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.     

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.     

Characterization and partial purification of an intestinal lipase.

The isoelectric points of the blood group $\text{A}{}^1\text{, A}{}^2$ and $\text{B}$ gene-associated glycosyltransferases in human ovarian cyst fluids were found by isoelectric focusing to be in the pH range 9.5–10. The $\text{A}{}^1$ and $\text{B}$ transferases in serum had isoelectric points similar to those of the enzymes in cyst fluids but $\text{A}{}^2$ transferases in serum had considerably lower isoelectric points, in the pH range 6–7. The difference in the pI values of the $\text{A}{}^1$ and $\text{A}{}^2$ transferases in the serum of a donor of the genotype $\text{A}{}^1\text{A}{}^2$ enabled the two enzymes to be preparatively separated by the isoelectric focusing technique. The dissimilarity in the pI values of the $\text{A}{}^2$ transferases in ovarian cyst fluids and serum samples indicates that the isoelectric point arises from a post-translational modification of the enzyme protein.     

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 reaction between the superoxide free radical and lipid hydroperoxide in sodium linoleate micelles

The oxidation of unsaturated fatty acid micelles by the superoxide free radical ($\text{O}{}^{\mathrm{?}}{}_2$), during γ irradiation in the presence of formate, is kinetically distinct from oxidation by hydroxyl free radicals (HO.). The evidence suggests that a direct reaction between ($\text{O}{}^{\mathrm{?}}{}_2$) and lipid hydroperoxide initiates a chain oxidation process in the micelles. While tetranitromethane, which reacts rapidly with ($\text{O}{}^{\mathrm{?}}{}_2$), protects the micelles from oxidation, active superoxide dismutase is no more effective than its apoprotein, due to lack of penetration of the micellar environment. We discuss these findings in the light of recent literature, and with reference to their possible significance for biological systems.     

Temperature dependence of N-phenyl-1-naphthylamine binding in egg lecithin vesicles

The temperature dependence of the binding of PhNapNH₂ ($\text{N-}\text{phenyl-1-naphthylamine}$) to vesicles of egg phosphatidylcholine has been determined. The Arrhenius plot of the association constant exhibits a discontinuity at 20.9 °C, some 30 °C above the broad phase transition region of the phospholipid. In the temperature range above 20 °C, $\text{ΔH}{}^0\text{= ?6100}\text{cal·mol}{}^{\mathrm{?1}}$ and $\text{ΔS}{}^0\text{= 9.7}\text{e. u.}$; in the temperature range below 20 °C, $\text{ΔH}{}^0\text{= 0}\text{cal · mol}{}^{\mathrm{?1}}$ and $\text{ΔS}{}^0\text{= 30.4}\text{e. u.}$ These values are consistent with the view that there are well ordered lipid-lipid bonds below 20 °C which are significantly less important above this temperature. The order in the temperature range of 5 to 20 °C, though significantly greater than that above 20 °C, is still significantly less than that in the crystalline state.     

Genetic changes in yeast cells cotransformed with mitochondrial DNA and plasmid YEp13 are not elicited by recombinant molecules made by covalent insertion of mitochondrial DNA into YEp13

$\text{Saccharomyces cerevisiae}$ strain DC5-E2 that lacks mtDNA ($\text{leu2 rho}{}^{\mathrm{o}}$) can be cotransformed with a mixture of mtDNA and the plasmid YEp13 (LEU2/2μ/pBR322) to produce Leu⁺ transormants which, on being mated to $\text{mit}{}^{\mathrm{?}}$ tester strains, generate respiratory competent diploids (such events are denoted marker rescue). In this work strain DC5-E2 was transformed with recombinant molecules consisting of a mtDNA segment including the $\text{oli2}$ gene inserted into YEp13. The Leu⁺ transformants made with the recombinant plasmids were unable to rescue $\text{mit}{}^{\mathrm{?}}$ testers carrying mutations in the $\text{oli2}$ region, in contrast to Leu⁺ cotransformants made with mixtures of YEp13 and $\text{oli2}$ mtDNA. We conclude that marker rescue events occur as a result of interactions between the mtDNA of the $\text{mit}{}^{\mathrm{?}}$ tester and the mtDNA sequences introduced by transformation. Such interactions cannot occur when the latter mtDNA is forced to replicate in covalent association with YEp13, probably in the nucleus.     

