Differential effects of spermine on phosphatidylinositol 3-kinase and phosphatidylinositol phosphate 5-kinase

The metabolism of phosphoinositides plays an important role in the signal transduction pathways. We report here that naturally occuring polyamines affect the activities of phosphatidylinositol (PI) 3-kinase and PI 4-phosphate (PIP) 5-kinase differently. While polyamines inhibited the PI 3-kinase activity, they stimulated the activity of PIP 5-kinase in the order of spermine > spermidine > putrescine. Spermine inhibited the PI 3-kinase activity in a concentration-dependent manner with an IC₅₀ of 100 μM. On the other hand, spermine (5 mH) stimulated the activity of PIP 5-kinase 2–3 fold. Kinetic studies of spermine-mediated inhibition of PI 3-kinase revealed that it was noncompetitive with respect to ATP. The effect of Mg²⁺ and PIP, concentration on kinase activity was sigmoidal, with spermine inhibiting PI 3-kinase activity at all PIP₂ concentrations. While 1 mH calcium stimulated PI 3-kinase activity at submaximal concentrations of Mg²⁺ (1.25 mH), inhibition was observed at optimal concentration of Mg²⁺(2 mM). We propose that spermine may modulate the cellular signal by virtue of its differential effects on phosphoinositide kinases.     

The regulatory role of Mg2+ on rabbit skeletal muscle phosphorylase kinase

The stimulation of phosphorylase kinase by Mg²⁺ was studied. Both the nonactivated and activated kinases are stimulated by Mg²⁺ at concentrations that are 100- to 200-fold greater than ATP. This stimulation is observed at both pH 6.8 and 8.2 and results in a 10-fold increase in the activity of the nonactivated kinase. Mg²⁺ stimulation is additive with that observed by calmodulin. Both the Ca²⁺-dependent and -independent activities of the kinase are stimulated by high [Mg²⁺]. Kinetically this stimulation can be explained by a decrease in the K_m for both phosphorylase b and ATP or an increase in V. The pH $\text{6.8}\text{8.2}$ ratio (0.06) is unaffected by [Mg²⁺] between 5 and 20 mm, but increases when [Mg²⁺] is less than 5 mm or greater than 20 mm. The stimulation by high [Mg²⁺] is explained by a direct effect of this cation on the kinase molecule rather than on its protein substrate, phosphorylase. This activating effect of high [Mg²⁺] does not result in any permanent change in the kinase molecule and can be readily reversed by diluting [Mg²⁺] to a low value.     

Analysis of chlorophyll fluorescence induction curves in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea as a function of magnesium concentration and NADPH-activated light-harvesting chlorophyll ab-protein phosphorylation

The effect of Mg²⁺ concentration and phosphorylation of light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ on various chlorophyll fluorescence induction parameters of isolated pea thylakoids has been studied. (1) Lowering the Mg²⁺ concentration from 3 to 0.4 mM decreases only the variable fluorescence (F_v) and the area above the induction curve while at the same time increasing the slow exponential component of the rise (β_max). (2) A further decrease in Mg²⁺ concentration from 0.4 to 0 mM decreases the initial (F₀) fluorescence level such that the ratio $\text{F}{}_{\mathrm{<mtext>v</mtext>}}\text{F}{}_{\mathrm{<mtext>m</mtext>}}$ increases slightly as does the area above the induction curve and β_max. (3) Thylakoid membranes, phosphorylated at 5 mM Mg²⁺, show an equal decrease in F_v and F₀, no change in the area above the induction curve and an increase in β_max. At 2 mM Mg²⁺, however, phosphorylation induced a more extensive quenching of F_v so that the $\text{F}{}_{\mathrm{<mtext>v</mtext>}}\text{F}{}_{\mathrm{<mtext>m</mtext>}}$ ratio was lowered and the area above the induction curve decreased while β_max increased. (4) When phosphorylated membranes were subsequently suspended in an Mg²⁺-free medium the effect on F₀ due to phosphorylation was found to be additive to that due to the absence of Mg²⁺. The effect of membrane phosphorylation on fluorescence is discussed in relation to the control of excitation energy distribution and shows that different mechanisms operate depending on the background Mg²⁺ levels. At high Mg²⁺ the phosphorylation seems to affect the absorption cross-section of Photosystem II while at lower Mg²⁺ levels there is an additional effect of increased spillover from Photosystem II to I.     

