Phosphatase synthesis in Klebsiella (Aerobacter) aerogenes growing in continuous culture

1. Phosphatase synthesis was studied in Klebsiella aerogenes grown in a wide range of continuous-culture systems. 2. Maximum acid phosphatase synthesis was associated with nutrient-limited, particularly carbohydrate-limited, growth at a relatively low rate, glucose-limited cells exhibiting the highest activity. Compared with glucose as the carbon-limiting growth material, other sugars not only altered the activity but also changed the pH–activity profile of the enzyme(s). 3. The affinity of the acid phosphatase in glucose-limited cells towards p-nitrophenyl phosphate (K_m 0.25–0.43mm) was similar to that of staphylococcal acid phosphatase but was ten times greater than that of the Escherichia coli enzyme. 4. PO₄³⁻-limitation derepressed alkaline phosphatase synthesis but the amounts of activity were largely independent of the carbon source used for growth. 5. The enzymes were further differentiated by the effect of adding inhibitors (F⁻, PO₄³⁻) and sugars to the reaction mixture during the assays. In particular, it was shown that adding glucose, but not other sugars, stimulated the rate of hydrolysis of p-nitrophenyl phosphate by the acid phosphatase in carbohydrate-limited cells at low pH values (<4.6) but inhibited it at high pH values (>4.6). Alkaline phosphatase activity was unaffected. 6. The function of phosphatases in general is discussed and possible mechanisms for the glucose effect are outlined.     

Delta endotoxin inhibits a phosphatase in midgut epithelial membranes of Heliothis virescens

A vanadate- and delta endotoxin-sensitive phosphatase copurified with plasma membranes and brush border membranes from the midgut epithelium of Heliothis virescens. Phosphatase activity was stimulated under alkaline conditions. Total phosphatase activity was inhibited 60% in the midgut membranes by 360 nM delta endotoxin with a K_i = 76 nM. Brush border membranes were inhibited 75% by 1.47 μM delta endotoxin with a K_i = 64.7 nM. Vanadate (200 μM) completely inhibited the toxinsensitive alkaline phosphatase. A 72 kDa protein was phosphorylated when membranes were incubated with Mg²⁺ and [³²P]orthophosphate, and phosphorylation was inhibited by both vanadate and delta endotoxin, suggesting that destabilization of this phosphoprotein was responsible for phosphatase inhibition.     

Phosphatase antagonist okadaic acid inhibits steady-state K+ currents in guard cells of Vicia faba

The effects of protein phosphatase inhibitors on steady-state K⁺ currents in the plasma membrane of Vicia faba guard cells were studied. Cells were impaled with double barrelled electrodes to monitor membrane voltage and K⁺ currents under voltage clamp. Okadaic acid (OA) (1 μM), a specific inhibitor of phosphatase 1 and 2A activity, blocks inward (l_K⁺_(in)) and outward (l_K⁺_(out)) rectifying K⁺ channels. Both currents decreased in parallel with a sigmoidal time course with 50% inhibition at about 8 min. With 0.2 μM OA inhibition became slower and more variable (4–34 min). Inhibition did not recover by washing cells ≤ 20 min in OA-free solution. In five out of seven cells OA also induced a rise in the background conductance, which lagged behind the inhibition of K⁺ current. Both decaying l_K*_(out) and rising leak conductance caused a depolarization. OA-induced inhibition of l_K⁺_(in) and l_K⁺_(out) was without a significant effect on the kinetics of voltage-dependent current activation and deactivation. In an alternative approach, guard cells were loaded from the voltage recording pipette with the non-specific phosphatase inhibitor naphthylphosphate. After an impalement of some minutes l_K⁺_(in) and l_K⁺_(out) were small or undetectable. In conclusion inward and outward K⁺ channels in guard cells have a common voltage-independent mode of control which is sensitive to phosphatase inhibitors. The known specificity of OA points to a mode of action in which a net increase of protein phosphorylation through inhibition of phosphatase 1 and/or 2A activity blocks conductance of both, l_K⁺_(in) and l_(out)     

Phosphatases; origin,characteristics and function in lakes

Phosphatases catalyze the liberation of orthophosphate from organic phosphorus compounds. The total phosphatase activity in lake water results from a mixture of phosphatases localized on the cell surfaces of algae and bacteria and from dissolved enzymes supplied by autolysis or excretion from algae, bacteria and zooplankton. External lake water phosphatases usually have pH optima in the alkaline region. Acid phosphatases generally seem to be active in the internal cell metabolism. The synthesis of external alkaline phosphatases is often repressed at high phosphate concentrations and derepressed at low phosphate concentrations. Phosphatase activity has therefore been used as a phosphorus deficiency indicator in algae and in natural plankton populations. The possibilities for this interpretation of phosphatase activity in lake water are limited, however, and this is discussed. The in situ hydrolysis capacity, i.e. the rate by which orthophosphate is released from natural substrates, is unknown. However, we advocate that this process is important and that the rate of substrate supply, rather than phosphatase activity, limits the enzymatic phosphate regeneration.     

