inhibition was on the exterior of the vesicle. These results suggest that calcium activates basal turnover and inhibits K+ stimulation of the H+,K+-ATPase by binding at a cytosolic divalent cation site. The pseudo-first order rate constant for phosphoenzyme formation from 5 microM [gamma-32P]ATP was at least 22 times slower in the presence of calcium (0.015 s-1) than magnesium (greater than 0.310 s-1). The Ca.EP (phosphoenzyme formed in the presence of Ca2+) formed dephosphorylated four to five times more slowly that the Mg.EP (phosphoenzyme formed in the presence of Mg2+) in the presence of 8 mm trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) or 250 microM ATP. Approximately 10% of the Ca.EP formed was sensitive to a 100 mM KCl chase compared with greater than 85% of the Mg.EP. By comparing the transient kinetics of the phosphoenzyme formed in the presence of magnesium (Mg.EP) and calcium (Ca.EP), we found two actions of divalent cations on dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanism of eosin Y action of activity of solubilized Ca2+, Mg2+- ATPase from smooth muscle sarcolemma

The eosin Y inhibitory effect on the activity of smooth muscle plasma membrane Ca(2+)-transporting ATPase was studied: effect of this inhibitor on the maximal initial rate of ATP-hydrolase reaction, catalyzed by Ca2+, Mg(2+)-ATPase, on the affinity of enzyme for the reaction reagents (Ca2+, Mg2+, ATP). Dependence of eosin Y inhibitory effect on some physicochemical factors of incubation medium was studied too. It was determined that eosin Y inhibited reversibly and with high specificity purified Ca2+, Mg(2+)-ATPase solubilized from myometrial cell plasma membrane (Ki--0.8 microM), decreased the turnover rate of this enzyme determined both by Mg2+, ATP and Ca2+. This inhibitor had no effect on the enzyme affinity for Ca2+, increased affinity for Mg2+ and decreased affinity for ATP. It was determined that inhibition of Ca2+, Mg(2+)-ATPase by eosin Y depended on pH and dielectric permeability of the incubation medium: increasing of pH from 6.5 to 8.0 reduced the apparent Ki, decreasing of dielectric permeability from 74.07 to 71.19 increased the apparent Ki.     

Kinetic mechanism of Fo x F1 mitochondrial ATPase: Mg2+ requirement for Mg x ATP hydrolysis

The initial rates of ATP hydrolysis catalyzed by Fo x F1 (bovine heart submitochondrial particles) preincubated in the presence of Pi for complete activation of the oligomycin-sensitive ATPase were measured as a function of ATP, Mg2+, and Mg x ATP concentrations. The results suggest the mechanism in which Mg x ATP complex is the true substrate of the ATPase and the second Mg2+ bound at a specific pH-dependent site is needed for the catalysis. Simple hyperbolic Michaelis--Menten dependences of the reaction rate on the substrate (Mg x ATP) and activating Mg2+ were found. In contrast to the generally accepted view, no inhibition of ATPase by free Mg2+ was found. Inhibition of the reaction by free ATP is due to a decrease of free Mg2+ needed for the catalysis. In the presence of both Ca2+ and Mg2+ the kinetics of ATP hydrolysis suggest that the Ca x ATP complex is neither hydrolyzed nor competes with Mg x ATP, and free Ca2+ does not affect the hydrolysis of Mg x ATP complex. A crucial role of free Mg2+ in the time-dependent inhibition of ATPase by azide is shown. The dependence of apparent Km for Mg x ATP on saturation of the Mg2+-specific site suggests the formal ping-pong mechanism in which bound Mg2+ participates in the overall reaction after dissociation of one product (most likely Pi) thus promoting either release of ADP (catalytic turnover) or slow isomerization of the enzyme--product complex (formation of the dead-end ADP(Mg2+)-inhibited enzyme). The rate of Mg x ATP hydrolysis only slightly depends on pH at saturating Mg2+. In the presence of limited amounts of free Mg2+ the pH dependence of the initial rate corresponds to the titration of a single group with pKa = 7.5. The simple competition between H+ and activating Mg2+ was observed. The specific role of Mg2+ as a coupling cation for energy transduction in Fo x F1-ATPase is discussed.     

Differences between microsomal and mitochondrial-matrix palmitoyl-coenzyme A hydrolase, and palmitoyl-L-carnitine hydrolase from rat liver.

