Catabolism of d-fructose and d-ribose by Pseudomonas doudoroffii

1. The 1-P-fructokinase (1-PFK) and 6-P-fructokinase (6-PFK) from Pseudomonas doudoroffii were partially purified by a combination of (NH₄)₂SO₄ fractionation and DEAE-Sephadex column chromatography. The pH optima of these enzymes were 9.0 and 8.5, respectively.
2. When the concentrations of the substrates of the 1-PFK reaction were varied, Michaelis-Menten kinetics were observed. The Kms for d-fructose-1-P (F-1-P) and ATP were 3.03×10^-4 M and 3.39×10^-4 M, respectively. Variation of MgCl₂ at fixed concentrations of F-1-P and ATP resulted in sigmoidal kinetics; about 10 mM MgCl₂ was necessary for maximal activity. Activity of 1-PFK was inhibited when the ratio of ATP: Mg⁺⁺ was higher than 0.5, suggesting that ATP: 2Mg⁺⁺ was the substrate and that free ATP was inhibitory. Although an absolute requirement for K⁺ or NH ₊ ⁴ could not be demonstrated, these cations stimulated the rate of the reaction. Activity of 1-PFK was not significantly affected by 3 mM AMP, cyclic-AMP, P_i, d-fructose-6-P (F-6-P), ADP, P-enolpyruvate (PEP), pyruvate, citrate, or l-glutamate.
3. Sigmoidal kinetics were observed for 6-PFK when the concentration of F-6-P was increased and the level of ATP was kept constant. Activity of 6-PFK was increased by ADP, inhibited by PEP, and unaffected by 3 mM AMP, cyclic-AMP, P_i, F-1-P, pyruvate, or citrate.

     

Fluxes and compartmentation of 3-O-methyl-D-glucose in Riccia fluitans

In thalli of the aquatic liverwort, Riccia fluitans, 3-O-methyl-D-glucose (3-OMG) is not metabolized. Intracellular compartmentation, accumulation and transmembrane fluxes of 3-OMG have been determined by compartmental analysis. A novel set of equations has been derived to extend this method to non-steady-state conditions of constant but unequal unidirectional fluxes. Efflux kinetics with 3-OMG and L-glucose revealed two intracellular flux compartments, presumably cytoplasm and vacuole; an additional quickly exchanging compartment (half-time approx. 1 min) has been assigned to the apoplast. With 1 mM 3-OMG given externally, cytoplasmic 3-OMG concentration (c _c) attains a quasi-steady state of about 10 mM lasting for >100 h, whereas the presumed vacuolar concentration (c _v) rises steadily, but does not reach flux equilibrium even after two weeks (c _v=46 mM). After 24 h incubation with 0.03 mM 3-OMG, c _c=1 mM approx., and c _v=3 mM approx., thus indicating accumulation by active hexose transport at both the plasmalemma and tonoplast. External D-glucose, but no D-mannitol, competitively inhibits 3-OMG uptake (cis-inhibition) and stimulates 3-OMG efflux at the plasmalemma by a factor up to 2.5. This trans-stimulation saturates half-maximally at 1.5 mM D-glucose. It clearly indicates a hexose carrier in the plasmalemma with one substrate-binding site for D-glocose and 3-OMG, alternately exposed to the cytoplasmic and outside compartment. The extent of the measured trans-stimulation can only be explained, if in the transport cycle the translocation of the empty substrate-binding site across the plasmalemma is rate-limiting.     

