Evidence for a quaternary structure change in the cooperative binding of cyanide to ferrihemoglobin A.

At pH 6.5 in a 0.05 $\text{M}$ bis-Tris-0.1 $\text{M}$ Cl^? buffer, tetra aquo ferrihemoglobin A (HbA⁺) binds CN^? with a Hill coefficient of n = 1.4. The Hill coefficient increases slightly and the average CN^? affinity decreases in the presence of excess spin labeled triphosphate (SLTP). This is probably the result of the finding that the SLTP exhibits a twofold higher affinity for HbA⁺ than for tetra cyano HbA⁺. Over the course of heme saturation with CN^?, a certain fraction of the SLTP is specifically released. This shows linkage between organic phosphate binding and heme ligation. These findings bear a marked resemblance to the ligand binding phenomena in hemoglobin A (HbA) and provide good evidence that under these experimental conditions, HbA⁺ is undergoing a quaternary conformation change as the hemes become saturated.     

Purification of plant calmodulin by fluphenazine-Sepharose affinity chromatography.

The calcium-dependent binding of phenothiazine drugs to calmodulin (Levin, R. M. and Weiss, B. (1977) Mol. Pharmacol. $\text{13}$, 690–697) has been utilized to develop a rapid purification procedure for calmodulin based on fluphenazine-Sepharose affinity chromatography. Calmodulin from plant, a fungus, porcine brain and the coelenterate, $\text{Renilla}\text{reniformis}$, were easily purified by the calcium-dependent binding of calmodulin to fluphenazine-Sepharose.     

Phospholipid metabolism of stimulated lymphocytes: Comparison of the activation of acyl-CoA: Lysolecithin acyltransferase with the binding of concanavalin A to thymocytes

Calf thymocytes were isolated and incubated with concanavalin A. The effect of the mitogen on the enzyme activity of membrane-bound lysolecithin acyltransferase (acyl-CoA: $\text{1-acylglycero-3-phosphorylcholine}\text{-O-}\text{acyltransferase}$, EC 2.3.1.23) was determined as also the binding of ¹²⁵I-labelled concanavalin A to intact cells and isolated membranes.The lysolecithin acyltransferase was found to be activated three times in microsomal membranes. The activation occurred directly after binding of concanavalin A and was temperature independent, since similar activities were found in cells treated with concanavalin A at 0 and 37 °C.The acyltransferase activation using increasing concentrations of concanavalin A revealed a different behaviour, as compared to the binding of concanavalin A. While the binding of concanavalin A to intact cells expressed a normal hyperbolic saturation function the activation process of the acyltransferase described a sigmoidal relationship. Corespondingly, the interaction coefficients for both functions were different (Sips coefficient for binding = 1.0 and Hill coefficient of the enzyme activation = 1.8).These results indicate that the acyltransferase activation is due to a cooperative interaction between the ligand-receptor complex and the 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.     

Binding of organic phosphates by human hemoglobin at alkaline pH-values

The hemoglobin-oxygen equilibrium binding curve was found to be sensitive to the addition of inositol hexaphosphate at pH 9.1. A solution of hemoglobin A in 0.050 $\text{M}$ sodium borate was half-saturated with oxygen at a partial pressure of 0.55mm Hg. Hemoglobin A in 0.050 $\text{M}$ sodium borate, 0.001 $\text{M}$ inositol hexaphosphate, pH 9.1 was half-saturated with oxygen at a partial pressure of 0.95mm Hg. The Hill plot was linear with a slope of 2.0 in the absence of phosphates. In the presence of inositol hexaphosphate the slope of the Hill plot increased from 1.0 to 2.36. The dependence of fractional saturation of hemoglobin with oxygen on concentration of inositol hexaphosphate was determined at partial pressures of oxygen of 0.46 and 1.07mm Hg.     

Specific activity of LHRH and TRH degrading enzymes in various tissues of normal and castrated male rats.

