Inactivation of membrane-bound (Na+ +K+)-ATPase of Yoshida sarcoma cells and cobra venom cytotoxin complex with the glycolipid components of the enzyme system.

The molecular mechanisms involved in the inactivation of (Na+ + K+)-stimulated ATPase of Yoshida sarcoma cells by a cytotoxic protein (P6) from cobra venom have been examined. The overall data obtained using purified (Na+ + K+)-stimulated ATPase of Yoshida sarcoma cells suggest that cytotoxin P6 combines with phosphatidyl serine and a glycolipid which are closely associated with (Na+ + K+)-stimulated ATPase which in turn may lead to the inactivation of the enzyme in this cell system.     

Inhibition of (Na+ + K+)-ATPase by ouabain: involvement of calcium and membrane proteins.

Treatment of plasma membrane isolated from murine plasmocytoma MOPC 173 with an EDTA-containing buffer resulted in a 300-fold increase in sensitivity of (Na+ + K+)-stimulated Mg2+-ATPase to ouabain. This phenomenon was associated with the solubilization by EDTA of phospholipid free proteins (approx. 30 000-34 000 daltons) from the cytoplasmic face of the plasma membrane and with removal of about 90% of the membrane bound Ca2+. The recovery of the original resistance to ouabain required specifically Ca2+ and was associated with a binding of the solubilized proteins to the membrane.     

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

Structural changes in the purified (Na+ + K+)-ATPase accompanying detergent inactivation were investigated by monitoring changes in light scattering, intrinsic protein fluorescence, and tryptophan to beta-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 (Na+ + K+)-ATPase and p-nitrophenylphosphate activity by digitonin (which occurs during the rapid phase of inactivation) is unlikey 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 (Na+ + K+)-ATPase or p-nitrophenylphosphatase activity and that the rate-limiting step in the slow inactivation phase involves dissociation of an essential lipid.     

Reversible inactivation of (Na+ + K+)-ATPase by use of a cleavable bifunctional reagent.

1. Purified (Na+ + K+)-ATPase, prepared from rabbit kidney outer medulla, is incubated with the bifunctional NH2-directed reagent dimethyl 3,3'-dithiobis-propionimidate. This results in a cross-link between the subunits of the enzyme and a simultaneous reduction of the (Na+ + K+)-ATPase and K+-stimulated p-nitrophenylphosphatase activities. 2. The most abundant cross-link product is a dimer of the two different subunits of the enzyme. 3. Reduction of the disulfide cross-link by dithioerythritol results in partial recovery of the original subunit structure of the enzyme and of the (Na+ + K+)-ATPase and K+-stimulated p-nitrophenylphosphatase activities. 4. These results suggest that a free mobility of the subunits of the (Na+ + K+)-ATPase system relative to each other is essential for proper functioning of both enzyme activities.     

Effects of polyamines on partial reactions of membrane (Na+ + K+)-ATPase.

Spermine and spermidine inhibit the (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) reaction so that the effect increases as the ionic content due to Na+ and K+ in the reaction is reduced. Several other amines inhibit (Na+ + K+)-ATPase to varying degress and methylglyoxal-bis-(guanylhydrazone) was the most potent inhibitor among those tested. The inhibition by polyamines of the ATPase is uncompetitive with respect to Mg2+ and ATP activation of the reaction. Various naturally occurring polyamines and other amines inhibited Na+ activation of (Na+ + K+)-ATPase as well as Na+-dependent phosphoenzyme formation in an apparently competitive manner with respect to Na+. Likewise, K+-activation of (Na+ + K+)-ATPase as well as K+-p-nitrophenyl phosphatase was inhibited in an apparently competitive manner with respect to K+. Both the cation charge and structure (e.g., aliphatic chain length) may contribute to the inhibitory effects of the amines; however, Na+ sites appear to be more sensitive to cation charge than the aliphatic chain length of the amine, whereas the opposite appears to be true for K+ sites. The results do not indicate a specific effect of polyamines on (Na+ + K+)-ATPase or its partial reactions.     