Multiple forms of alkaline phosphatase from Escherichia coli cells with repressed and derepressed biosynthesis of the enzyme.

Isolation of multiple forms of alkaline phosphatase from Escherichia coli cells with repressed and derepressed biosynthesis of the enzyme is reported. Three enzyme forms were isolated from cells with derepressed synthesis, and one form was isolated from cells with repressed enzyme synthesis. The multiple enzyme forms did not differ in pH optimum, thermostability, or the degree of inhibition with orthophosphate; however, they did differ in the relative rate of hydrolysis of different substrates. The addition of substrates to the cells during enzyme derepression resulted in changes of the ratio of the multiple forms.     

ATPase and phosphatase activities from human red cell membranes III. Stimulation of K+-activated phosphatase by phospholipase C

Summary Treatment of red cell membranes with pure phospholipase C inactivates (Na⁺+K⁺)-ATPase activity and Na⁺-dependent phosphorylation but increases K⁺-dependent phosphatase activity. When phospholipase A₂ replaces phospholipase C, all activities are lost. Activation of K⁺-dependent phosphatase by treatment with phospholipase C is caused by an increase in the maximum rate of hydrolysis ofp-nitrophenylphosphate and in the maximum activating effect of K⁺, the apparent affinities for substrate and cofactors being little affected. After phospholipase C treatment K⁺-dependent phosphatase is no longer sensitive to ouabain but becomes more sensitive to N-ethylmaleimide. In treated membranes Na⁺ partially replaces K⁺ as an activator of the phosphatase. Although ATP still inhibits phosphatase activity, neither ATP nor ATP+Na⁺ are able to modify the apparent affinity for K⁺ of K⁺-dependent phosphatase in these membranes.     

Acid phosphatase synthesis inAspergillus flavus

Proteins with phosphatase activity were produced during the growth ofAspergillus flavus in a phosphate-supplemented liquid synthetic medium. The best carbon and nitrogen sources for the synthesis of phosphatase were glucose and ammonium sulfate, respectively. The proteins were separated by molecular exclusion and ion exclusion chromatography (IEC) into three components one of which showed phosphatase activity. The molar mass of the enzyme was approximately 62 kDa. The purified enzyme exhibited an optimum activity at pH 4.0 and at 45°C. The activity of the enzyme was stimulated by Ca²⁺ and Mg²⁺ but inhibited by fluoride, iodoacetic acid, ethylenediaminetetraacetic acid and 2,4-dinitrophenol, and exhibited an apparentK _M of approximately 420 μmol/L.     

Vertical variations in kinetics of alkaline phosphatase and P species in sediments of a shallow Chinese eutrophic lake (Lake Donghu)

The vertical distribution of the variables relevant to P forms in sediments were studied in a shallow Chinese freshwater lake (Lake Donghu) in 1997, 1998, 1999 and 2000, to assess the contribution of enzyme to P availability in sediment cores. Sediment P was fractionationd into iron-bound P, calcium-bound P, acid soluble organic P (ASOP) and hot NaOH extractable residual organic P. The former two species made the largest contribution to the sediment P pool. All P species exhibited significantly higher concentrations in different depths at Station I, compared with those found at Station II, except for ASOP. Coupled with these lower ASOP concentrations, the V _max data of alkaline phosphatase, measured on the same samples, were significantly higher at station I. Taken together, ASOP were probably important in supplying the enzymatic substrate (Phosphatase Hydrolyzable Phosphorus, PHP) into interstitial water. Dissolved orthophosphate and PHP concentrations were highly heterogeneous , but peaked in subsurface, paralleled by higher V _max and lower K _m values of alkaline phosphatase, throughout the sediment core. Sediment in the eutrophic lake is not only enriched in available P (iron-bound P), or stores residual P, but also tends to release PHP, thereby inducing the production of alkaline phosphatase and releasing o-P into water column by enzymatic hydrolysis. The latter process may also occur in relatively deep sediment layers.     