Enthalpy and volume changes accompanying electron transfer from P-870 to quinones in Rhodopseudomonas sphaeroides reaction centers

A capacitor microphone was used to measure the enthalpy and volume changes that accompany the electron transfer reactions, $\text{PQ}{}_{\mathrm{A}}\text{→}\text{hv}\text{P}{}^{\mathrm{+}}\text{Q}{}^{\mathrm{?}}{}_{\mathrm{A}}\text{and PQ}{}_{\mathrm{A}}\text{Q}{}_{\mathrm{B}}\text{→}\text{hv}\text{P}{}^{\mathrm{+}}\text{Q}{}_{\mathrm{A}}\text{Q}{}^{\mathrm{?}}{}_{\mathrm{B}}$, following flash excitation of photosynthetic reaction centers isolated from Rhodopseudomonas sphaeroides. P is a bacteriochlorophyll dimer (P-870), and Q_A and Q_B are ubiquinones. In reaction centers containing only Q_A, the enthalpy of P⁺Q^?_A is very close to that of the PQ_A ground state (ΔH_r = 0.05 ± 0.03 eV). The free energy of about 0.65 eV that is captured in the photochemical reaction evidently takes the form of a substantial entropy decrease. In contrast, the formation of P⁺Q_AQ^?_B in reaction centers containing both quinones has a ΔH_r of 0.32 ± 0.02 eV. The entropy change must be near zero in this case. In the presence of o-phenanthroline, which blocks electron transfer between Q^?_A and Q_B, ΔH_r for forming P⁺Q^?_AQ_B is 0.13 ± 0.03 eV. The influence of flash-induced proton uptake on the results was investigated, and the ΔH_r values given above were measured under conditions that minimized this influence. Although the reductions of Q_A and Q_B involve very different changes in enthalpy and entropy, both reactions are accompanied by a similar volume decrease of about 20 ml/mol. The contraction probably reflects electrostriction caused by the charges on P⁺ and Q^?_A or Q^?_B.     

Characterization of the sub-transition of hydrated dipalmitoylphosphatidylcholine bilayers: X-ray diffraction study

The structural changes accompanying the recently described sub-transition of hydrated dipalmitoylphosphatidylcholine (Chen, S.C., Sturtevant, J.M. and Gaffney, B.J. (1980) Proc. Natl. Acad. Sci. USA 77, 5060–5063) have been defined using X-ray diffraction methods. Following prolonged storage at ?4°C the usual L_β′ gel form of hydrated dipalmitoylphosphatidylcholine (DPPC) is converted into a more ordered stable ‘crystal’ form. The bilayer periodicity is 59.1 Å and the most striking feature is the presence of a number of X-ray reflections in the wide angle region. The most prominent of these are a sharp reflection at $\text{1}\text{4.4}\text{A}\text{?}{}^{\mathrm{?1}}$ and a broader reflection at $\text{1}\text{3.9}\text{A}\text{?}{}^{\mathrm{?1}}$. This diffraction pattern is indicative of more ordered molecular and hydrocarbon chain packing modes in this low temperature ‘crystal’ bilayer form. At the sub-transition (T_rmsub = 15–20°C) an increase in the bilayer periodicity occurs ($\text{d=63.6}\text{A}\text{?}$) and a strong reflection at approx. $\text{1}\text{4.2}\text{A}\text{?}{}^{\mathrm{?1}}$ with a shoulder at approx. $\text{1}\text{4.1}\text{A}\text{?}{}^{\mathrm{?1}}$ is observed. This diffraction pattern is identical to that of the bilayer gel (L_β′) form of hydrated DPPC. Thus, the sub-transition corresponds to a bilayer ‘crystal’ → bilayer L_β′ gel structural rearrangement accompanied by a decrease in the lateral hydrocarbon chain interactions. Differential scanning calorimetry and X-ray diffraction show that on further heating the usual structural changes L_β′ → P_β′ and P_β′ → L_α occur at the pre- and main transitions, at approx. 35°C and 41°C, respectively.     

Reactivity of chemically generated superoxide radical anion with peroxides as determined by competition kinetics

A steady-state competition system has been developed to investigate the reactions of the superoxide radical anion ($\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$) with various peroxides, including the so-called Haber-Weiss reaction. Potassium superoxide dissolved in an oxygen-free solution of DMSO containing 18-dicyclohexyl-6-crown, is the source of $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$. High pressure liquid chromatography is used as an assay system for $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ reactivity, to detect and quantitate the yield of anthracene, formed as a major product in the reaction between $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ and 9,10-dihydroanthrancene. Decrease in anthracene yields, in the presence of peroxide, may be used to indicate a possible competing reaction between $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ and added peroxide. Complications involving peroxide-stimulated formation of anthraquinone derivatives are discussed. No evidence for a competing reaction between $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ and peroxide can be detected up to a 10-fold excess of peroxide over 9,10-dihydroanthracene.     