Activation of (Na+ + K+)-ATPase

Enzymes catalyze essential chemical reactions needed for living processes. (Na+ +K+)-ATPase (NKA) is one of the key enzymes that control intracellular ion homeostasis and regulate cardiac function. Little is known about activation of NKA and its biological impact. Here we show that native activity of NKA is markedly elevated when protein-protein interaction occurs at the extracellular DVEDSYGQQWTYEQR (D-R) region in the alpha-subunit of the enzyme. The apparent catalytic turnover of NKA is approximately twice as fast as the controls for both ouabain-resistant and ouabain-sensitive enzymes. Activation of NKA not only markedly protects enzyme function against denaturing, but also directly affects cellular activities by regulating intracellular Ca2+ transients and inducing a positive inotropic effect in isolated rat cardiac myocytes. Immunofluorescent labeling indicates that the D-R region of NKA is not a conventional digitalis-binding site. Our findings uncover a novel activation site of NKA that is capable of promoting the catalytic function of the enzyme and establish a new concept that activating of NKA mediates cardiac contraction.     

Low-affinity Na+ sites on (Na+ +K+)-ATPase modulate inhibition of Na+-ATPase activity by vanadate

Na+-ATPase activity is extremely sensitive to inhibition by vanadate at low Na+ concentrations where Na+ occupies only high-affinity activation sites. Na+ occupies low-affinity activation sites to reverse inhibition of Na+-ATPase and (Na+, K+)-ATPase activities by vanadate. This effect of Na+ is competitive with respect to both vanadate and Mg2+. The apparent affinity of the enzyme for vanadate is markedly increased by K+. The principal effect of K+ may be to displace Na+ from the low-affinity sites at which it activates Na+-ATPase activity.     

Solubilized (Na+ + K+)-ATPase from shark rectal gland and ox kidney--an inactivation study

The bi-exponential time-course of detergent inactivation at 37 degrees C of C12E8-solubilized (Na+ + K+)-ATPase from shark rectal glands and ox kidney was investigated. The data for shark enzyme, obtained at detergent/protein weight ratios between 2 and 16, are interpreted in terms of a simple model where the membrane bound enzyme is solubilized predominantly as (alpha-beta)2 diprotomers at low detergent concentrations and as alpha-beta protomers at high C12E8 (octaethyleneglycoldodecylmonoether) concentrations. It is observed that the protomers are inactivated 15-fold more rapidly than the diprotomers, and that the rate of inactivation of both oligomers is proportional to the detergent/protein ratio. Inactivation of kidney enzyme was biexponential with a very rapid inactivation of up to 40% of the enzyme activity. The observed rate of inactivation of the slower phase varied with the detergent/protein ratio, but the inactivation pattern for the kidney enzyme could not readily be accommodated within the model for inactivation of the shark enzyme. The rates of inactivation at 37 degrees C were about the same in KCl and NaCl, i.e., in the E2(K) and E1 X Na forms, for both enzymes.     

Radiation inactivation of (Na+ + K+)-ATPase. A small target size for the K+-occluding mechanism

Radiation inactivation of partially purified (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) from pig kidney outer medulla shows that the target size for Rb+ occlusion by the enzyme (in the absence of phosphorylation) is much smaller than the target size for p-nitrophenyl phosphatase activity, which is itself smaller than the reported target size for (Na+ + K+)-ATPase activity.     

Comparative temperature dependence of (Na+ + K+)-ATPase.

The temperature dependence of (Na+ + K+)-ATPase was measured, utilizing preparations of enzyme from heat and kidney of rats, hamsters, guinea pigs, ground squirrels, turtles, chickens, and ducks. The two hibernating species, hamsters and ground squirrels, were studied awake at normothermia and hibernating at 4 degrees C. The results for every species except the turtles showed the same temperature dependence established for (Na++K+)-ATPase from rabbit kidney with a quasi-linear dependence above 15 degrees C and little or no activity below 15 degrees C. Turtle enzymes showed a broad activity versus temperature curve with a fall-off at high and low temperatures. The data in all cases, including the turtle data, may be fitted by a previously described thermodynamic kinetic model. Further, the model will fith the turnover or decrease in enzyme activity at higher temperatures observed in a number of cases. These results do not support the widely imputed ion pumping role for (Na++K+)-ATPase.     

Radiation inactivation of (Na+ + K+)-ATPase a small target size for the K+-occluding mechanism

Radiation inactivation of partially purified (Na⁺ + K⁺)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) from pig kidney outer medulla shows that the target size for Rb⁺ occlusion by the enzyme (in the absence of phosphorylation) is much smaller than the target size for $\text{p-}\text{nitrophenyl phosphatase activity}$, which is itself smaller than the reported target size for (Na⁺ + K⁺)-ATPase activity.     

