Eosin,a fluorescent probe of ATP binding to the (Na+ + K+)-ATPase

(1) Eosin bound to the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in the presence of K⁺ has practically the same fluorescence as eosin without enzyme while in the presence of Na⁺ the fluorescence is higher, the excitation maximum is shifted from 518 to 524 nm, the emission maximum from 538 to 542 nm, and a shoulder appears at about 490 nm on the excitation curve. (2) The amount of eosin bound increases with the K⁺ concentration but with a low affinity. With equal concentrations of Na⁺ and K⁺ more is bound in the presence of Na⁺, and the difference between 150 mM Na⁺ and 150 mM K⁺ shows one high-affinity eosin binding site per ³²P-labelling site ($\text{K}{}_{\mathrm{D}}$ 0.45 μM). With lower concentrations of the cations there are between one and two Na⁺-dependent high-affinity eosin binding sites per ³²P-labelling site. (3) ATP (and ADP) prevents the hig-affinity Na⁺-dependent eosin binding and there is competition between eosin and ATP for the hydrolysis in the presence of Na⁺ (+Mg²⁺). (4) Eosin, like ATP, increases the Na⁺ relative to K⁺ affinity $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{= 150}\text{mM}$) for Na⁺ activation of hydrolysis and for Na⁺ protection against inactivation by $\text{N-}\text{ethylmaleimide}$. (5) The results suggest that the high affinity eosin binding site is an ATP binding site and that it is located on the enzyme in an environment with a low polarity, i.e., the conformational change induced by Na⁺ opens a high-affinity site for ATP while K⁺ closes the site (or decreases the affinity to a low level). The experiments suggest, furthermore, that the ATP which increases the Na⁺ relative to K⁺ affinity of the internal sites is not the ATP which is hydrolyzed, i.e., in a turnover cycle in the presence of $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}$ the system reacts with two different ATP molecules.     

Dopamine-stimulated cyclic AMP and binding of [3H]dopamine in acini from dog pancreas

Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [³H]dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1·10^?8 M and was half-maximal at $\text{7.9±3.4·10}{}^{\mathrm{?7}}\text{M}$. The increase at 1·10^?5 M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1·10^?9 M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC₅₀ value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1·10^?5 M dopamine was $\text{2.3±0.9·10}{}^{\mathrm{?6}}\text{M}$. Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [³H]Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37°C, it was rapid, reversible, saturable and stereospecific. The $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ value for high affinity binding sites was $\text{0.43±0.1·10}{}^{\mathrm{?7}}\text{M}$ and $\text{4.7±1.6·10}{}^{\mathrm{?7}}\text{M}$ for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was $\text{1.2±0.4·10}{}^{\mathrm{/t-7}}\text{M}$ noradrenaline, 2·10^/t-7 M epinine, $\text{4.1±1.8·10}{}^{\mathrm{/t-6}}\text{M}$ fluphenazine, $\text{8.0±1.6·10}{}^{\mathrm{/t-6}}\text{M}$ haloperidol, $\text{4.2±1.2·10}{}^{\mathrm{?6}}\text{M}$cis-flupenthixol, $\text{2.7±0.4·10}{}^{\mathrm{?5}}\text{M}$trans-flupenthixol, $\text{>1·10}{}^{\mathrm{?5}}\text{M}$ apomorphine, sulpiride, naloxone and isoproterenol.     

The effects of membrane lipid order and cholesterol on the internal and external cationic sites of the Na+−K+ pump in erythrocytes

Cholesterol depletion alters the apparent affinity of the internal cationic sites and the maximal translocation rate but not the affinity of the external cationic sites of the $\text{Na}{}^{\mathrm{+}}\text{?K}{}^{\mathrm{+}}$ pump in human erythrocytes. To test whether these effects were mediated by a direct cholesterol-internal site interaction or by a change in membrane lipid order, the effects of five fluidizing amphiphiles (chlorpromazine, imipramine, benzyl alcohol, sodium oleate and sodium benzenesulphonate) on the kinetic parameters of the $\text{Na}{}^{\mathrm{+}}\text{?K}{}^{\mathrm{+}}$ pump were determined. The cholesterol removal and all the agents used induced dose-response decreases in membrane lipid order as measured by fluorescence polarization or ESR. Positive and neutral amphiphiles mimicked the effects of cholesterol removal on the affinity of the internal sites of the pump and to a lesser extent on the maximal translocation rate. Anionic amphiphiles had no effect on internal sites, probably because they distributed preferentially within the outer leaflet on the membrane. These results indicate that cholesterol controls the affinity of the internal sites of the $\text{Na}{}^{\mathrm{+}}\text{?K}{}^{\mathrm{+}}$ pump by altering the membrane lipid order. In contrast, neither cholesterol depletion nor the agents used altered the affinity of the external sites of the $\text{Na}{}^{\mathrm{+}}\text{?K}{}^{\mathrm{+}}$ pump. This difference in sensitivity to membrane lipid order suggests that internal and external cationic sites, although borne by the same protein, are in different lipid environments.     

