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

(1) A (K+ + H+)-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 (K+ + H+)-ATPase to basal Mg2+-ATPase activity from 9 to 20. (2) The target size of (Na+ + K+)-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 X 10(11) is valid for membrane-bound ATPases. (3) The target size of (K+ + H+)-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 p-nitrophenylphosphatase activity has a target size of 295 kDa. (4) In the presence of added Mg2+ the target sizes of the (K+ + H+)-ATPase and its phosphatase activity are decreased by about 15%, while that for the (Na+ + K+)-ATPase is not significantly changed. This observation is discussed in terms of a Mg2+-induced tightening of the subunits composing the (K+ + H+)-ATPase molecule.     

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.     

The H+/ATP stoichiometry of the (H+ + K+)-ATPase of dog gastric microsomes

Gastric microsomal vesicles isolated from dog fundic mucosa were shown to be relatively ion tight and have a low level of proton permeability. The H⁺ translocase, basal ATPase and K⁺-activated ATPase activities of these vesicles were measured and the H⁺/ATP stoichiometry calculated using either the total K⁺-ATPase or the K⁺-stimulatable component (total K⁺-ATPase—basal ATPase). The former estimations consistently gave stoichiometric of approximately one, whereas the use of only the K⁺-stimulatable component gave widely differing values. Measurement of the dephosphorylation of the enzyme under basal conditions revealed both a labile and a stable phosphoenzyme component. The rate of decay of the labile component completely accounted for the basal ATPase activity observed. We conclude that the basal ATPase associated with our preparations is a spontaneous dephosphorylation of the phosphoenzyme occurring in the absence of K⁺ and that the H⁺/ATP stoichiometry of the gastric ATPase is one.     

Studies on (K+ + H+)-ATPase. V. Chemical composition and molecular weight of the catalytic subunit

(1) A $\text{(K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}\text{)-ATPase}$ preparation from porcine gastric mucosa is solubilized in sodium dodecyl sulfate, and is subjected to gel filtration. (2) A main subunit fraction is obtained, which is a protein carbohydrate lipid complex, containing 88% protein, 7% carbohydrate and 5% phospholipid. The detailed composition of the protein and carbohydrate moieties are reported. (3) Sedimentation analysis of the subunit preparation, after detergent removal, reveals no heterogeneity, but the subunits readily undergo aggregation. (4) Acylation of the subunit preparation with citraconic anhydride causes a clear shift of the band obtained after SDS gel electrophoresis, but the absence of broadening and splitting of the band pleads against subunit heterogeneity. (5) Treatment of the subunit preparation with dansyl chloride indicates that the NH₂ terminus is blocked, which favors the assumption of homogeneity of the protein. (6) Binding studies with concanavalin A indicate that at least 86% of the subunit preparation is composed of glycoprotein. (7) These findings, taken together, strongly suggest that there is a single subunit which is a glycoprotein and which represents the catalytic subunit of the enzyme. From sedimentation equilibrium analysis a molecular mass value of 119 kDa (S.E. 3, $\text{n = 6}$) is calculated for protein + carbohydrate and of 110 kDa (S.E. 3, $\text{n = 6}$) for protein only. (8) In combination with the molecular mass of 444 kDa (S.E. 10, $\text{n = 4}$) obtained for the intact enzyme by radiation inactivation we conclude that the enzyme appears to be composed of a homo-tetramer of catalytic subunits.     

Target molecular weight of the gastric (H+ + K+)-ATPase functional and structural molecular size

The state of assembly of the (H+ + K+)-ATPase in purified hog gastric mucosa membranes was studied by target size analysis applied to radiation-induced enzyme inactivation and polypeptide degradation data. Radiation inactivated the Mg2+-ATPase, K+-stimulated ATPase, and p-nitrophenyl phosphatase activities of the membrane preparation with a dose dependence characteristic of a target size of 270,000-daltons. Radiation also bleached the major 100,000-dalton sodium dodecyl sulfate-gel electrophoresis band of this preparation, indicating a radiation-induced degradation. This apparent polypeptide degradation exhibited a dose dependency corresponding to a target size of 250,000 daltons in situ. It is suggested that the gastric ATPase is a trimeric assembly of the 100,000-dalton polypeptides.     

