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.     

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.     

Kinetics of 42K and 86Rb loss from the crayfish retina in the dark and the effect of light on the rate of isotope loss

⁸⁶Rb and ⁴²K loss from non-illuminated crayfish retinas is described by a sum of two exponential functions with rate constants λ₁ ≈ 0.12 min^?1 and λ₂ ≈ 0.006 min^?1. ⁸⁶Rb and ⁴²K movements distinguish mainly by the λ₂ rate constants, $\text{λ}{}_2{}^{\mathrm{<mtext>Rb</mtext>}}\text{λ}{}_2{}^{\mathrm{<mtext>K</mtext>}}\text{= 0.7}$.Ouabain causes a delayed increase in the rate of isotope loss from non-illuminated retinas (λ ≈ 0.009 min^?1 after 40 min). Light stimuli evoke a temporary increase in the rate of both ⁸⁶Rb and ⁴²K loss by the same factor (2.7 under these conditions). In the presence of ouabain the light-induced isotope loss is abolished after 40 min if the retina is periodically illuminated but not if the retina is kept in the dark.     

Temperature-sensitive mutations in Drosophila melanogaster. 8. Temperature-sensitive periods of the lethal and morphological phenotypes of selected combinations of notch-locus mutations

Temperature-shift experiments were performed on five Notch-locus genotypes with temperature-sensitive phenotypes. The results show that temperature-sensitive periods (TSPs) for lethality may occur at any developmental stage: (1) $\text{N}{}^{\mathrm{g11}}\text{N}{}^{\mathrm{g11}}$;Dp^51b7 having a short embryonic TSP for lethality, (2) $\text{Ax}{}^{16172}\text{N}{}^{\mathrm{?40}}$ having a second-instar TSP for lethality, and (3) $\text{N}{}^{\mathrm{?103}}\text{fa}{}^{\mathrm{no}}$ with a long, possibly polyphasic, TSP, beginning in the embryonic stage and ending in the pupal stage. On the other hand, TSPs for adult morphological phenotypes appear to be restricted to the third larval instar: (1) $\text{Ax}{}^{16172}\text{N}{}^{\mathrm{?40}}$ having third-instar TSPs for wing vein gapping and ocellar bristle loss, and (2) $\text{N}{}^{\mathrm{?103}}\text{spl}$ having third-instar TSPs for eye facet disarray, wing notching, bristle number variation, and fusion of tarsal segments. The significance of these results is discussed in terms of the role of the Notch locus in development.     

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.     

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.     

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.     

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.     

Hydrolysis of a thiopeptide by cadmium carboxypeptidase A

Substitution of the active site zinc ion of carboxypeptidase A by cadmium yields an enzyme inactive towards ordinary peptide substrates. However, a substrate analog (BzGlyNHCH₂CSPheOH) containing a thioamide linkage at the scissile position is cleaved to the thioacid. The kinetic parameters and their pH dependencies are $\text{k}{}_{\mathrm{cat}}\text{K}{}_{\mathrm{m}}\text{= 5.04 × 10}{}^4\text{min}{}^{\mathrm{?1}}\text{M}{}^{\mathrm{?1}}$, decreasing with either acid or base (PK_E1 = 5.64, pK_E2 = 9.55), and k_cat = 1.02 × 10² min^?1, decreasing with acid (pK_ES = 6.61). The thiopeptide is less efficiently cleaved by native (zinc) carboxypeptidase A. This cadmium-sulfur synergism supports a mechanism wherein the substrate amide is activated by metal ion coordination to its (thio) carbonyl.     

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.     

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.     

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.     

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.     

The concept of high- and low-affinity reactions in bovine cytochrome c oxidase steady-state kinetics

(1) Analysis of the data from steady-state kinetic studies shows that two reactions between cytochrome c and cytochrome c oxidase sufficed to describe the concave Eadie-Hofstee plots ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{? 1 · 10}{}^{\mathrm{?8}}\text{M}$ and $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{? 2 · 10}{}^{\mathrm{?5}}\text{M}$). It is not necessary to postulate a third reaction of $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{? 10}{}^{\mathrm{?6}}\text{M}$. (2) Change of temperature, type of detergent and type of cytochrome c affected both reactions to the same extent. The presence of only a single catalytic cytochrome c interaction site on the oxidase could explain the kinetic data. (3) Our experiments support the notion that, at least under our conditions (pH 7.8, low-ionic strength), the dissociation of ferricytochrome c from cytochrome c oxidase is the rate-limiting step in the steady-state kinetics. (4) A series of models, proposed to describe the observed steady-state kinetics, is discussed.     

