Characterization of the β-adrenergic receptors of hamster white fat cells: Evidence against an important role for the α2-receptor subtype in the adrenergic control of lipolysis

The binding characteristics of the β-adrenergic antagonist, [³H]dihydroalprenolol, to hamster white adipocyte membranes were studied. This binding occurred at two classes of sites, one having high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 1.6±1.3}\text{nM}$) but low capacity ($\text{32±17}\text{fmol/mg}$ membrane protein) and one having low affinity but high binding capacity. While the binding at the high-affinity sites was competitively and stereoselectively displaced by both β-antagonists and β-agonists, competition at the low-affinity sites occurred only with β-antagonists and was non-stereoselective. Thus, the β-agonist (?)-isoproterenol was further used to define nonspecific binding. Under these conditions, saturation studies showed a single class of high-affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 1.6±0.5}\text{nM}$) binding sites with a binding capacity of $\text{53 ± 13}\text{fmol/mg}$ membrane protein (corresponding to $\text{4000 ± 980}$ sites per cell), and independent kinetic analysis provided a $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ value of 1.9 nM. Competition experiments showed that these binding sites had the characteristics of a $\text{β}{}_1\text{-}\text{receptor}$ subtype, yielding $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ values in good agreement with the $\text{K}{}_{\mathrm{<mtext>act</mtext>}}$ and the $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ values found for agonist-stimulation and for antagonist-inhibition of adenylate cyclase in membranes and of cyclic AMP accumulation and lipolysis in intact cells. Furthermore, the ability of β-agonists to compete with this binding was severely depressed by p[NH]ppG. These results thus support the contention that the specific [³H]dihydroalprenolol binding sites defined as the binding displaceable by (?)-isoproterenol represent the physiologically relevant β-adrenergic receptors of hamster white adipocytes. Finally, studies of the lipolytic response of these cells to (?)-norepinephrine showed that the inhibitory effect of the $\text{α}{}_2\text{-}\text{component}$ of this catecholamine was apparent only when the effects of endogenous adenosine were suppressed, a result which argues against an important regulatory role for the $\text{α}{}_2\text{-}\text{receptors}$ in the adrenergic control of lipolysis in hamster white adipocytes.     

Cotransport of phosphate and sodium by yeast

Phosphate uptake by yeast at pH 7.2 is mediated by two mechanisms, one of which has a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 30 μM and is independent of sodium, and a sodium-dependent mechanism with a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 0.6 μM, both $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values with respect to monovalent phosphate. The sodium-dependent mechanism has two sites with affinity for Na⁺, with affinity constants of 0.04 and 29 mM. Also lithium enhances phosphate uptake; the affinity constants for lithium are 0.3 and 36 mM. Other alkali ions do not stimulate phosphate uptake at pH 7.2. Rubidium has no effect on the stimulation of phosphate uptake by sodium.Phosphate and arsenate enhance sodium uptake at pH 7.2. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of this stimulation with regard to monovalent orthophosphate is about equal to that of the sodium-dependent phosphate uptake.The properties of the cation binding sites of the phosphate uptake mechanism and those of the phosphate-dependent cation transport mechanism have been compared. The existence of a separate sodium-phosphate cotransport system is proposed.     

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.     

Localization of the Na+-sugar cotransport system in a kidney epithelial cell line (LLC PK1)

Studies of the localization of the Na⁺-dependent sugar transport in monolayers of LLC PK₁ cells show that the uptake of a methyl α-d-glucoside, a nonmetabolizable sugar which shares the glucose-galactose transport system, occurs mainly from the apical side of the monolayer. Kinetics of [³H]phlorizin binding to monolayers of LLC PK₁ cells were also measured. These studies demonstrate the presence of two distinct classes of receptor sites. The class comprising high affinity binding sites had a dissociation constant ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}$) of 1.2 μM and a concentration of high affinity receptors of 0.30 μmol binding sites per g DNA. The other class involving low affinity sites had a $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of 240 μM with the number of binding sites equal to 12 μmol/g DNA. Phlorizin binding at high affinity binding sites is a Na⁺-dependent process. Binding at the low affinity sites on the contrary is Na⁺-independent. The mode of action of Na⁺ on the high affinity binding sites was to increase the dissociation constant without modifying the number of binding sites. The Na⁺ dependence and the matching of $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ for high affinity binding sites with the $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ of phlorizin for the inhibition of methyl α-d-glucoside strongly suggest that the high affinity phlorizin binding site is, or is part of the methyl α-d-glucoside transport system. Binding studies from either side of the monolayer also show that the binding of phlorizin at the Na⁺ dependent high affinity binding sites occurs mainly from the apical rather than the basolateral side. The specific location of the Na⁺-dependent sugar transport system in the apical membrane of LLC PK₁ cells is, therefore, another expression of the functional polarization of epithelial cells that is retained under tissue culture condition. In addition, since this sugar transport almost disappears after the cells are brought into suspension, it can be used as a marker to study the development of the apical membrane in this cell line.     