Studies on (K + H)-ATPase. V. Chemical composition and molecular weight of the catalytic subunit

(1) A (K⁺ + H⁺)-ATPase preparation from porcine gastric mucosa is solubilized in sodium dodecyl sulfate, and is subjected to gel filtration. (2) A main subunit fraction is obtained, which is a protein carbohydrate lipid complex, containing 88% protein, 7% carbohydrate and 5% phospholipid. The detailed composition of the protein and carbohydrate moieties are reported. (3) Sedimentation analysis of the subunit preparation, after detergent removal, reveals no heterogeneity, but the subunits readily undergo aggregation. (4) Acylation of the subunit preparation with citraconic anhydride causes a clear shift of the band obtained after SDS gel electrophoresis, but the absence of broadening and splitting of the band pleads against subunit heterogeneity. (5) Treatment of the subunit preparation with dansyl chloride indicates that the NH₂ terminus is blocked, which favors the assumption of homogeneity of the protein. (6) Binding studies with concanavalin A indicate that at least 86% of the subunit preparation is composed of glycoprotein. (7) These findings, taken together, strongly suggest that there is a single subunit which is a glycoprotein and which represents the catalytic subunit of the enzyme. From sedimentation equilibrium analysis a molecular mass value of 119 kDa (S.E. 3, n = 6) is calculated for protein + carbohydrate and of 110 kDa (S.E. 3, n = 6) for protein only. (8) In combination with the molecular mass of 444 kDa (S.E. 10, n = 4) obtained for the intact enzyme by radiation inactivation we conclude that the enzyme appears to be composed of a homo-tetramer of catalytic subunits.     

Studies on (K+ + H+)-ATPase V. Chemical composition and molecular weight of the catalytic subunit

(1) A (K+ + H+)-ATPase preparation from porcine gastric mucosa is solubilized in sodium dodecyl sulfate, and is subjected to gel filtration. (2) A main subunit fraction is obtained, which is a protein carbohydrate lipid complex, containing 88% protein, 7% carbohydrate and 5% phospholipid. The Detailed composition of the protein and carbohydrate moieties are reported. (3) Sedimentation analysis of the subunit preparation, after detergent removal, reveals no heterogeneity, but the subunits readily undergo aggregation. (4) Acylation of the subunit preparation with citraconic anhydride causes a clear shift of the band obtained after SDS gel electrophoresis, but the absence of broadening and splitting of the band pleads against subunit heterogeneity. (5) Treatment of the subunit preparation with dansyl chloride indicates that the NH2 terminus is blocked, which favors the assumption of homogeneity of the protein. (6) Binding studies with concanavalin A indicate that at least 86% of the subunit preparation is composed of glycoprotein. (7) These findings, taken together, strongly suggest that there is a single subunit which is a glycoprotein and which represents the catalytic subunit of the enzyme. From sedimentation equilibrium analysis a molecular mass value of 119 kDa (S.E. 3, n = 6) is calculated for protein + carbohydrate and of 110 kDa (S.E. 3, N = 6) for protein only. (8) In combination with the molecular mass of 444 kDa (S. E. 10, n = 4) obtained for the intact enzyme by radiation inactivation we conclude that the enzyme appears to be composed of a homo-tetramer of catalytic subunits.     

Studies on (K+ + H+)-ATPase III. Binding of adenylyl imidodiphosphate

1. Adenylyl imidodiphosphate (AMPPNP) binds to (K+ + H+)-ATPase from pig gastric mucosa with a dissociation constant (Kd) of 50 microM for the AMPPNP-enzyme complex. 2. Monovalent cations reduce the amount of AMPPNP bound in the following order of effectiveness Tl+ greater than K+ greater than Rb+ greater than Cs+ greater than Na+, Li+, choline+. 3. AMPPNP binding to the enzyme has a pH optimum at pH 7.0--7.5 in the absence of added ions, which is shifted to pH 8 upon addition of MgCl2. 4. Cyclodiaminotetraacetic acid (CDTA, Tris salt) inhibits binding of AMPPNP. This inhibition is not due to chelation of Mg2+. It may be due to direct binding of CDTA to the enzyme or to removal of stabilizing cations other than Mg2+. 5. Binding curves determined in the presence of various concentrations of Mg2+ show that at low Mg2+ concentrations (less than 0.5 mM), the apparent number of binding sites is reduced, while at higher Mg2+ concentrations (greater than or equal to 0.5 mM), the binding of AMPPNP is inhibited in a competitive way. 6. From these observations it is concluded that the enzyme has two binding sites for AMPPNP and only one for Mg-AMPPNP (or two with strong anti-cooperativity), and that Mg2+ inhibits binding of Mg-AMPPNP. This finding is interpreted in terms of a model involving a dimeric form of the enzyme.     

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.     

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.     