Stimulation of valyl- and isoleucyl-tRNA synthetase reactions by polyamines

The aminoacylation of tRNA catalysed by valyl-tRNA synthetase (EC 6.1.1.9) and isoleucyl-tRNA synthetase (EC 6.1.1.5) fromMycobacterium smegmatis is dependent on the presence of divalent metal ions. Polyamines alone, in the absence of metal ions, do not bring about aminoacylation. In the presence of suboptimal concentrations of Mg²⁺, polyamines significantly stimulate the reaction. Of the cations tested, only Mn²⁺, Co²⁺ and Ca²⁺ can partially substitute for Mg²⁺ in aminoacylation, and spermine stimulates aminoacylation in the presence of these cations also. At neutral pH, spermine deacylates nonenzymatically aminoacyl tRNA. AMP and pyrophosphate-dependent enzymatic deacylation of aminoacyl-tRNA (reverse reaction) is also stimulated by spermine. The inhibitory effect of high concentration of KC1 on aminoacylation is counteracted, by spermine. The low level of activity between pH 8.5–9.0 at 1.2 mM Mg²⁺ is restored to normal level on the addition of spermine. The inhibitory effect of high pH on aminoacylation in the presence of low concentration of Mg²⁺ is also prevntedvby spemine.     

Pathway for ATP synthesis by sarcoplasmic reticulum ATPase

Millisecond mixing and quenching experiments were performed in order to study the rate of phosphorylation by P_i of the Ca²⁺-dependent ATPase of sarcoplasmic reticulum vesicles. A rapid phosphoenzyme formation was observed when the vesicles were preincubated in the absence of Ca²⁺ prior to the addition of P_i and Mg²⁺ to the medium, the half-time being in the range of 6 to 10 ms. A lag phase and a 5- to 10-fold slower rate of phosphoenzyme formation were observed when the enzyme was preincubated with Ca²⁺ prior to the addition to the reaction mixture of P_i, Mg²⁺, and an excess of ethylene glycol bis(β-aminoethyl ether)N,N′-tetraacetic acid. The rate of phosphoenzyme hydrolysis was measured either by the addition of Ca²⁺ or, in the absence of Ca²⁺, by tracing the hydrolysis of radioactive phosphoenzyme upon the addition of nonradioactive P_i. In the presence of Ca²⁺, the rate of phosphoenzyme hydrolysis was found to be one order of magnitude slower than the rate of hydrolysis measured in the absence of Ca²⁺. Different rates of phosphoenzyme formation and cleavage were found depending on whether sarcoplasmic reticulum vesicles or purified Ca²⁺-dependent ATPase were used. A transient phosphorylation by P_i was observed when the enzyme was preincubated in the absence of Ca²⁺ and then added to a medium containing P_i, Mg²⁺, and excess of Ca²⁺. The enzyme was phosphorylated during the initial 100 ms, the phosphoenzyme formed being slowly hydrolyzed in the subsequent incubation intervals. In these conditions ATP synthesis was observed if ADP was added to the mixture 100 ms after starting the reaction. No transient phosphorylation by P_i was observed when the enzyme was preincubated with Ca²⁺. Synthesis of a small but significant amount of ATP was observed when the enzyme was preincubated in the absence of Ca²⁺ and then added to a medium containing P_i, ADP, Mg²⁺, and 20 mm CaCl₂. This was not observed when the enzyme was preincubated in the presence of Ca²⁺.     

