Sensitivity of the (Na+ + K+)-ATPase to state-dependent inhibitors: Effects of digitonin and triton X-100

Treatment of a purified (Na⁺ + K⁺)-ATPase preparation from dog kidney with digitonin reduced enzymatic activity, with the (Na⁺ + K⁺)-ATPase reaction inhibited more than the K⁺-phosphatase reaction that is also catalyzed by this enzyme. Under the usual assay conditions oligomycin inhibits the (Na⁺ + K⁺)-ATPase reaction but not the K⁺-phosphatase reaction; however, treatment with digitonin made the K⁺-phosphatase reaction almost as sensitive to oligomycin as the (Na⁺ + K⁺)-ATPase reaction. The non-ionic detergents, Triton X-100, Lubrol WX and Tween 20, also conferred sensitivity to oligomycin on the K⁺-phosphatase reaction (in the absence of oligomycin all these detergents, unlike digitonin, inhibited the K⁺-phosphatase reaction more than the (Na⁺ + K⁺)-ATPase reaction). Both digitonin and Triton markedly increased the K_0.5 for K⁺ as activator of the K⁺-phosphatase reaction, with little effect on the K_0.5 for K⁺ as activator of the (Na⁺ + K⁺)-ATPase reaction. In contrast, increasing the K_0.5 for K⁺ in the K⁺-phosphatase reaction by treatment of the enzyme with acetic anhydride did not confer sensitivity to oligomycin. Both digitonin and Triton also increased the inhibition of the K⁺-phosphatase reaction by ATP and decreased the inhibition by inorganic phosphate and vanadate. These observations are interpreted as digitonin and Triton favoring the E₁ conformational state of the enzyme (manifested by sensitivity to oligomycin and a greater affinity for ATP at the low-affinity substrate sites), as opposed to the E₂ state (manifested by insensitivity to oligomycin, greater sensitivity to phosphate and vanadate, and a lower K_0.5 for K⁺ in the K⁺-phosphatase reaction). In addition, digitonin blocked activation of the phosphatase reaction by Na⁺ plus CTP. This effect is consistent with digitonin dissociating the catalytic subunits of the enzyme, the interaction of which may be essential for activation by Na⁺ plus nucleotide.     

Disruption of the quaternary structure of (Na+ + K+)-dependent adenosine triphosphatase by Triton X-100.

(Na⁺ + K⁺)-dependent adenosine triphosphatase (NaK ATPase) consists of two polypeptide chains, a large polypeptide with a molecular weight of about 100,000, and a sialoglycoprotein with a molecular weight of about 40,000. In the presence of Triton X-100 both polypeptides react to form high molecular weight aggregates with apparent molecular weights of 168,000, 200,000 and 260,000. These aggregates arise as a result of disulfide bond formation which results from the autooxidation of sulfhydryl groups on the two polypeptides of NaK ATPase. These data are discussed in light of studies aimed at determining the size and subunit structure of membrane proteins with Triton X-100.     

Solubilization of guinea pig kidney (Na+ + K+)-ATPase with Lubrol W and Triton X-100

• 1.1. A comparison has been made of two non-ionic detergents, Lubrol W and Triton X-100 for the solubilization of (Na⁺ + K⁺)-ATPase (ATP phosphohydrolase, EC 3.6.1.3).
• 2.2. The optimum concentrations for maximum solubilization with Triton X-100 and Lubrol W were 0.05 and 0.1%, respectively.
• 3.3.|Monovalent cations, such as Na⁺ and K⁺, and ATP increased the specific activity of the enzyme solubilized by Triton X-100 and decreased that by Lubrol W.
• 4.4. Mg²⁺ inhibited solubilization with either of the two detergents.
• 5.5. Triton X-100 was found superior to LUbrol W in solubilization of (Na⁺ + K⁺)-ATPase.
• 6.6. A procedure for obtaining soluble enzyme preparation with specific activity ranging between 140 and 200 μmoles P_i per mg protein per h is described.
• 7.7. The soluble enzyme was more stable in the presence of Na⁺, K⁺ or ATP, but ATP was inferior to Na⁺ or K⁺.

     

Further studies on the activation of microsomal (Na+ + k+)-atpase by a leukocytic product.

Previous studies showed that microsomal (Na+ + K+)ATPase (ATP phosphohydrolase, EC 3.6.1.3) is activated by a proteinaeous material released by polymorphonuclear leukocytes. Investigations on the mode of action of the activator have been conducted by the siolation of 32P-labeled phosphoenzyme intermediates formed in the reaction of ATP and (Na+ + K)-ATPase, which has been postulated to occur through the formation and hydrolysis of acyl phosphate intermediates. The activator caused a concentration-dependent decrease in the recovery of phosphoenzyme intermediates that was not quantitatively altered by the Na+ or K+ concentration of the reaction mixture of by the presence of 1 mM oubain. A decline in phosphoenzyme intermediate recovery was promoted by the addition of the activator to preformed phosphoenzyme intermediates but not by activator that had been pretreated with protease or phenol. In addition, the activator caused a concentration-dependent stimulation of the p-nitrophenyl phosphatase and acetyl phosphatase activities of microsomal (Na+ + K+)-ATPase. It was proposed that the activator stimulates the dephosphorylation step of the (Na+ + K+)-ATPase reaction sequence.     

