Inhibition of Na+ and K+-stimulated ATPase of rabbit heart sarcolemma after, administration of heparin.

Intravenous heparin administration caused a marked inhibition of Mg⁺⁺-dependent (Na⁺+K⁺)-stimulated ATPase activity of sarcolemmal (SL) membranes prepared from rabbit heart, whereas basal Mg⁺⁺-ATPase was not affected. The inhibition depended on K⁺ concentration and was reversed only in the presence of albumin. Plasma free fatty acid (FFA) concentrations were raised in all animals, after heparin administration. The results obtained support the concept that FFA or other lipids originating in the plasma by the action of lipolytic enzymes released by heparin are involved in the mechanism of inhibition.     

Salinity-dependence of the properties of gill (Na++K+-ATPase in rainbow trout Oncorhynchus mykiss

• 1.1. The expected higher gill (Na⁺+K⁺)-ATPase activity in rainbow trout adapted to brackish water (BW) with respect to fresh water (FW) is accompanied by some changes in the enzyme kinetics while the enzyme sensitivity to ouabain is unaffected
• 2.2. Maximal activation is attained under the optimal conditions of 4 mM ATP, 7.5 mM Mg²⁺, 50 mM Na⁺, 2.5 mM K⁺, pH 7.0 in FW, and 3 mM ATP, 10 mM Mg²⁺, 100 mM Na⁺, 10 mM K⁺, pH 7.5 in BW.
• 3.3. The change of the enzyme activation kinetics by Mg²⁺, ATP, Na⁺ and K⁺ from simple saturation in FW to cooperativity in BW and other habitat-dependent variations including the pH alkaline shift in BW are hypothetically related to an adaptive significance to the different environmental salinity.
• 4.4. Gill total lipids and phospholipids are 30% lower in BW than in FW while their ratio is constant; some differences in gill total lipid fatty acid composition between FW and BW do not significantly affect the unsaturation parameters.

     

Conformational changes of membrane-bound (Na+−K+)-ATPase as revealed by antibody inhibition

Summary As different structural states of the (Na⁺–K⁺)-ATPase (EC 3.6.1.3) may lead to a changed reactivity to antibodies, the influence of Na⁺, K⁺, Mg⁺⁺, P_i and ATP on the reaction between highly purified (Na⁺–K⁺)-ATPase and antibodies directed against the membrane-bound enzyme was measured. The antigen antibody reaction was registered by measuring the antibody inhibition of (Na⁺–K⁺)-ATPase activity.In themembrane-bound but not in thesolubilized enzyme four different degrees of antibody inhibition were obtained at equilibrium of the antigen antibody reaction if different combinations of Na⁺, K⁺, Mg⁺⁺ and ATP were present during the incubation with the antibodies. Corresponding to the different degrees of inhibition, different rates of enzyme inhibition were measured. (a) The smallest degree of enzyme inhibition was obtained when (i) only Mg⁺⁺, (ii) Mg⁺⁺ and Na⁺ or (iii) Mg⁺⁺ and K⁺ were present during the antigen antibody reaction. (b) The enzyme activity was inhibited more strongly if Na⁺, Mg⁺⁺ and ATP were present together. (c) It was inhibited even more if only (i) Na⁺, (ii) K⁺, (iii) ATP or both (iv) ATP and Na⁺, (v) ATP and K⁺, (vi) ATP and Mg⁺⁺, or if (vii) no ATP and activating ions were present. (d) The highest degree of antibody inhibition was obtained if Mg⁺⁺, ATP and K⁺ were present together.In the presence of Mg⁺⁺ plus ADP and in the presence of Mg⁺⁺ plus the ATP analog adenylyl (--methylene) diphosphonate, Na⁺ and K⁺ did not influence the degree of antibody inhibition as they did in the presence of Mg⁺⁺ plus ATP. It was further found that the degree of antibody inhibition in the presence of Mg⁺⁺, ATP and K⁺ was affected by the sequence in which K⁺ and ATP were added to the enzyme prior to the addition of the antibodies.It is suggested that by antibody inhibition different conformations of the (Na⁺–K⁺)-ATPase could be detected. These conformations may possibly not occur in the solubilized enzyme and therefore do not seem to be necessarily linked to the intermediary steps of the ATP hydrolysis of the enzyme. The structural changes which are induced by Na⁺ and K⁺ in the presence of Mg⁺⁺ plus ATP are proposed to occur during the Na⁺–K⁺ transport.     

