Studies on (K+ + H+)-ATPase: VI. Determination of the molecular size by radiation inactivation analysis

(1) A $\text{(}\text{K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ containing membrane fraction, isolated from pig gastric mucosa, has been further purified by means of zonal electrophoresis, leading to a 20% increase in specific activity and an increase in ratio of $\text{(}\text{K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ to basal Mg²⁺-ATPase activity from 9 to 20. (2) The target size of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$, determined by radiation inactivation analysis, is 332 kDa, in excellent agreement with the earlier value of 327 kDa obtained from the subunit composition and subunit molecular weights. This shows that the Kepner-Macey factor of 6.4·10¹¹ is valid for membrane-bound ATPases. (3) The target size of $\text{(}\text{K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ is 444 kDa, which, in connection with a subunit molecular weight of 110000, suggests a tetrameric assembly of the native enzyme. The ouabain-insensitive K⁺-stimulated $\text{p-}\text{nitrophenylphosphatase}$ activity has a target size of 295 kDa. (4) In the presence of added Mg²⁺ the target sizes of the $\text{(}\text{K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ and its phosphatase activity are decreased by about 15%, while that for the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ is not significantly changed. This observation is discussed in terms of a Mg²⁺-induced tightening of the subunits composing the $\text{(}\text{K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ molecule.     

Regulation of rat brain (Na+ + K+)-ATPase activity by cyclic AMP

The interaction between the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ and the adenylate cyclase enzyme systems was examined. Cyclic AMP, but not 5′-AMP, cyclic GMP or 5′-GMP, could inhibit the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme present in crude rat brain plasma membranes. On the other hand, the cyclic AMP inhibition could not be observed with purified preparations of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme. Rat brain synaptosomal membranes were prepared and treated with either NaCl or cyclic AMP plus NaCl as described by Corbin, J., Sugden, P., Lincoln, T. and Keely, S. ((1977) J. Biol. Chem. 252, 3854–3861). This resulted in the dissociation and removal of the catalytic subunit of a membrane-bound cyclic AMP-dependent protein kinase. The decrease in cyclic AMP-dependent protein kinase activity was accompanied by an increase in $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity. Exposure of synaptosomal membranes containing the cyclic AMP-dependent protein kinase holoenzyme to a specific cyclic AMP-dependent protein kinase inhibitor resulted in an increase in $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme activity. Synaptosomal membranes lacking the catalytic subunit of the cyclic-AMP-dependent protein kinase did not show this effect. Reconstitution of the solubilized membrane-bound cyclic AMP-dependent protein kinase, in the presence of a neuronal membrane substrate protein for the activated protein kinase, with a purified preparation of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$, resulted in a decrease in overall $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity in the presence of cyclic AMP. Reconstitution of the protein kinase alone or the substrate protein alone, with the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ has no effect on $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity in the absence or presence of cyclic AMP. Preliminary experiments indicate that, when the activated protein kinase and the substrate protein were reconstituted with the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme, there appeared to be a decrease in the Na⁺-dependent phosphorylation of the Na⁺-ATPase enzyme, while the K⁺-dependent dephosphorylation of the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ was unaffected.     

Characterization of (Na+ + K+)-ATPase liposomes: II. Effect of α-subunit digestion on intramembrane particle formation and Na+,K+-transport

The effect of the protein structure of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ on its incorporation into liposome membranes was investigated as follows: the catalytic α-subunit of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ was split into low-molecular weight fragments by trypsin treatment and the digested enzyme was reconstituted at the same protein concentration as intact control enzyme. The reconstitution process was quantified by the average number of intramembrane particles appearing on concave and convex fracture faces after freeze-fracture of the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ liposomes. The number of intramembrane particles as well as their distribution on concave and convex fracture faces is not modified by the proteolysis. In contrast, the ATPase activity and the transport capacity of the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ decrease progessively with increasing incubation times in the presence of trypsin and are abolished when the original 100 000 molecular weight α-subunit is no longer visible by sodium dodecylsulfate gel electrophoresis. Apparently, functional ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ with intact protein structure and digested, non functional enzyme consisting of fragments of the α-subunit reconstitute in the same manner and to the same extent as judged by freeze-fracture analysis. We conclude that, while trypsin treatment modifies the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ molecule in a functional sense, it appears not to modify its interaction with the bilayer in producing intramembrane particles. On the basis of our results, we propose a lipid-lipid interaction mechanism for reconstitution of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$.     