(Na+ + K+)-ATPase: inactivation and degradation induced by oxygen radicals.

1. Purified (Na+ + K+)-ATPase was irreversibly inhibited upon exposure to hydrogen peroxide, the superoxide anion, and the hydroxyl radical. 2. Comparison of the SDS-gel electrophoretic patterns of the ATPase samples exposed to these oxidants revealed that inhibition occurred either without gross structural changes, or concomitant with fragmentation and cross-linking of the enzyme subunits. 3. The oxidant modified ATPase was also shown to be more susceptible to degradation by several proteolytic enzymes.     

Lack of immunological cross reactivity between the transport enzymes (Na+ + K+)-ATPase and (K+ + H+)-ATPase

Goat antisera against (Na⁺ + K⁺)-ATPase and its isolated subunits and against (K⁺ + H⁺)-ATPase have been prepared in order to test for immune cross-reactivity between the two enzymes, whose catalytic subunits show great chemical similarity. None of the (Na⁺ + K⁺)-ATPase antisera cross-reacted with (K⁺ + H⁺)-ATPase or inhibited its enzyme activity. The same was true for the (K⁺ + H⁺)-ATPase antiserum with regard to (Na⁺ + K⁺)-ATPase and its subunits and its enzyme activity. So not withstanding the chemical similarity of their subunits, there is no immunological cross-reactivity between these two plasma membrane ATPases.Number LIII in the series Studies on (Na⁺ + K⁺)-Activated ATPase.     

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.     

Minimal functional unit for transport and enzyme activities of (Na+ + K+)-ATPase as determined by radiation inactivation

Frozen aqueous suspensions of partially purified membrane-bound renal (Na⁺ + K⁺)-ATPase have been irradiated at –135°C with high-energy electrons. (Na⁺ + K⁺)-ATPase and K⁺-phosphatase activities are inactivated exponentially with apparent target sizes of $\text{184 ± 4 kDa and 125 ± 3 kDa}$, respectively. These values are significantly lower then found previously from irradiation of lyophilized membranes. After reconstitution of irradiated (Na⁺ + K⁺)-ATPase into phospholipid vesicles the following transport functions have been measured and target sizes calculated from the exponential inactivation curves: ATP-dependent Na⁺?K⁺ exchange, $\text{201 ± 4}\text{kDa}$; (ATP + P_i)-activated Rb⁺?Rb⁺ exchange, $\text{206 ± 7}\text{kDa}$ and ATP-independent Rb⁺?Rb⁺ exchange, $\text{117 ± 4}\text{kDa}$. The apparent size of the α-chain, judged by disappearance of Coomassie stain on SDS-gels, lies between 115 and 141 kDa. That for the β-glycoprotein, though clearly smaller, could not be estimated. We draw the following conclusions: (1) The simplest interpretation of the results is that the minimal functional unit for (Na⁺ + K⁺)-ATPase is αβ. (2) The inactivation target size for (Na⁺ + K⁺)-dependent ATP hydrolysis is the same as for ATP-dependent pumping of Na⁺ and K⁺. (3) The target sizes, for K⁺-phosphatase (125 kDa) and ATP-independent Rb⁺?Rb⁺ exchange (117 kDa) are indistinguishable from that of the α-chain itself, suggesting that cation binding sites and transport pathways, and the $\text{p-}\text{nitrophenyl}$ phosphate binding site are located exclusively on the α-chain. (4) ATP-dependent activities appear to depend on the integrity of an αβ complex.     

The action of trypsin on the gastric (H+ + K+)-ATPase.

The effect of trypsin on gastric (H+ + K+)-ATPase and K+-phosphatase was studied. Loss of both enzymic activities was biphasic, consisting of a fast and slow phase. Several peptides were produced from the original 105,000-dalton region of the sodium dodecyl sulfate electrophoretic separation, but only two, 87,000 and 47,000 daltons, were labeled following incubation with [gamma-33P]ATP. After a 30-min hydrolysis, 35% of the original peptide remained unaltered and appeared to be a glycoprotein. ATP and ADP abolished the second phase of tryptic inactivation of both activities and only two peptides, of 78,000 and 30,000 daltons, were found on the acrylamide gel in addition to the original 105,000-dalton region, neither of which was labeled by [gamma-33P]ATP. The protection was specific for these nucleotides, AMP, beta, gamma-methylene ATP, TTP, and pNPP being ineffective. Na+ and K+ at high concentrations reduced the rate of loss of activity but no change in the peptides produced was found. The level of phosphoenzyme was increased 2-fold by trypsin treatment, whereas the quantity of K+-sensitive phosphoenzyme remained relatively constant. Thus, the 105,000-dalton region is heterogeneous, consisting of a catalytic subunit (the active site is on a 47,000-dalton fragment), a glycoprotein, and another 105,000-dalton peptide. The action of trypsin is initially to prevent interconversion of a K+-insensitive to a K+-sensitive form of the phosphoenzyme, thus inhibiting hydrolysis.     

