Characterization of the (Na+ + K+)-ATPase in a membranous preparation from the optic ganglion of the squid (Loligo pealei)

(1) A membrane fraction enriched in $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ (EC 3.6.1.3) was obtained from optic ganglia of the squid (Loligo pealei) by density gradient fractionation of membranes followed by treatment with either SDS or Brij-58. The resulting membrane had an $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ specific activity of approx. 2 units/mg and was $\text{>95\%}$ ouabain-sensitive. (2) The $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ had a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP of $\text{0.42 ± 0.04}\text{mM}$ and a pH optimum of 7.0. It was inhibited by ouabain with a $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ of $\text{0.32 ± 0.04 μ}\text{M}$. (3) Optimum monovalent cation concentrations were: 240 mM NaCl, 60 mM KCl, tested with $\text{NaCl + KCl}\text{= 300}\text{mM}$. (4) The Mg²⁺ dependence of hydrolysis varied with the absolute ATP concentration. At 3 mM ATP, the$\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg²⁺ was $\text{0.86 ± 0.10}\text{mM}$, and at 6 mM ATP, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ was $\text{1.86 ± 0.44}\text{mM}$. High levels of Mg²⁺ caused inhibition of hydrolysis. (5) The interactions of Na⁺ and K⁺ were examined over a range of conditions. K⁺ levels caused modulations in the Na⁺ dependence in the range of 1–150 mM. (6) The $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ prepared from squid optic ganglion displays properties similar to those of the sodium pump in injected nerves.     

Kinetic studies on the inhibition of (Na+ + K+)-ATPase by diketocoriolin B

Diketocoriolin B, a sesquiterpene antitumor antibiotic, inhibits particulate $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{-ATPase}$ (ATP phosphohydrolase, EC 3.6.1.3) of Yoshida sarcoma cells competitively, with respect to ATP, and uncompetitively with respect to Na⁺ and K⁺. The inhibition is reduced by the addition of phosphatidylserine.Rat brain $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{-ATPase}$, which is solubilized by deoxycholate and requires phosphatidylserine for its activity, is also inhibited by diketocoriolin B competitively with respect to ATP and the inhibition was reversed by increasing the concentration of phosphatidylserine.However, several differences are found between the solubilized and particulate systems: (a) 2 moles of diketocoriolin B interact with the former, while only one mole interacts with the latter, (b) K⁺-dependent phosphatase activity of the former requires phospholipid and is sensitive to diketocoriolin B while the reverse is true with the latter.Based on these kinetic studies, it is supported that $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ has two binding sites for phospholipid, one being essential for K⁺-dependent phosphatase activity and when these two sites are filled with the appropriate phospholipids, ATP can bind to the enzyme.     

A high-affinity (Ca2+ + Mg2+)-ATPase in plasma membranes of rat ascites hepatoma AH109A cells

The activity of calcium-stimulated and magnesium-dependent adenosinetriphosphatase which possesses a high affinity for free calcium (high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$, EC 3.6.1.3) has been detected in rat ascites hepatoma AH109A cell plasma membranes. The high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ had an apparent half saturation constant of $\text{77 ± 31}\text{nM}$ for free calcium, a maximum reaction velocity of $\text{9.9 ± 3.5}\text{nmol}$ ATP hydrolyzed/mg protein per min, and a Hill number of 0.8. Maximum activity was obtained at 0.2 μM free calcium. The high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ was absolutely dependent on 3–10 mM magnesium and the pH optimum was within physiological range (pH 7.2–7.5). Among the nucleoside trisphosphates tested, ATP was the best substrate, with an apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 30 μM. The distribution pattern of this enzyme in the subcellular fractions of the ascites hepatoma cell homogenate (as shown by the linear sucrose density gradient ultracentrifugation method) was similar to that of the known plasma membrane marker enzyme alkaline phosphatase (EC 3.1.3.1), indicating that the ATPase was located in the plasma membrane. Various agents, such as K⁺, Na⁺, ouabain, KCN, dicyclohexylcarbodiimide and NaN₃, had no significant effect on the activity of high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$. Orthovanadate inhibited this enzyme activity with an apparent half-maximal inhibition constant of 40 μM. The high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ was neither inhibited by trifluoperazine, a calmodulin-antagonist, nor stimulated by bovine brain calmodulin, whether the plasma membranes were prepared with or without ethylene glycol $\text{bis}\text{(β-}\text{aminoethyl ether}\text{)-N,N,N′,N′-}\text{tetraacetic acid}$. Since the kinetic properties of the high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ showed a close resemblance to those of erythrocyte plasma membrane $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$, the high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ of rat ascites hepatoma cell plasma membrane is proposed to be a calcium-pumping ATPase of these cells.     

