Characterization of beta-adrenergic receptor and adenylate cyclase in canine cerebellum.

Binding of (?)-[${}^3\text{H}$]dihydroalprenolol to the synaptic membrane fractions of canine cerebellum was rapid and reversible with rate constants of 1.62 × 10⁸m^?1 min^?1 and 0.189 min^?1 for the forward and reverse reactions, respectively. The binding was of high affinity and saturable with an equilibrium dissociation constant (K_D) of 5 to 7 nm. Bound (?)-[${}^3\text{H}$]-dihydroalprenolol was displaceable with β-adrenergic agonists and antagonists, but not with a variety of other neuroactive substances such as acetylcholine, histamine, serotonin, dopamine, tyramine, (?)-phenylephrine, γ-aminobutyric acid, glycine, and glutamic acid. Adenylate cyclase of the membranes was stimulated at most three times by β-adrenergic agonists, but not significantly by the other neuroactive substances. Guanine nucleotides such as GTP and guanyl-5′-yl imidodiphosphate (Gpp(NH)p) were strictly required for β-adrenergic stimulation of adenylate cyclase with their optimum concentrations of 50 μm, although the nucleotides alone elevated virtually no basal activity. The affinities of β-adrenergic ligands including some stereoisomers for (?)-[${}^3\text{H}$]dihydroalprenolol binding sites were very similar to those for adenylate cyclase in the presence of GTP. Binding of β-adrenergic agonists to the membranes exhibited an apparent negative cooperativity as determined by displacement of (?)-[${}^3\text{H}$]dihydroalprenolol in the absence of purine nucleotides. This negative cooperativity was entirely abolished by addition of either GTP or Gpp(NH)p at 50 μm. Both (?)-isoproterenol-stimulated adenylate cyclase activity and binding of (?)-[${}^3\text{H}$]dihydroalprenolol were not affected by β₁-selective antagonists, (±)-atenolol, and (±)-practolol, at concentrations which completely inhibit peripheral β₁-responses in vitro, whereas β₂-selective agonists such as YM-08316 (BD-40A) and (±)-salbutamol not only stimulated adenylate cyclase but also competitively inhibited binding of (?)-[${}^3\text{H}$]dihydroalprenolol. These results indicate that canine cerebellar adenylate cyclase may be coupled specifically with β₂-adrenergic receptor.     

beta-Endorphin: characteristics of binding sites in the rat brain.

Stereospecific binding of human β-endorphin to rat membrane preparations is described for the first time using $\text{[}{}^3\text{H-Tyr}{}^{27}\text{]-β}{}_{\mathrm{h}}\text{-endorphin}$ as the ligand. The binding is time dependent and saturable with respect to β_h-endorphin with an apparent dissociation constant of 0.3 nM. Sodium ion (100 mM) elevates this value to 2.5 nM but has no effect on the total number of binding sites present in the membrane preparation. The ability of certain β-endorphin analogs, opiate agonists as well as antagonists to inhibit the binding of β_h-endorphin, is presented.     

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.     

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.     

Electrokinetic properties of (Na+, K+)-ATPase vesicles as studied by laser doppler spectroscopy

The technique of laser Doppler electrophoresis was applied for the study of the surface charge properties of (Na⁺,⁺)-ATPase containing microsomal vesicles derived from guinea-pig kidney. The influence of pH, the screening and binding of uni- and divalent cations and the binding of ATP show: (1) one net negative charge per protein unit with a $\text{p}\text{K = 3.9}$; (2) deviation from the Debye relation between surface potential and ionic strength for univalent cations, with no difference in the effect of Na⁺ and K⁺; (3) Mg²⁺ binds with an association constant of $\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 1.1 · 10}{}^2\text{M}{}^{\mathrm{?1}}$ while ATP binds with an apparent $\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 1.1 · 10}{}^4\text{M}{}^{\mathrm{?2}}$ for 1 mM Nacl, 0.2 mM KCI, 0.1 mM MgCl₂, 0.1 mM Tris-HCI (pH 7.3). The binding is weaker at higher Mg²⁺ concentrations. There is no ATP binding in the absence of Mg²⁺. In addition, the average vesicle size derived from the linewidth of the quasi-elastic light scattering spectrum is $\text{203.7 ± 15.2}\text{nm}$. In the presence of ATP a reduction in size is observed.     