DFT and docking studies of rhodostreptomycins A and B and their interactions with solvated/nonsolvated Mg2+ and Ca2+ ions

The interactions of L-aminoglucosidic stereoisomers such as rhodostreptomycins A (Rho A) and B (Rho B) with cations (Mg²⁺, Ca²⁺, and H⁺) were studied by a quantum mechanical method that utilized DFT with B3LYP/6-311G**. Docking studies were also carried out in order to explore the surface recognition properties of L-aminoglucoside with respect to Mg²⁺ and Ca²⁺ ions under solvated and nonsolvated conditions. Although both of the stereoisomers possess similar physicochemical/antibiotic properties against Helicobacter pylori, the thermochemical values for these complexes showed that its high affinity for Mg²⁺ cations caused the hydration of Rho B. According to the results of the calculations, for Rho A–Ca²⁺(H₂O)₆, ΔH = ?72.21 kcal?mol^?1; for Rho B–Ca²⁺(H₂O)₆, ΔH = ?72.53 kcal?mol^?1; for Rho A–Mg²⁺(H₂O)₆, ΔH = ?72.99  kcal?mol^?1 and for Rho B–Mg²⁺(H₂O)₆, ΔH = ?95.00  kcal?mol^?1, confirming that Rho B binds most strongly with hydrated Mg²⁺, considering the energy associated with this binding process. This result suggests that Rho B forms a more stable complex than its isomer does with magnesium ion. Docking results show that both of these rhodostreptomycin molecules bind to solvated Ca²⁺ or Mg²⁺ through hydrogen bonding. Finally, Rho B is more stable than Rho A when protonation occurs.

Modeling the scavenging activity of ellagic acid and its methyl derivatives towards hydroxyl, methoxy, and nitrogen dioxide radicals

The reaction mechanisms involved in the scavenging of hydroxyl (OH^·), methoxy (OCH₃ ^·), and nitrogen dioxide (NO₂ ^·) radicals by ellagic acid and its monomethyl and dimethyl derivatives were investigated using the transition state theory and density functional theory. The calculated Gibbs barrier energies associated with the abstraction of hydrogen from the hydroxyl groups of ellagic acid and its monomethyl and dimethyl derivatives by an OH· radical in aqueous media were all found to be negative. When NO₂ ^· was the radical involved in hydrogen abstraction, the Gibbs barrier energies were much larger than those calculated when the OH^· radical was involved. When OCH₃ ^· was the hydrogen-abstracting radical, the Gibbs barrier energies lay between those obtained with OH^· and NO₂ ^· radicals. Therefore, the scavenging efficiencies of ellagic acid and its monomethyl and dimethyl derivatives towards the three radicals decrease in the order OH^· >> OCH₃ ^· > NO₂ ^·. Our calculated rate constants are broadly in agreement with those obtained experimentally for hydrogen abstraction reactions of ellagic acid with OH· and NO₂· radicals.

The anaerobic metabolism of malate of Saccharomyces bailii and the partial purification and characterization of malic enzyme

1. The main pathway of the anaerobic metabolism of l-malate in Saccharomyces bailii is catalyzed by a l-malic enzyme.
2. The enzyme was purified more than 300-fold. During the purification procedure fumarase and pyruvate decarboxylase were removed completely, and malate dehydrogenase and oxalacetate decarboxylase were removed to a very large extent.
3. Manganese ions are not required for the reaction of malic enzyme of Saccharomyces bailii, but the activity of the enzyme is increased by manganese.
4. The reaction of l-malic enzyme proceeds with the coenzymes NAD and (to a lesser extent) NADP.
5. The K _m-values of the malic enzyme of Saccharomyces bailii were 10 mM for l-malate and 0.1 mM for NAD.
6. A model based on the activity and substrate affinity of malic enzyme, the intracellular concentration of malate and phosphate, and its action on fumarase, is proposed to explain the complete anaerobic degradation of malate in Saccharomyces bailii as compared with the partial decomposition of malate in Saccharomyces cerevisiae.

