A comparison between inosine- and guanosine-containing anticodons in ribosome-free codon-anticodon binding

Binding of the ribooligomers, AUC, AUU, AUA, AUCA, AUUA, and AUAA to the isoleucine-accepting tRNAs, tRNA${}_{\mathrm{<mtext>T.\; utilis</mtext>}}{}^{\mathrm{Ile}}$ (anticodon, -IAU-) and tRNA${}_{\mathrm{<mtext>E.\; coli</mtext>}}{}^{\mathrm{Ile}}$ (anticodon, -GAU-) was measured by equilibrium dialysis. With the aid of Scatchard plots, AUCA was shown to bind to one site per tRNA molecule, presumably the anticodon. AUA and AUAA did not measurably attach to either anticodon in the ribosome-free system. All other oligomers were bound to tRNA${}_{\mathrm{<mtext>E.\; coli</mtext>}}{}^{\mathrm{Ile}}$ about 5 to 13 times stronger than to tRNA${}_{\mathrm{<mtext>T.\; utilis</mtext>}}{}^{\mathrm{Ile}}$.     

The effect of formate on cytochrome aa3 and on electron transport in the intact respiratory chain

1. Formate inhibits cytochrome $\text{c}$ oxidase activity both in intact mitochondria and submitochondrial particles, and in isolated cytochrome $\text{aa}{}_3$. The inhibition increases with decreasing pH, indicating that HCOOH may be the inhibitory species.2. Formate induces a blue shift in the absorption spectrum of oxidized cytochrome $\text{aa}{}_3\text{(a}{}^{\mathrm{3+}}\text{a}{}_3{}^{\mathrm{3+}}$) and in the half-reduced species ($\text{a}{}^{\mathrm{2+}}\text{a}{}_3{}^{\mathrm{3+}}$). Comparison with cyanide-induced spectral shifts, towards the red, indicates that formate and cyanide have opposite effects on the $\text{aa}{}_3$ spectrum, both in the fully oxidized and the half-reduced states. The formate spectra provide a new method of obtaining the difference spectrum of $\text{a}{}_3{}^{\mathrm{2+}}$ minus $\text{a}{}_3{}^{\mathrm{3+}}$, free of the difficulties with cyanide (which induces marked high → low spin spectral shifts in cytochrome $\text{a}{}_3{}^{\mathrm{3+}}$) and azide (which induces peak shifts of cytochrome $\text{a}{}^{\mathrm{2+}}$ towards the blue in both α- and Soret regions).3. The rate of formate dissociation from cytochrome $\text{a}{}^{\mathrm{2+}}\text{a}{}_3{}^{\mathrm{3+}}\text{-}\text{HCOOH}$ is faster than its rate of dissociation from $\text{a}{}^{\mathrm{3+}}\text{a}{}_3{}^{\mathrm{3+}}\text{-}\text{HCOOH}$, especially in the presence of cytochrome $\text{c}$. The $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ for formate inhibition of respiration is a function of the reduction state of the system, varying from 30 mM (100% reduction) to 1 mM (100% oxidation) at pH 7.4, 30 °C.4. Succinate-cytochrome $\text{c}$ reductase activity is also inhibited by formate, in a reaction competitive with succinate and dependent on [formate]².5. Formate inhibition of ascorbate plus $\text{N,N,N′,N′-}\text{tetramethyl}\text{-p-}\text{phenyl-enediamine}$ oxidation by intact rat liver mitochondria is partially released by uncoupler addition. Formate is permeable through the inner mitochondrial membrane and no differences in ‘on’ or ‘off’ inhibition rates were observed when intact mitochondria were compared with submitochondrial particles.6. NADH-cytochrome $\text{c}$ reductase activity is unaffected by formate in submitochondrial particles, but mitochondrial oxidation of glutamate plus malate is subject both to terminal inhibition at the cytochrome $\text{aa}{}_3$ level and to a slow extra inhibition by formate following uncoupler addition, indicating a third site of formate action in the intact mitochondrion.     

