Stimulated P-nitrophenyl Phosphatase Research Articles

Characterization of the beta-subunit of the H(+)-K(+)-ATPase using an inhibitory monoclonal antibody

The gastric proton pump, H(+)-K(+)-ATPase, is composed of alpha- and beta-subunits. The 95-kDa alpha-subunit has been referred to as the catalytic subunit containing sites for ATP binding and phosphorylation. The beta-subunit is a glycoprotein with a 34-kDa core peptide that has a single transmembrane segment, a small cytoplasmic NH2-terminal, and a large extracellular COOH-terminal domain with seven potential N-linked glycosylation sites. To further study the beta-subunit, we developed monoclonal antibodies that identified a 52-kDa mannose-rich glycoprotein that was deglycosylated by endoglycosidase H such that six transient intermediates were identified, as well as a 34-kDa beta-subunit core peptide. These observations suggest that the beta-subunit is synthesized as a 52-kDa glycoprotein with seven N-linked precursor high-mannose oligosaccharides that mature into complex oligosaccharides. One antibody, 2G11, inhibits the K(+)-stimulated ATP hydrolysis as well as K(+)-stimulated p-nitrophenyl phosphatase (pNPPase) activity of the H(+)-K(+)-ATPase. Kinetic studies revealed that 2G11 inhibited maximum velocity (Vmax) of ATP hydrolysis by approximately 50% with no change in the Km for K+, whereas, for pNPPase both Vmax and Km were altered. These studies demonstrate a functional role for the beta-subunit in the H(+)-K(+)-ATPase activity, especially the K(+)-induced conformational states.

Inhibitory effect of tannic acid on gastric H+,K(+)-ATPase.

The effect of tannic acid on gastric H+,K(+)-ATPase was studied. Tannic acid dose-dependently inhibited pig gastric H+,K(+)-ATPase activity with an IC50 value of 2.9 x 10(-8) M. Tannic acid also inhibited K(+)-stimulated p-nitrophenyl phosphatase (K(+)-pNPPase) activity, which is found in gastric H+,K(+)-ATPase preparations, as well as H+,K(+)-ATPase activity, with an IC50 value of 4.1 x 10(-7) M. Kinetic studies showed that the inhibition of H+,K(+)-ATPase activity by tannic acid was competitive with respect to ATP and noncompetitive with respect to K+. These results show that tannic acid is a potent inhibitor of gastric H+,K(+)-ATPase; this may be related to its anti-secretory and anti-ulcerogenic effects.

Inhibition of gastric H+, K+-ATPase by chalcone derivatives, xanthoangelol and 4-hydroxyderricin, from Angelica keiskei Koidzumi

Two chalcone derivatives, xanthoangelol (1) and 4-hydroxyderricin (II) isolated from Angelica keiskei Koidzumi, inhibited pig gastric H+, K(+)-ATPase with IC50 values of 1.8 and 3.3 microM, respectively. The inhibition by I or II was competitive with respect to ATP and was non-competitive with respect to K+ I and II also inhibited K+, stimulated p-nitrophenyl phosphatase, with IC50 values of 1.3 and 3.5 microM, respectively. Proton transport in-vitro was inhibited by I or II, in a dose-dependent manner, 1 at 100 mg kg-1, i.p. significantly inhibited acid secretion and the formation of stress-induced gastric lesions. These results suggest that the antisecretory effect of 1 is due to the inhibition of gastric H+, K(+)-ATPase.