Interaction of substituted guanidines with the tetrodotoxin-binding component in Electrophorus electricus.

In efforts to understand the molecular properties of ion channels in biomembranes, we have investigated the interaction of substituted guanidines with the Na⁺ channel site in membranes isolated from Electrophorus electricus. This interaction was measured by equilibrium competitive binding studies with [${}^3\text{H}$]tetrodotoxin ([${}^3\text{H}$]TTX); TTX has been shown to bind specifically to the Na⁺ channel in electrically excitable membranes. Although guanidine and small substituted guanidines such as methylguanidine or aminoguanidine competed with [${}^3\text{H}$]TTX for the membrane binding site, the apparent K_I values for these derivatives were nearly seven orders of magnitude higher than the K_d for TTX. On the other hand, the binding of the guanidines was considerably enhanced by introducing a substituent aromatic ring or aliphatic chain. Detailed analysis of the binding of aliphatic guanidines of varying chain length clearly demonstrated the contribution made by hydrophobic interactions. These results suggest that the channel site may include a hydrophobic region in close proximity to the carboxylate previously postulated to be involved in TTX binding.     

Contrasting effects of maytansine and vinblastine on the alkylation of tubulin sulfhydryls

The ansa macrolide maytansine is a competitive inhibitor of vinblastine for binding to tubulin. Both drugs are potent inhibitors of microtubule assembly in vitro but maytansine, unlike vinblastine, is unable to induce tubulin aggregation or to stabilize colchicine binding. In this study, the effects of maytansine and vinblastine on the accessibility of tubulin's sulfhydryl groups were compared. It was found that 10 μm vinblastine inhibited the reaction of bovine brain tubulin with [¹⁴C]iodoacetamide by 45%. In contrast, maytansine, even up to 100 μm, had no effect on the reaction. However, when the two drugs were tested in combination, maytansine was a potent inhibitor of vinblastine's effect, consistent with the two drugs competing for the same or overlapping sites, but suggesting that the nature of the binding was different. In contrast, maytansine did not affect the suppression of alkylation induced by colchicine and podophylotoxin, consistent with these drugs binding to different sites. Maytansine and vinblastine were each able to increase the formation of $\text{β}{}^1$ by the bifunctional reagent, N,N′-ethylenebis-(iodoacetamide); $\text{β}{}^1$ is the designation for an electrophoretically faster migrating form of β-tubulin which apparently contains an intrachain crosslink. Thus, in at least the portion of the tubulin molecule involved in $\text{β}{}^1$ formation, the two drugs have similar effects. Since maytansine does not appear to suppress any competing alkylation reactions, it is possible that the enhancement of $\text{β}{}^1$ formation represents a genuine conformational effect. Since the sulfhydryl groups of tubulin may be involved in regulating microtubule assembly, it is likely that maytansine and vinblastine differ in the manner in which they inhibit microtubule assembly.     

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.     

Saturation transfer electron paramagnetic resonance on membrane bound proteins. II-Absence of rotational diffusion of the cholinergic receptor protein in Torpedo marmorata membrane fragments.

We have applied the technique of saturation transfer electron paramagnetic resonance to study the rotational diffusion of spin labeled membrane bound cholinergic receptors from Torpedo marmorata. Two different spin labels were used: a spin labeled maleimide derivative which binds covalently to proteins and a long chain spin labeled acylcholine which binds reversibly with a high affinity to the receptor protein. The maleimide spin label has a motion whose rotational correlation time is τ₂ > 10^?3 sec. The long chain spin labeled acylcholine indicates slightly more motion ($\text{τ}{}_2\text{? 10}{}^{\mathrm{?4}}\text{sec}$), but the nitroxide in this latter case is probably more loosely bound.     