     

Phosphoenolpyruvate synthetase inMethanobacterium thermoautotrophicum

The thermophilic autotrophMethanobacterium thermoautotrophicum assimilates CO₂ via a novel pathway rather than via the Calvin cycle. The central intermediate of this pathway is acetyl CoA which is reductively carboxylated to pyruvate. Cell extracts of the organism contained phosphoenolpyruvate synthetase with a specific activity of 100 nmol min^-1 mg^-1 protein (65°C). Pyruvate kinase and pyruvate, phosphate dikinase were not detected. Phosphoenolpyruvate synthetase was partially purified (50-fold) and the following reaction stoichiometry was established: $${\text{Pyruvate + ATP + H}}_{\text{2}} {\text{O }} \to {\text{ Phosphoenolpyruvate + AMP + P}}_{\text{i}} $$ The enzyme activity was depedent on free Mg²⁺ ions, NH ₄ ⁺ or K⁺ ions, and SH-groups. Mn²⁺, but not Ca²⁺, could partially substitute for Mg²⁺; Na⁺ could not substitute for K⁺ or NH ₄ ⁺ . The pH-optima,V _max-values and the apparentK _M-values for the substrates of the enzyme in both directions were determined. Thermodynamic, kinetic and regulatory features indicate that, in vivo, the enzyme functions in the direction of phosphoenolpyruvate synthesis from pyruvate. Not only is the synthesis of phosphoenolpyruvate via the PEP synthetase reaction energetically favorable; the enzyme also catalyzed this synthesis 100 times faster than the reverse reaction, the apparentK _M value for pyruvate (40 μM) being low and the apparentK _M value for phosphate (100 mM) being high. Furthermore, AMP, ADP, PP and α-ketoglutarate were inhibitors of PEP synthesis, indicating that the enzyme activity may be controlled in vivo. The role of phosphoenolpyruvate synthetase in autotrophic CO₂ assimilation pathway ofMethanobacterium, as expected from previous labelling studies, is confirmed.     

Cyclic 2,3-diphosphoglycerate metabolism in Methanobacterium thermoautotrophicum (strain ΔH): characterization of the synthetase reaction

The levels of cyclic 2,3-diphosphoglycerate (cDPG) in methanogenic bacteria are governed by the antagonistic activities of cDPG synthetase and cDPG hydrolase. In this paper we focus on the synthetase from Methanobacterium thermoautotrophicum. The cytoplasmic 150 kDa enzyme catalyzed cDPG synthesis from 2,3-diphosphoglycerate (apparent K_m=21 mM), Mg²⁺ (K_m=3.1 mM) and ATP (K_m=1–2 mM). In batch-fed cultures, the enzyme was constitutively present (6–6.5 nmol per min per mg protein) during the different growth phases. In continuous cultures, activity decreased in response to phosphate limitation. The synthetase reaction proceeded with maximal rate at pH 6 and at 65° C and was specifically dependent on high (>0.3M) K⁺ concentrations. The reaction conditions remarkably contrasted to those of cDPG degradation catalyzed by the previously described membrane-bound cDPG hydrolase.Abbreviations cDPG Cyclic 2,3-diphosphoglycerate - 2,3-DPG 2,3-Diphosphoglycerate - 2-PG 2-Phosphoglycerate - 3-PG 3-Phosphoglycerate     

Competitive blockage of the sodium channel by intracellular magnesium ions in central mammalian neurones

The aim of this study was to determine from macroscopic current analysis how intracellular magnesium ions, Mg _i ²⁺ , interfere with sodium channels of mammalian neurones. It is reported here that permeation across the sodium channel is voltage- and concentration-dependently reduced by Mg _i ²⁺ . This results in a general reduction of sodium membrane conductance and an outward sodium peak current at large positive potentials. 30 mM Mg _i ²⁺ leads to a negative shift of voltage dependence of sodium channel gating parameters, probably due to the surface potential change of the membrane. This shift alone is, however, insufficient to explain the reduction of outward sodium currents. The blockage by Mg _i ²⁺ is decreased upon increasing intracellular or extracellular Na⁺ concentration, which suggests that Mg?' interferes with sodium permeation by competitively occupying sodium channels. Using a kinetic model to describe the sodium permeation, the dissociation constant (at zero membrane potential) of Mg _i ²⁺ for the sodium channel has been calculated to be 8.65 ± 1.51 mM, with its binding site located at 0.26 ± 0.05 electrical distance from the inner membrane. This dissociation constant is smaller than that of Na _i ⁺, which is 83.76 ± 7.60 mM with its binding site located at 0.75 ± 0.23. The low dissociation constant of Mg _i ²⁺ reflects its high affinity for the sodium channel.     