Ouabain receptor interactions in (Na+ + K+)-ATPase preparations. II. Effect of cations and nucleotides on rate constants and dissociation constants

The action of ATP and its analogs as well as the effects of alkali ions were studied in their action on the ouabain receptor. One single ouabain receptor with a dissociation constant ($\text{K}{}_{\mathrm{<mtext>D</mtext>}}$) of 13 nM was found in the presence of ($\text{Mg}{}^{\mathrm{2+}}\text{+ P}{}_{\mathrm{i}}$) and ($\text{Na}{}^{\mathrm{+}}\text{+ Mg}{}^{\mathrm{2+}}\text{+ ATP}$). pH changes below pH 7.4 did not affect the ouabain receptor. Ouabain binding required Mg²⁺, where a curved line in the Scatchard plot appeared. The affinity of the receptor for ouabain was decreased by K⁺ and its congeners, by Na⁺ in the presence of ($\text{Mg}{}^{\mathrm{2+}}\text{+ P}{}_{\mathrm{i}}$), and by ATP analogs (ADP-C-P, ATP-OCH₃). Ca²⁺ antagonized the action of K⁺ on ouabain binding. It was concluded that the ouabain receptor exists in a low affinity (R_α) and a high affinity conformational state (R_β). The equilibrium between both states is influenced by ligands of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$. With 3 mM Mg²⁺ a mixture between both conformational states is assumed to exist (curved line in the Scatchard plot).     

Ligand-dependent reactivity of (Na+ + K+)-ATPase with showdomycin

Showdomycin inhibited pig brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ with pseudo first-order kinetics. The rate of inhibition by showdomycin was examined in the presence of 16 combinations of four ligands, i.e., Na⁺, K⁺, Mg²⁺ and ATP, and was found to depend on the ligands added. Combinations of ligands were divided into five groups in terms of the magnitude of the rate constant; in the order of decreasing rate constants these were: (1)$\text{Na}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}$, (2) $\text{Mg}{}^{\mathrm{2+}}$, $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{K}{}^{\mathrm{+}}$, $\text{K}{}^{\mathrm{+}}$ and none, (3) $\text{Na}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{,}\text{Na}{}^{\mathrm{+}}$, $\text{K}{}^{\mathrm{+}}\text{+}\text{Na}{}^{\mathrm{+}}$ and $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}$, (4) $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{ATP}\text{,}\text{K}{}^{\mathrm{+}}\text{+}\text{ATP}$ and $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}\text{, (5)}\text{K}{}^{\mathrm{+}}\text{+}\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}$ and ATP. The highest rate was obtained in the presence of Na⁺, Mg²⁺ and ATP. The apparent concentrations of Na⁺, Mg²⁺ and ATP for half-maximum stimulation of inhibition ($\text{K}{}_{0.5}{}^{\mathrm{<mtext>s</mtext>}}$) were 3 mM, 0.13 mM and 4μM, respectively. The rate was unchanged upon further increase in Na⁺ concentration from 140 to 1000 mM. The rates of inhibition could be explained on the basis of the enzyme forms present, including E₁, E₂, ES, E₁-P and E₂-P, i.e., E₂ has higher reactivity with showdomycin than E₁, while E₂-P has almost the same reactivity as E₁-P. We conclude that the reaction of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ proceeds via at least four kinds of enzyme form (E₁, E₂, E₁ · nucleotide and EP), which all have different conformations.     