Peptide receptors in human lung tumor cells in culture: vasoactive intestinal peptide (VIP) and secretin interaction with the Calu-1 and SW-900 cell lines

We have investigated the interaction of VIP and secretin with two human lung carcinoma cell lines in cultures, SW-900 and Calu-1. ¹²⁵I-labeled VIP binds to and is inactivated by SW-900 and Calu-1 cells in a time- and temperature-dependent manner. The rates of binding and of inactivation were higher at 30°C than at 15°C. At equilibrium, native VIP competitively inhibited the binding of ¹²⁵I-VIP in the 10^?10?10^?7M range, half-maximal inhibition being observed at 1.2 nM in SW-900 cells and at 1.1 nM VIP in Calu-1 cells. Scatchard analysis indicated two classes of binding sites with similar characteristics in both cell lines. SW-900 cells have 27 600 sites with a high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 0.34}\text{nM}$) and 1062 000 sites with a low affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 61.4}\text{nM}$). Calu-1 cells have 36 300 sites with a high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 0.33}\text{nM}$) and 1148 000 sites with a low affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 78.6}\text{nM}$). Secretin inhibited tracer binding but with a 5000 times lower potency than native VIP in both cell lines.     

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.     

Association of α2-macroglobulin-thrombin and α2-macroglobulin-plasmin complexes with isolated hepatocytes

¹²⁵I-labelled $\text{α}{}_2\text{-}\text{macroglobulin}$ complexed with thrombin or plasmin bound to hepatocytes in a concentration-and time-dependent manner. The apparent $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ values calculated from displacement experiments were 7.9 · 10^?8 M for $\text{α}{}_2\text{-}\text{macroglobulin-thrombin}$ and 8.5 · 10^?8 M for $\text{α}{}_2\text{-}\text{macroglobulin-plasmin}$. Association of these complexes was only partially reversible; after a 180 min incubation period, 50–60% of the bound radioactivity was internalized by the cells. $\text{α}{}_2\text{-}\text{Macroglobulin}$ itself bound also to hepatocytes, but the affinity of the $\text{α}{}_2\text{-}\text{macroglobulin}$ complexes was higher than that of the inhibitor alone, and $\text{α}{}_2\text{-}\text{macroglobulin}$ was not internalized, either. ¹²⁵I-labelled thrombin or plasmin bound to hepatocytes as well. These bindings were also concentration-dependent and could be decreased with an excess of unlabelled ligands. Binding rates and amounts of the bound proteinases were higher than those of their $\text{α}{}_2\text{-}\text{macroglobulin}$ complexes. The $\text{α}{}_2\text{-}\text{macroglobulin-thrombin}$ complex competed with the $\text{α}{}_2\text{-}\text{macroglobulin-plasmin}$ complex in binding to hepatocytes, whereas there was no competition between these complexes and the antithrombin III-thrombin complex. These results suggest that the binding sites of hepatocytes for $\text{α}{}_2\text{-}\text{macroglobulin-proteinase}$ and antithrombin III-proteinase complexes are different.     

The distribution of (Na++K+)-ATPase along the villus crypt-axis in the rabbit small intestine

The migration of intestinal epithelial cells from the crypts to the tips of villi is associated with progressive cell differentiation. The changes in Na⁺-pump levels during migration have been measured in epithelial cells isolated from rabbit small intestine. A significant proportion of ouabain-sensitive ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in the cell homogenates was latent but could be unmasked by detergent treatment. Highest detergent activation was observed in villus cells. The distribution of pumping sites was also assessed by measuring ouabain binding to intact cells. The kinetics of specific binding was consistent with the interaction of the cardiac glycoside with a single population of binding sites with an apparent $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of around 10^?7 M. Both enzyme assay and ouabain-binding measurements suggest that a 2–3-fold increase in the number of Na⁺-pumping sites accompanies cell differentiation in rabbit jejunal epithelium. This increase in pumping capacity might be an adaptation of the cells to their absorptive function.     