Premeiotic DNA synthesis in fission yeast

Sporulating and various non-sporulating strains of S. pombe, especially several mutants deficient in conjugation or meiosis, were compared with respect to DNA synthesis under sporulation conditions. Meiosis and sporulation were induced by a transfer to nitrogen-free medium. As synchronized mitotic division was observed in all the strains as a first response to the shift, reducing the DNA amount per cell from the replicated state in G2 to the unreplicated state in the G1 phase of the cell cycle. Cells of the heterothallic wild-type strains ($\text{h}{}^{\mathrm{+}}\text{h}{}^{\mathrm{+}}$ or $\text{h}{}^{\mathrm{?}}\text{h}{}^{\mathrm{?}}$) accumulated in G1 with respect to DNA synthesis when they were incubated separately. In a mixed culture of these strains a period of enhanced DNA synthesis was observed after the start of zygote formation. This period of synthesis was absent in mutant fus1, where only prezygotes accumulated. Hence we conclude that in zygotic meiosis the premeiotic DNA synthesis is confined to zygotes after conjugation has been completed. In the diploid sporulating wild-type strain ($\text{h}{}^{\mathrm{+}}\text{h}{}^{\mathrm{?}}$), capable of azygotic meiosis without prior conjugation, premeiotic DNA synthesis occurred between $\text{2}\text{1}\text{2}$ and 5 h after the shift to the sporulation medium. There was no significant premeiotic DNA synthesis observed in diploid cells of the meiosis-deficient mutants mei1 or mei3, whereas premeiotic DNA synthesis proceeded normally in mutant mei4, which is blocked at a stage after commitment to meiosis in opposition to both the other mutants.     

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.     

Nuclear magnetic resonance study of the rate of electron transfer between cytochrome c and iron hexacyanides

The rates of electron exchange between ferricytochrome c (C^III)3 and ferrocytochrome c (C^II) were observed as a function of the concentrations of ferrihexacyanide (Fe^III) and ferrohexacyanide (Fe^II) by monitoring the line widths of several proton resonances of the protein. Addition of Fe^II to C^III homogeneously increased the line widths of the two downfield paramagnetically shifted heme methyl proton resonances to a maximal value. This was interpreted as indicating the formation of a stoichiometric complex, C^III·Fe^II, in the over-all reaction:

$\text{C}{}^{\mathrm{III}}\text{+}\text{Fe}{}^{\mathrm{II}}\text{?}\text{k}{}_{\mathrm{?1}}\text{k}{}_1\text{C}{}^{\mathrm{III}}\text{·}\text{Fe}{}^{\mathrm{II}}\text{?}\text{k}{}_{\mathrm{?2}}\text{k}{}_2\text{C}{}^{\mathrm{II}}\text{·}\text{Fe}{}^{\mathrm{III}}\text{?}\text{k}{}_{\mathrm{?3}}\text{k}{}_3\text{C}{}^{\mathrm{III}}\text{+}\text{Fe}{}^{\mathrm{II}}$

Values for $\text{k}{}_1\text{k}{}_{\mathrm{?1}}\text{= 0.4 × 10}{}^3\text{m}{}^{\mathrm{?1}}\text{and}\text{k}{}_2\text{= 208}\text{s}{}^{\mathrm{?1}}$, respectively, were calculated from the maximal change in line width observed at pH 7.0 and 25 °C. Changes in the line width of C^III in the presence of Fe^II and either KCl or Fe^III suggest that complexation is principally ionic, that Fe^III and Fe^II compete for a common site. Addition of saturating concentrations of Fe^III to C^III produced only minor changes in the nuclear magnetic resonance spectrum of C^III suggesting that complexation occurs on the protein surface.Addition of Fe^III to C^II in the presence of excess Fe^II (to retain most of the protein as C^II) increased the line width of the methyl protons of ligated methionine 80. A value for k_?2 ≈ 2.08 × 10⁴ s^?1 was calculated from the dependence of linewidth on the concentration of Fe^II at 24 °C. These rates are shown to be consistent with the over-all rates of reduction and oxidation previously determined by stopped flow measurements, indicating that k₂ and k_?2 were rate limiting. From the temperature dependence the enthalpies of activation are 7.9 and 15.2 kcal/mol for k₂ and k_?2, respectively.     

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.     

Evidence for general catalysis and formation of nitrobenzene in the oxidation of phenylhydroxylamine in aqueous phosphate buffer

N-Phenylhydroxylamine is oxidized in aqueous phosphate buffer to nitrosobenzene, nitrobenzene, and azoxybenzene. Degradation is O₂ dependent and shows general catalysis by $\text{H}{}_2\text{PO}{}_4{}^{\mathrm{?}}$ (k₁ = 2.3 M^?2 sec^?1) and $\text{PO}{}_4{}^{\mathrm{?3}}$ (k₂ = 2.3 × 10⁵M^?2 sec^?1) or kinetically equivalent terms. Evidence is presented suggesting the intermediacy of a highly reactive species leading to these products.