The influence of spermine on the structural dynamics of yeast tRNAPhe

A tRNA^Phe derivative carrying ethidium at position 37 in the anticodon loop has been used to study the effect of spermine on conformational transitions of the tRNA. As previously reported (Ehrenberg, M., Rigler, R. and Wintermeyer, W. (1979) Biochemistry 18, 4588–4599) in the tRNA derivative the ethidium is present in three states (T₁–T₃) characterized by different fluorescence decay rates. T-jump experiments show two transitions between the states, a fast one (relaxation time 10–100 ms) between T₁ and T₂, and a slow one (100–1000 ms) between T₂ and T₃. In the presence of spermine the fast transition shows a negative temperature coefficient indicating the existence of a preequilibrium with a negative reaction enthalpy. Spermine shifts the distribution of states towards T₃, as does Mg²⁺, but the final ratio $\text{[}\text{T}{}_2\text{]}\text{[}\text{T}{}_1\text{]}$ obtained with spermine is higher than with Mg²⁺, which we tentatively interpret to mean that spermine stabilizes one particular conformation of the anticodon loop.     

Effect of spermine on peptide-bond formation,catalyzed by ribosomal peptidyltransferase

The effect of spermine on the binding of AcPhe-tRNA to poly(U)-programmed ribosomes (step 1) and on the puromycin reaction (step 2) has been studied in a cell-free system, derived from E. coli.In the absence of ribosomal wash (FWR fraction) and at suboptimal concentration of Mg⁺⁺ (6 mM), spermine stimulated the binding of AcPhe-tRNA at least five fold, while at 10 mM Mg⁺⁺ there was a three fold stimulation. The above stimulatory effect was decreased at 6 mM Mg⁺⁺, or was abolished at 10 mM Mg⁺⁺ by the presence of FWR during the binding. Beside the stimulatory effect, spermine enhanced the stability of initiation complex AcPhe-tRNA-poly(U)-ribosome.In step 2, spermine affected the final degree of puromycin reaction and the activity status of peptidyltransferase. Both stimulatory and inhibitory effects have been observed, depending on the experimental conditions followed during the binding of the donor and during the peptide bond formation.     

Activation state of ribulose bisphosphate carboxylase in soybean leaves

Conditions for extraction and assay of ribulose-1,5-bisphophate carboxylase present in an in vivo active form (initial activity) and an inactive form able to be activated by Mg²⁺ and CO₂ (total activity) were examined in leaves of soybean, Glycine max (L.) Merr. cv Will. Total activity was highest after extracts had preincubated in NaHCO₃ (5 millimolar saturating) and Mg²⁺ (5 millimolar optimal) for 5 minutes at 25°C or 30 minutes at 0°C before assay. Initial activity was about 70% of total activity. K_act (Mg²⁺) and K_act (CO₂) were approximately 0.3 millimolar and 36 micromolar, respectively. The carry-over of endogenous Mg²⁺ in the leaf extract was sufficient to support considerable catalytic activity. While Mg²⁺ was essential for both activation and catalysis, Mg²⁺ levels greater than 5 millimolar were increasingly inhibitory of catalysis. Similar inhibition by high Mg²⁺ was also observed in filtered, centrifuged, or desalted extracts and partially purified enzyme. Activities did not change upon storage of leaves for up to 4 hours in ice water or liquid nitrogen before homogenization, but were about 20% higher in the latter. Activities were also stable for up to 2 hours in leaf extracts stored at 0°C. Initial activity quickly deactivated at 25°C in the absence of high CO₂. Total activity slowly declined irreversibly upon storage of leaf homogenate at 25°C.     