Effect of mercuric chloride on the kinetics of cationic and substrate activation of the rat brain microsomal ATPase system

Mercuric chloride (HgCl 2), a neurotoxic compound, inhibited the adenosine triphosphatase (ATPase) system in a concentration-dependent manner. Hydrolysis of ATP was linear with time with or without HgCl 2 in the reaction mixtures. Higher inhibition of (Na +−K +)ATPase activity by HgCl 2 was observed in alkaline (8.0 to 9.0) pH and at lower temperatures (17 to 32°). Activation energy values were increased slightly in the presence of HgCl 2. Activation of (Na +−K +)ATPase by ATP in the presence of HgCl 2 showed a decrease in V max from 15.29 to 5.0 μmol of inorganic phosphate (P i)/mg protein/hr with no change in K m . Similarly, activation of K +-stimulated p-nitrophenyl phosphatase (K +-PNPPase) in the presence of HgCl 2 showed a decrease in V max from 3.26 to 1.35 μmol of p-nitrophenol (PNP)/mg protein/hr with no change in K m . K +-activation kinetic studies indicated that HgCl 2 decreased V max from 14.01 to 4.30 μmol P i/mg protein/hr in the case of (Na +-K +)ATPase and from 3.45 to 2.40 μmol PNP/mg protein/hr in the case of K +-PNPPase with no changes in K m . Na +-activation of (Na +-K +)ATPase in the presence of HgCl 2 showed a decrease in V max from 11.06 to 3.23 μmol P i/mg protein/hr and an increase in K m from 1.06 to 2.08 mM. Preincubation of microsomes with sulfhydryl (SH) agents dithiothreitol, cysteine and glutathione protected HgCl 2-inhibition of (Na +−K +)ATPase. The data suggest that HgCl 2 inhibited (Na +-K +)ATPase by interfering with the dephosphorylation of the enzymephosphoryl complex.

Coupled Na+/H+ exchange in rat parotid basolateral membrane vesicles.

pH gradient-dependent sodium transport in highly purified rat parotid basolateral membrane vesicles was studied under voltage-clamped conditions. In the presence of an outwardly directed H+ gradient (pHin = 6.0, pHout = 8.0) 22Na uptake was approximately ten times greater than uptake measured at pH equilibrium (pHin = pHout = 6.0). More than 90% of this sodium flux was inhibited by the potassium-sparing diuretic drug amiloride (K1 = 1.6 microM) while the transport inhibitors furosemide (1 mM), bumetanide (1 mM), SITS (0.5 mM) and DIDS (0.1 mM) were without effect. This transport activity copurified with the basolateral membrane marker K+-stimulated p-nitrophenyl phosphatase. In addition, 22Na uptake into the vesicles could be driven against a concentration gradient by an outwardly directed H+ gradient. pH gradient-dependent sodium flux exhibited a simple Michaelis-Menten-type dependence on sodium concentration consistent with the existence of a single transport system with KM = 8.0 mM at 23 degrees C. A component of pH gradient-dependent, amiloride-sensitive sodium flux was also observed in rabbit parotid basolateral membrane vesicles. These results provide strong evidence for the existence of a Na+/H+ antiport in rat and rabbit parotid acinar basolateral membranes and extend earlier less direct studies which suggested that such a transporter was present in salivary acinar cells and might play a significant role in salivary fluid secretion.

Half of the (Na+ + K+)-transporting-ATPase-associated K+-stimulated p-nitrophenyl phosphatase activity of gastric epithelial cells is exposed to the surface exterior

Ouabain inhibited 86RbCl uptake by 80% in rabbit gastric superficial epithelial cells (SEC), revealing the presence of a functional Na+,K+-ATPase [(Na+ + K+)-transporting ATPase] pump. Intact SEC were used to study the ouabain-sensitive Na+,K+-ATPase and K+-pNPPase (K+-stimulated p-nitrophenyl phosphatase) activities before and after lysis. Intact SEC showed no Na+,K+-ATPase and insignificant Mg2+-ATPase activity. However, appreciable K+-pNPPase activity sensitive to ouabain inhibition was demonstrated by localizing its activity to the cell-surface exterior. The lysed SEC, on the other hand, demonstrated both ouabain-sensitive Na+,K+-ATPase and K+-pNPPase activities. Thus the ATP-hydrolytic site of Na+,K+-ATPase faces exclusively the cytosol, whereas the associated K+-pNPPase is distributed equally across the plasma membrane. The study suggests that the cell-exterior-located K+-pNPPase can be used as a convenient and reliable 'in situ' marker for the functional Na+,K+-ATPase system of various isolated cells under noninvasive conditions.

