Histamine,theophylline and tryptamine transport through lipid bilayer membranes

Diffusion of histamine, theophylline and tryptamine through planar lipid bilayer membranes was studied as a function of pH. Membranes were made of egg phosphatidylcholine plus cholesterol (1 : 1 mol ratio) in tetradecane. Tracer fluxes and electrical conductances were used to estimate the permeabilities to nonionic and ionic species. Only the nonionic forms crossed the membrane at a significant rate. The membrane permeabilities to the nonionic species were: histamine, $\text{3.5 · 10}{}^{\mathrm{?5}}\text{cm}\text{· s}{}^{\mathrm{?1}}$; theophylline, $\text{2.9 · 10}{}^{\mathrm{?4}}\text{cm}\text{· s}{}^{\mathrm{?1}}$; and tryptamine, $\text{1.8 · 10}{}^{\mathrm{?1}}\text{cm}\text{· s}{}^{\mathrm{?1}}$. Chemical reactions in the unstirred layers are important in the transport of tryptamine and theophylline, but not histamine. For example, as pH decreased from 10.0 to 7.5 the ratio of nonionic (B) to ionic (BH⁺) tryptamine decreased by 300-fold, but the total tryptamine permeability decreased only 3-fold. The relative insensitivity of the total tryptamine permeability to the ratio, [B]/[BH⁺], is due to the rapid interconversion of B and BH⁺ in the instirred layers. Our model describing diffusion and reaction in the unstirred layers can explain some ‘anomalous’ relationships between pH and weak acid/base transport through lipid bilayer and biological membranes.     

The bimolecular decay rates of the flavosemiquinones of riboflavin,FMN and FAD

The bimolecular decay rates (2k) of the flavosemiquinones ($\text{FH}{}^{\mathrm{·}}\text{F}{}^{\mathrm{?}}$) of riboflavin, FMN and FAD have been determined using pulse radiolysis. The rates (defined as $\text{d}\text{[}\text{FH}{}^{\mathrm{·}}\text{F}{}^{\mathrm{?}}\text{]}\text{d}\text{t}\text{= ?2}\text{k}\text{[}\text{FH}{}^{\mathrm{·}}\text{F}{}^{\mathrm{?}}\text{]}{}^2$) for the neutral flavosemiquinones at zero ionic strength and pH 5.9 are (in units of mol^?1·dm³·s^?1): (1.2 ± 0.1)·10⁹, (5.0 ± 0.2)·10⁸ and (1.4 ± 0.1)·10⁸; and for the anionic flavosemiquinones at pH 11.2 (5.4 ± 0.9)·10⁸, (4.5 ± 0.3)·10⁷ and (8.5 ± 1.3)·10⁶, respectively. The kinetic salt effect has been used to formulate rate equations for each flavin to adjust for ionic strength effects.     

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.     

Effect of angiotensin II on artificial lipid membranes

(5-Isoleucine)-angiotensin II applied to black lipid membranes produced current fluctuations varying between $\text{Δ>G = 5 · 10}{}^{\mathrm{?11}}\text{Ω}{}^{\mathrm{?}}$ and 3.5 · 10^?10 Ω^?1. These fluctuations depend on the voltage and the hydrostatic pressure. The membrane resistance is lowered by $\text{Δ>R = 6.1 · 10}{}^7\text{Ω · cm}{}^2$. With (5-isoleucine, 8-leucine)-angiotensin II the jumps are of a single amplitude ($\text{Δ>G = 2 · 10}{}^{\mathrm{?10}}\text{Ω}{}^{\mathrm{?1}}$). In both cases water and ions are transported across the membrane.     

Osmotic permeability of Novikoff hepatoma cells

The osmotic permeability coefficient (P_f) for water movement across Novikoff hepatoma cells was found to be 82 ± 3 (S.E.) · 10^?5 cm · s^?1 at 20°C. The corresponding diffusional permeability coefficient for ³HHO (P_d) was 97 ± 10 (S.E.) · 10^?5 cm · s^?1, therefore the ratio $\text{P}{}_{\mathrm{<mtext>f</mtext>}}\text{P}{}_{\mathrm{<mtext>d</mtext>}}$ is close to unity. The apparent activation energy for water filtration was 10.4 ± 0.4 (S.E.) kcal · mol^?1. This value is significantly greater than the activation energy for the self diffusion of water. The product of the hydraulic permeability coefficient and the viscosity coefficient for water was temperature-dependent. However, the product of the hydraulic permeability coefficient and the viscosity coefficient for membrane lipid did not vary with temperature. These data are interpreted as evidence for water movement across a lipid membrane barrier rather than through aqueous channels.     