Modification of human prostatic acid phosphatase by potassium ferrate

Prostatic acid phosphatase (orthophosphoric-monoester phosphohydrolase, acid optimum, EC 3.1.3.2) reacts with potassium ferrate, K₂FeO₄ a potent oxidizing agent and an analogue of orthophosphate. Treatment of the enzyme with 10^?6m ferrate at pH 7.5 0 C leads to the immediate loss of 95% of the activity. Molybdate, the competitive inhibitor of prostatic phosphatase, partially protects the enzyme from inactivation. Ferrate inactivation at pH 7.5 is accompanied by the modification of 2 histidine, 4 lysine and 4 methionine residues. Histidine is protected by molybdate, whereas methionine is not and lysine is partly protected. Partial inactivation with ferrate leads to the retardation of the modified enzyme on Sephadex G-200 column, which is eluted in the position of the active monomeric unit.     

The K+- and HCO3−-stimulated phosphatase activities in renal microsomes of rats

The K⁺-stimulated phosphatase activity of microsomes from rat kidney was not inhibited by l-phenylalanine, but the HCO₃^?-stimulated phosphatase activity was markedly inhibited by l-phenylalanine. Valinomycin enhanced the HCO₃^?-stimulated phosphatase activity, but did not enhance the K⁺-stimulated phosphatase activity. Ouabain did not inhibit the HCO₃^?-stimulated phosphatase activity, but inhibited the K⁺-stimulated phosphatase activity.The renal K⁺-stimulated phosphatase activity was suppressed to 40% of the control values by adrenalectomy, but the renal HCO₃^?-stimulated phosphatase activity was little suppressed by adrenalectomy. The renal K⁺-stimulated phosphatase activity in intact and adrenalectomized rats was found to be significantly elevated, in a manner similar to the elevation of the renal (Na⁺ + K⁺)-ATPase activity by aldosterone treatment (P < 0.02).     

Glycerol-3-phosphatase and the control of glycerol metabolism in Dunaliella tertiolecta cells

Glycerol-3-phosphatase (EC 3.1.3.2.1) was studied by following the release of radioactive glycerol from L-(U-¹⁴C)glycerol-3-phosphate in Dunaliella tertiolecta enzyme extracts. The reaction showed a neutral pH optimum and had an absolute requirement for Mg²⁺. The substrate saturation curve was hyperbolic with an apparent K _m value for glycerol-3-phosphate of 0.7 mM in the absence of phosphate. Inorganic orthophosphate was a competitive inhibitor of the enzyme with an estimated K _j of 0.1 mM. The glycerol-3-phosphatase reaction was blocked nearly completely by millimolar Ca²⁺ concentrations. Ca²⁺ inhibition did not depend on the presence of calmodulin in the reaction medium. The characteristics of glycerol-3-phosphatase are discussed in relation to the regulation of the cyclic glycerol metabolism in Dunaliella cells during periods of osmotic stress.     

Measurement of the rate ATP synthesis in bovine anterior pituitary slices

A method is described which uses firely luciferase for the measurement of the specific activity of [^{3 2}P] orthophosphate in the γ-position in ATP. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) which uncouples respiration and oxidative phosphorylation and at 0.1 μ/ml reduced pituitary ATP concentration by 35%, decreased the rate of incorporation of [^{3 2}P] orthophosphate into ATP by 40%. Ouabain caused a 50% reduction in the specific activity of ATP after 30 min incubation. Four stimuli of growth hormone secretion, p-chloromercuribenzoate, prostaglandin E₂, 3-isobutyl-1-methylxanthine and Ba²⁺ did not alter the rate of incorporation of [^{3 2}P] orthophosphate into ATP; a fith stimulato, high K⁺, decreased the rate of incorporatio. These results suggest that overall. ATP utilisation is not greatly increased under conditions of stimulated secretion.     