Electron paramagnetic resonance study on calmodulin: conformational change and interaction with divalent cations

Biologically active spin labelled derivatives of calmodulin were prepared and used to study CA²⁺- and Mg²⁺-induced conformational changes of the protein. The rotational correlation time of the spin labelled residues increased upon addition of divalent cations. Two calcium ions per spin labelled calmodulin were found to induce a 75% conformational change, whereas four calcium ions were necessary for a maximum conformational change. The increase in rotational correlation time induced by Mg²⁺ is less pronounced. Two different covalently attached spin labels (iodoacetamide and maleimide) were compared and marked differences were found in their chemical stability. The binding of manganese ions to calmodulin could be observed directly from the electron paramagnetic resonance spectra of these paramagnetic ions. Two specific classes of binding sites, each binding two manganese ions with $\text{k}{}_{\mathrm{<mtext>D</mtext>}}\text{= 0.6 × 10}{}^{\mathrm{?6}}\text{m}\text{and}\text{k}{}_{\mathrm{D}}\text{= 3 × 10}{}^{\mathrm{?5}}\text{m}$, respectively, were determined. Further ion binding occurs at non-specific sites.     

Kinetic characterization and substrate requirement for the Ca2+ uptake system in platelet membrane

An ATP-dependent mechanism for Ca²⁺ uptake in human platelet membrane fractions has been identified and characterized. Ca²⁺ uptake into a membrane fraction is shown to be stimulated at low concentrations of ATP and Ca²⁺ and to require magnesium ions. Initial rate kinetics, using Eadie-Scatchard analysis, indicated a single class of calcium uptake sites in the presence of ATP, with a $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ for free [Ca²⁺] of 0.145 μM. Ca²⁺ uptake in the presence of several ATP concentrations demonstrates that ATP binds to at least two sites, representing high and low affinities of 3.21 and 80.1 μM, respectively. The neuroleptic drug fluphenazine inhibited ATP-stimulated calcium uptake ($\text{IC}{}_{50}\text{= 55 μ}\text{M}$), suggesting this ATP-dependent Ca²⁺ uptake system may provide a useful ion-transport model with which to study neuroleptic therapy in humans.     

[3H] BOC [NLE28,31]CCK27−33, a new highly labelle ligand for CCK receptors: Binding on brain and on pancreas

Preliminary results on the binding of [³H]Boc[Nle^28,31]CCK_27^?33, designated [³H]Boc[diNle]CCK₇, on mouse brain and rat pancreas membranes are presented. This new ligand for CCK receptors possesses a high specific activity (144 Ci/mmole), and binds in a saturable manner to mouse brain (Kd = 0.49 nM, Bmax = 49 fmoles/mg protein) and rat pancreas (Kd = 4.4 nM, Bmax = 696 fmoles/mg protein). Unlabelled Boc[diNle]CCK₇ displaces [¹²⁵I]CCK₈ from its binding sites on mouse brain membranes with a high affinity, slightly superior to that of CCK₈. The order of potencies to displace [³H]Boc[diNle]CCK₇ from its binding sites was the same on mouse brain and rat pancreas: $\text{[}{}^3\text{HBoc[diNle]CCK}{}_7\text{>}\text{CCK}{}_8\text{,}\text{Boc-CCK}{}_7\text{>}\text{non-sulfated CCK}{}_8$, the pancreas binding sites being more discriminative than the brain binding sites. Thus, [³H]Boc[diNle]CCK₇ is a very promising new probe for the characterization of CCK receptors and their interaction with different CCK fragments.     

Isolation of bovine cytochrome c1 as a single non-denatured subunit using gel filtration or high pressure liquid chromatography in deoxycholate

The non-denatured cytochrome $\text{c}{}_1$ subunit of bovine ubiquinone-cytochrome $\text{c}$ reductase was isolated using either gel filtration or high pressure liquid chromatography in 1% deoxycholate. The preparation was a single band on polyacrylamide gel electrophoresis in dodecyl sulfate, had a heme content of 31 nmol heme/mg protein, had an absorbance ratio $\text{A}{}_{417}\text{A}{}_{278}\text{= 2.65}$, a visible spectrum with maxima at 553, 530, 523.5, 417, 317, and 277 nm for the reduced protein, and an amino acid analysis identical to that previously reported for the isolated denatured protein. The Stokes' radius of this non-denatured deoxycholate solubilized protein was 34Å, indicating that the protein either is a dimer in deoxycholate, is asymmetric, or binds large amounts of detergent.     

