F.M.A.H. Schuurmans Stekhoven

Studies on (Na+ +K+) activated ATPase. XLI. Effects of N-ethylmaleimide on overall and partial reactions.

1. Preincubation with N-ethylmaleimide inhibits the overall activity of highly purified (Na+ +K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparations of rabbit kidney outer medulla. 2. This inhibition is decreased by addition of ATP or 4-nitrophenylphosphate under non-phosphorylating conditions, and also by addition of ADP or adenylylimidodiphosphate. 3. N-ethylmaleimide treatment leads to inhibition of K+-stimulated 4-nitrophenylphosphatase activity, Na+-stimulated ATPase activity, and phosphorylation by ATP as well as by inorganic phosphate. These inhibitions strictly parallel that of the overal (Na+ +K+)-ATPase reaction. 4. N-ethylmaleimide lowers the number of sites which are phosphorylated by inorganic phosphate, without affecting the dissociation constant of the enzyme-phosphate complex. 5. N-ethylmaleimide does not affect the relative stimulation by ATP of the K+-stimulated 4-nitrophenylphosphatase activity. 6. These effects of N-ethylmaleimide can be explained as a complete loss of active enzyme, either by reaction of N-ethylmaleimide inside the active center, or by alterations in the quaternary structure through reactions outside the active center.

Kinetics of Cu2+ inhibition of Na+K+-ATPase

The interaction of Cu2+ with enzymatic activity of rabbit kidney Na+/K(+)-ATPase was studied in media with buffered, defined free Cu2+ levels. The IC50-values are 0.1 mumol/l for Na+/K(+)-ATPase and 1 mumol/l for K(+)-pNPPase. Dithiothreitol (DTT) reverses the inhibitory effect of Cu2+ in vitro. Cu2+ exerts non-competitive effects on the enzyme with respect to Na+, K+, ATP or pNPP, but has a mixed-type inhibitory effect with respect to Mg2+. It is concluded that the appreciation of the inhibitory effect of Cu2+ on this enzyme requires carefully composed assay media that include a buffer for Cu2+, and that the IC50-values calculated according to this model indicate that Cu2+ may be more toxic than previously anticipated.

Studies on (Na++K+)-activated ATPase: XXXVII. Stabilization by cations of the enzyme-ouabain complex formed with Mg2+ and inorganic phosphate

Dissociation of the (Na+ + K+)-ATPase ouabain complex, formed in the presence of Mg2+ and inorganic phosphate (Complex II), is inhibited by Mg2+ (21-45%) and the alkali cations Na+ (25-59%) and K+ (27-75%) when kidney cortex tissue (bovine, rabbit, guinea pig) is the enzyme source. Choline chloride at 200 mM, equivalent to the highest concentration of NaCl tested, does not inhibit. Dissociation of Complex II from brain cortex (bovine, rat, rabbit) or heart muscle (rabbit) is much less inhibited: 0-11% by Na+ and 11-19% by K+. The degree of inhibition is not directly related to the size of the dissociation rate constant (k-) of the various complexes, but rather to the extent of interaction between the cation and ouabain binding sites for these tissues. Inhibition curves for Na+ and K+ are sigmoidal. Half-maximal inhibition for rabbit brain and kidney cortex is at 30-40 mM Na+ and 6-10 mM K+, and the maximally inhibitory concentrations are 50-150 and 15-20 mM, respectively. Maximal inhibition by Na+ or K+ for these tissues is the same. For guinea pig kidney cortex Na+ and K+ are almost equally effective, but 150 mM K+ or 200 mM Na+ are still not saturating, and inhibition curves indicate high- and low-affinity binding sites for the alkali cations. The inhibition curve for Mg2+ is not sigmoidal. In the kidney preparations Mg2+ inhibits half-maximally at 0.4-0.5 mM, maximally at 1-3 mM. Maximal inhibition by Mg2+ is higher than by Na+ or K+ for rabbit kidney cortex and lower for guinea pig kidney cortex. There is no competition or additivity among the cations, indicating the existence of different binding sites for Mg2+ and the alkali cations. Complex II differs in stability in the extent of inhibition, in the dependence of inhibition on the cation concentration and in the absence of antagonism between Na+ and K+, from the ouabain complex formed via phosphorylation by ATP (Complex I). This indicates that the phosphorylation states for the complexes are clearly different.