Phosphorylation Regulates an Inwardly Rectifying ATP-sensitive K<Superscript><Emphasis Type="Bold">+</Emphasis></Superscript>- Conductance in Proximal Tubule Cells of Frog Kidney

K⁺ channels in the renal proximal tubule play an important role in salt reabsorption. Cells of the frog proximal tubule demonstrate an inwardly rectifying, ATP-sensitive K⁺ conductance that is inhibited by Ba²⁺, G_Ba. In this paper we have investigated the importance of phosphorylation state on the activity of G_Ba in whole-cell patches. In the absence of ATP, G_Ba decreased over time; this fall in G_Ba involved phosphorylation, as rundown was inhibited by alkaline phosphatase and was accelerated by the phosphatase inhibitor F⁻(10 mM). Activation of PKC using the phorbol ester PMA accelerated rundown via a mechanism that was dependent on phosphorylation. In contrast, the inactive phorbol ester PDC slowed rundown. Inclusion of the PKC inhibitor PKC-ps in the pipette inhibited rundown. These data indicate that PKC-mediated phosphorylation promotes channel rundown. Rundown was prevented by the inclusion of PIP-2 in the pipette. PIP-2 also abrogated the PMA-mediated increase in rundown, suggesting that regulation of G_Ba by PIP-2 occurred downstream of PKC-mediated phosphorylation. G-protein activation inhibited G_Ba, with initial currents markedly reduced in the presence of GTPγs. These properties are consistent with G_Ba being a member of the ATP-sensitive K⁺ channel family.     

Participation of vacuoles in regulation of levels of K+, Mg2+ and orthophosphate ions in cytoplasm of the yeast Saccharomyces carlsbergensis

Intracellular distributions of K⁺, Mg²⁺ and orthophosphate under various conditions of cultivation or incubation of the yeast Saccharomyces carlsbergensis were studied by differential extraction of ion pools. The decisive role of vacuolar compartmentation of ions in regulation of K⁺, Mg²⁺ and orthophosphate levels in the yeast cytoplasm was shown. The content of intracellular K⁺ and Mg²⁺ in yeast increased or decreased primarily depending on the increase or decrease in the vacuolar ion pool. The levels of K⁺ and Mg²⁺ in the cytoplasm were practically unchanged. Vacuoles were involved in regulation of Mn²⁺ concentration in the cytoplasm of the yeast S. carlsbergensis accumulating this ion in the presence of glucose. Alongside the vacuolar compartmentation, the chemical compartmentation, i. e. formation of bound Mg²⁺, Mn²⁺ and K⁺ was, evidently, also involved in the control of ion levels in the cytoplasm. The orthophosphate level in the yeast cytoplasm was regulated by its accumulation in vacuoles and biosynthesis of inorganic polyphosphates in these organelles. The biosynthesis of low-molecular weight polyphosphates occurred parallel to the accumulation of Mg²⁺ or Mn²⁺ in vacuoles, thus confirming the availability of the other mechanism for the transport of these ions through the tonoplast differing from the transport mechanism through the plasmalemma.     

Purification and characterization of extracellular acid phosphatase of Tetrahymena pyriformis

An extracellular acid phosphatase secreted into the medium during growth of Tetrahymena pryiformis strain W was purified about 900-fold by (NH₄)₂SO₄ precipitation, gel filtration and ion exchange chromatography. The purified acid phosphatase was homogenous as judged by polycrylamide gel electrophoresis and was found to be a glycoprotein. Its carbohydrate content was about 10% of the total protein content. The native enzyme has a molecular weight of 120 000 as determined by gel filtration and 61 000 as determined by sodium dodecyl sulfate-polycrylamide gel electrophoresis. The acid phosphatase thus appears to consist of two subunits of equal size. The amino acid analysis revealed a relatively high content of asparic acid, glutamic acid and leucine. The purified acid phosphatase from Tetrahymena had a rather broad substrate specificity; it hydrolyzed organic phosphates, nucleotide phosphates and hexose phosphates, but had no diesterase activity. The K_m values determined with p-nitrophenyl phosphate, adenosine 5′-phosphate and glucose 6-phosphate were 3.1·10^?4 M, 3.9·10^?4 M and 1.6·10^?3 M, respectively. The optima pH for hydrolysis of three substrates were similar (pH 4.6). Hg²⁺ and Fe³⁺ at 5 mM were inhibitory for the purified acid phosphatase, and fluoride, L-(+)-tartaric acid and molybdate also inhibited its cavity at low concentrations. The enzyme was competitively inhibited by NaF (K_i=5.6·10^?4 M) and by L-(+)-tartaric acid (K_i = 8.5·10^?5 M), while it was inhibited noncompetitively by molybdate K_i = 5.0·10^?6 M). The extracellular acid phosphatase purified from Tetrahymena was indistinguishable from the intracellular enzyme in optimum pH, K_m, thermal stability and inhibition by NaF.     