Inhibition of anion transport in the red blood cell by anionic amphiphilic compounds I. Determination of the flufenamate-binding site by proteolytic dissection of the band 3 protein

Flufenamate, a non-steroidal anti-inflammatory drug, is a powerful inhibitor of anion transport in the human erythrocyte ($\text{I}{}_{50}\text{= 6·10}{}^{\mathrm{?7}}\text{M}$). The concentration dependence of the binding to ghosts reveals two saturable components. [¹⁴C]Flufenamate binds with high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}_1\text{= 1.2·10}{}^{\mathrm{?7}}\text{M}$) to 8.5·10⁵ sites per cell (the same value as the number of band 3 protein per cell); it also binds, with lower affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}_2\text{= 10}{}^{\mathrm{?4}}\text{M}$) to a second set of sites (4.6·10⁷ per cell). Pretreatment of cells with 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS), a specific inhibitor of anion transport, prevents [¹⁴C]flufenamate binding only to high affinity sites. These results suggest that high affinity sites are located on the band 3 protein involved in anion transport. Extracellular chymotrypsin and pronase at low concentration cleave the 95 kDa band 3 into 60 kDa and 35 kDa fragments without affecting either anion transport or [¹⁴C]flufenamate binding. Splitting by trypsin at the inner membrane surface of the 60 kDa chymotryptic fragment into 17 kDa transmembrane fragment and 40 kDa water-soluble fragment does not affect [¹⁴C]flufenamate binding. In contrast degradation at the outer membrane surface of the 35 kDa fragment by high concentration of pronase or papain decreases both anion transport capacity and number of high affinity binding sites for [¹⁴C]flufenamate. Thus it appears that 35 kDa peptide is necessary for both anion transport and binding of the inhibitors and that the binding site is located in the membrane-associated domain of the band 3 protein.     

An electron spin probe study of (Na+ + K+)-ATPase-Containing membranes

The modulating effect of membrane lipids on enzyme function has been described by several investigators. We have used the spin probe $\text{N-}\text{oxyl}\text{-4′,4′-}\text{dimethyloxazolidine}\text{-12-}\text{keto}$ methyl stearate (M 12-NSE) to study this interaction in ox brain membranes enriched with $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$. This methyl ester of stearic acid is practically insoluble in aqueous media, and consequently spectra of M 12-NSE-labelled preparations are free of “liquid lines”.At least two types of spectra may be obtained when ox brain microsomes are spin labelled with M 12-NSE, indicating the presence of two distinct binding sites. At one site the spin label is relatively unrestricted and gives rise to an isotropic spectrum. A second spectrum, which is obtained from spin label at another site, is similar to that which is observed after incorporation of M 12-NSE into phospholipid bilayers. This suggests that this latter site is within the core of the microsomal membrane.The two binding sites differ in their affinity for the spin probe. The low affinity site is both more abundant in crude preparations and is more easily removed by detergent treatment; spin labels at this site produce isotropic spectra. The high affinity sites are fewer in number and produce broad spectra. In addition these high affinity sites increase in concentration as the enzyme undergoes purification.The two sites are quite distinct in their sensitivity to ascorbic acid, the low affinity site showing a considerably greater rate of reduction by this agent.This study also demonstrates that the delipidation effects of sodium dodecyl sulfate and sodium deoxycholate on $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase-enriched}$ microsomes from ox brain are not identical.It is suggested that the two spin probe binding sites represent two different lipid domains, one of which is very closely associated with the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme and may reflect a protein-directed phospholipid specificity for this enzyme.     

