Joseph D. Robinson

Substrate sites of the (Na+ + K+)-dependent ATPase

Kinetic studies on a rat brain (Na+ + K+)-dependent ATPase (EC 3.6.1.3) preparation demonstrated high-affinity sites for ATP, with a Km near 1 mum, and low affinity sites for ATP, with a Km near 0.5 mM. In addition, the dissociation constant for ATP at the low affinity sites was approached through the ability of ATP to modify the rate of photo-oxidation of the enzyme in the presence of methylene blue; a value of 0.4 mM was obtained. The temperature dependence of the Km values in these two concentration ranges also differed markedly, and the estimated entropy of binding was +27 cal/degree per mol at the high affinity sites, whereas it was -20 cal/degree per mol at the low affinity sites. Moreover, the relative affinities of various congeners of ATP as of the K+ -dependent phosphatase reaction of the enzyme indicated an interaction at the low-affinity sites for ATP: ATP, ADP, CTP, and the [beta-gamma] -imido analog of ATP all competed with Ki values near those for the ATPase reaction at the low affinity sites. Conversely, the Km for nitrophenyl phosphate as a substrate for the phosphatase reaction was near its Ki as a competitor at the low-affinity sites of the ATPase reaction. These observations are incorporated into a reaction scheme with two classes of substrate sites on a dimeric enzyme, manifesting idverse enzymatic and transport characteristics.

Interactions between monovalent cations and the (Na+ + K+)-dependent adenosine triphosphatase

Abstract Kinetic properties of a (Na + + K + )-dependent ATPase preparation from rat brain were examined as functions of the concentrations of activating monovalent cations, in terms of: K 0.5 , the activator concentration for half-maximal velocity; n , the slope of the Hill plot; and V max , the maximal velocity corresponding to the level of complementary activator. As the level of NaCl was progressively raised, the kinetic response to K + showed corresponding increases in K 0.5 and n . By contrast, as the level of KCl was progressively raised, the response to Na + showed an initial decrease in n followed, at high KCl levels, by a subsequent increase; K 0.5 progressively increased. For both activators V max rose sharply with increasing levels of the complementary activator, and then appeared to plateau near 100 m m NaCl and 10 m m KCl. Substitution of NH 4 Cl or TlCl for KCl produced similar kinetic patterns, both with the (Na + + K + )-dependent ATPase and the associated K + -dependent phosphatase activity, although for both activities K 0.5 for Tl + and NH 4 + differed 40-fold, spanning the value for K + . The kinetic response of the phosphatase to Na + was not consistent with simple competition between Na + and K + for K + -sites. The data are considered in terms of specific kinetic models for the ATPase: the kinetics appear to require multiple sites for both Na + and K + with probable competition between Na + and K + and allosteric interactions; alternative reaction pathways may occur, dependent on the level of activation.

Structural changes in microsomal suspensions: III. Formation of lipid peroxides

Abstract Turbidity changes in suspensions of brain microsomes were induced by ascorbate, iron plus ascorbate, cysteine, and cysteine plus cystine. The changes were correlated with lipid peroxidation, which was measured either with thiobarbiturate or by oxygen consumption. The process was blocked by metal-binding compounds; of the metals tried only iron was capable of increasing lipid peroxidation with ascorbate or cysteine. Lipid peroxidation occurred in a similar fashion in suspensions of boiled microsomes. Determinations of the water content and of the relative ionic permeability of incubated microsomes suggested that lipid peroxidation resulted in an increased membrane permeability. Lipid peroxidation was accompanied by an increase in reactive sulfhydryl groups, and sulfhydryl-binding compounds could shorten the lag period before the lipid peroxidation and turbidity changes occurred.

Nucleotide and divalent cation interactions with the (Na+ + K+)-dependent ATPase

Abstract With a brain microsomal ( Na + + K + -dependent ATPase preparation (EC 3.6.1.3) the apparent K i for the MgATP complex was 0.48 mM. Free ATP was a competitive inhibitor with a K i of 4.8 mM, whereas the apparent affinity for free Mg2+, determined by a Be2+ inactivation technique, was 0.8 mM. Free Mg2+ was also a weak non-competitive inhibitor to MgATP, with a K 1 of about 40 mM. Mg2+ antagonized the effects of the modifier oligomycin, and oligomycin reduced the apparent affiniy for Mg2+, consistent with their favoring alternative allosteric states of the enzyme. Inhibition by CaCl2 was “mixed”, in keeping with competitive inhibition by CaATP toward MgATP together with competition by Ca2+ at Na+-sites, also demonstrated. With the associated K+-dependent nitrophenylphosphate activity CaCl2 was a conventional competitor toward MgCl2. Examination of the effects of free Mg2+ indicated that reaction schemes proposing a cyclical addition to and release from the enzyme of Mg2+ must incorporate a cyclical change in affinity of at least four orders of magnitude. An alternative scheme is suggested with multiple substrate sites of differing affinity, only half of which may be hydrolytically active at a given time.