The chlorophyll composition and Hill activity of the leaf, developing seed parts and pod have been studied in three species of legumes, $\text{Lathyrus latifolius}$, $\text{Pisum sativum}$ and $\text{Vicia faba}$. The studies indicate that in all the species, the level of chlorophyll, on mg/g fresh weight basis, is maximum in the leaf. However, Hill activity studies show that the cotyledonary chloroplasts in all the cases have a higher Hill activity than the leaf chloroplasts. Thus, the Hill activities of the cotyledonary chloroplasts are 340% of the leaf chloroplasts in $\text{Lathyrus}$$\text{latifolius}$, 144% of the leaf chloroplasts in $\text{Pisum}$$\text{sativum}$ and 200% of the leaf chloroplasts in $\text{Vicia}$$\text{faba}$.     

Evidence for cooperative Mn-ATP binding with Bacillus sp. glutamine synthetase

The binding of Mn-ATP with $\text{B. subtilis}$ glutamine synthetase, observed kinetically at 37°, pH 7.0, is cooperative (Hill $\text{n}\text{= 2.3, S}{}_{\mathrm{0·5}}\text{= 0.36mM)}$, a phenomenon overlooked in earlier studies. The Arrhenius plot is biphasic with a break at 26°C. Similar behavior is observed with the thermophilic $\text{B. stearothermophilus}$ enzyme, but is absent with the enzymes from $\text{E. coli}$, plant, and mammaliam sources under optimal assay conditions. The temperature dependence of the intrinsic fluorescence of the protein is also non-linear, and the intersection point of 18° shifts to 30° upon binding of substrates. These results are interpreted as indicating that $\text{Bacillus sp}$. enzymes can assume multiple, functionally important conformational states related to Mn-ATP binding at 37°. They also emphasize further that critical differences in mechanism exist among glutamine synthetases from different sources.     

The enhancement of the alkaline Bohr effect of some fish hemoglobins with adenosine triphosphate.

The oxygen equilibria of the hemoglobins of one holostean fish, the spotted gar (Lepisosteus osculatus), and of four teleost fish, the carpsucker (Carpiodes carpio), the small mouth buffalo fish (Ictiobus bubalus), the Rio Grande cichlid (Cichlasoma cyanoguttatum), and the redear sunfish (Lepomis microlophus), have been measured as a function of pH in the presence and absence of ATP. The oxygen equilibria of the teleost hemoglobins in the presence of 200 μm ATP can be superimposed within experimental error upon the data obtained in the absence of ATP by a simple downward shift of the pH scale by 0.5 unit. Thus the effects of proton and ATP binding appear equivalent: Both can be interpreted in terms of a two-state allosteric model in which binding occurs preferentially to the low-affinity T-state. The oxygen affinities of each of the teleost hemoglobins approach asymptotically a maximal value at high pH. Although these high affinities are associated with decreased cooperativity of oxygen binding, as reflected by the Hill coefficient n, the asymptotic value of n never appears lower than 1.2 to 1.4. This indicates that the data cannot be completely described in terms of a single high-affinity R-state in alkaline solution: At least two different conformations are required. The oxygen affinity of the spotted gar hemoglobin, like that of each of the teleost hemoglobins, reaches a maximal value (minimal value of log PO₂ for half-saturation) above pH 8, but unlike teleost hemoglobins, the Hill coefficient reaches maximal values of 2.6 to 2.7 at high pH. The data in the absence of ATP are superimposable on the data in its presence by a downward shift of the pH scale by 0.25 unit. The measurement of the Bohr effect ($\text{Δlog}\text{P}{}_{30}\text{ΔpH}$) in the presence and absence of ATP shows that the Bohr effect in each of the hemoglobins is substantially enhanced by organic phosphates, as it is in mammalian hemoglobins. The extent of the enhancement of the Bohr effect by 200 μm ATP for each of the hemoglobins is approximately the same in the range pH 6.7 to 7.3 (increase in $\text{Δlog}\text{P}{}_{50}\text{ΔpH}\text{~ 0.3}$). This is a direct consequence of the equivalence of the linked-function relationship to the effects of ATP and proton binding on oxygenation.     

Molting fluid chitinase: a homotropic allosteric enzyme.