The influence of temperature and gamma-aminobutyric acid on benzodiazepine receptor subtypes in the hippocampus of the rat

The influence of GABA on the affinity of flunitrazepam (FLU) for benzodiazepine receptor subtypes (type I and II) was studied by measurement of the competitive inhibition of [³H]FLU and [³H]propyl beta-carboline-3-carboxylate ([³H]PCC) binding. When assays were carried out at 0°C using a low concentration (0.040 nM) of [³H]PCC so that the type I receptors were selectively labelled, no significant effect of GABA (10^?4 M) on the $\text{FLU}\text{[}{}^3\text{H]}\text{PCC}$ competition curve was detected. In contrast, when assays were carried out at 0°C using [³H]FLU or a high concentration of [³H]PCC to achieve [³H]ligand receptor occupancy of both type I and type II receptors, GABA (10^?4 M) caused a significant increase in the affinity of FLU as measured by $\text{FLU}\text{[}{}^3\text{H]}\text{FLU}$ and $\text{FLU}\text{[}{}^3\text{H]}\text{PCC}$ competition experiments. Collectively, these data suggest that the influence of GABA on benzodiazepine receptor binding is mediated, primarily, by the type II receptor. It was also noted that the $\text{PCC}\text{[}{}^3\text{H]}\text{FLU}$ competition curve had a Hill coefficient of approximately 1 at 37°C as compared to the results of experiments at 0°C during which a Hill coefficient of approximately 0.7 was calculated.     

Chemical and spectroscopic conformation of an exogenous ligand bridge in half met hemocyanin.

Half $\text{met-N}{}_3{}^{\mathrm{?}}$ hemocyanin is shown to undergo a unique change at the Cu(II)?Cu(I) active site with temperature, exhibiting class II mixed valent properties at low temperature (The appearance of an intense near IR intervalence-transfer transition and a delocalized EPR spectrum). This requires a Cu(II)NNNCu(I) bridging geometry. The effects of CO coordination to half $\text{met-N}{}_3{}^{\mathrm{?}}$, combined with the presence of a low energy $\text{N}{}_3{}^{\mathrm{?}}\text{→ Cu(II)}$ charge transfer transition, demonstrate that azide is also bridging at room temperature. Finally, half $\text{met-N}{}_3{}^{\mathrm{?}}$ is found to be capable of coordination of a second $\text{N}{}_3{}^{\mathrm{?}}$ at the copper(II) site.     

Adriamycin inactivates cytochrome c oxidase by exclusion of the enzyme from its cardiolipin essential environment

The regulation of ligand binding to the muscarinic acetylcholine receptor in developing chick heart has been studied using the radiolabeled antagonist [³H]quinuclidinyl benzilate (QNB). In assays containing only buffer and a source of receptor protein, the antagonist radioligand bound to a single, high affinity state of the receptor. If Mg²⁺ and EDTA were added, [³H]QNB bound to a single, low affinity state. The guanine nucleotide analog, guanylylimidodiphosphate [Gpp(NH)p], reversed the effect of $\text{Mg}{}^{\mathrm{2+}}\text{EDTA}$ so that [³H]QNB again bound only to a single, high affinity state. Sodium could also reverse the effect of $\text{Mg}{}^{\mathrm{2+}}\text{EDTA}$ on antagonist binding but the effects of sodium and Gpp(NH)p on [³H]QNB binding were not additive.     

Hydrogen bonds in the tripeptide Pro-Leu-Gly-NH217O and 1H studies

¹⁷ONMR measurements of labeled Pro-Leu-Gly-NH₂ were carried out at different pH levels and in mixed solvents of water/acetonitrile. Complementary studies of the amide protons were carried out in acetonitrile-d₃. Only the prolyl $\text{C}\text{=}{}^{17}\text{O}$ group was sensitive to the pH level. Protonation of the amine group resulted in an upfield chemical shift of 18 ppm. The chemical shifts of each of the three oxygen sites was sensitive to the ratio water: acetonitrile. Solvent composition dependence of the chemical shift and linewidth suggests that the prolyl $\text{C}\text{=}{}^{17}\text{O}$ is involved in intramolecular hydrogen bond formation when Pro-Leu-Gly-NH₂ is dissolved in acetonitrile, while in water there is no intramolecular H bond.     