The interaction of ions with phosphatidylcholine bilayers

The interaction of lanthanides and other cations with phosphatidylcholine bilayers present as single bilayer vesicles in ²H₂O has been investigated in terms of stoichiometry, apparent binding constants and environmental conditions.Lanthanides are shown to form 2 : 1 (molar ratio) phosphatidylcholine to metal ion complexes.The apparent binding constant $\text{K}{}_{\mathrm{<mtext>b</mtext>}}$ varies as a function of the quantity of metal ion bound and as a function of the Cl^? concentration. The apparent binding constant at “zero loading” is $\text{K}{}_0\text{= 1.25 · 10}{}^4\text{L}{}^2\text{·}\text{M}{}^{\mathrm{?}}\text{at}\text{0.15}\text{M KCl}$. It decreases exponentially with increased “loading” expressed as the molar ratio of metal ion bound to effective phosphatidylcholine concentration and increases exponential with Cl^? concentration.The interaction of lanthanides and divalent cations such as Ca²⁺ and Mg²⁺ is independent of pH in the pH range 3–7⁺ and 3–10 respectively, but is sensitive to the nature of the anion. The presence of anions enhances the interaction with polyvalent cations, the chaotropic anions showing the largest effect. The order of enhancement is $\text{Cl}{}^{\mathrm{?}}\text{< Br}{}^{\mathrm{?}}\text{< NO}{}_3{}^{\mathrm{?}}\text{< SCN}{}^{\mathrm{?}}\text{< I}{}^{\mathrm{?}}\text{< ClO}{}_4{}^{\mathrm{?}}$. The nature of the monovalent counterion (cation) has little effect on the enhanced binding of lanthanides in the presence of the above anions.The affinity of other polyvalent cations for phosphatidylcholine bilayers has been determined by competition with lanthanides. The physiologically important divalent cations Ca²⁺ and Mg²⁺ both bind less strongly (by about an order of magnitude) to the lipid surface. The order of binding of cations reflects direct binding to the phosphodiester group, with UO₂²⁺ showing the highest affinity.     

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⁺.     

β-Endorphin: Characteristics of binding sites in the rabbit cerebellar and brain membranes

Specific binding of human β-endorphin to rabbit cerebellar and brain membranes was measured using $\text{[}{}^3\text{H}{}_2\text{-Tyr}{}^{27}\text{]-β}{}_{\mathrm{h}}\text{-endorphin}$ as the primary ligand. In both tissues binding was time dependent and saturable, with apparent equilibrium dissociation constants of 0.275 nM and 0.449 nM in the cerebellum and brain, respectively. The binding capacity of cerebellum is greater than that of brain. Kinetic studies showed that the association rate constants were 2.7 × 10⁷ M^?1min^?1 for cerebellum and 2.4 × 10⁷ M^?1min^?1 for brain. Dissociation of tritiated β_h-endorphin from both cerebellum and brain is not consistent with a first order decay from a single site. In the cerebellum, these is a time-dependent increase in slowly dissociating complex. The potency of several opioid peptides and opiates to inhibit the binding of tritiated β_h-endorphin was determined. Ligands with preference for μ, δ, and κ opiate receptor (morphine, Metenkephalin and ethylketocyclazocine) all have similar affinities toward β_h-endorphin sites in both brain and cerebellar membranes.     

Thiophosphorylation of (Na + K+)-ATPase yields an ADP-sensitive phosphointermediate

