In Vitro Effect of Ozagrel on Mushroom Tyrosinase

This investigation, in vitro, shows that ozagrel, an antithrombotic drug, inhibited both monophenolase and diphenolase activities of mushroom tyrosinase when l-tyrosine and l-DOPA were assayed spectrophotometrically, respectively. The IC₅₀ values, for monophenolase and diphenolase activities, were 1.35 and 3.45 mM, respectively. Ozagrel was estimated to be a reversible mixed-type inhibitor of diphenolase activity with the constants (K _S1, K _S2, K _i1, and K _i2) determined to be 2.21, 3.89, 0.454, and 0.799 mM, repectively. Increasing ozagrel concentrations provoked longer lag periods as well as a concomitant decrease in the monophenolase activity. Inhibition experiment demonstrated that ozagrel bound the enzyme at a site distincted from the substrate active site, but it bound to either E (Enzyme) or ES (Enzyme-Substrate) complex.     

PH dependence of the alpha-glucose 1,6-diphosphate inhibition of hexokinase II.

α-Glucose 1,6-diphosphate is a much better inhibitor of hexokinase II than 1,5-anhydroglucitol 6-phosphate or glucose 6-phosphate (Glc-6-P) at pH 6–7 and poorer at higher pH. Because the K_i of Glc-6-P is pH independent, the observed pH effects are attributed to the phosphate group at C-1 which is bound as a monoanion to a specific site but which is excluded as a dianion. None of the following kinetic properties of the hexokinase II reaction varies greatly with pH: V, K_m of glucose and K_m of ATP.     

Catalyses by polymer complexes. VII. Enhanced esterolytic reactivity of polymer-coenzyme A,glutathione complexes

The association of coenzyme A(CoASH) and glutathione (GSH) with the water-soluble polymers and their esterolytic reactivities were evaluated through the reaction with p-nitrophenyl acetate in the presence of cationic polymer micelles: partially laurylated poly(2-ethyl-1-vinylimidazole) and poly(4-vinylpyridine). The polymer micelles with high lauryl-group content (more than 12 mol%) markedly accelerated the reaction at very low concentrations of the polymer. Other polymers with no or small lauryl-group content only slightly enhanced the association and the reaction rate. From the rate-polymer concentration profiles, the association constants (K) and the rate constants for thiol coenzymes bound to the polymer (k′_a,bound) were determined: for polymers with more than 12 mol % lauryl-group content, K_CoASH = 1110–2270 M^?1, K_GSH = 170–503M^?1, k′_a,bound at pH 8.65 = 142–341M^?1 sec^?1. k′_a,bound were 20–340 times larger than that observed in the absence of the polymer. The logarithm of k′_a,bound was found to be correlated well with the polymer hydrophobicity, indicating that the hydrophobic environment of the polymer activated the bound thiol anions. On the other hand, the polymer hydrophobicity did not correlate with the association constant.     

Binding of water and electrolytes to proteins. An equilibrium dialysis study

The technique of equilibrium dialysis has been used to study water and salt binding to egg albumin, to human carbon monoxide hemoglobin, and to bovine serum albumin. The salts used were CsCl, KCl, NaCl, LiCl, Gu·HCl, NaBr, Cs₂SO₄, K₂SO₄, Na₂SO₄, Li₂SO₄, and Gu₂SO₄. The amount of water bound by proteins depends on the probe being used. Sulfates tend to bind to proteins and they also increase the water binding. At saturation, about 41 and 140 mol Gu·HCl bind to 1 mol egg albumin and to 1 mol carbon monoxide hemoglobin, respectively. Both proteins are dehydrated by Gu·HCl. The hydrodynamic hydration of egg albumin as determined by viscosity appers to increase as the relative viscosity of the medium increases.     