Potassium-activated phosphatase from human red blood cells

Summary The cell membrane K⁺-activated phosphatase activity was measured in reconstituted ghosts of human red cells having different ionic contents and incubated in solutions of varying ionic composition. When K⁺-free ghosts are suspended in K⁺-rich media, full activation of the phosphatase is obtained. Conversely, very little ouabainsensitive activity is detected in K⁺-rich ghosts suspended in K⁺-free media. These results, together with the fact that Na⁺ competitively inhibits the effects of K⁺ only when present externally, show that the K⁺ site of the membrane phosphatase is located at the outer surface of the cell membrane. The Mg⁺⁺ requirements for K⁺ activation of the membrane phosphatase are fulfilled by internal Mg⁺⁺. Addition of intracellular Na⁺ to ATP-containing ghosts raises the apparent affinity of the enzyme for K⁺, suggesting that the sites where ATP and Na⁺ produce this effect are located at the inner surface of the cell membrane. The asymmetrical features of the membrane phosphatase are those expected from the proposed role of this enzyme in the Na⁺–K⁺-ATPase system.The authors are established investigators of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.     

Sensitivity of mitochondrial Mg++ flux to reagents which affect K+ flux

Effects on Mg⁺⁺ transport in rat liver mitochondria of three reagents earlier shown to affect mitochondrial K⁺ transport have been examined. The sulfhydryl reactive reagent phenylarsine oxide, which activates K⁺ flux into respiring mitochondria, also stimulates Mg⁺⁺ influx. The K⁺ analog Ba⁺⁺, when taken up into the mitochondrial matrix, inhibits influx of both K⁺ and Mg⁺⁺. The effect on Mg⁺⁺ influx is seen only if Mg⁺⁺, which blocks Ba⁺⁺ accumulation, is added after a preincubation with Ba⁺⁺. Thus the inhibition of Mg⁺⁺ influx appears to require interaction of Ba⁺⁺ at the matrix side of the inner mitochondrial membrane. Added Ba⁺⁺ also diminishes observed rates of Mg⁺⁺ efflux but not K⁺ efflux. This difference may relate to a higher concentration of Ba⁺⁺ remaining in the medium in the presence of Mg⁺⁺ under the conditions of our experiments. Pretreatment of mitochondria with dicyclohexylcarbodiimide (DCCD), under conditions which result in an increase in the apparentK _m for K⁺ of the K⁺ influx mechanism, results in inhibition of Mg⁺⁺ influx from media containing approximately 0.2 mM Mg⁺⁺. The inhibitory effect of DCCD on Mg⁺⁺ influx is not seen at higher external Mg⁺⁺ (0.8 mM). This dependence on cation concentration is similar to the dependence on K⁺ concentration of the inhibitory effect of DCCD on K⁺ influx. Although mitochondrial Mg⁺⁺ and K⁺ transport mechanisms exhibit similar reagent sensitivities, whether Mg⁺⁺ and K⁺ share common transport catalysts remains to be established.Abbreviations used: DCCD, dicyclohexylcarbodiimide; PheAsO, phenylarsine oxide.     