Purification from brain of an intrinsic membrane protein fraction enriched in (Na+ + K+)-ATPase

A microsomal fraction from canine brain gray matter has been extracted with the detergent sodium dodecyl sulfate to partially purify the membrane bound $\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-stimulated}$ adenosine triphosphatase. Phospholipid, glycolipid, and a family of other glycoproteins are also enriched by the procedure; it is proposed that the product is an intrinsic membrane protein fraction. 6–8-fold purification of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ is obtained without solubilizing the enzyme and without irreversibly altering its turnover number. Final specific activities are 350–400 μmol of ATP hydrolyzed/h per mg protein. The stimulation and reversible inactivation of the $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ by dodecyl sulfate were examined for information relevant to the mechanism of action of the detergent.     

(Ca2+ + Mg2+)-ATPase in enriched sarcolemma from dog heart

An enriched fraction of plasma membranes was prepared from canine ventricle by a process which involved thorough disruption of membranes by vigorous homogenization in dilute suspension, sedimentation of contractile proteins and mitochondria at $\text{3000 × g}$ followed by sedimentation of a microsomal fraction at $\text{200 000 × g}$. The microsomal suspension was then fractionated on a discontinuous sucrose gradient. Particles migrating in the density range 1.0591–1.1083 were characterized by ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity and [³H]ouabain binding as being enriched in sarcolemma and were comprised of nonaggregated vesicles of diameter approx. 0.1 μm. These fractions contained $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ which appeared endogenous to the sarcolemma. The enzyme was solubilized using Triton X-100 and 1 M KCl and partially purified. Optimal Ca²⁺ concentration for enzyme activity was 5–10 μM. Both Na⁺ and K⁺ stimulated enzyme activity. It is suggested that the enzyme may be involved in the outward pumping of Ca²⁺ from the cardiac cell.     

Eosin,a fluorescent probe of ATP binding to the (Na+ + K+)-ATPase

(1) Eosin bound to the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in the presence of K⁺ has practically the same fluorescence as eosin without enzyme while in the presence of Na⁺ the fluorescence is higher, the excitation maximum is shifted from 518 to 524 nm, the emission maximum from 538 to 542 nm, and a shoulder appears at about 490 nm on the excitation curve. (2) The amount of eosin bound increases with the K⁺ concentration but with a low affinity. With equal concentrations of Na⁺ and K⁺ more is bound in the presence of Na⁺, and the difference between 150 mM Na⁺ and 150 mM K⁺ shows one high-affinity eosin binding site per ³²P-labelling site ($\text{K}{}_{\mathrm{D}}$ 0.45 μM). With lower concentrations of the cations there are between one and two Na⁺-dependent high-affinity eosin binding sites per ³²P-labelling site. (3) ATP (and ADP) prevents the hig-affinity Na⁺-dependent eosin binding and there is competition between eosin and ATP for the hydrolysis in the presence of Na⁺ (+Mg²⁺). (4) Eosin, like ATP, increases the Na⁺ relative to K⁺ affinity $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{= 150}\text{mM}$) for Na⁺ activation of hydrolysis and for Na⁺ protection against inactivation by $\text{N-}\text{ethylmaleimide}$. (5) The results suggest that the high affinity eosin binding site is an ATP binding site and that it is located on the enzyme in an environment with a low polarity, i.e., the conformational change induced by Na⁺ opens a high-affinity site for ATP while K⁺ closes the site (or decreases the affinity to a low level). The experiments suggest, furthermore, that the ATP which increases the Na⁺ relative to K⁺ affinity of the internal sites is not the ATP which is hydrolyzed, i.e., in a turnover cycle in the presence of $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}$ the system reacts with two different ATP molecules.     

Phosphatidylinositol as the endogenous activator of the (Na+ + K+)-ATPase in microsomes of rabbit kidney

Incubation of rabbit kidney microsomes with pig pancreatic phospholipase A₂ produced residual membrane preparations with very low $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity. The activity could be restored by recombination with lipid vesicles of negatively-charged glycerophospholipids. Vesicles of pure phosphatidylcholine and phosphatidylethanolamine were virtually inactive in this respect, but could reactivate in the presence of cholate.Incubation of the microsomes with a combination of phospholipase C (Bacillus cereus) and sphingomyelinase C (Staphylococcus aureus) resulted in 90–95% release of the phospholipids. The residual membrane contained only phosphatidylinositol and still showed 50–100% of the $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity.     