Energetics of coupled Na+ and Cl− entry into epithelial cells of bullfrog small intestine

Na⁺, K⁺ and Cl^? concentrations ($\text{c}{}_{\mathrm{j}}{}^{\mathrm{<mtext>i</mtext>}}$) and activities ($\text{a}{}_{\mathrm{j}}{}^{\mathrm{<mtext>i</mtext>}}$), and mucosal membrane potentials ($\text{E}{}_{\mathrm{<mtext>m</mtext>}}$) were measured in epithelial cells of isolated bullfrog (Rana catesbeiana) small intestine. Segments of intestine were stripped of their external muscle layers, and bathed (at 25°C and pH 7.2) in oxygenated Ringer solutions containing 105 mM Na⁺ and Cl^? and 5.4 mM K⁺. Na⁺ and K⁺ concentrations were determined by atomic absorption spectrometry and Cl^? concentrations by conductometric titration following extraction of the dried tissue with 0.1 M HNO₃. ¹⁴C-labelled inulin was used to determine extracellular volume. $\text{E}{}_{\mathrm{<mtext>m</mtext>}}$ was measured with conventional open tip microelectrodes, $\text{a}{}_{\mathrm{<mtext>Cl</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ with solid-state Cl^?-selective silver microelectrodes and $\text{a}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ and $\text{a}{}_{\mathrm{<mtext>K</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ with Na⁺- and K⁺-selective liquid ion-exchanger microelectrodes. The average $\text{E}{}_{\mathrm{<mtext>m</mtext>}}$ recorded was ?34 mV. $\text{c}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$, $\text{c}{}_{\mathrm{<mtext>K</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ and $\text{c}{}_{\mathrm{<mtext>Cl</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ were 51, 105 and 52 mM. The corresponding values for $\text{a}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$, $\text{a}{}_{\mathrm{<mtext>K</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ and $\text{a}{}_{\mathrm{<mtext>Cl</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ were 18, 80 and 33 mM. These results suggest that a large fraction of the cytoplasmic Na⁺ is ‘bound’ or sequestered in an osmotically inactive form, that all, or virtually all the cytoplasmic K⁺ behaves as if in free solution, and that there is probably some binding of cytoplasmic Cl^?. $\text{a}{}_{\mathrm{<mtext>Cl</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ significantly exceeds the level corresponding to electrochemical equilibrium across the mucosal and baso-lateral cell membranes. Earlier studies showed that coupled mucosal entry of Na⁺ and Cl^? is implicated in intracellular Cl^? accumulation in this tissue. This study permitted estimation of the steady-state transapical Na⁺ and Cl^? electrochemical potential differences (Δμ&#x0304;Na and Δμ&#x0304;Cl). Δμ&#x0304;Na (?7000 J · mol^?1; cell minus mucosal medium) was energetically more than sufficient to account for Δμ&#x0304;Cl (1000–2000 J · mol^?1).     

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.     

A proposed mechanism for light emission by bacterial luciferase involving dissociative electron transfer

A new mechanism that involves dissociative electron transfer in the energy transducing step is set forward for bacterial luciferase catalyzed light emission. The proposal involves (1) dissociation of the 4a-hydroperoxyflavin to a flavin radical and ${}^{\mathrm{?}}\text{O}{}_2{}^{\mathrm{?}}$, accounting for 570 and 620nm absorption, (2) ${}^{\mathrm{?}}\text{O}{}_2{}^{\mathrm{?}}$ addition to the aldehyde carbonyl to form a peroxyl radical, (3) abstraction of H from an enzyme thiol group to form RCH(OOH)OH, (4) thiyl radical abstraction of the H on C in RCH(OOH)OH, a step which can show a $\text{k}{}_{\mathrm{H}}\text{k}{}_{\mathrm{D}}$ of ca. 4, and (5) dissociative electron- transfer, a highly exothermic step that leads to a protonated flavin excited state, a carboxylic acid and water.     

Phosphorylation in isolated chloroplasts coupled to dichlorophenyldimethylurea-insensitive silicomolybdate reduction

1. The electron transport in isolated chloroplasts with silicomolybdate as electron acceptor has been reinvestigated. The silicomolybdate reduction has been directly measured as ΔA₇₅₀ or indirectly as O₂ evolution (in the presence or absence of ferricyanide).2. Silicomolybdate-dependent O₂ evolution is inhibited to a similar extent by 3-(3,4-dichlorophenyl) 1, 1-dimethylurea (DCMU) or dibromothymoquinone (DBMIB), indicating the existence of two different sites of silicomolybdate reduction: one before the DCMU block (i.e. at Photosystem II) and one after the DBMIB block (i.e. at Photosystem I).3. Silicomolybdate-dependent O₂ evolution is coupled to ATP synthesis with an $\text{ATP}\text{2}\text{e}{}^{\mathrm{?}}$ ratio of 1.0 to 1.1. The presence of ferricyanide inhibits this ATP synthesis ($\text{ATP}\text{2}\text{e}{}^{\mathrm{?}}$ ratio then is about 0.3).4. Silicomolybdate-dependent O₂ evolution is also coupled to ATP-synthesis in the presence of DCMU with an $\text{ATP}\text{2}\text{e}{}^{\mathrm{?}}$ ratio of 0.6–0.8 characteristic of Site II; in this case the electron transport itself is not affected by uncouplers or energy-transfer inbihitors.5. The data are interpreted as a further demonstration that the water-splitting reaction is responsible for the conservation of energy at Photosystem II.     