Cyclic AMP transport in human erythrocyte ghosts

10^?5 M cyclic AMP has high permeability in human erythrocyte ghosts ($\text{p = 0.061 · 10}{}^{\mathrm{?6}}\text{cm}\text{·}\text{s}{}^{\mathrm{?1}}$). Saturation of influx and efflux occurs. $\text{K}{}_{\mathrm{<mtext>z</mtext><mtext>t</mtext>}}{}^{\mathrm{<mtext>oi</mtext>}}\text{= 4.43}\text{mM}$. $\text{V}{}_{\mathrm{zt}}{}^{\mathrm{oi}}\text{= 259.6 μ}\text{M · min}{}^{\mathrm{?1}}$. $\text{K}{}_{\mathrm{<mtext>z</mtext><mtext>t</mtext>}}{}^{\mathrm{<mtext>io</mtext>}}\text{= 0.475 μ}\text{M}$. $\text{V}{}_{\mathrm{<mtext>z</mtext><mtext>t</mtext>}}{}^{\mathrm{<mtext>io</mtext>}}\text{= 28.3 μ}\text{M · min}{}^{\mathrm{?1}}$ at 30°C. Equilibrium exchange entry of cyclic AMP has similar kinetics to zero trans influx, though the system does show counterflow. Cythochalasin B is an apparent competitive inhibitor of cyclic AMP exit. ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 3.9 · 10}{}^{\mathrm{?7}}\text{M}$).Control experiments indicated that cyclic AMP remains intact during incubation with red blood cell ghosts and is contained within the intravesicular space during the transport experiments.     

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.     

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.     

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.     

Local measurement of lateral motion in erythrocyte membranes by photobleaching technique

The lateral diffusion coefficients ($\text{D}$) of the molecular fluorescence probe 3,3′-dioctadecylindocarbocyanine iodide (DII) in the membrane of discoid erythrocyte ghosts has been measured with the photobleaching technique between 7°C and 40°C. A fluorescence microscope which allows bleaching experiments within small local fields (approx. 1 μm²) at high magnification (X1600) has been used for these measurements. The diffusion coefficient increases from $\text{D = 9 · 10}{}^{\mathrm{?10}}\text{cm}{}^2\text{/s}$ to $\text{D = 7.5 · 10}{}^{\mathrm{?9}}\text{cm}{}^2\text{/s}$ from 7 to 40°C. An increase in membrane fluidity between 12°C and 17°C indicates a conformational change of the lipid bilayer moiety in this temperature region. The diffusion coefficient measured in the regions between the spicules of echinocytes is appreciably smaller than in the untransformed discoid ghosts. In the myelin tubes originating from cells, the lateral diffusion is somewhat larger (about a factor of 2) than in the non-transformed ghosts. With the fluorescence probe technique the rate of growth of myelin tubes of 0.3 μm diameter has been estimated.     

Kinetics of phospholipid exchange between bilayer membranes

In an accompanying publication by Duckwitz-Peterlein, Eilenberger and Overath ((1977) Biochim. Biophys. Acta 469, 311–325) it is shown that the exchange of lipid molecules between negatively charged vesicles consisting of total phospholipid extracts from Escherichia coli occurs by the transfer of single lipid monomers or small micelles through the water. Here a kinetic interpretation is presented in terms of a rate constant, $\text{k}{}_{\mathrm{?}}$, for the escape of lipid molecules from the vesicle bilayer into the water. The evaluated rate constants are $\text{k}{}_{\mathrm{?}}{}^{\mathrm{<mtext>P</mtext>}}\text{= (0.86 ± 0.05) · 10}{}^{\mathrm{?5}}\text{s}{}^{\mathrm{?1}}$ and $\text{k}{}_{\mathrm{?}}{}^{\mathrm{<mtext>E</mtext>}}\text{= (1.09 ± 0.13) · 10}{}^{\mathrm{?6}}\text{s}{}^{\mathrm{?1}}$ for phospholipid molecules with $\text{trans-}\text{Δ}{}^9\text{-hexadecenoate}$ and $\text{trans-}\text{Δ}{}^9\text{-octadecenoate}$, respectively, as the predominant acyl chain component. The rate constants are discussed in terms of the acyl chain and polar head group composition of the lipids.     