Palmitoyl-CoA hydrolase (EC 3.1.2.2) and palmitoyl-L-carnitine hydrolase (EC 3.1.1.28) activities from rat liver were investigated. 1. Microsomal and mitochondrial-matrix palmitoyl-CoA hydrolase activities had similar pH and temperature optima, although the activities showed different temperature stability. They were inhibited by Pb2+ and Zn2+. The palmitoyl-CoA hydrolase activities in microsomal fraction and mitochondrial matrix were differently affected by the addition of Mg2+, Ca2+, Co2+, K+ and Na+ to the reaction mixture. ATP, ADP and NAD+ stimulated the microsomal activity and inhibited the mitochondrial-matrix enzyme. The activity of both the microsomal and mitochondrial-matrix hydrolase enzymes was specific for long-chain fatty acyl-CoA esters (C12-C18), with the highest activity for palmitoyl-CoA. The apparent Km for palmitoyl-CoA was 47 microM for the microsomal enzyme and 17 microM for the mitochondrial-matrix enzyme. 2. The palmitoyl-CoA hydrolase and palmitoyl-L-carnitine hydrolase activities of microsomal fraction had similar pH optima and were stimulated by dithiothreitol, but were affected differently by the addition of Pb2+, Mg2+, Ca2+, Mn2+ and cysteine. The two enzymes had different temperature-sensitivities. 3. The data strongly suggest that palmitoyl-CoA hydrolase and palmitoyl-L-carnitine hydrolase are separate microsomal enzymes, and that the hydrolysis of palmitoyl-CoA in the microsomal fraction and mitochondria matrix was catalysed by two different enzymes.     

Cooperative effects of Ca2+ and Sr2+ on sarcoplasmic reticulum adenosine triphosphatase

The intrinsic fluorescence of purified Ca-ATPase from skeletal sarcoplasmic reticulum was measured in the presence of various concentrations of Ca2+, Sr2+, and Ba2+. Ca2+ and Sr2+ induce positive cooperative fluorescence enhancement, whereas Ba2+ does not change the fluorescence of ATPase. ATP does not seem to modify the kinetic parameters of Ca2+ and Sr2+ binding to ATPase. Nevertheless, p-nitrophenylphosphate hydrolysis, activated by Ca2+ or Sr2+ at various pHs, changes the affinity and the cooperative behavior for both cations and two components appear in the Hill plots. For Ca2+, nH of 1.6 to 3.5 were obtained, and 1.06 to 1.83 for Sr2+; nH changes of the second component seem to be pH dependent. Differences in the ratio between rates of Ca2+ transport and substrate hydrolysis by sarcoplasmic reticulum were found, i.e., two for ATP and one for p-nitrophenylphosphate. For Sr2+ this ratio was one for either ATP or p-nitrophenylphosphate.     

Purification and characterization of an extracellular β-xylosidase from the rumen anaerobic fungus Neocallimastix frontalis

The purification of beta-xylosidase (beta-D-xyloside xylohydrolase, EC 3.2.1.37) from Neocallimastix frontalis was performed by ammonium sulphate precipitation, ion exchange chromatography, gel filtration and preparative isoelectric focusing. The enzyme had a molecular mass of 180,000 Da, an isoelectric point at pH 4.35 and catalysed the hydrolysis of p-nitrophenyl-beta-D-xylopyranoside optimally at pH 6.5 and 35 degrees C with a Km of 0.33 mg ml-1. The enzymatic activity was strongly increased by the presence of Ca2+, Mn2+, Zn2+, Co2+ or Mg2+ and completely inhibited by Hg2+ and p-chloromercuribenzoate. The purified protein also had a low level of xylanase activity.     

Binding of divalent cation to phosphoenzyme of sodium- and potassium-transport adenosine triphosphatase.

In order to study the action of the divalent cation which is essential for phosphorylation of sodium- and potassium-transport adenosine triphosphatase, magnesium ion, the normal ligand, was replaced with calcium ion, which had properties diffeerent from those of Mg2+, Mn2+, Fe2+, Co2+, Ni2+, or Zn2+. Phosphorylation of the enzyme from ATP at pH 7.4 in the presence of Na+ and Ca2+ yielded a Ca.phosphoenzyme (60% of the maximal level) with a normal rate of dephosphorylation following a chase with unlabeled Ca.ATP (PK = 0.092S-1 at 0 degrees C). In contrast, after a chase by a chelator, namely ethylenediaminetetraacetic acid, 1,2-cyclohexylenedinitrilotetraacetic acid, or ethylene glycol bis-(beta-aminoethyl ether)N,N'-tetraacetic acid, dephosphorylation slowed within 5 s and half of the initial phosphoenzyme remained with a stability about 5-fold greater than normal. Three states of the phosphoenzyme were distinguished according to their relative sensitivity to ADP or to K+ added during a chase. Normally prepared Mg.phosphoenzyme was sensitive to K+ but not to ADP; Ca.phosphoenzyme was sensitive either to ADP or to K+; and the stabilized phosphoenzyme prepared from Ca.phosphoenzyme by addition of a chelator was sensitive neither to ADP nor to K+ nor to both together. Addition of Ca2+ to the stabilized phosphoenzyme restored the reactivity to that of Ca.phosphoenzyme. Addition of Mg2+ to the stabilized phosphoenzyme changed the reactivity to that of Mg.phosphoenzyme. Therefore, this unreactive, stabilized state of the phosphoenzyme appeared to be a divalent cation-free phosphoenzyme. With respect to sensitivity to ouabain, Ca.phosphoenzyme was as sensitive as Mg.phosphoenzyme but calcium-free phosphoenzyme was much less sensitive. It was concluded that the divalent cation required for phosphorylation normally remains tightly bound to the phosphoenzyme and is required for normal reactivity. Calcium ion was almost unique in dissociating relatively easily from the phosphoenzyme. Strontium ion appeared to act similarly to Ca2+.