Standardized method for the determination of human erythrocyte membrane adenosine triphosphatases

A standardized assay is described for the simultaneous determination of Mg²⁺-ATPase, Na⁺, K⁺-ATPase, and Ca²⁺-ATPase in human erythrocyte (RBC) membrane preparations. Membranes were prepared by lysis of RBCs in hypotonic buffer, and ATPase activity assays were based on the measurement of ³²P-labeled inorganic phosphate release from [γ-³²P]ATP. The results obtained by this method were compared with those of colorimetric determination of inorganic phosphate and of ATP hydrolysis with high-performance liquid chromatography. The activity of the three enzymes was measured in RBC membranes obtained from 30 normal subjects. Repeated sampling of individuals over a 4-month period showed that interindividual differences were substantial, but that in each individual enzymatic activity was maintained in a narrow range by presumed homeostatic mechanisms. Statistical analysis of the data showed no interdependence of the three enzymes; a correlation of activity with age, sex, or phase of the menstrual cycle was not apparent. The values obtained for the Ca²⁺-ATPase did not follow a normal distribution, and it is suggested that this enzyme has two phenotypic variants. The described method is sufficiently precise and economical to be recommended for adoption as standard procedure in clinical research.     

Localization of recently characterized membrane transport adenosine triphosphatases

Summary Na⁺, K⁺-ATPase is the best-known member of a family of membrane transport ATPases sharing a number of structural features. Methods recently developed for Na⁺, K⁺-ATPase localization may, therefore, be modifiable for use in studies of the distributions of related enzymes. Our experiences in the use of monophosphate and triphosphate substrate methods for localization of the gastric proton pump ATPase (H⁺, K⁺-ATPase) are described and the prospects for localizing other related enzymes by similar techniques are discussed.     

The (non)specificity in the lipid-requirement of calcium- and (sodium plus potassium)-transporting adenosine triphosphatases

The lipid requirement of alkali cation transporting ATPases has been investigated by studying the effect of well controlled modifications of the endogenous lipids on the activity of the enzymes. In principle, two different approaches can be distinguished. Firstly, complete delipidation with concomitant loss of activity followed by reconstitution of the system with well-defined lipids. A second approach involves specific enzymatic degradations or detergent mediated replacements, both selectively modifying the endogenous lipid complement. Each of these strategies has advantages and disadvantages, which have given rise to many conflicting opinions regarding the question whether a specificity in the lipid requirements of the various ATPases does exist. Only recently, it was established that the (Ca²⁺ + Mg²⁺)-ATPase in the sarcoplasmic reticulum does not have any particular specificity in its lipid requirement. Almost every amphiphile, including a variety of (non-ionic) detergents, can fulfil its needs in this respect. On the other hand, the analogous enzyme found in the erythrocyte membrane requires the presence of either mono- or diacylglycerophospholipids. In the native situation, however, it is only that fraction of the total $\text{glycero}$-phospholipid complement of the membrane that forms part of the inner half of the lipid bilayer that is actually involved. The most pronounced specificity in lipid requirement seems to exist for the (Na⁺ + K⁺)-ATPase system. Reactivation studies strongly suggest an absolute requirement for negatively charged phospholipids. Quite interestingly, individual differences may exist among various membrane species as to which of the endogenous negatively charged glycero-phospholipids is actually involved, e.g. phosphatidylserine in the erythrocyte membrane and phosphatidylinositol in rabbit kidney microsomes, respectively. Furthermore, it is suggested by experiments involving phospholipases that only a small fraction of the total activating phospholipid is involved; this fraction may be rather closely associated with the (Na⁺ + K⁺)- ATPase protein.     

Effects of inhibitors on the plasma membrane and mitochondrial adenosine triphosphatases of Neurospora crassa

A comparative study has been made of the effects of a variety of inhibitors on the plasma membrane ATPase and mitochondrial ATPase of Neurospora crassa. The most specific inhibitors proved to be vanadate and diethylstilbestrol for the plasma membrane ATPase and azide, oligomycin, venturicidin, and leucinostatin for mitochondrial ATPase. $\text{N,N′-}\text{Dicyclohexylcarbodiimide}$, octylguanidine, triphenylsulfonium chloride, and quercetin and related bioflavonoids inhibited both enzymes, although with different concentration dependences. Other compounds that were tested (phaseolin, fusicoccin, deoxycorticosterone, alachlor, salicyclic acid, $\text{N-1-}\text{napthylphthalamate}$, triiodobenzoic acid, cyclic AMP, cyclic GMP, theobromine, theophylline, and histamine) had no significant effect on either enzyme. Overall, the results indicate that the plasma membrane and mitochondrial ATPases are distinct enzymes, in spite of the fact that they may play related roles in H⁺ transport across their respective membranes.     

The localization and properties of membrane adenosine triphosphatases in the guinea-pig placenta

Summary The distribution and properties of cytochemically demonstrable phosphatases in the near-term guinea-pig placenta were examined using a strontium capture technique for sodium- and potassium-dependent adenosine triphosphatase (Na⁺, K⁺-ATPase) and a lead capture technique for magnesium-dependent adenosine triphosphatase (Mg²⁺-ATPase).Localizations with the strontium technique in the presence of an alkaline phosphatase inhibitor were mainly on the syncytiotrophoblast plasma membranes; the reaction was potassium-dependent and ouabain-sensitive. Reaction product using the lead capture method was found on both trophoblast and endothelial cell plasma membranes and was independent of magnesium and insensitive to p-hydroxymercuribenzoate (POHMB), an inhibitor of membrane ATPases. However, a very large proportion of this reaction could be blocked by an alkaline phosphatase inhibitor.It is concluded that the strontium capture technique gave a reliable localization for Na⁺, K⁺-ATPase. However, the lead capture method mainly demonstrated alkaline phosphatase, and does not offer a useful approach to specific ATPase studies in this particular system.     