Melittin-phospholipid interaction studied by employing the single tryptophan residue as an intrinsic fluorescent probe

The rotational correlation time of melittin, obtained from the nanosecond anisotropy of the emission from its single tryptophan residue, has been found to increase considerably in phosphate solution relative to that in aqueous solution, consistent with protein aggregation. The steady-state fluorescence spectra as well as the absorption spectra in phosphate solution exhibit a very good degree of similarity with those of the protein bound to egg phosphatidylcholine (PC) and distearoylphosphatidylcholine (DSPC) bilayer liposomes. The value of the second-order rate constant for dynamic quenching, $\text{k}{}_{\mathrm{<mtext>q</mtext>}}\text{= 1.4·10}{}^9\text{M}{}^{\mathrm{?1}}\text{·}\text{s}{}^{\mathrm{?1}}$, by acrylamide in 0.5 M phosphate solution is comparable to those for the protein-phospholipids complexes (1·10⁹ and 0.7·10⁹ M^?1·s^?1 for egg PC and DSPC, respectively). Similarities are also found in the nanosecond properties. There is a much stronger and quite similar dependence of the fluorescence spectra on time in the nanosecond range and of the fluorescence decay times on the emission wavelength in both cases as compared to the case in aqueous solution. These observations support the notion that melittin binds to the phospholipids in an aggregated form. The results suggest that the reduction in the $\text{k}{}_{\mathrm{<mtext>q</mtext>}}$ values of bound melittin relative to that in aqueous solution and the blue shift of the fluorescence spectrum (from 352 to 337 nm) are brought about by shielding of the tryptophan residue from the solvent through a combination of protein aggregation and enhancement of its α-helical content (suggested by published CD data). The magnitude of the $\text{k}{}_{\mathrm{<mtext>q</mtext>}}$ values for bound melittin, however, is still relatively high implying the occurrence of rather frequent encounters between the tryptophan residue and the hydrophilic acrylamide molecules. Thus, the residue is found not to penetrate deep into the phospholipid bilayer.     

Calcium ion-flux across phosphatidylcholine membranes mediated by ionophore A23187

The antibiotic A23187 carries Ca²⁺ across Müller-Rudin membranes made from $\text{1,2-}\text{dierucoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphocholine}$ and $\text{n-}\text{decane}$. The conductance of the membranes is not increased by the Ca²⁺-transport. The flux depends linearly on Ca²⁺ concentration and ionophore concentration (above pH 6). It increases with increasing pH, approximately by a factor of 4–5 between pH 6 and pH 8. Maximal Ca²⁺-fluxes of about $\text{10}{}^{\mathrm{?10}}\text{mol}\text{·}\text{cm}{}^{\mathrm{?2}}\text{·}\text{s}{}^{\mathrm{?1}}$ were found. A counter transport of H⁺ could not be detected.The complex formation between A23187 and Ca²⁺ in egg phosphatidylcholine vesicles was studied spectroscopically. The results are consistent with the formation of a 2 : 1 complex. Optical absorption measurements on single phosphatidylcholine membranes were used to calculate the concentration of membrane-bound ionophore A23187.     

Regulation of the Ca2+-pump by calmodulin in intact cells

ATP-enriched human red cells display high rates of Ca²⁺-dependent ATP hydrolysis (16 mmol·litre cells^?1·h^?1) with a high Ca²⁺ affinity ($\text{K}{}_{0.5}\text{～0.2 μ}\text{M}$). The finding suggests a mechanism for regulation of cell Ca²⁺ levels, involving highly-cooperative stimulation of active Ca²⁺ extrusion following binding of calmodulin to the $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$.     