Mitochondrial DNA: III. Electron microscopy of DNA released from mitochondria by osmotic shock

Abstract 1. 1. Isolated mitochondria were subjected to osmotic shock and the mitochondrial DNA released was studied by electron microscopy using the protein-monolayer technique. 2. 2. In shocked preparations of freshly isolated chick liver mitochondria, about 85 % of the long DNA visible was present as highly twisted circular molecules (average contour length ±S.D. = 5.2±0.4 μ ), the remainder as open or half-open circles (5.6±0.4 μ ). Storage of the mitochondria for up to 1 week prior to osmotic shock led to a decrease in the percentage of twisted circles with a concomitant increase in the percentage of open circles and linear molecules. 3. 3. The average number of cross-overs (±S.D.) in twisted circles at 20–22° was 33 (±7) for chick liver mitochondrial DNA (mol. wt. = 10–11·10 6 dalton) spread on H 2 O by osmotic shock, 35 (±6) for purified chick-liver mitochondrial DNA spread on 0.1 M ammonium acetate, and 13 (±2) for purified replicative form DNA of phage ΦX (mol. wt. = 3.4·10 6 dalton) spread on salt. 4. 4. Osmotic shock also released predominantly circular DNA from mitochondrial preparations of rat liver (average contour length 5.4 μ), carp liver (5.4 μ) and housefly flight muscle (5.2 μ). 5. 5. Heterogeneous linear DNA was obtained from Saccharomyces carlsbergensis mitochondria both by standard purification procedures and by osmotic shock. The conclusion is drawn that the intact mitochondrial DNA of yeast is substantially larger than 5 μ.

Studies on (Na+ + K+)-activated ATPase. XLIV. Single phosphate incorporation during dual phosphorylation by inorganic phosphate and adenosine triphosphate.

(Na+ + K+)-ATPase can be phosphorylated by its substrate ATP as well as by its product inorganic phosphate. The maximal capacity for phosphorylation by either of these two substances is one mol phosphate per mol enzyme. In order to investigate whether the enzyme molecule possesses only on phosphorylation site common to ATP and Pi, or two phosphorylation sites, one for ATP and one for Pi, dual phosphorylation of the enzyme has been carried out. Under conditions, which are maximally favourable for each type of phosphorylation, successive phosphorylation by Pi and ATP leads to a maximal incorporation of only one mol phosphate per mol enzyme. The phosphorylation capacity for ATP decreases by the same amount as the Pi-phosphorylation level increases, without an effect on the apparent affinity for ATP. The results can be explained by assuming either a single common phosphorylation site for Pi and ATP, or a conformational change of the enzyme following phosphorylation by Pi, which excludes phosphorylation by ATP.

Na+-like effect of imidazole on the phosphorylation of (Na+ + K+)-ATPase☆

A high basal level of phosphorylation (approx. 70% of the optimal Na+-dependent phosphorylation level) is observed in 50 mM imidazole-HCl (pH 7.0), in the absence of added Na+ and K+ and the presence of 10-100 microM Mg2+. In 50 mM Tris-HCl (pH 7.0) the basal level is only 5%, irrespective of the Mg2+ concentration. Nevertheless, imidazole is a less effective activator of phosphorylation than Na+ (Km imidazole-H+ 5.9 mM, Km Na+ 2 mM under comparable conditions). Imidazole-activated phosphorylation is strongly pH dependent, being optimal at pH less than or equal to 7 and minimal at pH greater than or equal to 8, while Na+-activated phosphorylation is optimal at pH 7.4. This suggests that imidazole-H+ is the activating species. Imidazole facilitates Na+-stimulated phosphorylation. The Km for Na+ decreases from 0.63 mM at 5 mM imidazole-HCl to 0.21 mM at 50 mM imidazole-HCl (pH 7; 0.1 mM Mg2+ in all cases). Imidazole-activated phosphorylation is more sensitive to inhibition by K+ (I50 = 12.5 microM) than Na+-activated phosphorylation (I50 = 180 microM). Mg2+ antagonizes activation by imidazole-H+ and also inhibition by K+. The Ki value for Mg2+ (approx. 0.3 mM) is the same for the two antagonistic effects. Tris buffer (pH 7.0) inhibits imidazole-activated phosphorylation with an I50 value of 30 mM in 50 mM imidazole-HCl (pH 7.0) plus 0.1 mM Mg2+. We conclude that imidazole-H+, but not Tris-H+, can replace Na+ as an activator of ATP-dependent phosphorylation, primarily by shifting the E2----E1 transition to the right, leading to a phosphorylating E1 conformation which is different from that in Tris buffer.

Studies on (NA+ + K+)-activated ATPase XLV. Magnesium induces two low-affinity non-phosphorylating nucleotide binding sites per molecule☆