Characterization of bio-related vanadium and zinc complexes containing tetradentate dithiolate-disulfide, -diamine and -amine-amide ligands

The reaction of [VCl₃(PMe₂Ph)₃] _{with HSS′S′SH (where the HS are thiophenolate and the S′ thioether functions, respectively), H2–1, yields [VCl(μ-SS′S′S)]2 (3) with one of the thiolate groups of each of the two ligands in the bridging mode. Reaction of Na2–1 with [VOCl2(thf)2] leads to a polymeric product of composition [VO(SS′S′S)]x (4). The products obtained from the reaction between [VOCl2(thf)2] and NaSNNSNa, Na2–2, (S is thiophenolate, N the amine function) depend on subtle changes in the diamine backbone of this ligand: If the amine functions are linked by -CH2CH2– (2a), the tetranuclear V^IV complex [V(SNNS)μ-O]4 (5) is formed alongside the V^III complex [VCl(SNNS)]. If the backbone is -CH(Me)CH(Me)- (2b), [VO(SNNS)] (7) and the dinuclear, asymmetrically oxo-bridged V^IV complex [{(SNN ^– S)(thf)V}μ-O{V(SNN ^– S)}] (8) are obtained. In 8, one amine of each of the two ligands is deprotonated to the amide group. In either case, the complexation is accompanied by oxidation of the thiolates to disulfides, leading to the generation of teraazatetrathio-cycloeicosanes (6a/b). Compounds 5 and 8·2THF have been structurally characterized by X-ray analyses. The connectivities have further been established for 3·2CH2Cl2 and for 6b, which exhibits the same conformation as formally characterized 6a. The cluster compound 5 is stabilized by an extended intramolecular N-H...O and N-H...S) hydrogen-bonding network. In 7·2THF, one of the THFs of crystallization is hydrogen-bonded to the NH of the penta-coordinated {VO(SNN ^– S)} moiety; further, there is an intramolecular hydrogen bond between one of the thiolates of this tetragonal-pyramidal half of the molecule and the NH of the octahedral {VO(SNN ^– S)thf} half. The generation of the ligand 2b from its precursor compound, the zinc complex [Zn(SNNS)] (9) leads to the structural characterization of 9·CH3OH with a large SZnS bite angle and a strong hydrogen bond between the methanolic OH and one of the thiolate sulfurs. The relevance of these compounds in biological systems is discussed.}     

Substrate specifity of the hexose carrier in the plasmalemma of Chenopodium suspension cells probed by transmembrane exchange diffusion

Substrate specifity of the proton-driven hexose cotransport carrier in the plasmalemma of photoautotrophic suspension cells of Chenopodium rubrum L. has been studies through the short-term perturbation of ¹⁴C-labelled efflux of 3-O-methyl-d-glucose. Efflux, occurring exclusively via carrier-mediated exchange diffusion, is trans-stimulated by the substrate and trans-inhibited by the glucose-transport inhibitors phlorizin (K _1/2=7.9 mM) and its aglucon phloretin (K _1/2=84 μM); with both inhibitors, 3-O-methyl-d-glucose efflux may be blocked completely. Trans-stimulation of efflux (up to fourfold) by a variety of the d-enantiomers of neutral hexoses, including glucose (K _1/2=48 μM), 3-O-methyl-d-glucose (K _1/2=139 μM), and fructose (K _1/2=730 μM), but not by, for instance, d-allose, and l-sorbose, shows that carrier-substrate interaction critically involves the axial position at C-1 and C-3, respectively. We suggest that substrate binding by the Chenopodium hexose carrier involves both hydrophobic interaction with the pyran-ring and hydrogen-ion bonding at C-1 and C-3 of the d-glucose conformation.     