The permeability of the lysosomal membrane to small ions

The permeability of the lysosomal membrane to small anions and cations was studied at 37°C and pH 7.0 in a lysosomal-mitochondrial fraction isolated from the liver of untreated rats. The extent of osmotic lysis following ion influx was used as a measure of ion permeancy. In order to preserve electroneutrality, anion influx was coupled to an influx of K⁺ in the presence of valinomycin, and cation influx was coupled to an efflux of H⁺ using the protonophore $\text{3-tert-}\text{butyl-5,2′-dichloro-4′-nitrosalicilylanilide}$. Lysosomal lysis was monitored by observing the loss of latency of two lysosomal hydrolases.The order of permeability of the lysosomal membrane to anions was found to be $\text{SCN}{}^{\mathrm{?}}\text{> I}{}^{\mathrm{?}}\text{> CH}{}_3\text{COO}{}^{\mathrm{?}}\text{> Cl}{}^{\mathrm{?}}\text{≈ HCO}{}^{\mathrm{?}}{}_3\text{≈ P}{}_{\mathrm{i}}\text{> SO}{}_4{}^{\mathrm{2?}}$ and that to cations $\text{Cs}{}^{\mathrm{+}}\text{> K}{}^{\mathrm{+}}\text{> Na}{}^{\mathrm{+}}\text{> H}{}^{\mathrm{+}}$. These orders are largely in agreement with the lyotropic series of anions and cations.The implications of these findings for the mechanism by means of which a low intralysosomal pH is produced and maintained are discussed.     

Bicarbonate ion-ATPase in rat liver cell fractions

The distribution of $\text{HCO}{}_3{}^{\mathrm{?}}\text{-}\text{ATPase}$ activity was studied in cell fractions prepared from homogenates of rat liver. The level of mitochondrial contamination in the microsomal fraction depended on the fractionation procedure and on the method of homogenization. With proper care, microsomes with undetectable mitochondrial contamination could be prepared. These microsomes had no detectable $\text{HCO}{}_3{}^{\mathrm{?}}\text{-ATPase}$ activity. Approximately 85 % of the total $\text{HCO}{}_3{}^{\mathrm{?}}\text{-ATPase}$ activity of the post 6000 x g · min supernatant was recovered in the mitochondrialfraction. The properties of this mitochondrial $\text{HCO}{}_3{}^{\mathrm{?}}\text{-}\text{ATPase}$ were not distinguishable from those of the various microsomal $\text{HCO}{}_3{}^{\mathrm{?}}\text{-}\text{ATPase}$ previously described by other investigators.     

Inhibition of anion transport in the red blood cell by anionic amphiphilic compounds I. Determination of the flufenamate-binding site by proteolytic dissection of the band 3 protein

Flufenamate, a non-steroidal anti-inflammatory drug, is a powerful inhibitor of anion transport in the human erythrocyte ($\text{I}{}_{50}\text{= 6·10}{}^{\mathrm{?7}}\text{M}$). The concentration dependence of the binding to ghosts reveals two saturable components. [¹⁴C]Flufenamate binds with high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}_1\text{= 1.2·10}{}^{\mathrm{?7}}\text{M}$) to 8.5·10⁵ sites per cell (the same value as the number of band 3 protein per cell); it also binds, with lower affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}_2\text{= 10}{}^{\mathrm{?4}}\text{M}$) to a second set of sites (4.6·10⁷ per cell). Pretreatment of cells with 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS), a specific inhibitor of anion transport, prevents [¹⁴C]flufenamate binding only to high affinity sites. These results suggest that high affinity sites are located on the band 3 protein involved in anion transport. Extracellular chymotrypsin and pronase at low concentration cleave the 95 kDa band 3 into 60 kDa and 35 kDa fragments without affecting either anion transport or [¹⁴C]flufenamate binding. Splitting by trypsin at the inner membrane surface of the 60 kDa chymotryptic fragment into 17 kDa transmembrane fragment and 40 kDa water-soluble fragment does not affect [¹⁴C]flufenamate binding. In contrast degradation at the outer membrane surface of the 35 kDa fragment by high concentration of pronase or papain decreases both anion transport capacity and number of high affinity binding sites for [¹⁴C]flufenamate. Thus it appears that 35 kDa peptide is necessary for both anion transport and binding of the inhibitors and that the binding site is located in the membrane-associated domain of the band 3 protein.     

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.     

Anomalies in 5'-end [32P] labelling of transfer RNA and partial sequence of a tRNAGlu from Scenedesmus obliquus

A chloroplast tRNA_m^Met species from $\text{Scenedesmus}\text{obliquus}$ is very poorly 5′-end [${}^{32}\text{P}$] labelled using [γ-³²P]ATP and T4 polynucleotide kinase. In sequencing the tRNA using standard 5′-labelled methods a very minor contaminating tRNA is preferentially labelled. The partial tRNA sequence determined by this method has an anticodon (CUC) for tRNA^Glu.     