(Ca + Mg)-stimulated ATPase activity of a rat brain microsomal preparation.

Abstract ATPase activity of freshly prepared brain microsomes was stimulated 20% when 0.1 mm CaCl2 was added in the presence of a “saturating” concentration of MgCl2 (4 m m ). This (Ca + Mg)-stimulated activity declined rapidly on storage. Treatment of the microsomes with 0.12% deoxycholate in 0.15 m KCl, followed by centrifugation and resuspension in sucrose, produced a preparation both stable on storage at −15 °C and with an increased stimulation in the presence of CaCl2. SrCl2 was more effective than CaCl2, but BaCl2 was a poor activator. KCl and NaCl stimulated the (Ca + Mg)-ATPase activity by reducing substrate (ATP) inhibition. The Km for ATP was 0.1 m m , a third that of the Mg-ATPase. CTP, ITP, and GTP could not substitute for ATP, although they were fair substrates for the Mg-ATPase. The energy of activation of the (Ca + Mg)-ATPase was 21 kcal, nearly twice that of the Mg-ATPase. After sucrose density-gradient centrifugation of the microsomal preparation, the (Ca + Mg)-ATPase activity was distributed with the (Na + K)-ATPase and not with the mitochondrial marker succinic dehydrogenase. Studies with ouabain, oligomycin, and azide distinguished the (Ca + Mg)-stimulated ATPase from (Na + K)- and mitochondrial ATPases. Sensitivity to ruthenium red suggested a link to Ca transport, although the microsomal 45Ca accumulating system was much more sensitive to the inhibitor than was this ATPase activity.

Phosphatase activity stimulated by Na+ plus K+: Implications for the (Na+ plus K+)-dependent adenosine triphosphatase☆

Abstract The effects of monovalent cations on the phosphatase activity of a partially purified (Na + plus K + )-dependent ATPase preparation were studied using as substrates p -nitrophenyl phosphate (NPP; NPPase activity) and acetyl phosphate (AcP; AcPase activity). K + -dependent NPPase activity was stimulated by Na + at low KCl concentrations but more markedly by Na + in the presence of CTP or AcP; the stimulation represented a decrease in the concentration of KCl for half maximal activation, K 0.5 . K + -dependent AcPase was also stimulated by Na + through a reduction in K 0.5 for KCl, and the K 0.6 for Na + activation was the same as that for the NPPase activity. Oligomycin and N -ethylmaleimide (NEM) inhibited activation of phosphatase activity by Na + and by Na + plus CTP or AcP but did not affect K + -dependent phosphatase activity in the absence of Na + . Moreover, NEM diminished the Na + -induced change in the K 0.5 of the AcPase for KCl. The data are considered in terms of K + -and (Na + plus K + )-activated pathways for the phosphatases with the latter being analogous to the (Na + plus K + )-activated ATPase pathway involving a Na + -dependent phosphorylation of the enzyme. In addition, the stimulation of NPPase activity after phosphorylation by AcP or CTP suggests that the hydrolytic site is not identical with (although sensitive to) the site for Na + -dependent phosphorylation.

Stimulation of the (Na+ + K+)-dependent adenosine triphosphatase by amino acids and phosphatidylserine: Chelation of trace metal inhibitors☆

Abstract Amino acids stimulated the (Na+ + K+)-dependent ATPase activity of a rabbit kidney preparation without affecting the Mg2+-ATPase activity; the most effective was histidine, producing a 2-fold increase in activity. Similar stimulation was produced by the well-known chelators EDTA, EGTA, and 8-hydroxyquinoline, and by the chelating phospholipid phosphatidylserine. In the presence of maximally effective concentrations of one agent, the other agents were unable to produce additional stimulation. It is suggested that the amino acids, phosphatidylserine, and the conventional chelators all stimulate the ATPase by a common mechanism: the removal of inhibitory trace metal (s). From measurements of the metal content of the enzyme preparation and experiments with extracted reagents it was concluded that the chelatable inhibitor was in the reagents used in the incubation medium rather than being endogenous to the enzyme; attempts to identify the inhibitor (s) were unsuccessful. The chelators also stimulated the K+-dependent phosphatase activity in the preparation but had no major effect on Na+-dependent incorporation of 32P from [32P]ATP. On monovalent cation activation the chelators appeared to relieve an uncompetitive inhibition of Na32 activation and a noncompetitive inhibition of K32 activation, also suggesting an action of the chelatable inhibitor on the later stages of the ATPase reaction sequence.