Assessment of nitrate leaching loss on a yield-scaled basis from maize and wheat cropping systems

Background and aims

It is so far a gap in knowledge to assess nitrate (NO₃ ^?) leaching loss linking with crop yield for a given cereal cropping system.

Methods

We conducted a meta-analysis on 32 published studies reporting both NO₃ ^? leaching losses and crop yields in the maize (N?=?20) and wheat (N?=?12) systems.

Results

On average, 22 % and 15 % of applied fertilizer N to wheat and maize systems worldwide are leached in the form of NO₃ ^?, respectively. The average area-scaled NO₃ ^- leaching loss for maize (57.4 kg N ha^?1) was approx. two times higher than for wheat (29.0 kg N ha^?1). While, if scaled to crop yields, the average yield-scaled NO₃ ^? losses were comparable between maize (5.40 kg N Mg^?1) and wheat (5.41 kg N Mg^?1) systems. Across all sites, the lowest yield-scaled NO₃ ^? leaching losses were observed at slightly suboptimal fertilization rates, corresponding to 90 % and 96 % of maximum maize or wheat yields, respectively.

Conclusions

Our findings suggest that small adjustments of agricultural N management practices can effectively reduce yield-scaled NO₃ ^? leaching losses. However, further targeted field experiments are still needed to identify at regional scale best agricultural management practices for reducing yield-scaled NO₃ ^? leaching losses in maize and wheat systems.     

Chronic mild salinity affects source leaves physiology and productivity parameters of rice plants (Oryza sativa L., cv. Taipei 309)

Aim

In rice, the top two leaves are the major carbohydrate source during grain filling. Physiological performance of these leaves under salinity may allow estimate stress-induced yield loss.

Methods

Greenhouse grown rice plants (cv. Taipei 309) were subjected to 10 and 20 mM NaCl stress levels from germination till maturity. Plant development was measured at the flowering stage and yield parameters were quantified after complete ripening of panicles.

Results

Gas exchange in the main source leaves were not significantly affected by any of the stress levels. However, growth parameters as well as total metabolizable carbohydrates content, chlorophyll content (CCI), maximal efficiency of PSII photochemistry in dark-adapted state (F _v/F _m) and lipid peroxidation were significantly affected. Rice yield, measured as total panicle production, declined to 78 and 21 % of controls in 10 and 20 mM NaCl stress, respectively. Stress-induced yield loss was positively related with the decline in CCI, F _v/F _m and K⁺/Na⁺ ratio as well as with the increase in lipid peroxidation and total soluble carbohydrate contents.

Conclusions

Though the stress levels used in this work are below what is considered the minimal critical threshold of toxicity for rice, they induce significant negative effects on plant development and yield, when present along the whole plant life cycle.     

Dark induction of nitrate reductase in the halophilic alga Dunaliella salina

The effect of nitrogen starvation on the NO₃-dependent induction of nitrate reductase (NR) and nitrite reductases (NIR) has been investigated in the halophilic alga Dunaliella salina. When D. salina cells previously grown in a medium with NH ₄ ⁺ as the only nitrogen source (NH ₄ ⁺ -cells) were transferred into NO ₃ ^? medium, NR was induced in the light. In contrast, when cells previously grown in N-free medium were transferred into a medium containing NO ₃ ^? , NR was induced in light or in darkness. Nitrate-dependent NR induction, in darkness, in D. salina cells previously grown at a photon flux density of 500 umol · m^?2 s^?1 was observed after 4 h preculture in N-free medium, whilst in cells grown at 100 umol · m^?2 s^?1 NR induction was observed after 7–8 h. An inhibitor of mRNA synthesis (6-methylpurine) did not inhibit NO ₃ ^? -induced NR synthesis when the cells, previously grown in NH ₄ ⁺ medium, were transferred into NO ₃ ^? medium (at time 0 h) after 4-h-N starvation. However, when 6-methylpurine was added simultaneously with the transfer of the cells from NH ₄ ⁺ to NO ₃ ^? medium (at time 0 h), NO ₃ ^? induced NR synthesis was completely inhibited. The activity of NIR decreased in N-starved cells and the addition of NO ₃ ^? to those cells greatly stimulated NIR activity in the light. The ability to induce NR in darkness was observed when glutamine synthetase activity reached its maximal level during N starvation. Although cells grown in NO ₃ ^? medium exhibited high NR activity, only 0.33% of the total NR was found in intact chloroplasts. We suggest that the ability, to induce NR in darkness is dependent on the level of N starvation, and that NR in D. salina is located in the cytosol. Light seems to play an indirect regulatory role on NO ₃ ^? uptake and NR induction due to the expression of NR and NO ₃ ^? -transporter mRNAs.     