Abstract ATP and adenylylimidodiphosphate ( Ado PP[ NH ]P ) bind to (Na + + K + )-ATPase in the absence of Mg 2+ (EDTA present) with a homogeneous but 15-fold different affinity, the K d values being 0.13 μM and 1.9 μM, respectively. The binding capacities of the two nucleotides are nearly equal and amount to 3.9 and 4 nmol/mg protein or 1.7 and 1.8 mol/mol (Na + + K + )-ATPase, respectively. The K d value for ATP is equal to the K m for phosphorylation by ATP (0.05–0.25 μM) and the binding capacity is equivalent to the phosphorylation capacity of 1.8 mol/mol (Na + + K + )-ATPase. Hence, the enzyme contains two high-affinity nucleotide binding and phosphorylating sites per molecule, or one per α-subunit. Additional low-affinity nucleotide binding sites are elicited in the presence of Mg 2+ , as shown by binding studies with the non-phosphorylating ( Ado PP [NH] P ). The K d and binding capacity for Ado PP [NH] P at these sites is dependent on the Mg 2+ concentration. The K d increases from 0.06 mM at 0.5 mM Mg 2+ to a maximum of 0.26 mM at 2 mM Mg 2+ and the binding capacity from 1.5 nmol/mg protein at 0.5 mM Mg 2+ to 3.3 nmol/mg protein at 4 mM Mg 2+ . Extrapolation of a double reciprocal plot of binding capacity vs. total Mg 2+ concentration yields a maximal binding capacity at infinite Mg 2+ concentration of 3.8 nmol/mg protein or 1.7 mol/mol (Na + + K + )-ATPase. The K d for Mg 2+ at the sites, where it exerts this effect, is 0.8 mM. The K d for the high-affinity sites increases from 1.5–1.9 μM in the absence of Mg 2+ to a maximum of 4.2 μM at 2 mM Mg 2+ concentration. The binding capacity of these sites (1.8 mol/mol enzyme) is independent of the Mg 2+ concentration. Hence, Mg 2+ induces two low-affinity non-phosphorylating nucleotide binding sites per molecule (Na + + K + )-ATPase in addition to the two high-affinity, phosphorylating nucleotide binding sites.

Eosin, a fluorescent marker for the high-affinity ATP site of (K+ + H+)-ATPase.

Abstract Eosin has been used as a fluorescent probe for studying conformational states in (K + + H + )-ATPase. The eosin fluorescence level is increased by Mg 2+ ( K 0.5 = 0.2 mM). This increase is counteracted by K + ( I 0.5 = 1.3 mM) and choline ( I 0.5 = 17.2 mM) and by ATP. Binding studies with eosin indicate that the increase and decrease in fluorescence is due to changes in binding of eosin to the enzyme. The Mg 2+ -induced specific binding has a K d of 0.7 μM and a maximal capacity of 3.5 nmol per mg enzyme, which is equivalent to 2.5 site per phosphorylation site. These experiments and the fact that eosin competitively inhibits (K + + H + )-ATPase towards ATP, suggest that eosin binds to ATP binding sites.

Studies on yeast mitochondria: I. Existence of three phosphorylation sites along the respiratory chain of isolated yeast mitochondria

Yeast mitochondria have been prepared from protoplasts of Saccharomyces carlsbergensis grown on a medium with glucose and Na-lactate as carbon sources. Specific activities, PO ratios, and respiratory control ratios were determined for the oxidation of ethanol, l-malate + pyruvate, sodium acetate + l-malate, citrate, succinate α-glycerophosphate, l-lactate, NADH, and NADPH. Phosphorous to oxygen ratios exceeding 2 were observed with ethanol, l-malate + pyruvate, and citrate as substrates, demonstrating the existence of at least three phosphorylation sites along the respiratory chain of yeast mitochondria. Externally added NADH and NADPH were oxidized with PO ratios of 2. Rotenone did not inhibit the oxidation of typical NAD-linked substrates like α-ketoglutarate, l-malate + pyruvate, and citrate; it only inhibited the oxidation of ethanol, probably through inhibition of alcohol dehydrogenase. Rotenone did not effect the PO ratios as measured with l-malate + pyruvate and citrate as substrates. Antimycin A inhibited almost completely the oxidation of l-malate + pyruvate, succinate. α-glycerophosphate, NADH, and NADPH, but l-lactate oxidation was only half inhibited.

Monoclonal antibody to phosphatidylserine inhibits Na+/K+-ATPase activity

A monoclonal IgG, directed to phosphatidylserine (PS1G3), partially (40-50%) inhibited Na+/K(+)-ATPase activity (forward running reaction cycle) without affecting the K0.5 values for Na+,K+ and MgATP. The Hill or interaction coefficients (nH) for Na+ and K+ for this reaction were reduced from 3.0 to 1.6 and from 1.6 to 0.8, respectively. The K(+)-stimulated p-nitrophenylphosphatase activity (p-NPPase), which is a partial reaction sequence of the Na+/K(+)-ATPase system (but in the backward running mode), was inhibited more strongly (about 70%) due to an increase in K+/substrate antagonism. In this system K0.5 and nH values for both p-nitrophenyl phosphate (p-NPP) and K+ were increased by the mAb. At the maximally inhibitory concentration of PS1G3 the Vmax of the p-NPPase was also reduced. Partial reactions, which were inhibited by PS1G3, are: (1) the Na(+)-activated phosphorylation (non-competitive vs. Na+), (2) the Rb+ occlusion (competitive vs. Rb+). Partial reactions not harmed by PS1G3 are: (3) the K(+)-dependent dephosphorylation, (4) the K(+)-dependent E1 + K+<-->E2K transition. We conclude that PtdSer is involved in cation occlusion, possibly by forming part of the access gate.