K+-stimulated phosphatase of microsomes from gastric mucosa

The light microsomal fraction was isolated from homogenates of rabbit and bullfrog gastric mucosa. On examination with the electron microscope, the light microsomes appear as tubular membranous structures with morphology and dimensions similar to the elements of the smooth-surfaced endoplasmic reticulum seen in intact oxyntic cells. A K⁺-stimulated, Mg⁺⁺-requiring p-nitrophenylphosphatase has been demonstrated in the gastric microsomes. Neither Na⁺ nor ouabain (10^?6–10^?3 M) altered the K⁺-stimulated phosphatase. SCN^? was not very effective as an inhibitor of the gastric microsomal phosphatase, in contrast to the effect of this anion on the ATPase activity; however, the gastric phosphatase as well as the ATPase are destroyed by phospholipase C, thus showing the lipoprotein nature of these enzymes. Kinetics of the K⁺ activation of the microsomal phosphatase suggest that the K⁺-PNPP complex is the active substrate for the enzymic reaction. Rb⁺, NH₄ ⁺ and Cs⁺ will substitute to some degree for K⁺ as an activator of the microsomal phosphatase. It is pointed out that K⁺ is an essential requirement for HCl secretion in intact gastric mucosa and the replacement of K⁺ with Rb⁺, Cs⁺ and NH₄⁺ is discussed. The K⁺-stimulated phosphatase presented in this paper may play a role in the H⁺ secretion process.     

Effects of nucleotides and Na + on ouabain activation of the phosphatase associated with Na + , K + -ATPase

Ouabain activation of the phosphatase associated with Na⁺,K⁺-ATPase is a time-dependent process which is stimulated by ATP and other nucleotides. Further stimulation by Na⁺ is observed under certain conditions. The stimulatory effect of ATP was found to be due to an increase in the affinity of the enzyme for ouabain. The time required for maximal ouabain activation to be achieved was decreased by ATP and further decreased by ATP + Na⁺.These conditions for maximal activation by ouabain are similar to those required for maximal ouabain binding and suggest that the same ouabain site is responsible for activation of Mg²⁺-dependent phosphatase and for inhibition of Na⁺,K⁺-ATPase and K⁺-phosphatase.     

Nature of the transport ATPase-digitalis complex. VI. Purification of and ouabain binding to a highly active cardiac Na+, K+-ATPase

A Na⁺,K⁺-ATPase has been isolated from canine heart with a specific activity as high as 200 μmoles of inorganic phosphate/mg protein/hour. Activity is not due to simple detergent activation since specific ouabain binding (i.e., [Mg⁺⁺,Na⁺,ATP] or [Mg⁺⁺,P_i]-ligand dependent) ranged from 200–450 pmoles/mg protein. Specific ouabain binding activities are up to ten times greater than heretofore reported.     

Modification of the erythrocyte membrane by sulfhydryl group reagents

Summary In this study, the consequences of modification of human erythrocyte membrane sulfhydryl groups by N-ethyl maleimide (NEM), 5,5dithiobis-(2-nitrobenzoic acid) (DTNB) andp-hydroxymercuriphenyl sulfonate (PHMPS) were investigated. These reagents differ in chemical reactivity, membrane penetrability and charge characteristics.Results of sulfhydryl modification were analyzed in terms of inhibitory effects on activities of five membrane enzymes; Mg⁺⁺- and Na⁺, K⁺-ATPase, K⁺-dependent and independentp-nitrophenyl phosphatase (NPPase) and DPNase. Structural considerations involved in the sulfhydryl-mediated inhibition were evaluated by studying the changes in susceptibility to sulfhydryl alteration produced by shearing membranes into microvesicles and by the addition of the membrane modifiers, Mg⁺⁺ and ATP.Conclusions from the data suggest that the effects of NEM appeared to result from modification of a single class of sulfhydryls; DTNB interacted with two different sulfhydryl classes. Increasing concentrations of PHMPS resulted in the sequential modification of many types of sulfhydryls, presumably as a result of increasing membrane structural disruption. DTNB and PHMPS caused solubilization of about 15% of membrane protein at concentrations giving maximal enzyme inhibition.In contrast to the usually observed parallels between Na⁺, K⁺-ATPase and K⁺-dependent NPPase, activities of Mg⁺⁺-ATPase, Na⁺, K⁺-ATPase and K⁺-dependent NPPase varied independently as a result of sulfhydryl modification. We suggest complex structural and functional relationships exist among these components of the membrane ATP-hydrolyzing system.Our studies indicate that the effects of sulfhydryl group reagents on these membrane systems should not be ascribed to sulfhydryl modificationper se, but rather to the resulting structural perturbations. These effects depend upon the structural characteristics of the particular membrane preparation studied and on the chemical characteristics of the sulfhydryl group reagent used.     