Temperature-dependence of activation and inhibition of rat-brain adenosine triphosphatase activated by sodium and potassium ions

1. The adenosine-triphosphatase activity of rat-brain microsomes was measured between 0° and 37°. The stimulatory effect of Na⁺ plus K⁺ on the Mg²⁺-dependent adenosine-triphosphatase activity decreased sharply with decreasing temperature and became negligible at 0°. An Arrhenius plot drawn from the experimental data showed two discontinuities: one at about 6° and the other at about 20°. 2. The increment in activity induced by Na⁺ plus K⁺ was more sensitive to oligomycin at lower than at higher temperatures, but the opposite was observed for ouabain. The action of oligomycin showed a biphasic character, since below a certain concentration it caused slight activation of Na⁺-plus-K⁺-activated adenosine triphosphatase. 3. Where oligomycin increased the activity of the enzyme, it also enhanced the accumulation of an acid-precipitable phosphorylated compound formed through the transfer of the γ-phosphate group of [³²P]ATP to the enzyme system. Stimulatory concentrations of oligomycin did not interfere with K⁺-mediated dephosphorylation of the intermediate, though high concentrations of oligomycin counteracted the effect of K⁺. 4. The temperature profile of K⁺-stimulated microsomal phosphatase qualitatively resembled that of microsomal adenosine triphosphatase.     

The localization and properties of membrane adenosine triphosphatases in the guinea-pig placenta.

The distribution and properties of cytochemically demonstrable phosphatases in the near-term guinea-pig placenta were examined using a strontium capture technique for sodium- and potassium-dependent adenosine triphosphatase (Na+, K+-ATPase) and a lead capture technique for magnesium-dependent adenosine triphosphatase (Mg2+-ATPase). Localizations with the strontium technique in the presence of an alkaline phosphatase inhibitor were mainly on the syncytiotrophoblast plasma membranes; the reaction was potassium-dependent and ouabain-sensitive. Reaction product using the lead capture method was found on both trophoblast and endothelial cell plasma membranes and was independent of magnesium and insensitive to p-hydroxymercuribenzoate (POHMB), an inhibitor of membrane ATPases. However, a very large proportion of this reaction could be blocked by an alkaline phosphatase inhibitor. It is concluded that the strontium capture technique gave a reliable localization for Na+, K+-ATPase. However, the lead capture method mainly demonstrated alkaline phosphatase, and does not offer a useful approach to specific ATPase studies in this particular system.     

Structure of the vacuolar adenosine triphosphatases

Vacuolar adenosine triphosphatases (V-ATPases) represent an important class of proton pumps found in endomembrane systems of eucaryotic cells, where they are involved in pH regulation. Progress has been made in the structure determination of this large, membrane-bound multisubunit enzyme complex. Electron microscopy of the V-ATPase has revealed a ball-and-stalk-like structure similar to F1F0-type ATP synthase, to which the V-ATPase is evolutionary related. Aside from the overall structural similarity of the V-ATPase and F-ATP synthase, a number of distinct structural differences exist between the two related enzymes, giving clues to their different function and regulation in the organism.     

Phosphorylation of erythrocyte membrane liberates calcium

Washed and permeabilized human erythrocyte ghosts were found to discharge calcium on treatment with ATP. Concomitantly, there was a decrease in phosphatidylinositol (PI) and an increase in phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2). These results support the hypothesis that an inositide shuttle, PI in equilibrium PIP in equilibrium PIP2, operates to maintain intracellular Ca2+ levels. The cation is thought to be sequestered in a cage formed by the head groups of two acidic phospholipid molecules, e.g., phosphatidylserine and phosphatidylinositol, with participation of both PO and fatty acid ester CO groups. These cages are stabilized by inter-headgroup hydrogen bonding. When the inositol group is phosphorylated in positions 4 and 5, inter-lipid hydrogen bonding is disrupted and the cage opens to release its Ca2+.     

Halobacterial adenosine triphosphatases and the adenosine triphosphatase from Halobacterium saccharovorum

Abstract Membranes prepared from various members of the genus Halobacterium contained a Triton X-100 activated adenosine triphosphatase. The enzyme from Halobacterium saccharovorum was unstable in solutions of low ionic strength (< 3 M NaCl) and maximally active in the presence of 3.5 M NaCl. A variety of nucleotide triphosphates was hydrolyzed. MgADP, the product of ATP hydrolysis, was not hydrolyzed and was a competitive inhibitor with respect to MgATP. The enzyme from H. saccharovorum was composed of at least 2 and possibly 4 subunits. The 83-kDa and 60-kDa subunits represented about 90% of total protein. The 60-kDa subunit reacted with dicyclohexylcarbodiimide (DCCD) when inhibition was carried out in an acidic medium. The significance of the two minor components (28 kDa and 12 kDa) is not established. The enzyme from H. saccharovorum , which differs from previously described halobacterial ATPases, possesses properties of an F₁F₀ as well as an E₁E₂ ATPase.     

The independence of membrane potential and potassium activation of the sodium pump in muscle

The membrane potential (E_m) of sartorius muscle fibers was made insensitive to [K⁺] by equilibration in a 95 mM K⁺, 120 mM Na⁺ Ringer solution. Under these conditions a potassium-activated, ouabain-sensitive sodium efflux was observed which had characteristics similar to those seen in muscles with E_m sensitive to [K⁺]. In addition, in the presence of 10 mM K⁺, these muscles were able to produce a net sodium extrusion against an electrochemical gradient which was also inhibited by 10^?4 M ouabain. This suggests that the membrane potential does not play a major role in the potassium activation of the sodium pump in muscles.