Respiration-driven proton translocation with nitrite and nitrous oxide in Paracoccus denitrificans

(1) $\text{H}$⁺/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O₂, NO^?₂ or N₂O. Under optimal H⁺-translocation conditions, the ratios $\text{H}{}^{\mathrm{+}}\text{O}$, $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were 6.0–6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as exogenous substrate, addition of NO^?₂ or N₂O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were ?0.84, ?2.33 and ?1.90, respectively. (3) The $\text{H}{}^{\mathrm{+}}\text{oxidant}$ ratios, mentioned in item 2, were not altered in the presence of $\text{valinomycin}\text{K}{}^{\mathrm{+}}$ and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O₂, NO^?₂ and N₂O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO^?₂, N₂O or O₂ pass two H⁺-translocating sites. The $\text{H}{}^{\mathrm{+}}\text{electron}$ acceptor ratios predicted by this scheme are in good agreement with the experimental values.     

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.     

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 non-covalent binding of benzo[a]pyrene and its hydroxylated metabolites to intracellular proteins and lipid bilayers

The non-covalent interactions of benzo[a]pyrene (BP) and several of its hydroxylated metabolites with ligandin, aminoazodye-binding protein A (Z-protein, fatty acid binding protein) and lecithin bilayers have been studied by equilibrium dialysis, an adsorption technique and fluorescence spectroscopy. Binding affinities expressed as $\text{v}\text{/c}$ (where $\text{v}$ = moles of BP or BP metabolite bound per mole of protein or lipid and c = unbound concentration), were measured at concentrations sufficiently low that there was no self-association of the unbound compounds as judged by their fluorescence characteristics. 3-Hydroxybenzo[a]pyrene (BP-3-phenol), 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene (BP-4,5-dihydrodiol) and 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (BP-7,8-dihydrodiol) bind more strongly ($\text{v}\text{/c = 10}{}^5\text{?5 · 10}{}^5\text{l}\text{·}\text{mol}{}^{\mathrm{?1}}$) to all three binders than does BP itself ($\text{v}\text{/c = 10}{}^4\text{?7 · 10}{}^4\text{l}\text{·}\text{mol}{}^{\mathrm{?1}}$). 9,10-Dihydro-9,10-dihydroxybenzo[a]pyrene (BP-9,10-dihydrodiol) binds to ligandin with an affinity similar to those of the other BP metabolites studied here, but binds much less strongly to both protein A and lecithin ($\text{v}\text{/c = 10}{}^4$ and 3 · 10⁴ l · mol^?1, respectively). The low affinity of BP-9,10-dihydrodiol for lecithin would account for earlier findings that on incubation of BP with isolated rat hepatocytes, this metabolite egressed from the cells to the extracellular medium much more readily than either BP-4,5-dihydrodiol or BP-7,8-dihydrodiol.Calculations based on these results suggest that within hepatocytes BP and its metabolites, including BP-9,10-dihydrodiol, will be found almost exclusively associated (>98%) with lipid membranes.     

A simple procedure for resolution of Escherichia coli RNA polymerase holoenzyme from core polymerase.

The association constant, K_A, for myosin subfragment-1 binding to actin was measured as a function of ionic strength [KCl, LiCl, and tetramethylammonium chloride (TMAC)]and temperature by the method of time-resolved fluorescence depolarization. The following thermodynamic values were obtained from solutions of 0.20 × 10^?6m S-1, 1.00 × 10^?6m actin in 0.15 m KCl, pH 7.0, at 25 °C: ΔG ° = ?39 ± 1 kJ M^?1, ΔH⁰ = 44 ± 2 kJ M^?1 and $\text{ΔS}{}^0\text{= 0.28 ± 0.01 kJ}\text{M}{}^{\mathrm{?1}}{}^0\text{K}{}^{\mathrm{?1}}$. For measurements in KCl (0.05 to 0.60 m), In $\text{K}{}_{\mathrm{a}}\text{= ?8.36 (KCl)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$. Thus, the binding is endothermic and strongly inhibited by high ionic strength. When KCl was replaced by LiCl or TMAC the ionic effects on the binding were cation specific. The nature of actin-(S-1) binding in the rigor state is discussed in terms of these results.     