Permeabilization of microorganisms by Triton X-100.

A simple permeabilization procedure has been developed which allows the reliable determination of enzyme activitiesin situ in a variety of different microorganisms. Permeabilization is obtained by freezing cell suspensions in the presence of a low concentration of the anionic detergent Triton X-100. After thawing, the cells can be used directly in the enzyme assays. The procedure has been optimized using the yeastSaccharomyces cerevisiae. Yeast cells are completely permeabilized by Triton X-100 concentrations of 0.05% (v/v), and permeabilization is independent of cell age and cell concentration. The treatment makes the cells freely diffusible for macromolecules up to molecular weights around 70,000. Cytoplasmic and mitochondrial amino acid biosynthetic enzymes as well as aminoacyl-tRNA synthetases could be readily measured in treated cells. The method has been successfully applied to the determination of enzyme activities in other fungi as well as in gram-positive and gramnegative bacteria.     

Effect of sodium deoxycholate on intestinal glucose absorption and (Na+-K+)-atpase in the rat

The effects of deoxycholate on glucose transport and intestinal (Na+-K+)-ATPase activity have been investigated in the rat jejunum in vivo using a perfusion technique.     

Dependence of renal (Na+ + k+)-adenosine triphosphatase activity on thyroid status.

In thyroidectomized rats, a single injection of L-2,,5,2'-triiodothyronine (T3) (50mug/100 g body weight) elicited at 45% increase in (Na+ + k+)-dependent adenosine triphosphatase (NaK-ATPase) activity of the membrane-rich fraction of renal cortex at the optimal time of response, 48 h after injection. Three successive doses of T3 (50 mug/100 g body weight), given on alternate days, increased NaK-ATPase by 67% in the renal cortex but had no significant effect on the outer medulla or the papilla. Moreover, T3 had no effect on Mg2+-dependent adenosine trisphatase (MgATPase) in cortex, cedulla, or papilla. Three doses of T3 (50 mug/100 g body weight) given on alternate days to thyroidectomized rats elecited a 134, 79, and 46% increase in Vmax for ATP, Na4, and K+, respectively. There were no changes in the Km for ATP or the K1/2 values for Na+ and K+. Two methods were used to estimate the effect of T3 on the number of NaK-ATPase units (assumed to represent the number of Na+ pump sites); rat renal plasma membrane fractions were incubated with [gamma-32P]ATP, Mg2+, and Na+; the 32P-labeled membrane protein yeild was quantitatively dependent on Na+ and was hydrolyzed on addition of K+. There was a linear correlation between the specific activity of NaK-ATPase (Vmax) and the amount of phosphorylated intermediate formed, in renal cortical membrane fractions from thyroidectomized rats given T3 or the diluent. There was also a linear correlation between the specific activity of NaK-ATPase (Vmax) and the amount of [3H]ouabain specifically bound (Na+-, Mg2+-, APT-dependent) to the NaK-ATPase preparation. Injection of T3 resulted in a 70% increase in NaK-ATPase activity, a 79% increase in formation of the phosphorylated intermediate, and a 65% increase in the [H]ouabain specifically bound to the NaK-ATPase system. The T3-dependent increases in Vmax for ATP, Na+, and K+ and the proportionate increases in the phosphorylated intermediate and in the amount of [3H]ouabain bound indicate that T3 increases the number of NaK-ATPase units and that this increase accounts for the increase in NaK-ATPase activity.     

Immunoprecipitation of Triton X-100-solubilized Mycoplasma mycoides proteins.

Mycoplasma mycoides subsp. mycoides (PG1 and strain Y) proteins were solubilized in Triton X-100, and the antigenic proteins were precipitated from this complex mixture by addition of antiserum and then separated by two-dimensional gel electrophoresis. Of the 300 proteins solubilized, about 10 were precipitated. Proteins of PG1, a slow-growing, small colony (SC) strain, were precipitated by antiserum to PG1 and by antiserum to strain Y, a fast-growing, large colony (LC) strain. Similarly, strain Y proteins were precipitated by antiserum to PG1 and by antiserum to strain Y. The few proteins precipitated in this way gave similar patterns after two-dimensional gel electrophoresis indicating that many of the dominant protein antigens of PG1 and strain Y are shared. Antiserum to Mycoplasma mycoides subsp. capri (PG3) also precipitated some proteins of strain Y. Antiserum to Mycoplasma gallisepticum gave no reaction with any M. mycoides antigens. It was concluded that, in addition to the polysaccharide antigens, there are proteins in M. mycoides that are antigenic and that some of these are found in both the SC and LC strains of subsp. mycoides and also in subsp. capri.     