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.     

Characterization of a β-actinin-like protein in purified non-muscle cell membranes. Its activity on (Na+ + K+)-ATPase

Treatment by EDTA of purified plasma membranes from MF₂S cells (a variant of the murine plasmacytoma MOPC 173) solubilized proteins and increased by a 1000-fold the sensitivity of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ to ouabain. When added back with Ca²⁺ to treated plasma membranes, these EDTA-solubilized proteins restored the initial sensitivity of the enzyme to its inhibitor. We report the purification of a protein of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 32 000, isolated from the EDTA-treated membrane supernatant. This protein was purified by a one-step procedure involving a preparative polyacrylamide gel electrophoresis without detergent. In the presence of Ca²⁺ it was able to restore the original sensitivity to ouabain of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from EDTA-treated membrane. This protein was shown to be similar to the β-actinin described by Maruyama by the following criteria: (1) molecular weight and amino acid composition; (2) cross-reactivity with their respective antisera; (3) in the presence of Ca²⁺ the same quantitative biological activity on ouabain sensitivity of the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$. A possible interaction between β-actinin, calmodulin and membrane-bound $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ is discussed.     

Characterization of (Na+ + K+)-ATPase liposomes: I. Effect of enzyme concentration and modification on liposome size,intramembrane particle formation and Na+,K+-transport

Rabbit renal ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ (EC 3.6.1.3) was purified and incorporated into phosphatidylcholine liposomes. Freeze-fracture analysis of the reconstituted system reveals intramembrane particles formed by ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ molecules which are randomly distributed on concave and convex fracture faces. The reconstituted ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ performs active Na⁺,K⁺-transport. The distribution of particles as well as the rate of active transport are directly proportional to the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ protein concentration used for reconstitution, while the total amount of sodium and potassium ions exchanged by ATP per volume vesicle suspension reaches maximum when each vesicle contains on the average more than two particles. ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ pretreated with ouabain or vanadate yields the same particle density and vesicle size as control enzyme. However, detergent-denatured enzyme loses its ability to form intramembrane particles or to increase the vesicle size indicating that the lipids surrounding the protein part of the molecule are essential for the reconstitution process. The vesicle diameter increases as a function of the number of particles per vesicle. Histograms of the size distribution become wider with increasing intramembrane particle density and tend to show more than one maximum.     

Phosphorylated intermediates of (Ca2+ + Mg2+)-ATPase and alkaline phosphatase in renal plasma membranes

Renal basal-lateral and brush border membrane preparations were phosphorylated in the presence of [γ-³²P]ATP. The ³²P-labeled membrane proteins were analysed on SDS-polyacrylamide gels. The phosphorylated intermediates formed in different conditions are compared with the intermediates formed in well defined membrane preparations such as erythrocyte plasma membranes and sarcoplasmic reticulum from skeletal muscle, and with the intermediates of purified renal enzymes such as $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ and alkaline phosphatase. Two Ca²⁺-induced, hydroxylamine-sensitive phosphoproteins are formed in the basal-lateral membrane preparations. They migrate with a molecular radius $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ of about 130 000 and 100 000. The phosphorylation of the 130 kDa protein was stimulated by La³⁺-ions (20 μM) in a similar way as the $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ from erythrocytes. The 130 kDa phosphoprotein also comigrated with the erythrocyte $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$. In addition in the same preparation, another hydroxylamine-sensitive 100 kDa phosphoprotein was formed in the presence of Na⁺. This phosphoprotein comigrates with a preparation of renal $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$. In brush border membrane preparations the Ca²⁺-induced and the Na⁺-induced phosphorylation bands are absent. This is consistent with the basal-lateral localization of the renal Ca²⁺-pump and Na⁺-pump. The predominant phosphoprotein in brush border membrane preparations is a 85 kDa protein that could be identified as the phosphorylated intermediate of renal alkaline phosphatase. This phosphoprotein is also present in basal-lateral membrane preparations, but it can be accounted for by contamination of those membranes with brush border membranes.     