[3H] CGP 39653 Binding to the Agonist Site of the N-Methyl- D-Aspartate Receptor Is Modulated by Mg2+ and Polyamines Independently of the Arcaine-Sensitive Polyamine Site

Abstract: This study investigated the binding of [³H] CGP 39653, a novel high-affinity antagonist of the N-methyl-D- aspartate (NMDA) recognition site of the NMDA receptor complex. [³H] CGP 39653 bound to the NMDA receptor in well washed rat brain membranes with an affinity of about 15 nM. Other NMDA site drugs inhibited [³H] CGP 39653 binding with the following order of potency: DL-(tetrazol-5- yl)glycine > glutamate > CGS 19755 > DL-2-amino-5- phosphonovalerate (DL-AP5) > NMDA. Glycine and 5, 7- dichlorokynurenate partially inhibited binding. The poly-amines spermine and spermidine increased [³H] CGP 39653 binding (EC₅₀ values of 10 and 22 μM, respectively). This effect was mimicked by arcaine, 1, 5-diethylaminopiperidine, diaminodecane, diethylenetriamine, and Mg²⁺. The increase in [3H] CGP 39653 was a result of an increased affinity of the binding site for the ligand with very little effect on binding site density. Spermine and Mg²⁺also increased the affinity of the antagonists DL-AP5 and CGS 19755, but had only minor effects on the affinity of glutamate and NMDA. Arcaine did not reverse the enhancement of [3H] CGP 39653 binding by spermine, spermidine, or Mg²⁺. Channel-blocking dissociative anesthetics, including dizocilpine and ketamine, did not alter basal or Mg²⁺-stimulated [3H] CGP 39653 binding. Spermine did not alter either the enhancement of [³H]- dizocilpine by glutamate or the inhibition of [³H]dizocilpine by DL-AP5 or CGS 19755. These studies show that poly-amines and divalent cations selectively enhance the affinity of antagonists for the agonist binding site on the NMDA receptor complex. However, this effect is mediated by a site independent of the primary polyamine site defined using [³H] dizocilpine binding.     

Enhancement of UDP-galactose:mucin galactosyltransferase activity by spermine

A microsomal preparation prepared from the mucosal lining of canine trachea catalyzed the transfer of galactose from its uridine diphosphate derivative to sialidase-treated ovine submaxillary mucin. Maximal incorporation occurred at 30 mm mn²⁺. When the concentration of mn²⁺ in the reaction mixture was reduced to 2.5 mm, approximately two-thirds of the enzymatic activity was lost, but full activity could be restored by the addition of 1 mm spermine. Under these conditions spermine did not affect the K_m for UDP-galactose, but lowered the K_m for sialidase-treated ovine submaxillary mucin and Mn²⁺ by a factor of 10. The effect of spermine was abolished with increasing concentrations of Mn²⁺, and in the absence of the metal, enzymatic activity was lost and could not be restored by the addition of spermine. Spermidine also stimulated activity at low levels of Mn²⁺, but to a lesser degree than spermine. A slight stimulatory effect was consistently derived from putrescine as well, while cadaverine, putreanine, and monoamines were ineffectual. Spermine had a similar effect on the enzymatic transfer of GalNAc to a protein core acceptor but had little or no effect on the enzymatic transfer of sialic acid to sialidase-treated ovine submaxillary mucin, galactose to N-acetylglucosamine, or fucose to sialidase-galactosidase-treated fetuin. Similar results were obtained with enzyme preparations prepared from canine submaxillary glands. Other polycationic compounds such as protamine, histone, and polylysine also stimulated enzymatic activity at suboptimal concentrations of mn²⁺.     

Evidence for an Amiloride-Inhibited Mg/2H Antiporter in Lutoid (Vacuolar) Vesicles from Latex of Hevea brasiliensis

Lutoids represent a lysosomal microvacuolar compartment of rubber-tree (Hevea brasiliensis) latex. We observed acidification of isolated vesicles after imposing an outward Mg²⁺ diffusion gradient and dissipation of a preformed pH gradient in the presence of exogenous Mg²⁺. These results suggest the presence of a Mg²⁺/H⁺ antiporter. The maximum Mg²⁺/H⁺ exchange rate was observed at pH 8.5. The K_m values for Mg²⁺ (2.6 mm) were identical for both influx and efflux experiments. When membrane potential was clamped at zero with K⁺ and valinomycin, the response of the membrane potential probe oxonol VI showed that the Mg²⁺/H⁺ exchange was electroneutral. Mg²⁺/H⁺ exchange was inhibited by amiloride and imipramine. Both the inhibiting concentration range and the K_m for Mg²⁺ are similar to those reported for the Mg²⁺/2Na⁺ antiporter in animals cell. These data are consistent with the existence of a Mg²⁺/2H⁺ antiporter in a plant tonoplast.     