The molting fluid of the tobacco hornworm has chitinase activity which shows allosteric behavior with chitin. A parabolic curve is obtained on a double reciprocal plot of $\text{1}\text{v}$ vs. $\text{1}\text{[S]}$, and a sigmoid curve results when v (N-acetylglucosamine produced) is plotted against [S] (chitin concentration in terms of N-acetylglucosamine concentration). The Hill coefficient with insect chitin is 1.95 ± 0.08. Allostery of the chitinase enables the insect to exert additional control over the integrity of structure of its cuticle.     

Phlorizin as a probe of the small-intestinal Na+,d-glucose cotransporter. A model

(1)‘Uptake’ of phlorizin by intestinal brush border membrane vesicles is stimulated, much as that of d-glucose, by the simultaneous presence of Na_out⁺ and $\text{Δψ?0}$. However, phlorizin contrary to d-glucose, fulfills all criteria of a non-translocated ligand (i.e., of a fully competitive inhibitor) of the Na⁺,d-glucose cotransporter. (2) The stoicheiometry of Na⁺/phlorizin binding is 1, as shown by a Hill coefficient of approx. 1 in the Na_out⁺-dependence of phlorizin binding. (3) The preferred order of binding at $\text{Δψ?0}$ is Na⁺ first, phlorizin second (4) The velocity of association of phlorizin to the cotransporter, but not the velocity of its dissociation therefrom, responds to Δψ. These observations while agreeing with the effect of $\text{Δψ?0}$ on the $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of phlorizin binding in the steady-state time range, also confirm that the mobile part of the cotransporter bears a negative charge of 1. (5) A model is proposed describing the Na⁺,Δψ-dependent interaction of phlorizin with the cotransporter and agreeing with a more general model of Na⁺,d-glucose cotransport. (6) The $\text{k}{}_{\mathrm{<mtext>on</mtext>}}$, $\text{k}{}_{\mathrm{<mtext>off</mtext>}}$ and $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ constants of phlorizin interaction with the Na⁺,d-glucose cotransporter are smaller in the kidney than in the small-intestinal brush border membrane, which results in a number of quantitative differences in the overall behaviour of the two systems.     

β-Adrenergic receptors of brain cells. Membrane integrity implies apparent positive cooperativity and higher affinity

β-Adrenergic receptors were studied in intact cells of chick, rat and mouse embryo brain in primary cultures, by the specific binding of [³H]dihydro-l-alprenolol ([³H]DHA). The results were compared to the receptor binding of broken cell preparations derived from the cell cultures or from the forebrain tissues used for the preparation of the cultures. Detailed analysis of [³H]DHA binding to living chick brain cells revealed a high-affinity, stereoselective, β-adrenergic-type binding site. Equilibrium measurements indicated the apparent positive cooperativity of the binding reaction. By direct fitting of the Hill equation to the measured data, values of B_max = 12.01 fmol/10⁶ cells (7200 sites/cell), K_d = 60.23 pM and the Hill coefficient n = 2.78 were found. The apparent cooperative character of the binding was confirmed by the kinetics of competition with l-alprenolol, resulting in maximum curves at low ligand concentrations. The rate constants of the binding reaction were estimated as k₊ = 8.31·10⁷ M^?1 · min^?1 and k_? = 0.28 min^?1 from the association results, and k_? = 0.24 min^?1 from the dissociation data. The association kinetics supported the cooperativity of the binding, providing a Hill coefficient n = 1.76; K_d, as $\text{(k}{}_{\mathrm{?}}\text{/k}{}_{\mathrm{+}}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>n</mtext>}}$ was found to be 101 pM. Analysis of the equilibrium binding of [³H]DHA to rat and mouse living brain cells resulted in values of B_max = 13.04 fmol/10⁶ cells (7800 sites/cell), K_d = 43.85 pM and n = 2.52, and B_max = 8.08 fmol/10⁶ cells (4800 sites/cell), K_d = 46.70 pM and n = 1.63, respectively, confirming the apparent cooperativity of the β-receptor in mammalian objects, too. The [³H]DHA equilibrium binding to broken cell preparations of either chick, rat or mouse brain cultures or forebrain tissues was found to be non-cooperative, with a Hill coefficient n = 1, K_d in the range 1–2 nM, and a B_max of 10³–10⁴ sites/cell. Our findings demonstrate that cell disruption causes marked changes in the kinetics of the β-receptor binding and in the affinity of the binding site, although the number of receptors remains unchanged.     