Effect of triiodothyronine on the synthesis and degradation of renal cortical (Na+ + k+)-adenosine triphosphatase.

The present studies concern the roles of synthesis and degradation of the large subunit of (Na+ + k+)-adenosine triphosphatase (NaK-ATPase) in the response to triiodothyronin (T3). Single doses of either the diluent of T3 (50 mug/100 g body weight) were given to two pairs of surgically thyroidectomized rats. Twenty hours after injection, the rats received 3H- or 35S-labeled methionine administered as a constant injusion into the tail vein for 1 h. The kidneys were removed either 8 h or 20 h after infusion and the eight kidneys were divided into pairs, as follows. I, 3H (diluent)/35S (T3); II, 35S (diluent)/3H (T3); III, 3H (diluent)/35S (diluent); IV, 3H (T3)/35S (T3). Partially purified NaK-ATPase was prepared from the pooled homogenates and prepared from the pooled homogenates and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAG-electrophoresis). The large subunit of NaK-ATPase was identified by (Na+ + mg2+)-dependent and K+-sensitive incorpotation of 32P from [gamma-32P]ATP. This component had an estimated molecular weight of 92,000 and migrated as a single peptide in gels of varying total carylamide concentration, with respect to: (1) Coomassie blue staining, (b) (Na+ + Mg2+)-dependent, K+-sensitive incorporation of 32P from [gamma-32-P]ATP, and (c) T3-dependent enhanced incorporation of labeled methionine. T3 augmented incorporation of labeled methionine into the large subunit by 44% 8 h after infusion of the amino acid and by 61% 20 h after infusion. Incorporation of methionine into two adjacent polypeptides in the SDS gels was unaffected by thyroid status. The effect otical NaK-ATPase was assessed by a double label technique. Pairs of thyroidectomized rats were injected with either the diluent or 50 mug of T3/100 g body weight at 48-h after the first injection (diluent or T3, i.e. Day "zero"). Kidney cortices were processed on either Day 4 or Day 6; the partially purified NaK-ATPase fraction was prepared, labeled with [gamma-32P]ATP, and analyzed by SDS-PAG-electrophoresis. The degredation rate constants of the large subunit were similar; 0.145 and 0.124 day-1 for the hypothyroid and T3-treated groups, respectively. Thus, the T2-dependent increase in incorporation of labeled methionine into the large subunit appears to result from enhanced synthesis and this increase is sufficient to account for the entire increase in both the number of the activity of the NaK-ATPase units.     

Lowry determination of protein in the presence of Triton X-100.

The Lowry procedure has been modified for use in the presence of Triton X-100 (TX-100) by the addition of 10% sodium dodecyl sulfate. The method is applicable to samples containing 40–120 μg protein.     

Effects of Triton X-100 on acid phosphatases with different substrate specificities

Summary The effect of Triton X-100 on the activities of acid phosphatases from wheat germ, potato and human prostate was tested using -glycerophosphate, p-nitro-phenyl phosphate and naphthol AS BI phosphate as substrates. There was little effect on -glycerophosphatase activity at the concentrations of Triton X-100 tested. However at low concen trations of the detergent there was a stimulation of the activities of p-nitrophenyl phosphatase and naphthol phosphatase which were inhibited with the higher concentrations. Triton X-100 was found to enhance colour production between naphthol AS BI and fast red violet LB.Further evidence is presented confirming the presence of more than one acid phosphatase from each of the sources employed.     

Effects of pH on the reactivation of human spermatozoa demembranated with Triton X-100

The aim of this work was to study the role of different parameters involved in the motility of human spermatozoa. Human spermatozoa were totally demembranated with 0.05% Triton X-100, and the demembranation was checked using electron microscopy. We have shown that, with a concentration of ATP-Mg lower than 2 mM, a pH effect was observed with a dose-dependent motility reactivation at pH 7.1, with 14% +/- 2.0% motile cells at 1 mM ATP-Mg and a straight line velocity (VSL) of 12.0 +/- 1.4 microns/sec. However, at pH 7.8, more than 65% of the spermatozoa were reactivated with as low as 0.02 mM ATP-Mg and 77.8% +/- 2.5% of them were motile at 1 mM ATP-Mg and had a VSL of 23.4 +/- 3.9 microns/sec. The depletion of free calcium by the addition of 0.5 mM EGTA in the reactivation medium (RM) improved the percentage of motile cells and the VSL most markedly at low ATP-Mg and low pH. If no MgSO4 was added in RM, cells were not motile at pH 7.8, but 30-40% reactivated at pH 7.1. If 5 mM Ca2+ was added to the RM, up to 88% of the cells became reactivated at both pHs, but the beat frequencies were very low, suggesting different mechanisms of reactivation when Mg2+ or when Ca2+ is present in the RM.     