Interactions of lectins with (Na+ + K+)-ATPase of eel electric organ

Interaction of lectins with a detergent-solubilized ATPase from eel electric organ was studied. Concanavalin A, which binds to α-mannosides, altered the rate of enzyme migration in agar and inhibited the formation of an antigen-antibody precipitate; other lectins had no such effects. Concanavalin A similar amounts partially inhibited $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$; this inhibition was reversible by α-methylglucoside. There was no corresponding effect of concanavalin A on the potassium $\text{p-}\text{nitrophenyl-phosphatase}$. Concanavalin A also did not interfere with ouabain binding. Thus, concanavalin A binds to an antigenic region also involved in Na⁺ and/or ATP binding, but does not interact with a K⁺ site.     

Inhibition of the (Na+ + K+)-dependent ATPase by inorganic phosphate

Inhibition of the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-dependent}$ ATPase by inorganic phosphate, P_i, was examined in terms of product inhibition of the various activities catalyzed by an enzyme preparation from rat brain, and considered in terms of the specific transport processes of the membrane Na⁺,K⁺-pump that these activities reflect. The K⁺-dependent phosphatase activity of the enzyme was most sensitive to P_i, and inhibition was competitive toward the substrate, nitrophenyl phosphate, as would be expected if P_i were released from the same enzyme form that bound substrate. However, this enzymatic activity does not seem to represent a transport process, and thus a cyclical discharge of K⁺ may not be involved. The Na⁺-dependent exchange activity was unaffected by P_i, in accord with the absence of P_i release in the reaction sequence. For the corresponding Na⁺/Na⁺ exchange function of the pump, which reportedly does not involve ATP hydrolysis either, prior release of P_i obviously cannot be required for Na⁺ discharge. With the Na⁺-dependent ATPase activity, measured using micromolar concentrations of ATP, P_i inhibited, but far less than with the phosphatase activity, and inhibition was not competitive toward ATP. Moreover, inhibition decreased as the Na⁺ concentration was raised from 10 to 100 mM. This elevated concentration of Na⁺ also led to substrate inhibition. For this ATPase activity, and the corresponding transport process, uncoupled Na⁺ efflux, the findings suggest that Na⁺ discharge follows P_i release, in contrast to Na⁺/Na⁺ exchange. The $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-dependent}$ ATPase activity, measured with millimolar concentrations of ATP and reflecting the coupled Na⁺,K⁺-transport function, was similarly sensitive to P_i, and again inhibition was not competitive toward ATP. However, in this case inhibition did not increase as the Na⁺ concentration was lowered. For this activity, and the associated transport process, the site of Na⁺ discharge in the overall reaction sequence remains unresolved.     

Structural changes in (Na+ + K+)-ATPase accompanying detergent inactivation

Structural changes in the purified $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ accompanying detergent inactivation were investigated by monitoring changes in light scattering, intrinsic protein fluorescence, and tryptophan to β-parinaric acid fluorescence resonance energy transfer. Two phases of inactivation were observed using the non-ionic detergents, digitonin, Lubrol WX and Triton X-100. The rapid phase involves detergent monomer insertion but little change in protein structure or little displacement of closely associated lipids as judged by intrinsic protein fluorescence and fluorescence resonance energy transfer. Lubrol WX and Triton X-100 also caused membrane fragmentation during the rapid phase. The slower phase of inactivation results in a completely inactive enzyme in a particle of 400 000 daltons with 20 mol/mol of associated phospholipid. Fluorescence changes during the course of the slow phase indicate some dissociation of protein-associated lipids and an accompanying protein conformational change. It is concluded that non-parallel inhibition of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ and $\text{p-}\text{nitrophenylphosphate}$ activity by digitonin (which occurs during the rapid phase of inactivation) is unlikely to require a change in the oligomeric state of the enzyme. It is also concluded that at least 20 mol/mol of tightly associated lipid are necessary for either $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ or $\text{p-}\text{nitrophenylphosphatase}$ activity and that the rate-limiting step in the slow inactivation phase involves dissociation of an essential lipid.     