The insect brain (Na+ + K+)-ATPase Binding of ouabain in the hawk moth, Manduca sexta

(1) A quantitative study has been made of the binding of ouabain to the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in homogenates prepared from brain tissue of the hawk moth, Manduca sexta. The results have been compared to those obtained in bovine brain microsomes. (2) The insect brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ will bind ouabain either in the presence of Mg²⁺ and P_i, (‘Mg²⁺, P_i’ conditions) or in the presence of Na⁺, Mg²⁺, and an adenine nucleotide (‘nucleotide’ conditions) as is the case for the bovine brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$. The binding conditions did not alter the total number of receptor sites measured at high ouabain concentrations in either tissue. (3) Potassium ion decreases the affinity (increases the $\text{K}{}_{\mathrm{<mtext>D</mtext>}}$) of ouabain to the M. sexta brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ under both binding conditions. However, ouabain binding is more sensitive to K⁺ inhibition under the nucleotide conditions. In bovine brain ouabain binding is equally sensitive to K⁺ inhibition under the both conditions. (4) The enzyme-ouabain complex has a rate of dissociation that is 10-fold faster in the M. sexta preparation than in the bovine brain preparation. Because of this, the M. sexta ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ has a higher $\text{K}{}_{\mathrm{<mtext>D</mtext>}}$ for ouabain binding and is less sensitive to inhibition by ouabain than the bovine brain enzyme. (5) This data supports the hypothesis that two different conformational states of the M. sexta ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ can bind ouabain.     

Evidence for essential disulfide bonds in the β-subunit of (Na+ + K+)-ATPase

$\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ from dog kidney lost its activity when heated at 55°C in the presence of 0.3 M 2-mercaptoethanol. Either heat treatment alone or addition of reducing agent at around 25°C caused little inactivation. One disulfide bond per protomer (mol. wt. 146000) was reduced in the inactivated sample but in active samples no reduction occurred. Neither K⁺-dependent phosphatase activity nor phosphoenzyme formation in the presence of Na⁺ was detected in the inactivated sample, suggesting that the disulfide bond was essential for the catalytic cycle of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$. This essential disulfide bond belonged to the β-subunit, the glycoprotein component of the enzyme, indicating that the β-subunit may be an integral component of the $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ system.     

Comparison of red cell and kidney (Na+ + K+)-ATPase at 0°C

Human red cell and guinea pig kidney $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ were phosphorylated at 0°C. Using concentrations of ATP ranging from 10^?6 to 10^?8 M, ATP-dependent regulation of reactivity is observed with red cell but not kidney $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ at 0°C. In particular, with the red cell enzyme only, the following are observed: (i) the ratio of enzyme-bound ATP (E·ATP, measured by the pulse-chase method of Post, R.L., Kume, S., Tobin, T., Orcutt, B. and Sen, A.K. (1969) J. Gen. Physiol. 54, 306s-326s) to steady-state level of total phosphoenzyme (EP) decreases with decrease in ATP concentration and (ii) the apparent turnover of phosphoenzyme (ratio of Na⁺-stimulated ATP hydrolysis to level of total EP at steady state) also varies as a function of ATP concentration. In addition, when EP is formed at very low ATP (0.02 μM), and then EDTA is added, rapid disappearance of a fraction of EP occurs, presumably due to ATP resynthesis, only with the red cell enzyme. These differences in behaviour of the red cell and kidney enzymes are explained on the basis of the observed predominance of K⁺-insensitive EP in red cell, but K⁺-sensitive EP in kidney $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ at 0°C.     

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.     