Occurrence of passive furosemide-sensitive transmembrane potassium transport in cultured cells

Furosemide ($\text{1 · 10}{}^{\mathrm{?4}}\text{M}$) inhibits a proportion of the total passive (ouabain-insensitive) K⁺ influx into primary chick heart cell cultures (85%), BC₃H1 cells (75%), MDCK cells (40%) and HeLa cells (57%). This action of furosemide upon K⁺ influx is independent of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{pump}$ inhibition since the furosemide-sensitive component of the K⁺ influx is identical in the presence and absence of ouabain ($\text{1 · 10}{}^{\mathrm{?3}}\text{M}$). For HeLa cells the passive, furosemide-sensitive component of K⁺ influx is markedly dependent upon the external K⁺, Na⁺ and Cl^? content. Acetate, iodide and nitrate are ineffective as substitutes for Cl^?, whereas Br^? is partially effective. Partial Cl^? replacement by NO₃^? gave an apparent affinity of 100 mM [Cl]. Na⁺ replacement by choline⁺ abolishes the furosemide-sensitive component, whereas Li⁺ replacement reduces this component by 48%. Partial Na⁺ replacement by choline⁺ gives an apparent affinity of 25 mM [Na⁺]. Variation in the external K⁺ content gives an affinity for the furosemide-sensitive component of approx. 1.0 mM. Furosemide inhibition of the passive K⁺ inflúx is of high affinity, half-maximal inhibition being observed at $\text{5 · 10}{}^{\mathrm{?6}}\text{M}$ furosemide. Piretanide ($\text{1 · 10}{}^{\mathrm{?4}}\text{M}$) and phloretin ($\text{1 · 10}{}^{\mathrm{?4}}\text{M}$) inhibit the same component of passive K⁺ influx as furosemide; ethacrynic acid and amiloride ($\text{both}\text{1 · 10}{}^{\mathrm{?4}}\text{M}$) partially so. The stilbene, SITS ($\text{1 · 10}{}^{\mathrm{?6}}\text{M}$), was ineffective as an inhibitor of the furosemide-sensitive component.     

Structure and action of heteronemertine polypeptide toxins Binding of cerebratulus lacteus toxin B-IV to axon membrane vesicles

The binding of the crustacean selective protein neurotoxin, toxin B-IV, from the nemertine Cerebratulus lacteus to lobster axonal vesicles has been studied. A highly radioactive, pharmacologically active derivative of toxin B-IV has been prepared by reaction with Bolton-Hunter reagent. Saturation binding and competition of ¹²⁵I-labeled toxin B-IV by native toxin B-IV have shown specific binding of ¹²⁵I-labeled toxin B-IV to a single class of binding sites with a dissociation constant of 5–20 nM and a binding site capacity, corrected for vesicle sidedness, of 6–9 pmol per mg membrane protein. This compares to a value of 3.8 pmol [³H]saxitoxin bound per mg in the same tissue. Analysis of the kinetics of toxin B-IV association ($\text{k}{}_{\mathrm{+1}}\text{=7.3·10}{}^5\text{M}{}^{\mathrm{?1}}\text{·s}{}^{\mathrm{?1}}$) and dissociation ($\text{k}{}_{\mathrm{?\; 1}}\text{=2·10}{}^{\mathrm{?3}}\text{s}{}^{\mathrm{?1}}$) shows a nearly identical $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of about 3 nM. There is no competition of toxin B-IV binding by purified toxin from Leiurus quinquestriatus venom while Centruroides sculpturatus Ewing toxin I appears to cause a small enhancement of toxin B-IV binding.     

Ouabain receptor interactions in (Na+ + K+)-ATPase preparations. II. Effect of cations and nucleotides on rate constants and dissociation constants