Insulin reversal of biochemical changes in hearts from diabetic rats.

Reversal of myocardial biochemical changes with insulin treatment (4 and 8 wk) was studied in 8 and 12 wk streptozotocin (STZ)-diabetic rats. STZ-induced diabetes was characterized by elevations in blood glucose, serum cholesterol, and triglycerides and depressed serum insulin levels. Insulin treatment for 4 and 8 wk completely restored the serum alterations to control values. The polyuria, polydipsia, and polyphagia were also markedly diminished by the insulin treatment. Diabetic rats had pronounced decreases in body, heart, and left ventricular weights, all of which were completely reversed by the insulin treatment. Hydroxyproline accumulation in diabetic rat hearts was only reversed by the 8-wk and not by the 4-wk insulin treatment. STZ produced a significant depletion of left ventricular magnesium content as well as depression of K+-stimulated sarcoplasmic reticulum and myofibrillar ATPase activities. Both the 4- and 8-wk insulin treatment produced a complete recovery of the myocardial magnesium content. No significant changes in sarcolemmal Na+-K+-ATPase and K+-stimulated p-nitrophenyl phosphatase activities were observed in diabetic animals compared with control. The decreased latency of the lysosomal hydrolase, N-acetyl-beta-glucosaminidase, and the increased collagen deposition observed in the diabetic hearts were only partially reversed by the 4-wk insulin treatment, but completely reversed by the 8-wk treatment period.

Isolation of plasma membrane, golgi apparatus, and endoplasmic reticulum fractions from single homogenates of mouse liver.

Procedures to isolate plasma membrane, Golgi apparatus, and endoplasmic reticulum from a single homogenate of mouse liver are described. Fractions contain low levels of contaminating membranes as determined from morphometry and analyses of marker enzymes. The method requires only 2-3 gm of liver as starting material and yields approximately 0.7, 0.7, and 0.5 mg protein/gm liver, respectively, for endoplasmic reticulum, Golgi apparatus, and plasma membrane. Golgi apparatus fractions show high levels of galactosyltransferase activity and consist of cisternal stacks and associated secretory vesicles and tubules. Endoplasmic reticulum fractions are enriched in both glucose-6-phosphatase and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-cytochrome c reductase and contain membrane vesicles with attached ribosomes. K+-stimulated p-nitrophenyl phosphatase and (Na+K+) adenosine triphosphatase activity are enriched in the plasma membrane fraction. This fraction consists of membrane sheets, many with junctional complexes, and bile canaliculi that are representative of the total hepatocyte plasma membrane. The fractionation procedure is designed to utilize small amounts of tissue (e.g., with liver slices), to reduce the total time required for fractionation, and to permit comparisons of constituents of plasma membrane, Golgi apparatus, and endoplasmic reticulum prepared from the same starting homogenates.

Effects of chlordecone and its structural analogs on p-nitrophenyl phosphatase

The effects of chlordecone, mirex, and four photoderivatives of mirex on K + -stimulated p-nitrophenyl phosphatase (KPNPPase) in rat brain synaptosomes were determined. Additionally, the effect of chlordecone on the substrate and K + activation kinetics and the inhibition of KPNPPase at 17, 27, and 37 °C were determined. The sensitivity of KPNPPase to various chemicals was highly variable. Chlordecone was the most effective inhibitor with an IC 50 of 6.2. 10 −6M. All other chemicals showed a maximum inhibition of 20% at 20 μM concentration. The order of inhibition at 20 μM concentration was chlordecone (100%) > 8-monohydro mirex (20%) > 2,8-dihydromirex (10%) > mirex (10%) > 10-monohydro mirex (9%) > 5,10-dihydro mirex (2%). Double reciprocal plots of the data obtained on the effect of chlordecone on the substrate and K + activation kinetics showed that the V max and K m were decreased with respect to paranitrophenyl phosphate, while it was competitive inhibition with respect to K + activation. Chlordecone inhibited KPNPPase more at 37 °C than at 27 °C and 17 °C suggesting a positive temperature effect. These results suggest that chlordecone is the most effective inhibitor among its structural analogs tested on KPNPPase in rat brain synaptosomes.