Characterization of a recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus that isomerizes l-fucose, d-arabinose, d-altrose, and l-galactose

A recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus was purified as a single 68 kDa band with an activity of 76 U mg^?1. The molecular mass of the native enzyme was 204 kDa as a trimer. The maximum activity for l-fucose isomerization was at pH 7 and 75°C in the presence of 1 mM Mn²⁺. Its half-life at 70°C was 6.1 h. For aldose substrates, the enzyme displayed activity in decreasing order for l-fucose, with a k _cat of 11,910 min^?1 and a K _m of 140 mM, d-arabinose, d-altrose, and l-galactose. These aldoses were converted to the ketoses l-fuculose, d-ribulose, d-psicose, and l-tagatose, respectively, with 24, 24, 85, 55% conversion yields after 3 h.     

Use of cytosolic metabolite patterns to estimate free magnesium in normoxic myocardium

Cytosolic free magnesium (Mg_f) is considered relatively constant. To test this concept, Mg_f was estimated during hyperkalemic ventricular akinesis, normal and maximum adrenergic stimulation, and sulfate loading of the normoxic perfused guinea-pig heart. The Mg_f estimates utilized a new sliding scale derived from the Mg²⁺-dependence of glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase (GAPDH/PGK). The pseudo constant K_GAPDH′·K_PGK′ was measured as ([creatine phosphate][3-phosphoglycerate][lactate]K_LDH/([creatine][P_i[glyceraldehyde 3-phosphate][pyruvate]K_CK), which varied with magnesium due to K_CK (CK, LDH = creatine kinase, lactate dehydrogenase). However, the correct magnesium dependencies of the true constants K_GAPDH·K_PGK and K_CK were taken from the literature. The [Mg²⁺] at which pseudo K_GAPDH′·K_PGK′ equalled true K_GAPDH·K_PGK was the best estimate of Mg_f. Mg_f fell to ≈0.13 mM in hyperkalemic arrest from a control of ≈0.6 mM, rising to ≈0.85 mM only during maximum adrenergic stress. Mg_f increased further to ≈1.3 mM during sulfate loading which induced ATP catabolism. Mg_f and ATP were reciprocally related. Thus; (1) myocardial free [Mg²⁺] judged from GAPDH/PGK mass-action relations changed appreciably only under extreme physiological states; (2) ATP was a major chelator of Mg²⁺ in perfused myocardium, i.e., acute ATP pool size reduction may be associated with increments in Mg_f.     

Effect of Mg2+ concentrations on phosphorylation/activation of phosphorylase b kinase by cAMP/Ca2+-independent,autophosphorylation-dependent protein kinase