Characterization of the Formation and Distribution of Photosynthetic Products by Sedum praealtum Chloroplasts

Photoassimilation of ¹⁴CO₂ by intact chloroplasts from the Crassulacean acid metabolism plant Sedum praealtum was investigated. The main water-soluble, photosynthetic products were dihydroxyacetone phosphate (DHAP), glycerate 3-phosphate (PGA), and a neutral saccharide fraction. Only a minor amount of glycolate was produced. A portion of neutral saccharide synthesis was shown to result from extrachloroplastic contamination, and the nature of this contamination was investigated with light and electron microscopy. The amount of photoassimilated carbon partitioned into starch increased at both very low and high concentrations of orthophosphate. High concentrations of exogenous PGA also stimulated starch synthesis.

DHAP and PGA were the preferred forms of carbon exported to the medium, although indirect evidence suported hexose monophosphate export. The export of PGA and DHAP to the medium was stimulated by high exogenous orthophosphate, but depletion of chloroplastic reductive pentose phosphate intermediates did not occur. As a result only a relatively small inhibition in the rate of CO₂ assimilation occurred.

The rate of photoassimilation was stimulated by exogenous PGA, ribose 5-phosphate, fructose 1,6-bisphosphate, fructose 6-phosphate, and glucose 6-phosphate. Inhibition occurred with phosphoenolpyruvate and high concentrations of PGA and ribose 5-phosphate. PGA inhibition did not result from depletion of chloroplastic orthophosphate or from inhibition of ribulose 1,5-bisphosphate carboxylase. Exogenous PGA and phosphoenolpyruvate were shown to interact with the orthophosphate translocator.

     

Binding to the high-affinity substrate site of the (Na+ + K+)-dependent ATPase

The (Na⁺ + K⁺)-dependent ATPase exhibits substrate sites with both high affinity (K _m near 1 µM) and low affinity (K _m near 0.1 mM) for ATP. To permit the study of nucleotide binding to the high-affinity substrate sites of a canine kidney enzyme preparation in the presence as well as absence of MgCl₂, the nonhydrolyzable - imido analog of ATP, AMP-PNP, was used in experiments performed at 0–4°C by a centrifugation technique. By this method theK _D for AMP-PNP was 4.2 µM in the absence of MgCl₂. Adding 50 µM MgCl₂, however, decreased theK _D to 2.2 µM; by contrast, higher concentrations of MgCl₂ increased theK _D until, with 2 mM MgCl₂, theK _D was 6 µM. The half-maximal effect of MgCl₂ on increasing theK _D occurred at approximately 1 mM. This biphasic effect of MgCl₂ is interpreted as Mg²⁺ in low concentrations favoring AMP-PNP binding through formation at the high-affinity substrate sites of a ternary enzyme-AMP-PNP-Mg complex; inhibition of nucleotide binding at higher MgCl₂ concentrations would represent Mg²⁺ acting through the low-affinity substrate sites. NaCl in the absence of MgCl₂ increased AMP-PNP binding, with a half-maximal effect near 0.3 mM; in the presence of MgCl₂, however, NaCl increased theK _D for AMP-PNP. KCl decreased AMP-PNP binding in the presence or absence of MgCl₂, but the simultaneous presence of a molar excess of NaCl abolished (or masked) the effect of KCl. ADP and ATP acted as competitors to the binding of AMP-PNP, although a substrate for the K⁺-dependent phosphatase reaction also catalyzed by this enzyme,p-nitrophenyl phosphate, did not. This lack of competition is consistent with formulations in which the phosphatase reaction is catalyzed at the low-affinity substrate sites.     

Protein phosphatases: possible bisphosphonate binding sites mediating stimulation of osteoblast proliferation