The effect of hormones on metal and metal-ATP interactions with fat cell adenylate cyclase.

1-adrenaline, ACTH and glucagon activate the adenylate cyclase of rat adipocytes by decreasing its S_0.5(Mg²⁺) (concentration yielding 0.5 V_max) from its basal value of 11.5 to 1.2, 0.3 and 1.8 mM and by increasing its K_i(ATP^4?) from 0.03 to 0.25; 0.62 and 0.16 mM respectively. The kinetic properties of the enzyme are regulated by its state of saturation with ATP^4? or Mg²⁺; its saturation with ATP^4? and citrate^3? suppressed its basal and hormone-dependent activities. The hormone-dependent decrease in K_m and increase in V_max of the enzyme occur when shifting from suboptimal low concentrations of hormone and Mg²⁺ to optimal conditions, i.e., high concentration of hormone and low concentration of Mg²⁺. The increase in the state of saturation of the enzyme with Mg²⁺ decreases the hormone-dependent effects on V_max and results in identical values of K_m (0.14 mM) for its basal and 1-adrenaline dependent activities. CaCl₂ saturation curves at 5 mM ATP with either 5, 10 or 20 mM MgCl₂ show that the substitution of 5 mM MgCl₂ by 10 mM and 20 mM MgCl₂ increased the K_i(Ca²⁺) of the enzyme from 0.19 to 0.49 and 0.94 mM but decreased its K_i(CaATP) from 0.42 to 0.19 and 0.14 mM respectively. Only when the concentration of MgCl₂ exceeded that of ATP did 1-adrenaline and ACTH activate the enzyme by increasing its K_i(Ca²⁺), although only ACTH increased its K_i(CaATP). An increase in energy charge would decrease the intracellular concentrations of Mg²⁺ and Ca²⁺ because ATP^4? has stronger binding constants for Mg²⁺ and Ca²⁺ than ADP^3? and AMP^2?. Hence, the reported properties of the enzyme suggests that changes in energy charge may allow for metabolic feedback control of the hormonal responsiveness of the Mg²⁺, Ca²⁺, ATP^4? -sensitive adenylate cyclase.     

The response to ribulose bisphosphate4− (RuBP4−) and RuBP-Mg2− in catalysis by structurally divergent RuBP carboxylase/oxygenases

Free ribulose bisphosphate (RuBP^4?) rather than its magnesium complex (RuBP-Mg^2?) was the apparent substrate for spinach ribulose bisphosphate carboxylase/oxygenase. The apparent K_m for total RuBP (pH 8.0 at 30° C) increased with increasing Mg²⁺ concentrations from 11.6 μM at 13.33 mM Mg²⁺ to 32.6 μM at 40.33 mM Mg²⁺. Similarly the apparent K_m for RuBP-Mg^2? complex increased with increasing Mg²⁺ from 9.4 μM at 13.33 mM Mg²⁺ to 29.7 μM at 40.33 mM Mg²⁺. However, the K_m values for uncomplexed RuBP^4? were independent of the (saturating) concentration of Mg²⁺ (K_m=2.2 μM). The V_max did not vary with the changing concentrations of Mg²⁺. In contrast, the K_m for total RuBP remained constant with varying Mg²⁺ concentrations (K_m=59.5 μM) for the enzyme from R. rubrum. The apparent K_m for the RuBP-Mg^2? complex decreased with increasing Mg²⁺ concentrations from 16.0 μM at 7.5 mM Mg²⁺ to 5.9 μM at 27.5 mM Mg²⁺. The initial velocity for the C. vinosum enzyme was also found to be independent of the (saturating) concentration of Mg²⁺ when total RuBP was varied in the assay. Thus the response to total RuBP by these two bacterial enzymes, which markedly differ in structure, was closely similar.     