Simultaneous Binding of Basic Peptides at Intracellular Sites on a Large Conductance Ca2+-activated K+ Channel : Equilibrium and Kinetic Basis of Negatively Coupled Ligand Interactions

The homologous Kunitz inhibitor proteins, bovine pancreatic trypsin inhibitor (BPTI) and dendrotoxin I (DTX-I), interact with large conductance Ca²⁺-activated K⁺ channels (maxi-K_Ca) by binding to an intracellular site outside of the pore to produce discrete substate events. In contrast, certain homologues of the Shaker ball peptide produce discrete blocking events by binding within the ion conduction pathway. In this study, we investigated ligand interactions of these positively charged peptide molecules by analysis of single maxi-K_Ca channels in planar bilayers recorded in the presence of DTX-I and BPTI, or DTX-I and a high-affinity homologue of ball peptide. Both DTX-I (K _d, 16.5 nM) and BPTI (K _d, 1,490 nM) exhibit one-site binding kinetics when studied alone; however, records in the presence of DTX-I plus BPTI demonstrate simultaneous binding of these two molecules. The affinity of BPTI (net charge, +6) decreases by 11.7-fold (K _d, 17,500 nM) when DTX-I (net charge, +10) is bound and, conversely, the affinity of DTX-I decreases by 10.8-fold (K _d, 178 nM) when BPTI is bound. The ball peptide homologue (BP; net charge, +6) exhibits high blocking affinity (K _d, 7.2 nM) at a single site when studied alone, but has 8.0-fold lower affinity (K _d, 57 nM) for blocking the DTX-occupied channel. The affinity of DTX-I likewise decreases by 8.4-fold (K _d, 139 nM) when BP is bound. These results identify two types of negatively coupled ligand–ligand interactions at distinct sites on the intracellular surface of maxi-K_Ca channels. Such antagonistic ligand interactions explain how the binding of BPTI or DTX-I to four potentially available sites on a tetrameric channel protein can exhibit apparent one-site kinetics. We hypothesize that negatively coupled binding equilibria and asymmetric changes in transition state energies for the interaction between DTX-I and BP originate from repulsive electrostatic interactions between positively charged peptide ligands on the channel surface. In contrast, there is no detectable binding interaction between DTX-I on the inside and tetraethylammonium or charybdotoxin on the outside of the maxi-K_Ca channel.     

Activation mechanisms of PIP5K isozymes by the small GTPase ARF6

The membrane phospholipid, phosphatidylinositol 4-phosphate 5-kinase (PIP5K), plays important roles in a wide variety of signal transduction systems through its product phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). To date, three mammalian PIP5K isozymes, α, β and γ have been identified and each isozyme plays specific physiological functions. In this study, we refer to human PIP5Kα, β, and γ as PIP5K_A, _B, and _C, respectively. In the present study, we demonstrated that PIP5K_B, but not PIP5K_C, was directly activated by ARF6 in vitro, and provide a working hypothesis for the novel activation mechanism of PIP5K_C by ARF6 in the cell. In an in vitro system, PIP5K_B, which was expressed in and purified from mammalian cells, was significantly activated by ARF6. Although, in contrast, PIP5K_C was not activated by ARF6 under the same conditions, its kinase core domain (KCD) fragment, which is highly conserved among PIP5K isozymes, preserved the lipid kinase activity and was activated by ARF6. In addition, it was found that ARF6 bound to the KCD and that the N-terminal domain deletion mutant of PIP5K_C was activated by ARF6. These results suggest that the N-terminal domain of PIP5K_C masks its KCD, thereby preventing the interaction of PIP5K_C with and its activation by ARF6. Under certain physiological setting(s), the conformation of PIP5K_C would be changed to unmasked open form, and thereby PIP5K_C would be able to interact with and activated by ARF6.     

Kinetic constants for the inhibition of camel retinal acetylcholinesterase by the carbamate insecticide lannate

We have designed this study to determine various kinetic parameters of camel retinal membrane‐bound acetylcholinesterase (AChE; EC 3.1.1.7) inhibition by carbamate insecticide lannate [methyl N‐{{(methylamino)carbonyl}oxy} ethanimidothioate]. All these kinetic constants were derived by simple graphical methods. The value of kinetic parameters was estimated as follows: 0.061 (μM)⁻¹, 1.14 (μM)⁻¹, 0.216 μM, 0.016 min⁻¹, 0.0741 (μM min)⁻¹, 0.746 μM, and 4.42 μM for velocity constant (K_v), new inhibition constant (K_nic), dissociation constant (K_d), carbamylation rate constant (k_2c), overall carbamylation rate constant (k′2 ), 50% inhibition constant (K_I50), and 99% inhibition constant (K_I99), respectively. These unique methods may be used to estimate such kinetic parameters for time‐dependent inhibition of enzymes by variety of chemicals, insecticides, herbicides, and drugs. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 41–46, 1999     