Characteristics of β-adrenergic-agonist binding to rat adipocyte membranes: Evidence that (±)-[3H]hydroxybenzylisoproterenol interacts selectively with the adipocyte β-adrenergic receptors

The binding characteristics of the β-adrenergic agonist $\text{(±)-[}{}^3\text{H]hydroxybenzylisoproterenol}$ to rat adipocyte membranes were studied. Binding was rapid, reaching equilibrium within 10 min at 37°C (second order rate constant k₁=1.37·10⁷·M^?1·min^?1). Dissociation of specific binding by 0.5 mM (?)-isoproterenol suggested dissociation from two different sites with respective dissociation rate constants k₂ of 0.106·min^?1 and 0.011·min^?1.[³H]Hydroxybenzylisoproterenol binding was saturable (B_max=690±107 fmol/mg protein), yielding curvilinear Scatchard plots. Computer modeling of these data were consistent with the existence of two classes of [³H]hydroxybenzylisoproterenol binding sites, one having high affinity (K_D=3.5±0.7 nM) but low binding capacity (10% of the total sites) and one haveing low affinity (K_D=101±20 nM) but high binding capacity (90% of the sites). Adrenergic ligands competed with [³H]hydroxybenzylisoproterenol binding with the following order of potency=(?)-propranolol>(?)-isoproterenol>(?)-norepinephrine≈ (?)-epinephrine>>(+)-isoproterenol=(+)-propranolo, which is consistent with binding to β₁-adrenergic receptors. Competition curves of [³H]hydroxybenzylisoproterenol binding by the β-agonist (?)-isoproterenol were shallow and modeled to two affinity states of binding, whereas, competition curves by β-antagonist (?)-propranolol were steeper with Hill number near to one. Gpp[NH]p severely reduced [³H]hydroxybenzyl-isoproterenol binding, an effect which apparently resulted from the reduction of the number of both the high and low affinity sites. In membranes which had been previously exposed to (?)-isoproterenol, then number of [³H]hydroxybenzylisoproterenol binding sites was reduced by 50%, an effect which apparently resulted from the loss of part of both the high and low affinity state binding sites. Finally, the ability of (?)-isoproterenol to stimulate adenylate cyclase correlate closely with the ability of (?)-isoproterenol to displace [³H]hydroxybenzylisoproterenol binding. Comparison of these findings with the binding characteristics of the β-antagonist [³H]dihydroalprenolol to rat adipocyte membranes, led to conclude that [³H]hydroxybenzylisoproterenol can be successfully used to label the β-adrenergic receptors of rat fat cells and suggests that it might be a better ligand than [³H]dihydroalprenolol in these cells.     

Acid dissociation of cyclohexaamylose and cycloheptaamylose

Acid dissociation constants of aqueous cyclohexaamylose (6-Cy) and cycloheptaamylose (7-Cy) have been determined at 10–47 and 25–55°C, respectively, by pH potentiometry. Standard enthalpies and entropies of dissociation derived from the temperature dependences of these pK_a's are ΔH⁰ = 8.4 ± 0.3 kcal mol^?1, $\text{Δ}\text{S}{}^0\text{= ?28. ± 1}\text{cal mol}{}^{\mathrm{?1}}{}^0\text{K}{}^{\mathrm{?1}}$ for 6-Cy and ΔH⁰ = 10.0 ± 0.1 kcal mol^?1, $\text{Δ}\text{S}{}^0\text{= ?22.4 ±0.3}\text{cal mol}{}^{\mathrm{?1}}{}^0\text{K}{}^{\mathrm{?1}}$ for 7-Cy. Intrinsic ¹³C nmr resonance displacements of anionic 6- and 7-Cy were measured at 30°C in 5% D₂O ($\text{v}\text{v}$). These results indicate that the dissociation of 6- and 7-Cy involves both C2 and C3 2⁰-hydroxyl groups. The thermodynamic and nmr parameters are discussed in terms of interglucosyl hydrogen bonding.     