We investigated the existence of a bisphosphonate (BP) target site in osteoblasts. Binding assays using [³H]-olpadronate ([³H]OPD) in whole cells showed the presence of specific, saturable and high affinity binding for OPD (K_d = 1.39 ± 0.33 μM) in osteoblasts. [³H]OPD was displaced from its binding site by micromolar concentrations of lidadronate, alendronate and etidronate (K_d = 1.42 ± 0.15 μM, 2.00 ± 0.2 μM and 2.4 ± 0.4 μM, respectively), and by millimolar concentrations of the non-permeant protein phosphatase (PP) substrates p-nitrophenylphosphate and α-naphtylphosphate. PP inhibitors orthovanadate, NaF or vpb(bipy) did not displace [³H]OPD.As expected, specific OPD binding was detected in the plasma membrane of ROS 17/2.8 cells, although significant BP binding was also found intracellularly. Moreover, OPD increased DNA synthesis in these cells with a temporal profile similar to the protein tyrosine phosphatase (PTP) inhibitors, Na₃VO₄ and vpb(bipy); but different from a general PP inhibitor (NaF). The stimulatory effect of OPD and PTP inhibitors on osteoblast proliferation was inhibited by the protein tyrosine kinase inhibitors genistein and geldanamycin. These results provide new evidence on the existence of a BP target in osteoblastic cells, presumably a PTP, which may be involved in the stimulatory action of BPs on osteoblast proliferation.     

Divalent cations and the phosphatase activity of the (Na + K)-dependent ATPase

Phosphatase activity of a kidney (Na + K)-ATPase preparation was optimally active with Mg²⁺ plus K⁺. Mn²⁺ was less effective and Ca²⁺ could not substitute for Mg²⁺. However, adding Ca²⁺ with Mg²⁺ or substituting Mn²⁺ for Mg²⁺ activated it appreciably in the absence of added K⁺, and all three divalent cations decreased apparent affinity for K⁺. Inhibition by Na⁺ decreased with higher Mg²⁺ concentrations, when Ca²⁺ was added, and when Mn²⁺ was substituted for Mg²⁺. Dimethyl sulfoxide, which favorsE ₂ conformations of the enzyme, increased apparent affinity for K⁺, whereas oligomycin, which favorsE ₁ conformations, decreased it. These observations are interpretable in terms of activation through two classes of cation sites. (i) At divalent cation sites, Mg²⁺ and Mn²⁺, favoring (under these conditions)E ₂ conformations, are effective, whereas Ca²⁺, favoringE ₁, is not, and monovalent cations complete. (ii) At monovalent cation sites divalent cations compete with K⁺, and although Ca²⁺ and Mn²⁺ are fairly effective, Mg²⁺ is a poor substitute for K⁺, while Na⁺ at these sites favorsE ₁ conformations. K⁺ increases theK _m for substrate, but both Ca²⁺ and Mn²⁺ decrease it, perhaps by competing with K⁺. On the other hand, phosphatase activity in the presence of Na⁺ plus K⁺ is stimulated by dimethyl sulfoxide, by higher concentrations of Mg²⁺ and Mn²⁺, but not by adding Ca²⁺; this is consistent with stimulation occurring through facilitation of an E₁ to E₂ transition, perhaps an E₁-P to E₂-P step like that in the (Na + K)-ATPase reaction sequence. However, oligomycin stimulates phosphatase activity with Mg²⁺ plus Na⁺ alone or Mg²⁺ plus Na⁺ plus low K⁺: this effect of oligomycin may reflect acceleration, in the absence of adequate K⁺, of an alternative E₂-P to E₁ pathway bypassing the monovalent cation-activated steps in the hydrolytic sequence.     

(Na + K)-ATPase activity of crude homogenates of rat skeletal muscle as estimated from their K-dependent 3-O-methylfluorescein phosphatase activity

A highly sensitive fluorimetric assay using 3-O-methylfluorescein phosphate as substrate was used in the determination of K⁺-dependent phosphatase activity in preparations of rat skeletal muscle. The gastrocnemius muscle was chosen because of mixed fibre composition. Crude, detergent treated homogenate was used so as to avoid loss of activity during purification. K⁺-dependent phosphatase activities in the range 0.19–0.37 μmol · (g wet weight)⁻¹ · min⁻¹ were obtained, the value decreasing with age and K⁺-deficiency. Complete inhibition of the K⁺-dependent phosphatase was obtained with 10⁻³ M ouabain. Using a KSCN-extracted muscle enzyme the intimate relation between K⁺-dependent phosphatase activity and (Na⁺ + K⁺)-activated ATP hydrolysis could be demonstrated. A molecular activity of 620 min⁻¹ was estimated from simultaneous determination of K⁺-dependent phosphatase activity and [³H]ouabain binding capacity using the partially purified enzyme preparation. The corresponding enzyme concentration in the crude homogenates was calculated and corresponded well with the number of [³H]ouabain binding sites measured in intact muscles or biopsies hereof.