The chemistry of vitamin B12. The coordination of biologically important molecules

The following equilibrium constants (given as logK in units of m⁻¹) were determined for the substitution of co-ordinated H₂O in aquocobalamin by glycine (bound through N) 5.8, cysteine (bound through S) 6.0 or 8.3, depending on the value chosen for the pK of the thiol group, and phenolate 2.9. The spectrum of the phenolate cobalamin shows an additional intense absorption band at 468nm with a molar extinction coefficient of 1.1×10⁴, which is assigned to a charge transfer from the phenolate to the cobalt ion. Equilibrium constants have also been determined for the equilibria between adenylcobamide cyanide and CN⁻, HO⁻ and H⁺, which show that the adenine is more easily displaced by CN⁻ and HO⁻ than is 5,6-dimethylbenziminazole in vitamin B₁₂, but can be protonated by acid while still remaining co-ordinated to the cobalt. It is shown that in the binding of corrinoids to proteins and polypeptides the formation of hydrogen bonds is far more important than co-ordination by the metal.     

Active Detergent-solubilized H+,K+-ATPase Is a Monomer

The H⁺,K⁺-ATPase pumps protons or hydronium ions and is responsible for the acidification of the gastric fluid. It is made up of an α-catalytic and a β-glycosylated subunit. The relation between cation translocation and the organization of the protein in the membrane are not well understood. We describe here how pure and functionally active pig gastric H⁺,K⁺-ATPase with an apparent Stokes radius of 6.3 nm can be obtained after solubilization with the non-ionic detergent C₁₂E₈, followed by exchange of C₁₂E₈ with Tween 20 on a Superose 6 column. Mass spectroscopy indicates that the β-subunit bears an excess mass of 9 kDa attributable to glycosylation. From chemical analysis, there are 0.25 g of phospholipids and around 0.024 g of cholesterol bound per g of protein. Analytical ultracentrifugation shows one main complex, sedimenting at s_20,w = 7.2 ± 0.1 S, together with minor amounts of irreversibly aggregated material. From these data, a buoyant molecular mass is calculated, corresponding to an H⁺,K⁺-ATPase α,β-protomer of 147.3 kDa. Complementary sedimentation velocity with deuterated water gives a picture of an α,β-protomer with 0.9–1.4 g/g of bound detergent and lipids and a reasonable frictional ratio of 1.5, corresponding to a Stokes radius of 7.1 nm. An α₂,β₂ dimer is rejected by the data. Light scattering coupled to gel filtration confirms the monomeric state of solubilized H⁺,K⁺-ATPase. Thus, α,β H⁺,K⁺-ATPase is active at least in detergent and may plausibly function as a monomer, as has been established for other P-type ATPases, Ca²⁺-ATPase and Na⁺,K⁺-ATPase.     

Chelate effect and cooperativity effect in metal-ligand and macromolecule-ligand equilibria. I. Chemical potential changes and cooperativity-chelation parameters

Chelate and cooperativity effects both in the field of complexes formed in solution by metal ion with ligands and in the field of binding between protein and ligands were examined on the basis of thermodynamic arguments.The analysis was carried out by means of the formation function $\text{n}$ = ? ln Σ_M/? ln[A] where Σ_M is a partition function having free metal or macro-molecule as basis reference level and A is a ligand. The chemical potential changes due to cooperativity and chelation are calculated from differences between areas of the diagram $\text{n}$ = f(ln[A]). The chemical potentials are: Δμ°_γ = -RT ln K_γ (homotropic co-operativity), Δμ°_γ′ = -RT ln K_γ′ (heterotropic co-operativity), Δμ°_? = -RT ln K_? (homotropic chelation), Δμ°_?′ = -RT ln K_?′ (heterotropic chelation). The cooperativity and chelation parameters K_γ, K_γ′, K_?, K_?′ are related to each other by other parameters K_η = K_?·K_γ and K_η′= K_?′·K_γ′. All these dimensionless parameters are derived as ratios of experimental equilibrium constants. Therefore a corresponding consistent chemical potential scale can be obtained from experimental data for all these effects, leading to quantitative comparisons between cooperative and chelate effects, either homotropic or heterotropic.Thermodynamic function changes in metal-ligand complexes can also be compared on this same scale with the energetic changes in protein-ligand complexes.     