Yeast mitochondrial calcium uptake: Regulation by polyamines and magnesium ions

Spermine, spermidine, and magnesium ions modulate the kinetic parameters of the Ca²⁺ transport system ofEndomyces magnusii mitochondria. Mg²⁺ at concentrations up to 5 mM partially inhibits Ca²⁺ transport with a half-maximal inhibiting concentration of 0.5 mM. In the presence of 2 mM MgCl₂, theS _0.5 value of the Ca²⁺ transport system increases from 220 to 490 µM, which indicates decreased affinity for the system. Spermine and spermidine exert an activating effect, having half-maximal concentrations of 12 and 50 µM, respectively. In the case of spermine, theS _0.5 value falls to 50–65 µM, which implies an increase in the transport system affinity for Ca²⁺. Both Mg²⁺ and spermine cause a decrease of the Hill coefficient, giving evidence for a smaller degree of cooperativity. Spermine and spermidine enable yeast mitochondria to remove Ca²⁺ from the media completely. In contrast, Mg²⁺ lowers the mitochondrial buffer capacity. When both Mg²⁺ and spermine are present in the medium, their effects on theS _0.5 value and the free extramitochondrial Ca²⁺ concentration are additive. The ability of spermine and Mg²⁺ to regulate yeast mitochondrial Ca²⁺ transport is discussed.     

The influence of spermine on the structural dynamics of yeast tRNA

A tRNA^Phe derivative carrying ethidium at position 37 in the anticodon loop has been used to study the effect of spermine on conformational transitions of the tRNA. As previously reported (Ehrenberg, M., Rigler, R. and Wintermeyer, W. (1979) Biochemistry 18, 4588–4599) in the tRNA derivative the ethidium is present in three states (T₁–T₃) characterized by different fluorescence decay rates. T-jump experiments show two transitions between the states, a fast one (relaxation time 10–100 ms) between T₁ and T₂, and a slow one (100–1000 ms) between T₂ and T₃. In the presence of spermine the fast transition shows a negative temperature coefficient indicating the existence of a preequilibrium with a negative reaction enthalpy. Spermine shifts the distribution of states towards T₃, as does Mg²⁺, but the final ratio obtained with spermine is higher than with Mg²⁺, which we tentatively interpret to mean that spermine stabilizes one particular conformation of the anticodon loop.     

Some characteristics of cotton pyrophosphatase activation by magnesium

The effect of Mg²⁺ ions on inducing pyrophosphatase activity of germinating cotton (Gossypium hirsutum L.) seeds was investigated. The presence of Mg²⁺ ions in the germination medium markedly shortened time for the attainment of the pyrophosphatase maximum activity (T _max). In the absence of Mg²⁺ ions in the nutrient medium, T _max comprised 6.0–6.5 days, whereas in the presence of 3–5 mM Mg²⁺, T _max was decreased to 3–4 days. An increase in the concentration of Mg²⁺ ions in the medium up to 5 mM resulted in an increase in pyrophosphatase activity. The effect of Mg²⁺ ions on the activity of a purified pyrophosphatase preparation isolated from three-day-old cotton seedlings was investigated. Mg²⁺ ions did not affect the rate of attainment of a maximum pyrophosphatase activity, but decreased the value of the Michaelis-Menten constant.     