New experimental approach to the estimation of rate of electron transfer from the primary to secondary acceptors in the photosynthetic electron transport chain of purple bacteria

A method for calculating the rate constant ($\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$) for the oxidation of the primary electron acceptor (A₁) by the secondary one (A₂) in the photosynthetic electron transport chain of purple bacteria is proposed.The method is based on the analysis of the dark recovery kinetics of reaction centre bacteriochlorophyll (P) following its oxidation by a short single laser pulse at a high oxidation-reduction potential of the medium. It is shown that in Ectothiorhodospira shaposhnikovii there is little difference in the value of $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ obtained by this method from that measured by the method of Parson ((1969) Biochim. Biophys. Acta 189, 384–396), namely: $\text{(4.5±1.4) · 10}{}^3\text{s}{}^{\mathrm{?1}}$ and $\text{(6.9±1.2) · 10}{}^3\text{s}{}^{\mathrm{?1}}$, respectively.The proposed method has also been used for the estimation of the $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ value in chromatophores of Rhodospirillum rubrum deprived of constitutive electron donors which are capable of reducing P⁺ at a rate exceeding this for the transfer of electron from A₁ to A₂. The method of Parson cannot be used in this case. The value of $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ has been found to be $\text{(2.7±0.8) · 10}{}^3\text{s}{}^{\mathrm{?1}}$.The activation energies for the A₁ to A₂ electron transfer have also been determined. They are 12.4 kcal/mol and 9.9 kcal/mol for E. shaposhnikovii and R. rubrum, respectively.     

The characterization of energized and partially de-energized (respiration-independent) β-galactoside transport into escherichia coli

Unidirectional fluxes of [¹⁴C]lactose by whole cells of Escherichia coli under highly energized and partially de-energized (in the presence of CN^?) conditions are analyzed kinetically.When the cells are energized, the value for $\text{V}$ influx is $\text{0.45 ± 0.01}\text{mM}$ internal concentration increment/s and $\text{K}{}_{\mathrm{t}}$ is $\text{0.26 ± 0.03}\text{mM}$. At an external concentration of 0.61 mM the steady-state internal concentration is 0.25 M, reached after about 1h. The maximum steady-state concentration ratio is $\text{2 · 10}{}^3$.The efflux process under these conditions is non-saturable, being linearly dependent upon internal concentration over the range 25–250 mM with a first-order rate constant of $\text{8.8 ± 0.2 · 10}{}^{\mathrm{?4}}\text{s}{}^{\mathrm{?1}}$.The transport in the presence of CN^? is active, with a maximum concentration ratio (internal concentration/external concentration) of 104, and the uptake is mimicked by anoxia (< 70 ppm O₂).The effects of CN^? are to lower the $\text{V}$ for influx and to change the efflux from a non-saturable to a saturable process with a value for $\text{K}{}_{\mathrm{t}}$ (60 mM) intermediate between that for energized efflux ($\text{> 250}\text{mM}$) and influxe (0.3–0.6 mM), the latter value not changing appreciably. Partial de-energization thus affects both the influx and efflux processes.     

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.     

Electron transfer from pyridinyl radicals, hydrated electrons, CO2.- and O2.- to bacterial cytochrome P450.

Several rate constants for one-electron reduction of cytochrome P450 are more rapid in the absence than in the presence of the specific substrate. The respective values for methyl viologen, nicotinamide adenine dinucleotide and the 1-methyl-4-(and -3-)carbamidopyridinium radicals are 2.6, 3.4, 6 and 35 × 10⁷ M^?1 s^?1 without camphor, and 0.15, 0.1, 1.8 and 110 × 10⁷ M^?1 s^?1 for the camphor complex. Hydrated electrons react with cytochrome P450 with a rate constant of 3.0 × 10¹⁰ M^?1 s^?1 whether camphor is bound or not, but little of the reduction takes place at the haem iron. No reduction of the haem iron by $\text{CO}{}_2{}^{\mathrm{?-}}$ or $\text{O}{}_2{}^{\mathrm{?-}}$ could be detected, whether camphor is bound or not.