Selectivity of interaction of univalent cations with mammalian ribosomes studied by equilibrium dialysis in the presence of the K+ analogue, 204Tl+

The interaction of K⁺ with mammalian ribosomes was studied by equilibrium dialysis and compared with that of other univalent cations. The heavy K⁺ analogue, Tl⁺, binds more firmly than K⁺ to ribosomes and, unlike K⁺, has a practically useful isotope. With ²⁰⁴Tl⁺ as a marker of K⁺-selective binding the ribosome-cation interaction could be followed down to levels below 0.1 average Tl⁺-occupied site per ribosome. The Tl⁺/ribosome ratio varied with the free Tl⁺ concentration in a multiple way. At high Tl⁺ saturation Tl⁺ was easily displaced by Mg²⁺. With decreasing Tl⁺ saturation the competitive activity of Mg⁺⁺ was strikingly reduced, indicating that Tl⁺ and Mg⁺⁺ compete with different efficiency for different classes of sites.The experiments on univalent cations were performed at 1.5 mM Mg²⁺ under two complementary conditions: (1) Ribosomes were pretreated with 5 × 10^?2, 5 × 10^?3, and 5 × 10^?4 M LiNO₃, NaNO₃, KNO₃, and CsNO₃, and then equilibrated with different concentrations of ²⁰⁴TlNO₃ in the same buffers. (2) Ribosomes were pretreated with 10^?2, 10^?4, and 10^?6 M ²⁰⁴TlNO₃, and then equilibrated with different concentrations of LiNO₃, NaNO₃, KNO₃, and CsNO₃ (displacement experiments). At high Tl⁺ saturation Na⁺ and Li⁺ were about as active as K⁺ and Cs⁺ in competing with ²⁰⁴Tl⁺. With decreasing Tl⁺ saturation a differentiation occurred in favor of K⁺ and Cs⁺, with some preference for K⁺. It is concluded that ribosomes contain a limited number of sites with pronounced ion specificity. Of physiological cations K⁺ is most firmly bound to these sites.     

Probing of the porcine serum lipoprotein surfaces by Mn(II) binding: an e.s.r. study

Mn(II) ions were used for probing the surfaces of porcine LDL₁, LDL₂ and HDL. From the intensity of the e.p.r. lines corresponding to the unbound Mn(II) the percentage of the ions bound to the lipoprotein surface is determined. From the titration curves the binding parameters, dissociation constant. K_d, and the number of binding sites, n, in all the three lipoproteins studied have been derived. There are at least two types of binding sites in each lipoprotein class. The ”weak’ binding sites are charaterized by approximately the same value of $\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{(≈ 6.2 × 10}{}^{\mathrm{?3}}\text{mol l}{}^{\mathrm{?1}}$ and different values for n (n = 114 for LDL₁, n = 135 for LDL₂ and n = 28 for HDL). Similarly, for the ”strong’ binding sites K_d ≈ 1.6 × 10^?4 mol l^?1 and the number of binding sites is 15, 20 and 5 for LDL₁, LDL₂ and HDL respectively. It is concluded that the binding sites are probably located in the protein part of the lipoproteins and that they are mainly associated with the negatively charged amino acids.     

ELECTROKINETICS : XVII. SURFACE CHARGE AND ION ANTAGONISM

1. The question of the critical pore diameter for streaming potential is discussed. 2. The surface charge is calculated for cellulose in contact with solutions of K₃PO₄, K₂CO₃, K₂SO₄, KCl, and ThCl₄. 3. The surface charge of cellulose in contact with a solution of 2 x 10^–4 N NaCl is calculated as a function of temperature and is found to show a sharp break at 39°. This is interpreted in terms of the change of the specific heat of water. 4. A marked ion antagonism is found in NaCl:KCl, KCl:MgCl₂, NaCl:MgCl₂, NaCl:CaCl₂, KCl:CaCl₂, CaCl₂:MgCl₂ mixtures when the surface charge is calculated as a function of concentration.     