Effects of Triton X-100 on proton transfer and in the photocycle of archaerhodopsin 4

Archaerhodopsin 4 (AR4), a retinal-containing membrane protein, exhibits a reversed order of proton release and uptake at neutral pH, as compared to the well-known bacteriorhodopsin (BR). In a preceding report, we stated that Triton X-100 solubilized the claret membrane containing AR4 (CM) into monomeric proteins and altered the time order in AR4 at neutral pH. The present study examined the mechanism underlying this phenomenon. We employed a photoelectrochemical cell suitable for observation of the proton pumping behaviors of both the membrane patch and detergent-solubilized proteins over a wide pH range. The pK(a) values of the proton release complex (PRC) in the initial state and the M state were determined with this device. The pK(a) of PRC of monomeric AR4 decreased to a value lower than 7.0 in the photocycle, allowing early proton release at neutral pH. The pK(a) of PRC in the initial state was also strongly affected by solubilization.     

Triton X-100 solubilization of mitochondrial inner and outer membranes

Rat liver mitochondrial inner and outer membranes were subjected to the solubilizing effect of the nonionic detergent Triton X-100 under various conditions. After centrifugation, the supernatants (containing the solubilized fraction) and pellets were characterized chemically and/or ultrastructurally. The detergent seems to act by inducing a phase transition from membrane lamellae to mixed protein-lipid-detergent micelles. Different electron-micro-scopy patterns are shown by the inner membranes after treatment with different amounts of surfactant, whereas the corresponding images from outer membranes vary but slightly. Selective solubilization of various components is observed, especially in the case of the inner membrane. Some membrane lipids (e.g., cardiolipin) are totally solubilized at detergent concentrations when others, such as sphyngomyelin, remain in the membrane. Other inner-membrane components (flavins, cytochromes, coenzymeQ) show different solubilization patterns. This allows the selection of conditions for optimal solubilization of a given membrane component with some degree of selectivity. The influence of Triton X-100 on various mitochondrial inner-membrane enzyme activities was studied. The detergent seems to act especially through disruption of the topology of the functional complexes, although the activity of the individual enzymes appears to be preserved. Relatively simple enzyme activities, such as ATPase, are more or less solubilized according to the detergent concentration, whereas the more complex succinate-cytochromec reductase activity practically disappears even at low Triton X-100 concentrations.     

Interaction of Triton X-100 with the pigment-protein complexes of photosynthetic membranes.

The interaction of the non-ionic detergent Triton X-100 with photosynthetic membrane components of Pisum sativum (pea) is described. The detergent affected both the wavelength and the intensity of the 77K fluorescence-emission peaks of both Photosystem I and Photosystem II preparations, in addition to the effects on whole thylakoids recently described by Murphy & Woodrow [(1984) Biochem. J. 224, 989-993]. Below its critical micellar concentration, Triton X-100 had no effect on 77K fluorescence emissions even after prolonged incubations of up to 30 min. Above the critical micellar concentration of about 0.16 mg X ml-1, Triton X-100 caused a dramatic increase in the intensity of the 680 nm emission. The intensity of the 680 nm fluorescence emission continued to increase as more Triton X-100 was added, until limiting concentrations of detergent were reached. These limiting concentrations were proportional to the amount of membrane present and generally occurred at Triton X-100/chlorophyll (w/w) ratios of 100-200:1. In all cases the detergent effect was seen within 10 min, and is often considerably faster, with longer detergent treatments causing no further effects. The data are discussed in terms of a three-stage mechanism for detergent solubilization of membrane components.     

Triton X-100 alters the resistance level of methicillin-resistant Staphylococcus aureus to oxacillin

Abstract We used population analysis to examine the effects of Triton X-100 on the level of resistance to oxacillin of 18 methicillin-resistant Staphylococcus aureus . In the presence of 0.02% Triton X-100, 17 formerly methicillin-resistiant strains exhibited enhanced sensitivity to oxacillin. One homogeneous isolate, KSAF1 was barely affected by the Triton X-100. Sensitivities of lysostaphin, 51 kDa N -acetylglucosaminidase and 62 kDa N -acetylmuramoyl-l-alanine amidase to heat-inactivated cells were not affected when the bacteria were grown in 0.02% Triton X-100. Our data, together with those of a previous study, suggested that Triton X-100 alters the resistance level of methicillin-resistant S. aureus by influencing a factor(s) other than PBPs, bacteriolytic enzymes, or femAB products.