A new assay for ATP sulfurylase based on differential solubility of the sodium salts of adenosine 5′-phosphosulfate and sulfate

A new assay for ATP sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) has been devised, the key feature of which is the quantitative precipitation of the unreacted substrate as Na₂SO₄, in ethanol:water (5:1), leaving the product adenosine 5′-phosphosulfate (APS) in solution with an 80% yield. This separation procedure, coupled with the use of $\text{[}{}^{35}\text{S]SO}{}_4{}^{\mathrm{2?}}$, makes it possible to assay the synthesis of picomole amounts of [³⁵S]APS and thereby determine accurately the initial velocity of the forward reaction catalyzed by ATP sulfurylase. Extracts from tobacco cells synthesized [³⁵S]APS and a negligible amount of one unknown sulfur compond, but they did not synthesize 3′-phosphoadenosine 5′-[³⁵S]phosphosulfate (PAPS) under the assay conditions adopted, so that the ³⁵S remaining in solution after ethanol precipitation of $\text{[}{}^{35}\text{S]SO}{}_4{}^{\mathrm{2?}}$ is a valid assay of the ATP sulfurylase reaction in these extracts. This assay is simple, rapid, and suitable for assaying multiple samples. Extracts of four additional species of plants were incubated under the same conditions, and each catalyzed the synthesis of [³⁵S]APS according to this assay. The assay is suitable for use with the enzyme from any organism, provided that the product APS is not acted upon by another enzyme.     

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.     

Kinetics of bidirectional active sodium fluxes in the toad bladder

The kinetics of isotopic Na⁺ flows was studied in urinary bladders of toads from the Dominican Republic. Initial studies of the potential dependence of passive serosal to mucosal ²²Na⁺ efflux demonstrated the absence of isotope interaction and/or other coupling with passive Na⁺ flow. The electrical current $\text{I}$ and mucosal to serosal ²²Na⁺ influx were then measured with transmembrane potential clamped at $\text{Δψ = 0, 25, 50, 75 or 100}\text{mV}$. Subsequent elimination of active Na⁺ transport mucosal amiloride permitted calculation of the rates of active Na⁺ transport $\text{J}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>a</mtext>}}$ and active and passive influx and $\text{J}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>a</mtext>}}$ and $\text{J}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>p</mtext>}}$. The results indicate that for Dominican toad bladders mounted in chambers only Na⁺ contributes significantly to transepithelial active ion transport; hence $\text{J}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>a</mtext>}}\text{= J}{}^{\mathrm{<mtext>a</mtext>}}$. $\text{J}{}^{\mathrm{<mtext>a</mtext>}}$ was abolished at $\text{Δψ = E = 96.3 ± 1.9}\text{(S.E.) mV}$. As Δψ approached $\text{E}$, active efflux $\text{J}{}^{\mathrm{<mtext>a</mtext>}}$ became demonstrable. At $\text{Δ = 100}\text{mV}\text{,}\text{J}{}^{\mathrm{<mtext>a</mtext>}}$ exceeded $\text{J}{}^{\mathrm{<mtext>a</mtext>}}$, so that $\text{J}{}^{\mathrm{<mtext>a</mtext>}}$ was negative. Experimental values of $\text{J}{}^{\mathrm{<mtext>a</mtext>}}$ agreed well with theoretical values predicted by a thermodynamic formulation: $\text{J}{}_{\mathrm{<mtext>exp</mtext>}}{}^{\mathrm{<mtext>a</mtext>}}\text{= 0.985}\text{J}{}_{\mathrm{<mtext>theor</mtext>}}{}^{\mathrm{<mtext>a</mtext>}}\text{(r = 0.993)}$. The dependence of $\text{J}{}^{\mathrm{<mtext>a</mtext>}}$ on Δψ is curvilinear.     