Fluorescent labeling of (Na + K)-ATPase by 5-iodoacetamidofluorescein

5-Iodoacetamidofluorescein (5-IAF) covalently labels dog kidney (Na⁺ + K⁺)-ATPase with approximately 2 moles incorporated per mole of enzyme. ATPase and K⁺-phosphatase activities are fully retained after reaction, and the kinetic parameters for Na⁺, K⁺, Mg²⁺, ATP and p-nitrophenyl phosphate are likewise not significantly affected. The fluorescence of the bound 5-IAF is increased by ATP, Na⁺, and Mg²⁺, and decreased by K⁺. These fluorescence changes likely reflect ligand-induced stabilization of the E₁ or E₂ states of the enzyme.     

Heme-linked protonation of HCN, CO, NO and O2 complexes of reduced horseradish peroxidases.

Small reversible changes in the absorption spectra of HCN, CO, NO and O₂ complexes of ferrous diacetyldeuteroperoxidase A, not hitherto observed, were attributed to proton dissociation of a distal amino acid residue. From spectrophotometric titration data the pK_a was measured as 5.5 (HCN), 5.6 (ligand free), 6.0 (CO), 6.55 (NO) and 8.0 (O₂). The value of 8.0 for the pK_a of the O₂ complex was also obtained from a curve of pH dependence of proton uptake in the reaction of the ferrous enzyme with O₂. Absorption bands in the visible region were shifted to longer wavelengths in the order of CO to NO to O₂ which is the decreasing order of the energy of π¹ level of these diatomic ligands.The pK_a values for CO complexes of ferroperoxidases, isoenzymes A and (B+C) were varied with substituents at the 2 and 4 positions of deuterohemin IX, and the $\text{Δp}\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{Δp}\text{K}{}_3$ ratio was about 0.3 in both series of isoenzyme preparations, where pK₃ is a measure of basicity of pyrrole nitrogen.The present data support the previous conclusion (Yamada and Yamazaki (1974) Arch. Biochem. Biophys., 165, 728) that the pK_a for ferroperoxidases, measured from small reversible changes in the absorption spectra, represents a proton dissociation constant of a distal amino acid residue and that there is hydrogen bonding between the residue and a ligand atom directly bound to the iron atom.     

Molecular properties of the red cell calcium pump: I. Effects of calmodulin,proteolytic digestion and drugs on the kinetics of active calcium uptake in inside-out red cell membrane vesicles

In calmodulin-stripped inside-out human red cell membrane vesicles /IOV/ ATP + Mg²⁺-dependent active calcium uptake is stimulated by the addition of calmodulin. Calmodulin increases the maximum calcium transport rate /V_max/, decreases K_Ca, and does not affect K_ATP of calcium uptake. The action of both membrane bound and external calmodulin is competitively inhibited by phenothiazines. Drugs reacting with SH groups of proteins reversibly inhibit calcium pumping by decreasing V_max and not affecting K_Ca and K_ATP. The relative magnitude of calmodulin stimulation of calcium transport is unaltered by SH reagents.Mild proteolytic digestion of IOVs stimulates active calcium uptake and mimics the effects of calmodulin on the kinetic parameters — that is converts the system to a “high calcium-affinity” state. Proteolysis eliminates calcium-dependent calmodulin binding to IOV membranes and any further stimulation of calcium uptake by calmodulin. Based on these results the presence of a calmodulin-binding regulatory subunit of the red cell calcium pump at the internal membrane surface is postulated.     

Kinetic studies of a (Na++ K++ Mg2+)ATPase in sugar beet roots. III. A proposed model for the (Na++ K+) activation and its significance for field properties