Properties of [3H]diazepam binding sites on rat blood platelets

Intact rat blood platelets are shown to possess benzodiazepine binding sites of the peripheral type, binding of [³H]diazepam being strongly inhibited by Ro5-4864 (K_i = 3.6 ± 0.5 nM) but only weakly inhibited by clonazepam (K_i = 35.1 ± 18.2 μM). Binding of [³H]diazepam is specific and saturable. Scatchard analysis reveals a single class of binding sites with K_D = 14.7 ± 1.0 nM and B_max = 564 ± 75 fmoles/10⁸ platelets. The Hill coefficient is 0.94, indicating a lack of binding site heterogeneity or negative cooperativity. Binding reaches equiliibrium at 6 min, with k₊₁ = 2.9 × 10⁷ M^?1 min^?1, and is rapidly reversible ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext><mtext>\; =\; 2.2\; </mtext><mtext>min</mtext>}}$ with K_?1 = 0.315 min^?1. K_D derived from the rate constants agrees with that estimated by Scatchard analysis. K_D of the crude membrane fraction of platelets is also close to that of intact platelets. Binding of [³H]diazepam is linear with platelet number (between 0.25–2 × 10⁸ platelets), is temperature sensitive with maximum binding at 0°C, and has a broad optimal pH range between pH 5–9.     

A reappraisal of the relationship of phosphate-acceptor protein to parvalbumins.

A phosphate-acceptor protein thought to be related to parvalbumins was described from dogfish muscle (Blum, H.E. $\text{et al.}$, 1974 Proc. Nat. Acad. Sci. USA $\text{71}$, 2198–2202). Further examination of this material indicated that the fraction obtained contained mainly classical parvalbumin, contaminated by less than 5% of a true phosphate-acceptor protein of MW ca 18 000 that accompanies parvalbumin throughout its purification. No such acceptor could be found in hake subjected to identical purification procedures. It is concluded that a phosphate-acceptor protein such as found in dogfish muscle bears no relation to parvalbumins.     

Specific in vivo binding of 77Br-p-bromospiroperidol in rat brain: A potential tool for gamma ray imaging

The binding of the gamma labeled neuroleptic, ⁷⁷Br-$\text{p}$-bromosprioperidol, in the rat brain was examined $\text{in vivo}$. This binding parallels the binding of ³H-spiroperidol, in that binding is especially high in dopaminergically innervated areas, is saturable, and is displaced by high doses of unlabeled spiroperidol (1–5). Thus, ⁷⁷Br-$\text{p}$-bromospiroperidol is a suitable ligand for use in gamma ray imaging techniques for $\text{in vivo}$ monitoring of receptor binding.     

Partial purification and properties of a non-ligandin (3H) 3-methylcholanthrene binding protein from liver cytosol

(³H) 3-Methylcholanthrene binds $\text{in vivo}$ to a macromolecule in addition to the previously reported binding to ligandin in liver cytosol. The properties of this second molecule are identical to those of the glucocorticosteroid receptor (Binder II) through 400 fold purification over the cytosol proteins (elution position from DEAE-Sephadex A-50 columns, molecular weight by gel filtration and pI value by isoelectrofocusing). The carcinogen, probably a metabolite, binds very strongly or covalently to the macromolecule $\text{in vivo}$, but non-covalently $\text{in vitro}$ in the absence of microsomes. Large amounts of unlabeled carcinogen administered $\text{in vivo}$ do not compete significantly with subsequent (³H) dexamethasone binding to the hormone receptor fraction $\text{in vitro}$. Methylcholanthrene and dexamethasone do not compete for binding sites $\text{in vitro}$ on isolated unlabeled Binder II leading to the conclusion that the glucocorticosteroid receptor and the methylcholanthrene binding protein are distinct entities.     