Characterization of α2-adrenergic receptors in human platelets by binding of a radioactive ligand [3H]yohimbine

[³H]Yohimbine, a potent α₂-adrenergic antagonist, was used to label the α₂-adrenergic receptors in membranes isolated from human platelets. Binding of [³H]yohimbine to platelet membranes appears to have all the characteristics of binding to α₂-adrenergic receptors. Binding reached a steady state in 2–3 min at 37°C and was completely reversible upon the addition of excess phentolamine or yohimbine (both at 10^?5 M;$\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 2.37}\text{min}$). [³H]Yohimbine bound to a single class of noncooperative sites with a dissociation constant of 1.74 nM. At saturation, the total number of binding sites was calculated to be 191 fmol/mg protein. [³H]Yohimbine binding was stereo-specifically inhibited by epinephrine: the (?) isomer was 11-times more potent than the (+) isomer. Cathecholamine agonists competed for the occupancy of the [³H]yohimbine-binding sites with an order of potency: clonidine > (?)-epinephrine > (?)-norepinephrine >> (?)-isoproterenol. The potent α₂-adrenergic antagonist, phentolamine, competed for the sites whereas the β-antagonist, (±)-propanolol, was a very weak inhibitor. 0.1 mM GTP reduced the bindng affinity of the agonists, while producing no change in antagonist-binding affinity. Dopamine and serotonine competed only at very high concentrations. Similarly, muscarinic cholinergic ligands were also poor inhibitors of [³H]yohimbine binding. These results suggest tht [³H]yohimbine binding to human platelet membranes is specific, rapid, saturable, reversible and, therefore, can be successfully used to label α₂-adrenergic receptors.     

Studies on the mechanism of electron transport in the bc1-segment of the respiratory chain in yeast. II. The binding of antimycin to mitochondrial particles and the function of two different binding sites

1. In mitochondrial particles antimycin binds to two separate specific sites with dissociation constants $\text{K}{}_{\mathrm{<mtext>d</mtext><mtext>1</mtext>}}\text{≦ 4 · 10}{}^{\mathrm{?13}}\text{M}$ and $\text{K}{}_{\mathrm{<mtext>d</mtext><mtext>2</mtext>}}\text{= 3 · 10}{}^{\mathrm{?9}}\text{M}$, respectively.2. The concentrations of the two antimycin binding sites are about equal. The absolute concentration for each binding site is about 100 – 150 pmol per mg of mitochondrial protein.3. Antimycin bound to the stronger site mainly inhibits NADH- and succinate oxidase. Binding of antimycin to the weaker binding site inhibits the electron flux to exogenously added cytochrome c after blocking cytochrome oxidase by KCN.4. Under certain conditions cytochrome b and c₁ are dispensible components for antimycin-sensitive electron transport.5. A model of the respiratory chain in yeast is proposed which accounts for the results reported here and previously. (Lang, B., Burger, G. and Bandlow, W. (1974) Biochim. Biophys. Acta 368, 71–85).     

A stereochemical study on the metabolism of isobutyrate in Pseudomonas putida

Ammonium[2-³H,1-¹⁴C]isobutyrate was converted by Pseudomonas putida ATCC 21244 into S(+)-β-hydroxyisobutyric acid (β-HIBA) with loss of the α-tritium atom. The recovered isobutyrate had the same ${}^3\text{H}{}^{14}\text{C}$ as the starting material. Ammonium (2S)-[3-¹³C]isobutyrate was synthesized and converted by P. putida into β-HIBA. The ¹³C-nmr of the corresponding methyl ester benzoate showed ¹³C enrichment in the hydroxymethyl carbon atom. The results therefore indicate that isobutyrate metabolism in this organism proceeds via an unsaturated intermediate (probably methacrylyl-CoA) formed by dehydrogenation of the 2-pro-S-methyl group of the precursor (isobutyryl-CoA). Hydration of the intermediate proceeds with addition of a proton at C-2 from the same side as the hydrogen removed in the dehydrogenation.