A microsomal (Na⁺+ K⁺+ Mg²⁺)ATPase preparation from sugar beet roots was used. The activation by simultaneous addition of Na⁺ and K⁺ at different levels was examined in terms of steady state kinetics. The observed data can be summarized in the following way: 1. The apparent affinity between the enzyme and the substrate MgATP depends on the ratio between Na⁺ and K⁺. At low Na⁺ concentration (below 5 mM), the apparent K_m decreases with increasing concentrations of K⁺ (1–20 mM). At 5 mM Na⁺, the K⁺ level does not change the apparent K_m, while at Na⁺ levels above 10 mM, the apparent K_m between enzyme and substrate increases with increasing concentration of K⁺. 2. When the MgATP concentration is kept constant, homotropic cooperativity (concerning one type of ligand) and heterotropic cooperativity (concerning different types of ligands) exist in the activation by Na⁺ and K⁺. The Na⁺ binding is cooperative with different K_m values and Hill coefficients (n) in the presence of low and high concentration of K⁺. At low Na⁺ level (< 5 mM). a negative cooperativity exists for Na⁺ (n_Na < 1) which is more pronounced in the presence of high [K⁺]. When the concentration of Na⁺ is raised the negative cooperativity disappears and turns into a positive one (n_Na > 1). Only K⁺ binding in the presence of low [Na⁺] shows cooperativity with a Hill coefficient that reflects changes from negative to positive homotropic cooperativity with increasing concentrations of K⁺ (n_K < 1 → n_K > 1). In the presence of [Na⁺] > 10 mM, the changes in n_k are insignificant. 3. A model is proposed in which one or two different K sites and one or two Na sites control the catalytic activity, with multiple interactions between Na⁺, K⁺ and MgATP. 4. In the presence of Na⁺ (< 10 mM), K⁺ is probably bound to two K sites, one of which translocates K⁺ through the membrane by an antiport Na⁺/K⁺ mechanism. This could be connected with an elevated K⁺ uptake in the presence of Na⁺ and could therefore explain some field properties of sugar beets.     

Different domain organization of two main conformational states of Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase

The Na⁺/K⁺-ATPase generates an electrochemical gradient of Na⁺ and K⁺, which is necessary for the functioning of animal cells. During the catalytic act, the enzyme passes through two principal conformational states, E₁ and E₂. To assess the domain organization of the protein in these conformations, thermal denaturation of Na⁺/K⁺-ATPases from duck salt gland and from rabbit kidney has been studied in the absence and in the presence of Na⁺ or K⁺, which induce the transition to E₁ or E₂. The melting curves for the ion-free forms of the two ATPases have different shapes: the rabbit protein shows one transition at 56.1°C, whereas the duck protein shows two transitions, at 49.8 and 56.9°C. Addition of Na⁺ or K⁺ ions abolishes the difference in thermal behavior between these enzymes, but through opposite effects. The melting curves for the E₂ conformation (K⁺ bound) in both cases exhibit a single peak of heat absorption at ∼63°C. For the E₁ conformation (Na⁺ bound), each melting curve has three peaks, indicating denaturation of three domains. The difference in the domain organization of Na⁺/K⁺-ATPase in the E1 and E2 states may account for the different sensitivity to temperature, proteolysis, and oxidative stress observed for the two enzyme conformations.     

Design and synthesis of thiazolylhydrazone derivatives as inhibitors of chitinolytic N-acetyl-β-d-hexosaminidase

N-acetyl-β-d-hexosaminidase (Hex) is potential target for pesticide design. Here, a series of thiazolylhydrazone derivatives were designed, synthesized and evaluated as competitive inhibitors of OfHex1, a Hex from the agricultural pest Ostrinia furnacalis. The derivative 3k, with a (benzyloxy)methyl group at the N3 atom, demonstrated greater potency with a K_i of 10.2?µM. Molecular docking analysis indicated that the (benzyloxy)methyl group of 3k was bound to a previously unexplored pocket formed by Loop478-496. Then further optimization around naphthalene ring led to find the more potency substituent phenyl. The derivative 7, with phenoxyethyl group at R₁ and a phenyl group at R₂, demonstrated an augmented potency with a K_i of 2.1?µM. Molecular docking analysis indicated that 7 was bound to the active pocket of OfHex1 more favorably than 3k. This work suggests a novel scaffold for developing specific Hex inhibitors.     

Cat Heart Muscle in Vitro : VI. Potassium exchange in papillary muscles

The exchange of cell K with K⁴², J _K, has been measured in cat right ventricular papillary muscle under conditions of a steady state with respect to intracellular K concentration. Within the limits of the measurement, all of cell K exchanged at a single rate. Cells from small cats are smaller and have larger surface/volume ratios than cells from large cats. The larger surface/volume ratio results in larger flux values. J _K increases in an approximately linear manner as the external K concentration is increased twentyfold, from 2.5 to 50 mM, at constant intracellular K concentration. The permeability for K ions, P _K, calculated from the influx and membrane potential, remains very nearly constant over this range of external K concentrations. J _K is not affected by replacement of O₂ by N₂, or by stimulated contractions at 60 per minute, but K influx decreases markedly in 10^-5 M and 10^-8 M ouabain.