Induction of a relatively fast transbilayer movement of phosphatidylcholine in vesicles. A 13C NMR study

[$\text{N-}{}^{13}\text{CH}{}_3$] Phosphatidylcholines are introduced into the outer monolayer of phosphatidylcholine vesicles with the phosphatidylcholine exchange protein from bovine liver. The transbilayer distribution of the [$\text{N-}{}^{13}\text{CH}{}_3$] phosphatidylcholine is measured with ¹³C NMR. The transbilayer movements of $\text{[N-}{}^{13}\text{CH}{}_3\text{]-}\text{dioleoyl}$ phosphatidylcholine and [$\text{N-}{}^{13}\text{CH}{}_3$] dimyristoyl phosphatidylcholine at 30°C in vesicles composed of these phosphatidylcholines are extremely slow processes with estimated half-times of days. [$\text{N-}{}^{13}\text{CH}{}_3$] Dioleoyl phosphatidylcholine introduced into dimyristoyl phosphatidylcholine vesicles migrates from the outer to the inner monolayer with a half-time of less than 12 h. The data suggest that differential changes in the lateral packing of the two monolayers might be a driving force for transbilayer transport of phospholipids.     

In vitro reaction of radioactive 7beta, 8alpha, --dihydroxy--9alpha, 10alpha-epoxy--7,8,9,10--tetrahydrobenzo (A) pyrene and 7beta,8alpha--dihydroxy--9beta, 10beta--epoxy--7,8,9,10--tetrahydrobenzo (A) pyrene with DNA.

The $\text{in vitro}$ reaction of bacteriophage T7-DNA with the radioactive diastereomeric benzo(a)pyrene-diol-epoxides, (±) [${}^3\text{H}{}_9$, ${}^3\text{H}{}_{10}$]-7β,8α-dihydroxy-9α,10β-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, and (±) [${}^3\text{H}{}_9$, ${}^3\text{H}{}_{10}$]-7β,8α-dihydroxy-9β,19β-epoxy-7,8,9,10-tetrahydrobenzo(1)pyrene, was investigated. Chromatographic analysis of digests of the DNA allowed the distinction of characteristic deoxynucleoside adduct peaks for the two benzo(a)pyrene-diol-epoxides. Our results, together with data from the literature, allow the identification of these adducts as mostly N₂-(10-7β,8α,9α-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyreney1)deoxyguanosine and N₂-(10-7β,8α,9β-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyreney1)deoxyguanosine, respectively. DNA-benzo(a)pyrene adducts with the same chromatographic properties were formed in mouse embryo fibroblasts upon treatment with benzo(a)pyrene.     

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.     

Studies of the nucleotide-binding sites on the mitochondrial F1-ATPase through the use of a photoactivable derivative of adenylyl imidodiphosphate

(1) $\text{N-4-}\text{Azido}\text{-2-}\text{nitrophenyl}\text{-γ-[}{}^3\text{H]aminobutyryl-Ado}\text{PP[}\text{NH}\text{]P(}\text{NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P)}$ a photoactivable derivative of 5-adenylyl imidodiphosphate (AdoPP[NH]P), was synthesized. (2) Binding of ³H]NAP₄-AdoPP[NH]P to soluble ATPase from beef heart mitochrondria (F₁) was studied in the absence of photoirradiation, and compared to that of $\text{[}{}^3\text{H]Ado}\text{PP[}\text{NH}\text{]P}$. The photoactivable derivative of AdoPP[NH]P was found to bind to F₁ with high affinity, like AdoPP[NH]P. Once $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ had bound to F₁ in the dark, it could be released by AdoPP[NH]P, ADP and ATP, but not at all by NAP₄ or AMP. Furthermore, preincubation of F₁ with unlabeled AdoPP[NH]P, ADP, or ATP prevented the covalent labeling of the enzyme by $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ upon photoirradiation. (3) Photoirradiation of F₁ by $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ resulted in covalent photolabeling and concomitant inactivation of the enzyme. Full inactivation corresponded to the binding of about 2 mol $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}\text{mol F}{}_1$. Photolabeling by NAP₄-AdoPP[NH]P was much more efficient in the presence than in the absence of MgCl₂. (4) Bound $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ was localized on the α- and β-subunits of F₁. At low concentrations (less than 10 μM), bound $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ was predominantly localized on the α-subunit; at concentrations equal to, or greater than 75 μM, both α- and β-subunits were equally labeled. (5) The extent of inactivation was independent of the nature of the photolabeled subunit (α or β), suggesting that each of the two subunits, α and β, is required for the activity of F₁. (6) The covalently photolabeled F₁ was able to form a complex with aurovertin, as does native F₁. The ADP-induced fluorescence enhancement was more severely inhibited than the fluorescence quenching caused by ATP. The percentage of inactivation of F₁ was virtually the same as the percentage of inhibition of the ATP-induced fluorescence quenching, suggesting that fluorescence quenching is related to the binding of ATP to the catalytic site of F₁.     