Inhibition of sodium and potassium adenosine triphosphatase by 2‘,3‘-O-(2,4,6-trinitrocyclohexadienylidene) adenine nucleotides. Implications for the structure and mechanism of the Na:K pump.

Trinitrophenyl derivatives of adenine nucleotides (TNP-nucleotides: 2',3'-O-2,4,6-trinitrocyclohexadienylidene complexes at neutral or basic pH) are potent inhibitors of (Na,K)-ATPase activity. The inhibitory potency of the derivatives tested followed the sequence: TNP-ADP greater than TNP-ATP greater than TNP-AMP much greater than TNP-IMP greater than TNP-adenosine. In the presence of Na+ plus K+, high and low affinity activation of ATPase activity by ATP was observed. Under these conditions, TNP-ATP inhibited (Na,K)-ATPase activity competitively with respect to ATP at the kinetically defined "low affinity ATP site." In the presence of Na+ alone, only high affinity activation by ATP was observed. Under these conditions, TNP-ATP inhibited (Na)-ATPase and enzyme phosphorylation by competing with ATP at the kinetically defined "high affinity ATP site." The Ki values for inhibition were similar to the KD values determined by direct TNP-ATP binding measurements, indicating that the same TNP-ATP site is involved in the inhibition of (Na,K)-ATPase and (Na)-ATPase activities. We conclude that high and low affinity ATP "sites" are interconvertible (i.e. they represent two forms of the same site) and do not co-exist independently. TNP-ATP also inhibited competitively the K+-stimulated p-nitrophenyl phosphatase activity and enzyme phosphorylation by Pi, suggesting that the catalytic site for these substrates is associated with the TNP-ATP site. A kinetic model for (Na,K)-ATPase turnover based on a single ATP site which changes affinity during turnover is presented. The model was analyzed by the King-Altman (1956) J. Phys. Chem. 60, 1375-1378) method to obtain the steady state equation for the rate of ATP hydrolysis as a function of ATP concentration. Computer simulations using published values of the rate constants of intermediate steps suggest that the model is adequate to describe the observed dependence of enzyme activity on ATP concentration and the inhibition by TNP-ATP. The implications of these results on the structure and mechanism of the (Na,K) pump are discussed.

Preparation and properties of highly enriched cardiac sarcolemma from isolated adult myocytes.

Isolated myocytes were prepared from adult canine hearts using a combined technique of myocardial perfusion followed by incubation with collagenase. More than 60% of the cells routinely excluded trypan blue dye. Disruption of the myocytes was accomplished using high pressure nitrogen cavitation. After differential and sucrose gradient centrifugation, the peak sarcolemmal fraction averaged 100-fold enrichment in ouabain-inhibited K+-stimulated p-nitrophenyl phosphatase and 82-fold in ouabain-inhibited (Na+,K+)-ATPase. These sarcolemmal membranes are enriched in phospholipid phosphorus (1.98 mumol/mg of protein) and more than 4-fold in sphingomyelin and cholesterol. Polyacrylamide gels revealed three major protein peaks at 50,000, 91,000, and 140,000 apparent molecular weights. This work demonstrates the feasibility of preparing highly pure cardiac sarcolemma from isolated adult myocytes. The problem of cellular cross-contamination due to heterogeneity of cell types in whole myocardial tissue has been circumvented. The level of enrichment exceeds all reported preparations of cardiac sarcolemma from whole myocardium and cultured myocytes. This preparation should prove to be useful as an in vitro model for studies of physiological, pharmacological, and pathological perturbations of sarcolemmal structure and function.

The isolation and partial characterization of the plasma membrane from Trypanosoma brucei.