Adenosine triphosphate-dependent calcium accumulation by brain microsomes☆

A microsomal fraction from rat brain accumulated calcium by a temperature-dependent process requiring both ATP and Mg2+. After brief incubations the concentration of calcium in the microsomes reached levels several hundred times that in the medium. The addition of oxalate, phosphate, or acetate did not augment the accumulation. Univalent cations reduced the uptake, and ouabain was not inhibitory. A variety of enzyme inhibitors, including sulfhydryl reagents, amytal, and oligomycin, decreased calcium accumulation. Procaine reduced uptake by one-third, but a series of neurotropic drugs had little effect. The relationship between calcium accumulation and ATPase activity as well as the state of the accumulated calcium remained unclear. A possible homeostatic role in neural metabolism is suggested.

Fluoride and beryllium interact with the (Na + K)-dependent ATPase as analogs of phosphate

Fluoride irreversibly inhibits the (Na + K)-ATPase, and this inactivation requires divalent cations (Mg2+, Mn2+, or Ca2+), is augmented by K+, but is diminished by Na+ and by ATP. Prior incubation with the aluminum chelator deferoxamine markedly slows inactivation, whereas adding 1 µM AlCl3 speeds it, consistent with AlF−4 being the active species. Prior incubation of the enzyme with vanadate also blocks inactivation by fluoride added subsequently. Fluoride stimulates ouabain binding to the enzyme, and thus the analogy between AlF−4 and both orthophosphate and orthovanadate is reflected not only in the similar dependence on specific ligands for their enzyme interactions and their apparent competition for the same sites, but also in their common ability to promote ouabain binding. Beryllium also irreversibly inhibits the enzyme, and this inactivation again requires divalent cations, is augmented by K+, but is diminished by Na+ and by ATP. Similarly, prior incubation of the enzyme with vanadate blocks inactivation by beryllium added subsequently. Inactivation by beryllium, however, does not require a halide, and, unlike inactivation by fluoride, increases at basic pHs. These observations suggest that beryllium, as beryllium hydroxide complexes, acts as a phosphate analog, similar to AlF−4 and vanadate.

Differential modification of the (Na+ + K+)-dependent ATPase by dimethylsulfoxide

1. 1. Dimethylsulfoxide reversibly inhibited the (Na+ + K+)-dependent ATPase activity of a brain microsomal enzyme preparation, but stimulated the associated K+-dependent phosphatase activity; this disparate effect was not caused by two other lipophilic agents, Lubrol-W and propanol. 2. 2. For the ATPase dimethylsulfoxide reduced the concentration for half-maximal activation, K0,5, for NaCl, but increased it for KCl; in both cases V was decreased. Since both V and Km for ATP were also decreased in parallel, it thus appeared that dimethylsulfoxide was an uncompetitive inhibitor to ATP and Na+, perhaps acting to modify a stage following their reaction with the enzyme, and thereby altering the apparent affinity for K+. 3. 3. With the phosphatase, however, dimethylsulfoxide reduced K0,5 for KCl and increased V. But in the presence of CTP and NaCl, which have been shown to modify the phosphatase reaction to resemble more closely the ATPase, dimethylsulfoxide then increased K0,5 for KCl although V was still increased. The stimulation of the phosphatase could be accounted for by a decrease in Km for the substrate, nitrophenylphosphate. 4. 4. The effects on apparent affinity for K+ thus represent an action of dimethylsulfoxide dependent both on the route of substrate entry (Na+-dependent for the ATPase, K+-dependent for the phosphatase) and the reaction history (prior interaction with nucleotide plus Na+, or not). If dimethylsulfoxide acts by selecting or modifying intermediary reaction conformations (or their analogues), then the variable cation affinity shown for such intermediate stages may bear on the cyclical changes in affinity proposed to accompany the hydrolytic process.