Interaction of lymphocytes and macrophage cell line cells (M1 cells). I. Functional maturation and appearance of Fc receptors im M1 cells

M₁ cells, which are cell line cells established from myeloid leukemia cells of the SL strain mouse, can differentiate from blast cells ($\text{M}{}_1{}^{\mathrm{?}}$) to mature macrophages ($\text{M}{}_1{}^{\mathrm{+}}$) within 48 hr, when they are cultured with conditioned medium (CM) obtained from murine embryonic fibroblasts. While $\text{M}{}_1{}^{\mathrm{?}}$ cells have no phagocytic activity nor Fc receptor (FcR), $\text{M}{}_1{}^{\mathrm{+}}$ cells possess both characteristics. The appearance of FcR is temperature-dependent and inhibited by a metabolic inhibitor, cycloheximide. FcR on $\text{M}{}_1{}^{\mathrm{+}}$ cells is resistant to trypsin and pronase. $\text{M}{}_1{}^{\mathrm{+}}$ cells improve the viability of macrophage-depleted SL splenic lymphocytes and restore the in vitro secondary plaque forming cell response of macrophage-depleted spleen cells to particulate and soluble antigens. $\text{M}{}_1{}^{\mathrm{?}}$ cells lack this macrophage-substituting capacity. Mm₁ cells, mutant cells from M₁ cells, having FcR and higher phagocytic activity than $\text{M}{}_1{}^{\mathrm{+}}$ cells, are also devoid of this capacity.     

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.     

Characterization of the (Na+ + K+)-ATPase in a membranous preparation from the optic ganglion of the squid (Loligo pealei)

(1) A membrane fraction enriched in $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ (EC 3.6.1.3) was obtained from optic ganglia of the squid (Loligo pealei) by density gradient fractionation of membranes followed by treatment with either SDS or Brij-58. The resulting membrane had an $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ specific activity of approx. 2 units/mg and was $\text{>95\%}$ ouabain-sensitive. (2) The $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ had a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP of $\text{0.42 ± 0.04}\text{mM}$ and a pH optimum of 7.0. It was inhibited by ouabain with a $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ of $\text{0.32 ± 0.04 μ}\text{M}$. (3) Optimum monovalent cation concentrations were: 240 mM NaCl, 60 mM KCl, tested with $\text{NaCl + KCl}\text{= 300}\text{mM}$. (4) The Mg²⁺ dependence of hydrolysis varied with the absolute ATP concentration. At 3 mM ATP, the$\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg²⁺ was $\text{0.86 ± 0.10}\text{mM}$, and at 6 mM ATP, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ was $\text{1.86 ± 0.44}\text{mM}$. High levels of Mg²⁺ caused inhibition of hydrolysis. (5) The interactions of Na⁺ and K⁺ were examined over a range of conditions. K⁺ levels caused modulations in the Na⁺ dependence in the range of 1–150 mM. (6) The $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ prepared from squid optic ganglion displays properties similar to those of the sodium pump in injected nerves.     

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.     

Properties of bilayer membranes in the presence of dipicrylamine. A comparative study by optical absorption and electrical relaxation measurements

In order to test the question if a pool of lipophilic ions may exist in black lipid membranes which cannot be detected by electrical relaxation measurements we have performed simultaneously measurements of the optical absorption of a lipophilic ion. The absorbance of membrane-bound dipicrylamine at 410 nm was measured with a sensitive spectrophotometer which can detect absorbance changes $\text{? 4 · 10}{}^{\mathrm{?5}}$. A minimal concentration of about 6 · 10¹¹ dipicrylamine ions per cm² of the membrane could be detected with this instrument. The dipicrylamine concentration in the membrane obtained with the optical method $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>opt</mtext>}}$ is compared with the concentrations $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>el</mtext>}}$ obtained from simultaneous electrical relaxation measurements. $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>opt</mtext>}}$ and $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>el</mtext>}}$ agreed at low dipicrylamine concentrations (10^?8–10^?7 M in the aqueous phase) and showed saturation at higher concentrations (up to 5 · 10^?6 M). In the saturation range $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>opt</mtext>}}$ was maximally four times higher than $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>el</mtext>}}$. The significance of this difference is discussed together with general aspects of the saturation phenomenon.