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.     

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

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

Damping of oscillations in the semiquinone absorption in reaction centers after successive flashes. Determination of the equilibrium between QA−QB and QAQB−

A quantitative model for the damping of oscillations of the semiquinone absorption after successive light flashes is presented. It is based on the equilibrium between the states Q_A^?Q_B and Q_AQ_B^?. A fit of the model to the experimental results obtained for reaction centers from Rhodopseudomonas sphaeroides gave a value of $\text{α =}\text{[}\text{Q}{}_{\mathrm{<mtext>A</mtext>}}{}^{\mathrm{?}}\text{Q}{}_{\mathrm{<mtext>B</mtext>}}\text{]}\text{([}\text{Q}{}_{\mathrm{<mtext>A</mtext>}}{}^{\mathrm{?}}\text{Q}{}_{\mathrm{<mtext>B</mtext>}}\text{] + [}\text{Q}{}_{\mathrm{<mtext>A</mtext>}}\text{Q}{}_{\mathrm{<mtext>B</mtext>}}{}^{\mathrm{?}}\text{])}\text{= 0.065 ± 0.005}$ (T = 21°C, pH 8).     

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.     

Membrane lipid dynamics and enzymic activity in bovine adrenal cortex microsomes

The lipid dynamics of the adrenocortical microsomal membranes was studied by monitoring the fluorescence anisotropy and excited state lifetime of a set of anthroyloxy fatty acid probes (2-, 7-, 9- and 12-(9-anthroyloxy)-stearic acid (AP) and 16-(9-anthroyloxy)palmitic acid (AS). It was found that a decreasing polarity gradient from the aqueous membrane interface to the membrane interior, was present. This gradient was not modified by the proteins, as evidenced by comparison of complete membranes and derived liposomes, suggesting that the anthroyloxy probes were not in close contact with the proteins. An important change of the value of the mean rotational relaxation time as a function of the position of the anthroyl ring along the acyl chain was evidenced. In the complete membranes, a relatively more fluid medium was evidenced in the C₁₆ as compared to the C₂ region, while the rotational motion appeared to be the most hindered at the C₇–C₉ level. In the derived liposomes, a similar trend was observed but the mobility was higher at all levels. The decrease of the mean rotational relaxation time was more important for 12-AS and 16-AP. Temperature dependence of the mean rotational relaxation time of 2-AS, 12-AS and 16-AP in the complete membranes revealed the existence of a lipid reorganization occurring around 27°C and concerning mainly the C₁₆ region. The extent to which the acyl chain reacted to this perturbation at the C₁₂ level depended on pH. The presence of proteins increased the apparent magnitude of this reorganization and also modified the critical temperature from approx. 23°C in the derived liposomes to approx. 27°C in the complete membranes. Thermal dependence of the maximum velocity of the $\text{3-}\text{oxosteroid}\text{Δ}{}^5\text{?Δ}{}^4\text{-}\text{isomerase}$, the second enzyme in the enzymatic sequence, responsible for the biosynthesis of the $\text{3-}\text{oxo}\text{-Δ}{}^4\text{-}\text{steroids}$ in the adrenal cortex microsomes, was studied. The activation energy of the catalyzed reaction was found to be low and constant ($\text{2–5}\text{kcal}\text{·}\text{mol}{}^{\mathrm{?1}}$) in the temperature range 16–40°C at pH 7.5, 8.5 and 9, corresponding to the minimum, intermediate and maximum rate, respectively. A drastic increase of the activation energy ($\text{20}\text{kcal}\text{·}\text{mol}{}^{\mathrm{?1}}$) was observed at temperature below 16°C at pH 7.5. A correlated change of the $\text{p}\text{K}{}_{\mathrm{<mtext>ES</mtext>}}{}^{\mathrm{<mtext>app</mtext>}}$ as function of temperature was detected; at 36°C $\text{p}\text{K}{}_{\mathrm{<mtext>ES</mtext>}}{}^{\mathrm{<mtext>app</mtext>}}\text{= 8.3}$ while at 13°C the value shifted to 8.7. The pH range of the group ionization was narrower at 13°C. In contrast with the behaviour of the $\text{3β-}\text{hydroxy}\text{-Δ}{}^5\text{-}\text{steroid dehydrogenase}$, the $\text{3-}\text{oxosteroid}\text{Δ}{}^5\text{?Δ}{}^4\text{-}\text{isomerase}$ was apparently unaffected by the lipid reorganization at 27°C. It is suggested that this enzyme possesses a different and more fluid lipid environment than the bulk lipids.     

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.