ATP/ADP exchange activity of gastric (H+ + K+)-ATPase

The ATP/ADP exchange is shown to be a partial reaction of the $\text{(}\text{H}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ by the absence of measurable nucleoside diphosphokinase activity and the insensitivity of the reaction to $\text{P}{}^1$, $\text{P}{}^5$ -di(adenosine-5′) pentaphosphate, a myokinase inhibitor. The exchange demonstrates an absolute requirement for Mg²⁺ and is optimal at an ADP/ATP ratio of 2. The high ATP concentration ($\text{K}{}_{0.5}\text{= 116 μ}\text{M}$) required for maximal exchange is interpreted as evidence for the involvement of a low affinity form of nucleotide site. The ATP/ADP exchange is regarded as evidence for an ADP-sensitive form of the phosphoenzyme. In native enzyme, pre-steady state kinetics show that the formation of the phosphoenzyme is partially sensitive to ADP while modification of the enzyme by pretreatment with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of Mg²⁺ results in a steady-state phosphoenzyme population, a component of which is ADP sensitive. The ATP/ADP exchange reaction can be either stimulated or inhibited by the presence of K⁺ as a function of pH and Mg²⁺.     

Characterization of partially purified (Na+ + K+)-ATPase from porcine lens

The partial purification of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from pig lens has been achieved by treatment with deoxycholate followed by density gradient centrifugation. The specific activity of the final preparation, ranging from 300 to 500 nmol/h per mg protein, is increased approx. 100-fold compared to the homogenate. A parallel increase in $\text{p-}\text{nitrophenylphosphatase}$ activity is also observed. Sodium dodecyl sulfate (SDS) gel electrophoresis reveals six major protein bands, one of which is the 93 kDa α subunit of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ which can be phosphorylated by reaction with [γ-³²P]ATP. A second band contains a glycoprotein which displays an apparent molecular weight of 51 000 and thus appears to be the β subunit of the enzyme. The enzyme is sensitive to ouabain with the $\text{I}{}_{50}$ for $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ and $\text{p-}\text{nitrophenylphosphatase}$ inhibition being 1.2 and 1.3 μM, respectively. Several agents which inhibit $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from other tissues such as oligomycin, Ca²⁺, vanadate, $\text{N-}\text{ethylmaleimide}$, $\text{p-}\text{chloromercuribenzenesulfonic acid}$ (PCMBS) and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) also inhibit the lens enzyme. Monovalent cations other than K⁺ are partially effective in activating the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}$)-ATPase and $\text{p-}\text{nitrophenylphosphatase}$ activities. The K⁺ congeners were relatively more effective in supporting $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ compared to $\text{p-}\text{nitrophenylphosphatase}$ activity. Other kinetic properties of the lens enzyme are also comparable to those of the enzyme from other tissues. Utilizing the partially purified membrane bound enzyme, discontinuities in Arrhenius plots of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity, $\text{p-}\text{nitrophenylphosphatase}$ activity and fluoresence polarization of the fluidity probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), are observed near the physiological temperature of lens. The possible significance of these observations for the mechanism of cataract formation are discussed.     

Electrokinetic properties of (Na+, K+)-ATPase vesicles as studied by laser doppler spectroscopy

The technique of laser Doppler electrophoresis was applied for the study of the surface charge properties of (Na⁺,⁺)-ATPase containing microsomal vesicles derived from guinea-pig kidney. The influence of pH, the screening and binding of uni- and divalent cations and the binding of ATP show: (1) one net negative charge per protein unit with a $\text{p}\text{K = 3.9}$; (2) deviation from the Debye relation between surface potential and ionic strength for univalent cations, with no difference in the effect of Na⁺ and K⁺; (3) Mg²⁺ binds with an association constant of $\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 1.1 · 10}{}^2\text{M}{}^{\mathrm{?1}}$ while ATP binds with an apparent $\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 1.1 · 10}{}^4\text{M}{}^{\mathrm{?2}}$ for 1 mM Nacl, 0.2 mM KCI, 0.1 mM MgCl₂, 0.1 mM Tris-HCI (pH 7.3). The binding is weaker at higher Mg²⁺ concentrations. There is no ATP binding in the absence of Mg²⁺. In addition, the average vesicle size derived from the linewidth of the quasi-elastic light scattering spectrum is $\text{203.7 ± 15.2}\text{nm}$. In the presence of ATP a reduction in size is observed.