Production of Alkaline Lipase by Corynebacterium paurometabolum, MTCC 6841 Isolated from Lake Naukuchiatal, Uttaranchal State, India

A moderately psychrophilic bacterium Corynebacterium paurometabolum MTCC 6841 (gram positive, short rod type) producing extracellular alkaline lipase was isolated from Lake Naukuchiatal, Uttaranchal, India. The bacterium was able to grow within a broad range of pH (5–10). Soyabean oil and olive oil served as the best carbon sources for lipase production. The bacterium preferred inorganic nitrogenous compounds, NaNO₃ and KNO₃, over organic nitrogenous compound for its growth. Maximum lipase production occurred at 25°C and 8.5 pH. The enzyme activity was found to be maximum at the same values of temperature and pH. The enzyme was reasonably stable in the presence of various organic solvents. No significant effect of Ca⁺, Cu⁺⁺, Fe⁺⁺, Na⁺, K⁺, Mg⁺⁺, Mn⁺, NH4⁺, Co⁺⁺ ions over enzyme activity was detected. Treatment with EDTA reduced the activity to nearly one half.     

Molecular properties and sensitivity to cations of β-Galactosidase from Streptococcus thermophilus with four enzyme substrates

Summary -Galactosidase (E.C. 3.2.1.23) from an autolytic strain of Streptococcus thermophilus was purified to near homogeneity (466 U/mg protein). The quaternary structure of the -galactosidase was complex, the enzyme apparently existing in three forms in solution (as determined by HPLC ion-exchange or gel permeation chromatography). One form of the enzyme was stable but a second form dissociated with time in a temperature- and concentration-dependent manner to give the stable form. A single subunit was identified with a molecular weight of 116 000.Activation of enzyme activity by cations (Mg²⁺, K⁺ and Na⁺) was complex and varied markedly according to whether o-nitrophenyl--d-galactopyranoside (ONPG), d-nitrophenyl--d-galactopyranoside (PNPG), 4-methylumbelliferyl--d-galactopyranoside (4MeUmG) or lactose was the enzyme substrate. With all substrates there was synergistic activation with either Mg⁺⁺ and K⁺ or Mg⁺⁺ and Na⁺. Na⁺ was the better activator with either ONPG or 4MeUmG as the substrate while K⁺ was the better activator of lactose and PNPG hydrolysis. With either ONPG or 4MeUmG as substrate, and in the presence of both Mg²⁺ and K⁺, Na⁺ further enhanced activity. In contrast, Na⁺ was a competitive inhibitor of the Mg²⁺ and K⁺ activated reaction with either lactose or PNPG as the substrate. Analysis of the effect of the cations on the kinetics of lactose hydrolysis showed they all acted by increasing the binding of lactose of the enzyme as well as by increasing the maximum activity. Weak competitive inhibition of activity (with lactose) by galactose was found with a K_i of 350 mM galactose.     

Early changes of 'leak flux' and the cation content of lymphocytes by concanavalin A.

The leak fluxes of Na⁺, K⁺, Mg⁺⁺ and Ca⁺⁺ in mouse thymocytes are increased by Concavaline A (Con A), within minutes after mitogen addition. The intracellular Mg⁺⁺ and K⁺ concentrations were decreased and the Na⁺ and Ca⁺⁺ contents were increased by Con A in mouse thymocytes and spleen cells.     