Thiophosphorylation of (Na + K+)-ATPase yields an ADP-sensitive phosphointermediate

(1) Treatment of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ from rabbit kidney outer medulla with the $\text{γ-}{}^{35}\text{S}$ labeled thio-analogue of ATP in the presence of $\text{Na}{}^{\mathrm{+}}\text{+ Mg}{}^{\mathrm{2+}}$ and the absence of K⁺ leads to thiophosphorylation of the enzyme. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for [γ-S]ATP is 2.2 μM and for Na⁺ 4.2 mM at 22°C. Thiophosphorylation is a sigmoidal function of the Na⁺ concentration, yielding a Hill coefficient $\text{n}\text{H}\text{= 2.6}$. (2) The thio-analogue ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>\; =\; 35\; \mu M</mtext>}}$) can also support overall $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity, but $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ at 37°C is only $\text{1.3 γ}\text{mol}\text{· (mg protein)}{}^{\mathrm{?}}\text{·}$ h^?1 or 0.09% of the specific activity for ATP ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>\; =\; 0.43\; mM</mtext>}}$). (3) The thiophosphoenzyme intermediate, like the natural phosphoenzyme, is sensitive to hydroxylamine, indicating that it also is an acylphosphate. However, the thiophosphoenzyme, unlike the phosphoenzyme, is acid labile at temperatures as low as 0°C. The acid-denatured thiophosphoenzyme has optimal stability at pH 5–6. (4) The thiophosphorylation capacity of the enzyme is equal to its phosphorylation capacity, indicating the same number of sites. Phosphorylation by ATP excludes thiophosphorylation, suggesting that the two substrates compete for the same phosphorylation site. (5) The (apparent) rate constants of thiophosphorylation (0.4 s^?1 vs. 180 s^?1), spontaneous dethiophosphorylation (0.04 s^?1 vs. 0.5 s^?1) and K⁺-stimulated dethiophosphorylation (0.54 s^?1 vs. 230 s^?1) are much lower than those for the corresponding reactions based on ATP. (6) In contrast to the phosphoenzyme, the thiophosphoenzyme is ADP-sensitive (with an apparent rate constant in ADP-induced dethiophosphorylation of 0.35 s^?1, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$$\text{ADP = 48 μM at 0.1 mM ATP}$) and is relatively K⁺-insensitve. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for K⁺ in dethiophosphorylation is 0.9 mM and in dephosphorylation 0.09 mM. The thiophosphoenzyme appears to be for 75–90% in the ADP-sensitive E₁-conformation.     

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.     

Studies on (K+ + H+)-ATPase. I. Essential arginine residue in its substrate binding center

1. A membrane vesicle fraction containing a high (K+ + H+)-ATPase activity was isolated from porcine gastric mucosa. The enzyme has a pH optimum of 7.0 and is stimulated by T1+, K+, Rb+ and NH4+ with KA values of 0.13, 2.7, 7.6 and 26 mM, respectively, at this pH. 2. Incubation of the isolated membrane fraction with butanedione leads to inactivation of the (K+ + H+)-ATPase activity. The pH-dependence of the (K+ + H+)-ATPase activity. The pH-dependence of the inactivation and the reversibility of the reaction, observed after removal of excess butanedione and borate, indicate that modification of arginine is involved. 3. The inactivation of (K+ + H+)-ATPase activity by butanedione is time-dependent and follows second-order kinetics. From the dependence of the inactivation rate on the reagent concentration it appears that a single arginine residue is involved in the inactivation of the (K+ + H+)-ATPase activity. 4. ATP, deoxy-ATP, ADP and adenylyl imidodiphosphate (AMPPNP), but not CTP, GTP and ITP which are poor substrates, protect the enzyme against butanedione inactivation, suggesting that the essential arginine residue is located in the ATP binding centre. 5. In the presence of Mg2+ the butanedione inactivation is increased, and the protection by ATP, deoxy-ATP and ADP (but not that by AMPPNP) is less pronounced. This suggests that Mg2+ induces a conformational change in the enzyme, exposing the arginine group and coinciding with phosphorylation and subsequent release of ADP from its binding site.