In a previous report (Yu and Yang,Biochem. Biophys. Res. Commun. 207, 140–147 (1995)], phosphorylase b kinase from rabbit skeletal muscle was found to be phosphorylated and activated by a cyclic nucleotide- and Ca²⁺-independent protein kinase previously identified as an autophosphorylation-dependent multifunctional protein kinase (autokinase) from brain and liver (Yanget al, J. Biol. Chem. 262, 7034–7040, 9421–9427 (1987)]. In this report, the effect of Mg²⁺ ion concentration on the auto-kinase-catalyzed activation of phosphorylase b kinase is investigated. The levels of phosphorylation and activation of phosphorylase b kinase catalyzed by auto-kinase are found to be dependent on the concentration of Mg²⁺ ion used. Phosphorylation of phosphorylase b kinase at high Mg²⁺ ion (>9 mM) is 2–3 times higher than that observed at low Mg²⁺ ion (1 mM) and this results in a further 2- to 3-fold activation of the enzyme activity at high Mg²⁺ ion. Analysis of the phosphorylation stoichiometry ofα andβ subunits of phosphorylase b kinase at different Mg²⁺ ion concentrations further reveals that the phosphorylation level of theβ subunit remains almost unchanged, whereas the phosphorylation level of theα subunit increases dramatically and correlates with the increased enzyme activity. In similarity with theβ subunit, phosphorylations of myelin basic protein and histone 2A by auto-kinase are also unaffected by Mg²⁺ ion. Taken together, the results provide initial evidence that Mg²⁺ ion may specifically render thea subunit a better substrate for auto-kinase to cause further phosphorylation/activation of phosphorylase b kinase, representing a new mode of control mechanism for the regulation of auto-kinase involved in the phosphorylation and concurrent activation of phosphorylase b kinase.     

Activation of the human red cell calcium ATPase by calcium pretreatment

Some kinetic parameters of the human red cell Ca²⁺-ATPase were studied on calmodulin-free membrane fragments following preincubation at 37°C. After 30 min treatment with EGTA(1 mm) plus dithioerythritol (1 mm), a V _max of about 0.4 μmol P_i/mg × hr and a K _s of 0.3 μm Ca²⁺ were found. When Mg²⁺ (10 mm) or Ca²⁺(10 μm) were also added during preincubation, V _maxbut not Kwas altered. Ca²⁺ was more effective than Mg²⁺, thus increasing V _max to about 1.3 μmol P_i/mg × hr. The presence of both Ca²⁺ and Mg²⁺ during pretreatment decreasedKto 0.15 μm, while having no apparent effect on V _max. Conversely, addition of ATP (2 mm) with either Ca²⁺ or Ca²⁺ plus Mg²⁺increased V_max without affecting K. Preincubation with Ca²⁺ for periods longer than 30 min further increased V_maxand reduced Kto levels as low as found with calmodulin treatment. The Ca²⁺ activation was not prevented by adding proteinase inhibitors (iodoacetamide, 10 mm; leupeptin, 200 μm; pepstatinA, 100 μm; phenylmethanesulfonyl fluoride, 100 μm). The electrophoretic pattern of membranes preincubated with or without Mg²⁺, Ca²⁺ or Ca²⁺ plus Mg²⁺ did not differ significantly from each other. Moreover, immunodetection of Ca²⁺-ATPase by means of polyclonal antibodiesrevealed no mobility change after the various treatments. The above stimulation was not altered by neomycin (200 μm), washing with EGTA (5 mm) or by both incubating and washing with delipidized serum albumin (1 mg/ml), or omitting dithioerythritol from the preincubation medium. On the other hand, the activation elicited by Ca²⁺ plus ATP in the presence of Mg²⁺ was reduced 25–30% by acridine orange (100 μm), compound 48/80 (100 μm) or leupeptin (200 μm) but not by dithio-bis-nitrobenzoic acid (1 mm). The fluorescence depolarization of 1,6-diphenyl-and l-(4-trimethylammonium phenyl)-6-phenyl 1,3,5-hexatriene incorporated into membrane fragments was not affected after preincubating under the different conditions. The results show that proteolysis, fatty acid production, an increased phospholipid metabolism or alteration of membrane fluidity are not involved in the Ca²⁺ effect. Ca²⁺ preincubation may stimulate the Ca²⁺-ATPase activity by stabilizing or promoting the E₁ conformation.