Whole sheets of plasma membrane, each with their attached flagellum, were purified from Trypanosoma brucei. The method devised for their isolation included a new technique of cell breakage that used a combination of osmotic stress followed by mechanical sheer and avoided the problem of extreme vesiculation as well as the trapping of organelles in cell 'ghosts'. The purified membranes all contained the pellicular microtubular array. The antigenic surface coat was completely released from the plasma membrane during the isolation procedure. The membranes had a very high cholesterol/phospholipid ratio (1.54). A large proportion (42%) of the cellular DNA was recovered in the plasma-membrane fraction unless a step involving deoxyribonuclease treatment, which decreased the DNA content to less than 13%, was included before secrose-density gradient centrifugation. This step also aided the separation of plasma membranes from other cellular components. The ouabain-sensitive Na+ + K+-stimulated adenosine triphosphatase and adenylate cyclase co-purified with the plasma membranes. Although 5'-nucleotidase was thought to be a plasma-membrane component, it was easily detached from the membrane. The purified membranes were essentially free of L-alanine-alpha-oxoglutarate aminotransferase, L-asparte-alpha-oxoglutarate aminotransferase, malate dehydrogenase, oligomycin-sensitive adenosine triphosphatase, glucose 6-phosphatase, Mg2+-stimulated p-nitrophenyl phosphatase and catalase.

Reaction of (Na-K)ATPase with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole: evidence for an essential tyrosine at the active site.

The reaction of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole [NBD-Cl] with purified eel electrophax Na+ and K+ stimulated adenosine triphosphatase [(Na-K)ATPase] has been monitored by changes in the (Na-K)ATPase activity, the K+ stimulated p-nitrophenyl phosphatase [PNPase] activity, and the protein ultraviolet absorption spectrum. The NBD-Cl reacts with two tyrosine residues per mol of enzyme (approximately 6-7 nmol/mg of protein), as judged by changes in protein absorption spectra and incorporation of [14C]NBD-Cl. The modified tyrosine groups are located on the Mr = 95 000 polypeptide chain and react at different rates. Only one tyrosine modification is necessary for complete inhibition of (Na-K)ATPase activity, although both must be modified for complete inhibition of PNPase activity. Reversal of these modifications by 2-mercaptoethanol restores 65% of both activities. Na+ increases the rate of tyrosine modification, K+ decreases the rate, and ATP affords the more reactive tyrosine group complete protection. NBD-Cl modification of approximately 6-7 nmol of tyrosine groups/mg of protein results in a large decrease in ATP affinity as judged by equilibrium binding. These results are compared with similar results obtained from NBD-Cl modification of the coupling factors of oxidative phosphorylation and photophosphorylation. A model is presented suggesting an asymmetric arrangement of two 95 000 polypeptide chains with a single tyrosine residue at the ATP site.

Inhibition of renal (Na+K+)-ATPase and cation transport by an antibody against NaI extracted kidney plasma membranes

1. An antiserum was raised in rabbits against NaI extracted plasma membranes of rat kidney tubule cells. The IgG fraction of the antiserum was used for studies on (Na+K+)-ATPase and cation transport. 2. Immunodiffusion of the IgG against Triton X 100 solubilized subcellular fractions resulted in a precipitation line with basal lateral membranes and endoplasmic reticulum but not with brush borders. 3. The antibody inhibited (Na+K+)-ATPase activity of basal lateral membranes by 83%. Mg-ATPase was not affected. Kinetic analysis with Na+, K+ and Mg·ATP revealed noncompetitive inhibition. Hill constants were unchanged, excluding inhibition by a conformational change. 4. K+-stimulated p-nitrophenyl phosphatase was inhibited to the same degree as was (Na+K+)-ATPase. 5. In renal epithelial cell cultures total22Na efflux and total42K-uptake were diminished by approximately 30% in the presence of the antibody. 6. It is concluded that an antibody can be raised inhibiting both (Na+K+)-ATPase and cation transport from the outside of the cell if a completely disintegrated membrane fraction is used for immunization and if the antibody is tested against antigen of the same organ from the same species. 7. The experiments do not exclude the presence of an additional (Na+K+)-ATPase antibody that is capable to inhibit the enzyme in vitro at the inside of the plasma membrane.