(1) Treatment of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ from rabbit kidney outer medulla with the $\text{γ-}{}^{35}\text{S}$ labeled thio-analogue of ATP in the presence of $\text{Na}{}^{\mathrm{+}}\text{+ Mg}{}^{\mathrm{2+}}$ and the absence of K⁺ leads to thiophosphorylation of the enzyme. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for [γ-S]ATP is 2.2 μM and for Na⁺ 4.2 mM at 22°C. Thiophosphorylation is a sigmoidal function of the Na⁺ concentration, yielding a Hill coefficient $\text{n}\text{H}\text{= 2.6}$. (2) The thio-analogue ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>\; =\; 35\; \mu M</mtext>}}$) can also support overall $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity, but $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ at 37°C is only $\text{1.3 γ}\text{mol}\text{· (mg protein)}{}^{\mathrm{?}}\text{·}$ h^?1 or 0.09% of the specific activity for ATP ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>\; =\; 0.43\; mM</mtext>}}$). (3) The thiophosphoenzyme intermediate, like the natural phosphoenzyme, is sensitive to hydroxylamine, indicating that it also is an acylphosphate. However, the thiophosphoenzyme, unlike the phosphoenzyme, is acid labile at temperatures as low as 0°C. The acid-denatured thiophosphoenzyme has optimal stability at pH 5–6. (4) The thiophosphorylation capacity of the enzyme is equal to its phosphorylation capacity, indicating the same number of sites. Phosphorylation by ATP excludes thiophosphorylation, suggesting that the two substrates compete for the same phosphorylation site. (5) The (apparent) rate constants of thiophosphorylation (0.4 s^?1 vs. 180 s^?1), spontaneous dethiophosphorylation (0.04 s^?1 vs. 0.5 s^?1) and K⁺-stimulated dethiophosphorylation (0.54 s^?1 vs. 230 s^?1) are much lower than those for the corresponding reactions based on ATP. (6) In contrast to the phosphoenzyme, the thiophosphoenzyme is ADP-sensitive (with an apparent rate constant in ADP-induced dethiophosphorylation of 0.35 s^?1, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$$\text{ADP = 48 μM at 0.1 mM ATP}$) and is relatively K⁺-insensitve. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for K⁺ in dethiophosphorylation is 0.9 mM and in dephosphorylation 0.09 mM. The thiophosphoenzyme appears to be for 75–90% in the ADP-sensitive E₁-conformation.     

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.     

Sodium dependence of maximum flux, JM, and Km of amino acid transport in Ehrlich ascites cells

The Michaelis-Menten parameters, $\text{J}{}_{\mathrm{<mtext>M</mtext>}}$ and $\text{K}{}_{\mathrm{m}}$ of the initial 1-min fluxes of uptake of l-phenylalanine and of α-aminoisobutyric acid were determined for extracellular concentrations of Na⁺ ranging from 0.5 to 110 mequiv/l for Ehrlich ascites tumor cells. The maximal initial flux, $\text{J}{}_{\mathrm{<mtext>M</mtext>}}$, decreased with decrease in extracellular Na⁺ for both α-aminoisobutyric acid and phenylalanine but the $\text{K}{}_{\mathrm{m}}$ for α-aminoisobutyric acid increased markedly as the Na⁺ concentration fell whereas the $\text{K}{}_{\mathrm{m}}$ for phenylalanine decreased. Cycloleucine behaved like phenylalanine.The data provides strong evidence that the Na⁺-independent flux of phenylalanine is an exchange diffusion flux that can be varied by changing the intracellular level of amino acids such as phenylalanine. For phenylalanine, cyclolcucine, and methionine this exchange diffusion flux appears to be additive with the Na⁺-dependent initial flux. α-Aminoisobutyric acid also has an exchange diffusion that is Na⁺-independent but it has a high $\text{K}{}_{\mathrm{m}}$ and is not additive with the Na⁺-dependent flux.     

Kinetic studies on the inhibition of (Na+ + K+)-ATPase by diketocoriolin B

Diketocoriolin B, a sesquiterpene antitumor antibiotic, inhibits particulate $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{-ATPase}$ (ATP phosphohydrolase, EC 3.6.1.3) of Yoshida sarcoma cells competitively, with respect to ATP, and uncompetitively with respect to Na⁺ and K⁺. The inhibition is reduced by the addition of phosphatidylserine.Rat brain $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{-ATPase}$, which is solubilized by deoxycholate and requires phosphatidylserine for its activity, is also inhibited by diketocoriolin B competitively with respect to ATP and the inhibition was reversed by increasing the concentration of phosphatidylserine.However, several differences are found between the solubilized and particulate systems: (a) 2 moles of diketocoriolin B interact with the former, while only one mole interacts with the latter, (b) K⁺-dependent phosphatase activity of the former requires phospholipid and is sensitive to diketocoriolin B while the reverse is true with the latter.Based on these kinetic studies, it is supported that $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ has two binding sites for phospholipid, one being essential for K⁺-dependent phosphatase activity and when these two sites are filled with the appropriate phospholipids, ATP can bind to the enzyme.