Structure and Dynamics of Double Helices in Solution: Modes of DNA Bending

Abstract

The long range structure of DNA restriction fragments has been analysed by electro-optical measurements. The overall rotation time constants observed in a low salt buffer with monovalent ions is shown to decrease upon addition of Mg²⁺ or spermine. Since the circular dichroism and also the limiting value of the linear dichroism remain almost constant under these conditions, the effect is attributed to a change of the long range structure. According to a weakly bending rod model, the persistence length decreases from about 600 Å in the absence of Mg²⁺ or spermine to about 350 Å in the presence of these ions. The persistence length measured in the presence of Mg²⁺ is almost independent of temperature in the range of 10 to 40 °C. The nature of DNA bending is analysed by measurements of bending amplitudes and time constants from dichroism decay curves. The observed absence of changes in the bending amplitudes upon addition of Mg²⁺ or spermine, even though addition induces changes of the persistence length by a factor of 2, is hardly consistent with simple thermal bending. The combined results, including the remarkably small temperature dependence of persistence length and bending amplitude, can be explained by the existence of two bending effects: inherent curvature of DNA dominates at low temperature, whereas thermal bending prevails at high temperature. Analysis of bending amplitudes from dichroism decay curves according to an arc model provides an approximate measure for the degree of bending in restriction fragments. The model is consistent with the observed chain length dependence of bending amplitudes and provides an approximate curvature corresponding to a radius of ab out400Å. Thus the curvature observed in restriction fragments is similar to that observed for high molecular DNA condensed into toroids by addition of ions like spermine.

Particularly strong bending of DNA is induced by [CO(NH₃)₆]³⁺, indicated by an apparent persistence length of 200 Å and an increased bending amplitude together with a reduced limit value of the linear dichroism. This effect is attributed to the high charge density of this ion and potential site binding.     

Divalent metal ion effects on a mutant histidinol phosphate phosphatase from Salmonella typhimurium

The effect of divalent metal ions on the activity of a mutant histidinol phosphate phosphatase has been studied. The enzyme was isolated from strain TA387, a mutant of Salmonella typhimurium with a nonsense lesion near the midpoint of the bifunctional hisB gene. Mn²⁺, Mg²⁺, Co²⁺, and Zn²⁺ shift the optimal pH of phosphatase activity to 6.5 while Be²⁺ and Ca²⁺ have no effect on the shape of the pH profile. In the absence of divalent metal ions, the pH optimum is 7.5. Four Me²⁺ ions, Mn²⁺, Co²⁺, Zn²⁺, and Fe²⁺ decreased the K_m of histidinol phosphate at pH 6.5 from 5.5 mm (without Me²⁺) to 0.14 mm. Ni²⁺ and Be²⁺ increased the K_m to 22.2 and 25.0 mm, respectively, and Ca²⁺ and Mg²⁺ had an intermediate effect. Changes in maximal velocity were substantially less, only about 2-fold changes being observed. It was shown that the maximal velocity at optimal pH was the same in the absence and presence of Mn²⁺. Kinetic analysis indicated that there was a rapid equilibrium-ordered addition of Mn²⁺ to the enzyme before the addition of the substrate, histidinol phosphate. A k_imn²⁺ of 4.3 μm was calculated for the metal ion activation at both pH 6.5 and 7.5. Addition of ethyl-enediaminetetracetate (EDTA) strongly inhibited the phosphatase; inhibition could be reversed by addition of several Me²⁺ ions, Mg²⁺ being the most efficient followed by Mn²⁺. Prolonged incubation with EDTA led to irreversible inactivation.     

Purification and properties of ATPase from an alkalophilic Bacillus

ATPase was purified from an alkalophilic Bacillus. The enzyme has a molecular weight of 410,000 and consists of five types of subunits of molecular weights of 60,000 (α), 58,000 (β), 34,000 (γ), 14,000 (δ), and 11,000 (?). The subunit structure is suggested to be α₃β₃γδ?. The enzyme is activated by Mg²⁺ and Ca²⁺. The pH optima of the enzyme with 0.1 and 2.0 mm Mg²⁺ are 9 and 6, and those with 1 and 10 mm Ca²⁺ are 8–9 and 7, respectively. Ca²⁺-ATPase hydrolyzes only ATP, whereas Mg²⁺-ATPase hydrolyzes GTP and, to a lesser extent, ATP. The values of V and K_m of the enzyme with ATP in the presence of 10 mm Ca²⁺ or 0.6 mm Mg²⁺ at pH 7.2 are 17 or 0.5 units/mg protein and 1.2 or 0.3 mm, respectively. The enzyme with Mg²⁺ is appreciably activated by HCO^?₃. Relationship of the ATPase to the active transport system in the bacterium is suggested.