The zinc-stimulated acid and neutral p-nitrophenyl phosphatase activities of chick liver and duodenum

1. 1.|Some properties of the soluble Zn 2+-stimulated p-nitrophenyl phosphatases active at acid and neutral pH have been studied with (NH 4) 2SO 4-precipitated preparations from the soluble cytoplasm fraction of homogenates from chick liver, duodenum and metanephros. Analogous Zn 2+-stimulated phosphatase activity was not found in the particulate fraction from these tissues or in any fraction from homogenates of chick brain or heart muscle. 2. 2.|The pH optima of these soluble, Zn 2+-stimulated enzymes were at pH 4.9, 6.2 and 6.7, respectively, for duodenum, liver and metanephros. The enzymes responsible for the low level of soluble activity without added Zn 2+ or with added Mg 2+ showed maximal activity at pH 5.4–5.8 in all three tissues. 3. 3.|The soluble Zn 2+-stimulated p-nitrophenyl phosphatase enzymes of liver and duodenum were similar in most other properties examined. With preparations from both tissues, a marked (10- to 20-fold) stimulation of p-nitrophenyl phosphatase activity was elicited only by Zn 2+, but Mg 2+, Mn 2+ and Co 2+ caused some stimulation. The optimal concentration of Zn 2+ varied over the range 3–10 mM, depending on pH and type of buffer used. A marked and specific stimulatory effect of Zn 2+ was seen only for hydrolysis of p-nitrophenyl phosphate, of the many phosphate compounds tested. Hydrolysis of ATP by these preparations was moderately stimulated by both Mg 2+ and Zn 2+. Indirect evidence suggests that the Zn 2+-stimulated hydrolysis of p-nitrophenyl phosphate was not due to the inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1), fructose-1,6-diphosphatase (fructose-1,6-diphosphate 1-phosphohydrolase, EC 3.1.3.11), or ATPase (ATP phosphohydrolase, EC 3.6.1.3) present in these preparations.

The Effects of 2H2O on Sodium plus Potassium Ion-stimulated Adenosine Triphosphatase of Rat Brain

Abstract Deuterated water (2H2O) was found to inhibit the Mg2+-dependent Na+ + K+-stimulated adenosine triphosphatase of rat brain microsomal membrane preparations. At a concentration of 95% 2H2O, the inhibition observed varied between 30 and 52%; no inhibition of the basic Mg2+-stimulated component in the ATPase was observed. The inhibition of the Na+ + K+-stimulated ATPase was not time dependent and was completely reversed on washing the enzyme free of 2H2O. Kinetic analysis of the effects of 2H2O on the various parameters which influence the activity of the Na+ + K+-stimulated ATPase revealed that the inhibition was apparently noncompetitive with respect to ATP, competitive with respect to Na+, and uncompetitive with respect to K+. The K+-stimulated p-nitrophenyl phosphatase activity associated with the ATPase was stimulated by 2H2O in a competitive manner with respect to K+. Based on these data it is suggested that Na+ activates the Na+ + K+-stimulated ATPase prior to its phosphorylation by ATP and that the presence of H2O at the Na+ site has an important functional role for the activation of the enzyme by Na+. A second locus of action of 2H2O is suggested to be on E2-P (the presumed conformation of the phosphorylated enzyme which is hydrolyzed in the presence of K+) prior to the action of K+ on it. Possibly this results in a change in this phosphorylated form so that its affinity for K+ is increased.

The Purification of Bovine Thyroid Plasma Membranes and the Properties of Membrane-bound Adenyl Cyclase