The action of ATP and its analogs as well as the effects of alkali ions were studied in their action on the ouabain receptor. One single ouabain receptor with a dissociation constant ($\text{K}{}_{\mathrm{<mtext>D</mtext>}}$) of 13 nM was found in the presence of ($\text{Mg}{}^{\mathrm{2+}}\text{+ P}{}_{\mathrm{i}}$) and ($\text{Na}{}^{\mathrm{+}}\text{+ Mg}{}^{\mathrm{2+}}\text{+ ATP}$). pH changes below pH 7.4 did not affect the ouabain receptor. Ouabain binding required Mg²⁺, where a curved line in the Scatchard plot appeared. The affinity of the receptor for ouabain was decreased by K⁺ and its congeners, by Na⁺ in the presence of ($\text{Mg}{}^{\mathrm{2+}}\text{+ P}{}_{\mathrm{i}}$), and by ATP analogs (ADP-C-P, ATP-OCH₃). Ca²⁺ antagonized the action of K⁺ on ouabain binding. It was concluded that the ouabain receptor exists in a low affinity (R_α) and a high affinity conformational state (R_β). The equilibrium between both states is influenced by ligands of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$. With 3 mM Mg²⁺ a mixture between both conformational states is assumed to exist (curved line in the Scatchard plot).     

Ligand binding properties of the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase labelled with N-cyclohexyl-N′-(4-dimethylamino-α-naphthyl)carbodiimide

The $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ of rabbit sarcoplasmic reticulum, when labelled at two Ca²⁺-protected sites with $\text{N-}\text{cyclohexyl}\text{-N′-(4-}\text{dimethylamino}\text{-α-}\text{naphthyl}\text{)}\text{carbodiimide}$ (NCD-4) retains Ca²⁺ binding capacity at the sites with $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ values of approx. 3 μM and 0.12 mM as assessed by fluorescence titration. The sites correspond to the two high-affinity Ca²⁺ binding sites present in the native ATPase. The NCD-4 labelled ATPase exhibits slow conformational changes at each site on addition of Ca²⁺. It retains the ability to form phosphoenzyme, and can most likely translocate Ca²⁺.     

The dehalogenation of iodouracil by cysteine. Intramolecular general-acid catalysis of cysteine addition to 5-iodouracil

The rate-determining step of the cysteine-catalyzed deiodination of 5-iodouracil is the formation of 5-iodo-6-cysteinyl-5,6-dihydrouracil. The rate of the reaction depends upon the concentration of un-ionized 5-iodouracil and the following ionic species of cysteine; ^?OOC(NH₃⁺)CHCH₂S^?. Unlike the reaction of 2-mercapto-ethanol with 5-iodouracil, the cysteine reaction is not subject to catalysis by imidazolium ion and tris(hydroxymethyl)aminomethane hydrochloride. When the rates of cysteine reacting with 5-iodouracil are measured in both H₂O and D₂O, a large kinetic isotope effect is observed ($\text{k}{}_2{}^{\mathrm{<mtext>H</mtext><mtext>20</mtext>}}\text{k}{}_2{}^{\mathrm{<mtext>D</mtext><mtext>20</mtext>}}\text{= 4.10}$), thus implicating the protonated α amino group of cysteine as an intramolecular general acid catalyst for the reaction. These results and possible mechanisms for the actual dehalogenation of the intermediate 5-iodo-6-cysteinyl-5,6-dihydrouracil are discussed in terms of a possible mechanism for enzymatic halopyrimidine dehalogenation.     

Differential decrease of the central beta-adrenergic receptor in the rat after subchronic infusion of desipramine and clenbuterol

The effect of subchronic infusion of desipramine, a norepinephrine uptake inhibitor, and clenbuterol, a beta-adrenergic agonist, on the central beta receptor of the rat was determined using in vitro [³H]dihydroalprenolol binding. Desipramine produced significant decreases of the receptor in neocortex and hippocampal formation, and clenbuterol effected such decreases in corpus striatum and cerebellum. Both drugs caused a marked decrease in the activity of isoproterenol-sensitive adenylate cyclase in neocortex. The alpha₂ receptor of neocortex and cerebellum was unchanged by either drug as assessed by in vitro$\text{[}{}^3\text{H}\text{]p-}\text{aminoclonidine}$ binding. The results are discussed in terms of the different mechanisms of action of desipramine and clenbuterol, and the efficacy of these two drugs in the treatment of depression.     