Solubilization and anionic regulation of cerebral sedative/convulsant receptors labeled with [35S] tert-butylbicyclophosphorothionate (TBPS)

Binding activity for the cage convulsant [³⁵S]-$\text{tert}$-butylbicyclophosphorothionate, which appears to label a site closely associated with the chloride ionophore of the GABA_A/benzodiazepine receptor complex has been solubilized from rat cerebral cortex using the zwitterionic detergent CHAPS. Of several detergents screened, only CHAPS and CHAPSO were capable of solubilizing the binding activity with good recovery. The pharmacologic specificity of soluble [³⁵S]-$\text{tert}$-butylbicyclophosphorothionate binding is very similar to the membrane state. In both the membrane and soluble state, [³⁵S]-$\text{tert}$-butylbicyclophosphorothionate binding is enhanced by anions which support inhibitory post-synaptic potentials (“Eccles anions”), suggesting that [³⁵S]-$\text{t}$-butylbicyclophosphorothionate may label chloride channels thought to be involved in these potentials. Since this solubilization procedure also preserves GABA and benzodiazepine binding and their regulation by drugs such as barbiturates, purification and isolation of the macromolecular complex including chloride channel and GABA-benzodiazepine sites may be feasible.     

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.     

Receptor-like stereospecific binding of a pyrethroid insecticide to mouse brain membranes

A heterogeneous particulate fraction of mouse brain homogenates binds NRDC 157 (3-phenoxybenzyl [1$\text{R}$,$\text{cis}$]-3-(2,2-dibromovinyl)-2,2- dimethylcyclopropanecarboxylate), a potent pyrethroid insecticide, stereospecifically and with high affinity. Stereospecific binding is a minor component of total binding (2.8%); the remainder of observed binding is predominantly nonspecific and unsaturable. Stereospecific binding is half-saturated at 4×10^?8$\text{M}$ and fully saturated at concentrations in excess of 1×10^?7$\text{M}$. The stereospecific binding capacity of this preparation was 200–250 pmoles of NRDC 157 per gram equivalent of brain tissue (2.3–2.8 pmol/mg protein). This binding site may represent the neural receptor involved in the stereospecific toxic action of pyrethroids.     

Pharmacologic identification of muscarinic receptors in the pylorus of the cat by binding of [3H]-quinuclidinyl benzilate

Muscarinic receptors in the smooth muscle of the cat pylorus (pyloric sphincter) were identified by binding of the ligand (±) [³H]-quinuclidinyl benzilate ([³H]-QNB). Receptor related binding of [³H]-QNB reached steady-state in thirty minutes at 37°C, was saturable, showed pharmacologic specificity and was stereoselective. An apparent equilibrium dissociation constant, K_D, of 1.9 ± 0.3 nM and maximum receptor concentration of 122 ± 13 femtomoles per mg of protein (means ± S.E.M.) were determined from Scatchard plots of [³H]-QNB binding. Hill coefficients of 0.99 and 1.01 indicated the absence of cooperative interactions. The muscarinic antagonists atropine and propantheline inhibited binding with IC₅₀ values in the nanomolar range, whereas bethanechol was over four orders of magnitude less potent. Noncholinergic agents had little or no effect on [³H]-QNB binding. The $\text{levo}$ isomer of QNB was about seventy times more effective at inhibiting binding than its $\text{dextro}$ isomer while $\text{dextro}$ benzetimide was greater than two thousand fold more active than $\text{levo}$ benzetimide. The isomers of another anticholinergic compound, tropicamide, also competed for [³H]-QNB binding sites in a stereoselective manner, the $\text{levo}$ isomer being eighty-five times more potent than the $\text{dextro}$ isomer.     

Oxygen binding of the heme-containing subunits of the extracellular hemoglobin of Lumbricus, terrestris

Fully oxygenated heme-containing subunits of the extracellular hemoglobin of $\text{Lumbricus}\text{,}\text{terrestris}$ were isolated by gel filtration in 0.05 M sodium borate, pH 9.0 at 7 ± 1°C. The polypeptide chain composition of the isolated subunits was determined by SDS PAGE. Most of the subunits exhibited reversible oxygenation curves with oxygen affinities higher than the intact hemoglobin. The Hill plots were nonlinear for $\text{Lumbricus}$ hemoglobin and its subunits: the latter exhibited substantial cooperativities as evidenced by Hill constants at half-saturation in the range 2.0 to 2.8.