Abstract Bovine thyroid membranes have been purified about 100-fold on the basis of thyrotropin- or F--stimulated adenyl cyclase activity. These activities copurified with Na+-K+-requiring adenosine triphosphatase, K+-stimulated p-nitrophenyl phosphatase, and 5'-nucleotidase. The adenyl cyclase of these preparations is stimulated over a wide range of bovine thyrotropin concentrations, by human chorionic thyrotropin, and sometimes by prostaglandin E1, but not by long acting thyroid stimulator. The adenyl cyclase is stable in the frozen state but loses thyrotropin and F- responsiveness rapidly at 43°. Maximal activity is obtained with a Mg++:ATP ratio of 2 and when the ATP concentration exceeds 2 mm. The thyrotropin response is enhanced by K+ ion whereas the F- response is not affected. Li+ ion inhibits strongly, especially when the Mg++ concentration is limiting. Na+ is weakly inhibitory. Both F-- and thyrotropin-stimulated cyclase activity are strongly inhibited by N-ethylmaleimide, p-chlormercuribenzoate, sodium dodecyl sulfate, filipin, sodium tetraphenylboron, valinomycin, and monensin A. Another class of agents, which includes the phenothiazines, thymol, cobramine B, and gramicidin S, inhibits thyrotropin stimulation of the enzyme but enhances the F- response. It is concluded that the thyrotropin and the adenyl cyclase (F- activation) are distinct entities, and it is suggested that prostaglandin E1 uses a different receptor than thyrotropin.

The Effects of Long Chain Fatty Acids on Sodium Plus Potassium Ion-stimulated Adenosine Triphosphatase of Rat Brain

Abstract Long chain fatty acids inhibit the Mg2+-dependent Na+ + K+-stimulated ATPase of rat brain microsomal membrane preparations without any significant effect on the basic Mg2+-stimulated component. The magnitude of inhibition increases with the chain length, reaching a maximum with myristate followed by a decline with higher fatty acids. Myristate (5 x 10-5 m) produces about 45% inhibition of the Na+ + K+-stimulated ATPase. Unsaturated fatty acids are more inhibitory than their corresponding saturated congeners; e.g. at 5 x 10-5 m, oleate produces 66% inhibition compared with 15% by stearate. The inhibitory effects are not due to the binding of Mg2+ by fatty acids as the effects are not reversed by increasing the Mg2+ concentration in the reaction. The inhibition is freely reversible and does not appear to be time dependent. Kinetic analysis of the action of myristate on the Na+ + K+-stimulated ATPase indicates that the inhibition is uncompetitive with respect to ATP and competitive with respect to K+. The primary site of action of myristate appears to be an inhibition of K+ activation of the enzyme. The Na+ activation of the enzyme is also inhibited, but only when the concentration of Na+ is very low; in the presence of higher concentrations of Na+, the action of myristate can be interpreted as a second (in addition to Na+) competitive inhibitor of K+. K+-stimulated p-nitrophenyl phosphatase is also inhibited in a competitive fashion by myristate. The myristate inhibition of the Na+ + K+-stimulated ATPase is as well elicited in the presence of ouabain or oligomycin. The effects of these mixed inhibitors are of a cumulative nature suggesting separate binding sites for each of the inhibitors.

Interaction of solvents with membranal and soluble potassium ion-dependent enzymes.

The effects of dimethyl sulphoxide and glycerol on ox brain microsomal Na(+)+K(+)-stimulated adenosine triphosphatase (EC 3.6.1.3), K(+)-stimulated p-nitrophenyl phosphatase and K(+)-dependent muscle pyruvate kinase (EC 2.7.1.40) were studied. Dimethyl sulphoxide at concentrations below 20% (v/v) was found to stimulate the p-nitrophenyl phosphatase and pyruvate kinase by increasing their affinity for K(+) but to inhibit the Na(+)+K(+)-stimulated adenosine triphosphatase. The latter enzyme activity was also inhibited by glycerol, which like dimethyl sulphoxide, stimulated the K(+)-activated p-nitrophenyl phosphatase at a wide range of concentrations. The solvent effects were promptly reversed by dilution. Similarity was found between glycerol and dimethyl sulphoxide, on one hand, and ATP, on the other, in their stimulatory effect and their ability to increase the ouabain- and oligomycin-sensitivity of the K(+)-stimulated p-nitrophenyl phosphatase. However, only the solvents, not the ATP, increased the binding of K(+) by the microsomes. From the above findings it is suggested that solvents may act on K(+)-dependent enzymes by altering the state of solvation of the activating cation as well as by changing the enzyme structure.