Author index

The ionic influence and ouabain sensitivity of lymphocyte Mg²⁺-ATPase and $\text{Mg}{}^{\mathrm{2+}}\text{-(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-activated}$ ATPase were studied in intact cells, microsomal fraction and isolated plasma membranes. The active site of 5′-nucleotidase and Mg²⁺-ATPase seemed to be localized on the external side of the plasma membrane whereas the ATP binding site of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ was located inside the membrane.Concanavalin A induced an early stimulation of Mg²⁺-ATPase and $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ both on intact cells and purified plasma membranes. In contrast, 5′-nucleotidase activity was not affected by the mitogen. Although the thymocyte Mg²⁺-ATPase activity was 3–5 times lower than in spleen lymphocytes, it was much more stimulated in the former cells (about 40 versus 20 %). $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity was undetectable in thymocytes. However, in spleen lymphocytes $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity can be detected and was 30 % increased by concanavalin A. Several aspects of this enzymic stimulation had also characteristic features of blast transformation induced by concanavalin A, suggesting a possible role of these enzymes, especially Mg²⁺-ATPase, in lymphocyte stimulation.     

The insect brain (Na+ + K+)-ATPase Binding of ouabain in the hawk moth, Manduca sexta

(1) A quantitative study has been made of the binding of ouabain to the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in homogenates prepared from brain tissue of the hawk moth, Manduca sexta. The results have been compared to those obtained in bovine brain microsomes. (2) The insect brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ will bind ouabain either in the presence of Mg²⁺ and P_i, (‘Mg²⁺, P_i’ conditions) or in the presence of Na⁺, Mg²⁺, and an adenine nucleotide (‘nucleotide’ conditions) as is the case for the bovine brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$. The binding conditions did not alter the total number of receptor sites measured at high ouabain concentrations in either tissue. (3) Potassium ion decreases the affinity (increases the $\text{K}{}_{\mathrm{<mtext>D</mtext>}}$) of ouabain to the M. sexta brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ under both binding conditions. However, ouabain binding is more sensitive to K⁺ inhibition under the nucleotide conditions. In bovine brain ouabain binding is equally sensitive to K⁺ inhibition under the both conditions. (4) The enzyme-ouabain complex has a rate of dissociation that is 10-fold faster in the M. sexta preparation than in the bovine brain preparation. Because of this, the M. sexta ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ has a higher $\text{K}{}_{\mathrm{<mtext>D</mtext>}}$ for ouabain binding and is less sensitive to inhibition by ouabain than the bovine brain enzyme. (5) This data supports the hypothesis that two different conformational states of the M. sexta ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ can bind ouabain.     

Assembly of the vesicular stomatitis virus envelope: incorporation of viral polypeptides into the host plasma membrane

Incorporation of viral polypeptides into the host plasma membrane is an essential step in the formation of the lipoprotein envelope of vesicular stomatitis virus. A quantitative study of this process was carried out using a double-isotope labeling procedure. Infected cells were incubated for two hours with ¹⁴C-labeled amino acids, pulse-labeled with [³H]leucine and incubated for various times with an excess of non-radioactive leucine. The ${}^3\text{H}{}^{14}\text{C}$ ratio was determined for each viral polypeptide in isolated plasma membranes and in the whole cell by polyacrylamide gel electrophoresis. It was found that [³H]leucine-labeled viral polypeptides could be detected in the plasma membranes immediately following a 30-second pulse but that the ${}^3\text{H}{}^{14}\text{C}$ ratios of polypeptides in the plasma membrane did not reach the ${}^3\text{H}{}^{14}\text{C}$ ratios in the whole cells until the end of a two-minute chase period. The addition of puromycin to the cultures at the end of the pulse period did not affect subsequent incorporation of [³H]leucine-labeled polypeptides into the plasma membrane. The incorporation of various amino acid analogs into the viral polypeptides did not affect the efficiency with which they were incorporated into the plasma membranes. It is proposed that viral polypeptides are selected for incorporation into the plasma membrane from a small interior pool of completed molecules.     

Activation of heart sarcolemmal Ca2+/Mg2+ ATPase by cyclic AMP-dependent protein kinase.

The sarcolemmal membrane obtained from rat heart by hypotonic shock-LiBr treatment method was found to incorporate ³²P from [γ-³²P] ATP in the absence and presence of cyclic AMP and protein kinase. The phosphorylated membrane showed an increase in Ca²⁺ ATPase and Mg²⁺ ATPase activities without any changes in Na⁺K⁺ ATPase activity. The observed increase in $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ ATPase activity was found to be associated with an increase in V_max value of the reaction whereas K_a value for $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ was not altered. These results provide information concerning biochemical mechanism for increased calcium entry due to hormones which are known to elevate cyclic AMP levels in myocardium and produce a positive inotropic effect.