Estimation of ouabain specifically bound to dog renal (Na+ + K+)-ATPase in vivo.

Suspensions of viable renal cortical cells hydrolyzed a synthetic ester substrate (alpha-N-tosyl-L-arginine methyl ester, Tos-Arg-OMe) and generated kinins from a kininogen substrate. This kallikrein-like esterase activity increased linearly with cell number, or time of exposure to substrate. No radiolabelled substrate or product was found within the cells. Most of the activity appeared to be on cell surfaces as supernatant media had less than 20% of the Tos-Arg-OMe esterase activity on the cell suspensions. Cell surface Tos-Arg-OMe esterase activity was inhibited by aprotinin, benzamidine, pentamidine, and a tris-amidine derivative (alpha,alpha',alpha'-tris(3-amidinophenoxy)mesitylene). Preincubation of cells with phospholipase A2 increased renal cell surface esterase activity up to 76% while only slightly increasing supernatant activity. In contrast, preincubation with deoxycholate caused clearing of suspensions and a marked increase in supernatant esterase activity. Renal cell kininogenase (EC 3.4.21.8) activity was inhibited by preincubation with aprotinin, the tris-amidine derivative, or anti-rat urinary kallikrein antibody. Kallikrein elaborated by renal cells formed a single precipitin line with an antibody to rat urinary kallikrein but the two enzymes were not immunologically identical. We conclude that kallikrein's active sites are facing the external environment of renal cortical cells in suspension with access to substrates, inhibitors, and antibody.     

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 (Na+ + K+)-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 (Na+ + K+)-ATPase is obtained without solubilizing the enzyme and without irreversibly altering its turnover number. Final specific activities are 350--400 mumol of ATP hydrolyzed/h per mg protein. The stimulation and reversible inactivation of the (Na+ + K+)-ATPase by dodecyl sulfate were examined for information relevant to the mechanism of action of the detergent.     

An analysis of biphasic time courses: the inactivation of (Na+ + K+)-ATPase and Ca2+-ATPase by ATP-analogs

The inactivation of (NA+ + K+)-ATPase and Ca2+-ATPase brought about by the substitution of ATP by covalently binding analogs is studied. Most of the analogs cause biphasic courses of inactivation. The families of time courses obtained for different concentrations of the analog exhibit a characteristic feature that is common to both ATPases. The times of transition from one branch to the other of the biphasic curves are practically independent of the concentration of the analog. An analysis of the eigenvalues from different reaction models shows that for these time evolutions the enzyme exists necessarily in two states, only one of which is active for the analog. As a preliminary attempt, the models have been fitted to the experimental data of three different sets of families of curves. It is demonstrated that a two-sites model of inactivation of (Na+ + K+)-ATPase postulated in the literature cannot be valid.     

Na+ currents generated by the purified (Na+ + K+)-ATPase on planar lipid membranes

Purified (Na+ + K+)-ATPase from pig kidney was attached to black lipid membranes and ATP-induced electric currents were measured as described previously by Fendler et al. ((1985) EMBO J. 4, 3079-3085). An ATP concentration jump was produced by an ultraviolet-light flash converting non-hydrolysable caged ATP to ATP. In the presence of Na+ and Mg2+ this resulted in a transient current signal. The pump current was not only ATP dependent, but also was influenced by the ATP/caged ATP ratio. It was concluded that caged ATP binds to the enzyme (and hence inhibits the signal) with a Ki of approx. 30 microM, which was confirmed by enzymatic activity studies. An ATP affinity of approx. 2 microM was determined. The addition of the protonophore 1799 and the Me+/H+ exchanger monensin made the bilayer conductive leading to a stationary pump current. The stationary current was strongly increased by the addition of K+ with a K0.5 of 700 microM. Even in the absence of K+ a stationary current could be measured, which showed two Na+-affinities: a high-affinity (K0.5 less than or equal to 1 mM) and a low-affinity (K0.5 greater than or equal to 0.2 M). In order to explain the sustained electrogenic Na+ transport during the Na+-ATPase activity, it is proposed, that Na+ can replace K+ in dephosphorylating the enzyme, but binds about 1000-times weaker than K+. The ATP requirement of the Na+-ATPase was the same (K0.5 = 2 microM) with regard to the peak currents and the stationary currents. However, for the (Na+ + K+)-ATPase the stationary currents required more ATP. The results are discussed on the basis of the Albers-Post scheme.