Identification of Ca2+-pump-related phosphoprotein in plasma membrane vesicles of Ehrlich ascites carcinoma cells

Plasma membrane vesicles of Ehrlich ascites carcinoma cells have been isolated to a high degree of purity. In the presence of Mg²⁺, the plasma membrane preparation exhibits a Ca²⁺-dependent ATPase activity of 2 μmol P_i per h per mg protein. It is suggested that this $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ activity is related to the measured Ca²⁺ transport which was characterized by $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for ATP and Ca²⁺ of $\text{44 ± 9 μ}\text{M}$ and $\text{0.25 ± 0.10 μ}\text{M}$, respectively. Phosphorylation of plasma membranes with [γ-³²P]ATP and analysis of the radioactive species by polyacrylamide gel electrophoresis revealed a Ca²⁺-dependent hydroxylamine-sensitive phosphoprotein with a molecular mass of 135 kDa. Molecular mass and other data differentiate this phosphoprotein from the catalytic subunit of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ and from the catalytic subunit of $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ of endoplasmic reticulum. It is suggested that the 135 kDa phosphoprotein represents the phosphorylated catalytic subunit of the $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ of the plasma membrane of Ehrlich ascites carcinoma cells. This finding is discussed in relation to previous attempts to identify a Ca²⁺-pump in plasma membranes isolated from nucleated cells.     

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.     

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.     

Effects of oligomycin and quercetin on the hydrolytic activities of the (Na+ + K+)-dependent ATPase

Quercetin inhibited a dog kidney ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ preparation without affecting $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP or $\text{K}{}_{0.5}$ for cation activators, attributable to the slowly-reversible nature of its inhibition. Dimethyl sulfoxide, a selector of E₂ enzyme conformations, blocked this inhibition, while the K⁺-phosphatase activity was at least as sensitive to quercetin as the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity, all consistent with quercetin favoring E₁ conformations of the enzyme. Oligomycin, a rapidly-reversible inhibitor, decreased the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP and the $\text{K}{}_{0.5}$ for cation activators, and its inhibition was also diminished by dimethyl sulfoxide. Although oligomycin did not inhibit the K⁺-phosphatase activity under standard assay conditions, a reaction presumably catalyzed by E₂ conformations, its effects are nevertheless accommodated by a quantitative model for that reaction depicting oligomycin as favoring E₁ conformations. The model also accounts quantitatively for effects of both dimethyl sulfoxide and oligomycin on $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for substrate, and $\text{K}{}_{0.5}$ for K⁺, as well as for stimulation of phosphatase activity by both these reagents at low K⁺ but high Na⁺ concentrations.     

Immunoreactivity of subunits of the (Na+ + K+)-ATPase Cross-reactivity of the α,α+ and β forms in different organs and species

The immunologic cross-reactivity of the α and α+ forms of the large subunit and the β subunit of the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from brain and kidney preparations was examined using rabbit antiserum prepared against the purified holo lamb kidney enzyme. As previously reported by Sweadner ((1979) J. Biol. Chem. 254, 6060–6067) phosphorylation of the large subunit of the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in the presence of Na⁺, Mg²⁺, and [$\text{γ-}{}^{32}\text{P}\text{]}\text{ATP}$ revealed that dog and, very likely, rat brain contain two forms of the large subunit (designated α and α+) while dog, rat, and lamb kidney contain only one form (α). The cross-reactivity of the α and α+ forms in these preparations was investigated by resolving the subunits by SDS-polyacrylamide gel electrophoresis. The separated polypeptides were transferred to unmodified nitrocellulose paper, and reacted with rabbit anti-lamb kidney serum, followed by detection of the antigen-antibody complex with ¹²⁵I-labeled protein A and autoradiography. By this method, the α and α+ forms of rat and dog brain, as well as the α form found in kidney, were shown to cross-react. In addition, membranes from human cerebral cortex were shown to contain two immunoreactive bands corresponding to the α and α+ forms of dog brain. In contrast, the brain of the insect Manduca sexta contains only one immunoreactive polypeptide with a molecular weight intermediate to the α and α+ forms of dog brain. The β subunit from lamb, dog and rat kidney and from dog and rat brain cross-reacts with anti-lamb kidney ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ serum. The mobility of the β subunit from dog and rat brain on SDS-polyacrylamide electrophoresis gels is greater than the mobility of the β subunit from lamb, rat or dog kidney.     