The phosphatase activity of the plasma membrane Ca pump. Activation by acidic lipids in the absence of Ca increases the apparent affinity for Mg

The purified PMCA supplemented with phosphatidylcholine was able to hydrolyze pNPP in a reaction media containing only Mg²⁺ and K⁺. Micromolar concentrations of Ca²⁺ inhibited about 75% of the pNPPase activity while the inhibition of the remainder 25% required higher Ca²⁺ concentrations. Acidic lipids increased 5-10 fold the pNPPase activity either in the presence or in the absence of Ca²⁺. The activation by acidic lipids took place without a significant change in the apparent affinities for pNPP or K⁺ but the apparent affinity of the enzyme for Mg²⁺ increased about 10 fold. Thus, the stimulation of the pNPPase activity of the PMCA by acidic lipids was maximal at low concentrations of Mg²⁺. Although with differing apparent affinities vanadate, phosphate, ATP and ADP were all inhibitors of the pNPPase activity and their effects were not significantly affected by acidic lipids. These results indicate that (a) the phosphatase function of the PMCA is optimal when the enzyme is in its activated Ca²⁺ free conformation (E2) and (b) the PMCA can be activated by acidic lipids in the absence of Ca²⁺ and the activation improves the interaction of the enzyme with Mg²⁺.     

Further Studies on the Roles of Sodium and Potassium in the Generation of the Electro-Olfactogram : Effects of mono-, di-, and trivalent cations

In the negative EOG-generating process a cation which can substitute for Na⁺ was sought among the monovalent ions, Li⁺, Rb⁺, Cs⁺, NH₄⁺, and TEA⁺, the divalent ions, Mg⁺⁺, Ca⁺⁺, Sr⁺⁺, Ba⁺⁺, Zn⁺⁺, Cd⁺⁺, Mn⁺⁺, Co⁺⁺, and Ni⁺⁺, and the trivalent ions, Al⁺⁺⁺ and Fe⁺⁺⁺. In Ringer solutions in which Na⁺ was replaced by one of these cations the negative EOG's decreased in amplitude and could not maintain the original amplitudes. In K⁺-Ringer solution in which Na⁺ was replaced by K⁺, the negative EOG's reversed their polarity. Recovery of these reversed potentials was examined in modified Ringer solutions in which Na⁺ was replaced by one of the above cations. Complete recovery was found only in the normal Ringer solution. Thus, it was clarified that Na⁺ plays an irreplaceable role in the generation of the negative EOG's. The sieve hypothesis which was valid for the positive EOG-generating membrane or IPSP was not found applicable in any form to the negative EOG-generating membrane. The reversal of the negative EOG's found in K⁺- , Rb⁺- , and Ba⁺⁺-Ringer solutions was attributed to the exit of the internal K⁺. It is, however, not known whether or not Cl^- permeability increases in these Na⁺-free solutions and contributes to the generation of the reversed EOG's.     

In-vitro-studies on the influence of cations, neurotransmitters and tubocurarine on calcium-ganglioside-interactions.

The influence of K⁺, Na⁺, Mg⁺⁺, Li⁺, a serotonin, acetylcholine and tubocurarine on calcium-ganglioside-interactions was studied by way of equilibrium dialysis using ⁴⁵Ca as tracer. Experiments were carried out at 22 °C and 4 °C, respectively. The concentrations of the substances were in the range of physiologically relevant conditions. Cations caused a release of Ca⁺⁺ from calcium-ganglioside-complexes in the sequence of their molar efficiency: Mg⁺⁺ ≈ Li⁺ > K⁺ ≈ Na⁺. Tubocurarine, serotonin and acetylcholine also affected calcium-ganglioside-interactions. Ca⁺⁺ was displaced from ganglioside most effectively by tubocurarine, followed by serotonin, whereas acetylcholine competed considerably more weakly.     

Mechanism of action of cesalin, an antitumor protein.

A protein, cesalin, isolated from $\text{Caesalpinia}$$\text{gilliesii}$ is cytotoxic to KB cells in tissue culture. It has been shown to bind to the plasma membrane of this cell line and to inhibit Na⁺, K⁺-ATPase (ATP phosphohydrolase EC 3.6.1.3). Similar studies with HTC cells show no cytotoxicity or inhibition of plasma membrane Na⁺, K⁺-ATPase. The Na⁺, K⁺-ATPase of human erythrocytes and rat brain and kidney tissues are not inhibited. 5′-Nucleotidase and Mg⁺⁺-ATPase are not inhibited by cesalin in any cells tested.     

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.     