Plasma membrane NADH dehydrogenase and Ca2+-dependent potassium transport in erythrocytes of several animal species

Ca²⁺-dependent K⁺ transport and plasma membrane NADH dehydrogenase activities have been studied in several ‘high-K⁺’ (human, rabbit and guinea pig) and ‘low-K⁺’ (dog, cat and sheep) erythrocytes. All the species except sheep showed Ca²⁺-dependent K⁺ transport. NADH-ferricyanide reductase was detected in all the species and showed positive correlation with the flavin contents of the membranes. NADH-cytochrome $\text{c}$ reductase was very low or absent in dog, sheep and guinea pig membranes. No correlation was found between NADH dehydrogenase and Ca²⁺-dependent K⁺ channel activities in the species studied. Nor were any of the above activities correlated with $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity.     

An electron spin probe study of (Na+ + K+)-ATPase-Containing membranes

The modulating effect of membrane lipids on enzyme function has been described by several investigators. We have used the spin probe $\text{N-}\text{oxyl}\text{-4′,4′-}\text{dimethyloxazolidine}\text{-12-}\text{keto}$ methyl stearate (M 12-NSE) to study this interaction in ox brain membranes enriched with $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$. This methyl ester of stearic acid is practically insoluble in aqueous media, and consequently spectra of M 12-NSE-labelled preparations are free of “liquid lines”.At least two types of spectra may be obtained when ox brain microsomes are spin labelled with M 12-NSE, indicating the presence of two distinct binding sites. At one site the spin label is relatively unrestricted and gives rise to an isotropic spectrum. A second spectrum, which is obtained from spin label at another site, is similar to that which is observed after incorporation of M 12-NSE into phospholipid bilayers. This suggests that this latter site is within the core of the microsomal membrane.The two binding sites differ in their affinity for the spin probe. The low affinity site is both more abundant in crude preparations and is more easily removed by detergent treatment; spin labels at this site produce isotropic spectra. The high affinity sites are fewer in number and produce broad spectra. In addition these high affinity sites increase in concentration as the enzyme undergoes purification.The two sites are quite distinct in their sensitivity to ascorbic acid, the low affinity site showing a considerably greater rate of reduction by this agent.This study also demonstrates that the delipidation effects of sodium dodecyl sulfate and sodium deoxycholate on $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase-enriched}$ microsomes from ox brain are not identical.It is suggested that the two spin probe binding sites represent two different lipid domains, one of which is very closely associated with the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme and may reflect a protein-directed phospholipid specificity for this enzyme.     

Isolation of a rat liver plasma membrane fraction of probable canalicular origin Preparative technique,enzymatic profile,composition, and solute transport

A technique currently used for isolation of brush border membranes from renal and intestinal epithelium that involves vigorous tissue homogenization and sedimentation of non-luminal membranes in the presence of Mg²⁺ has been adapted to rat liver. Liver plasma membranes so prepared consisted almost exclusively of vesicles by electron microscopy, showed some contamination with endoplasmic reticulum and minimal contamination with mitochondria or Golgi by marker enzymes, were highly enriched in alkaline phosphatase, Mg²⁺-ATPase, and 5′-nucleotidase activity compared with homogenate, and showed little enrichment in (Na⁺,K⁺)-ATPase. Comparison of this enzymatic profile with cytochemical studies localizing (Na⁺,K⁺)-ATPase and alkaline phosphatase to the sinusoidal/lateral and canalicular membranes, respectively, suggested that these membranes were predominantly of canalicular origin. They had a lower ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ specific activity, lower lipid content, and higher cholesterol to phospholipid molar ratio than a conventional plasma membrane preparation believed to be enriched in canaliculi. Moreover, it was possible to measure movement of d-[³H]glucose into an osmotically sensitive space bounded by these membrane vesicles.