Glucagon stimulation of hepatic Na+, K+-ATPase

Summary In the perfused rat liver administration of glucagon was shown to result in a transiently increased uptake of K⁺, indicating the possible involvement of the Na⁺, K⁺-ATPase. Direct measurement of the activity of Na⁺, K⁺-ATPase revealed a two-fold stimulation of the enzyme by glucagon. The effect of glucagon on the activity of the enzyme was immediate. Simultaneously with the increase in the activity of the Na⁺, K⁺-ATPase, the activity of Mg²⁺-ATPase decreased. In order to evaluate whether the activation of the Na⁺, K⁺-ATPase by glucagon is related to the metabolic effects of the hormone, experimental conditions known to interfere with the activity of the enzyme were employed and glucagon stimulation of Ca²⁺-efflux, mitochondrial metabolism and gluconeogenesis were measured. K⁺-free perfusate, high K⁺ perfusate or ouabain interfered to varying degrees with the glucagon stimulation of these responses. The combination of K⁺-free perfusate and ouabain almost completely abolished the glucagon stimulation of all three parameters. These results demonstrate the glucagon stimulation of Na⁺, K⁺-ATPase and raise the possibility that the activation of the enzyme by glucagon might be a necessary link for the manifestation of its metabolic effects.     

Further observations on effects of cations on enzyme induction in marine bacteria

Summary Induction of resting cells ofPseudomonas natriegens, nov. spec. to the oxidation of L-arabinose and mannitol, and an additional marine isolate (L5) to oxidation of lactose and mannitol, was found dependent on the presence of Na⁺ with marine levels of Mg⁺⁺ and K⁺ included in the suspending media. However, omission of Na⁺ and Mg⁺⁺, and increase of the concentration of K⁺ in the suspending media, permitted rapid utilization of these substrates (but not of glucuronate) by resting cells and greatly accelerated the rates of induced enzyme formation. Inclusion of Mg⁺⁺ in the suspending media suppressed the rates of induction and oxidation stimulated by elevation of the K⁺ concentration. Incubation of resting cells of isolate L5 in an assay medium containing 0.26 M KCl preserved the ability of the organisms to produce a significant level of the enzymes for oxidation of mannitol, even after 8 hrs aging of the cells in the cold. Chloramphenicol inhibited the synthesis of induced oxidative enzymes in both these marine isolates suspended in high K⁺ medium, and the extent of inhibition was proportional to the time of addition of the antibiotic. Interruption of induction of isolate L5 cells to mannitol by washing within 30 min removed the stimulatory effect of the elevated level of K⁺, whereas longer periods of incubation before interruption yielded cells more fully induced. A greater amount of β-galactosidase was produced by resting cells of the marine isolate L5 incubated with the inducer in media with elevated K⁺ concentrations than by those cultured on lactose or induced in the resting state in the presence of Na⁺. Moreover, inclusion of an elevated K⁺ concentration in the suspending media stimulated the production of L-arabinose isomerase in resting cells ofP. natriegens. The requirement for Na⁺ for the growth of these bacteria, however, was not replaceable by elevation of the K⁺ concentration of culture media. Supported by grant no.G-15838 from the National Science Foundation and equipment loan contract NR-103-398 with the Office of Naval Research. Contribution no. 35 from the University of Georgia Marine Institute at Sapelo Island.     

The effect of metal ions on the alkaline phosphatase of Rhizobium leguminosarum

The alkaline phosphatase (EC 3.1.3.1.) from Rhizobium leguminosarum WU235 has been purified. The enzyme is a non-specific phosphomonoesterase, has a molecular weight of 78,500 and a sub-unit molecular weight of 39,400. Magnesium and zinc ions are implicated in the structure of the enzyme; atomic absorption analysis gave 1.9 g-atoms Mg²⁺ and 1.9–5.1 g-atoms Zn²⁺ per mole of enzyme. In addition high concentrations of Mg²⁺ markedly stimulate the enzyme. The phosphatase is inhibited by Li⁺ and Na⁺ and stimulated by K⁺, Rb⁺ and Cs⁺, which suggests that the enzyme is K⁺ activated.