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.     

Characterization of (Na+ + K+)-ATPase liposomes: II. Effect of α-subunit digestion on intramembrane particle formation and Na+,K+-transport

The effect of the protein structure of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ on its incorporation into liposome membranes was investigated as follows: the catalytic α-subunit of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ was split into low-molecular weight fragments by trypsin treatment and the digested enzyme was reconstituted at the same protein concentration as intact control enzyme. The reconstitution process was quantified by the average number of intramembrane particles appearing on concave and convex fracture faces after freeze-fracture of the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ liposomes. The number of intramembrane particles as well as their distribution on concave and convex fracture faces is not modified by the proteolysis. In contrast, the ATPase activity and the transport capacity of the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ decrease progessively with increasing incubation times in the presence of trypsin and are abolished when the original 100 000 molecular weight α-subunit is no longer visible by sodium dodecylsulfate gel electrophoresis. Apparently, functional ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ with intact protein structure and digested, non functional enzyme consisting of fragments of the α-subunit reconstitute in the same manner and to the same extent as judged by freeze-fracture analysis. We conclude that, while trypsin treatment modifies the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ molecule in a functional sense, it appears not to modify its interaction with the bilayer in producing intramembrane particles. On the basis of our results, we propose a lipid-lipid interaction mechanism for reconstitution of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$.     

Controlled alkaline hydrolysis of steroidal α-bromoketones: New conditions and synthesis of 2α-hydroxy-3-ones

Controlled alkaline hydrolysis of 16α-bromo-17-keto steroids $\text{1}$, $\text{5}$ and $\text{7}$ with potassium carbonate and tetra-n-butylammonium hydroxide (n-Bu₄NOH) and synthesis of 2α-hydroxy-3-ones $\text{11}$, $\text{13}$ and $\text{16}$ by the controlled hydrolysis of the corresponding 2α-bromo-3-ones $\text{9}$, $\text{12}$ and $\text{15}$ are described. Treatm carbonate in aqueous acetone or with n-Bu₄NOH in aqueous dimethylformamide (DMF) gave 16α-hydroxy-17-ones $\text{3}$, $\text{6}$ and $\text{8}$ in 85–90% yield, respectively. 2α-Hydroxy-3-ones $\text{11}$, $\text{13}$ and $\text{16}$ were obtained by hydrolysis of the corresponding bromoketones $\text{9}$, $\text{12}$ and $\text{15}$ in high yields using the above conditions or sodium hydroxide in pyridine or DMF, respectively. Deuterium labeling experiments suggested that equilibration between the 2α-bromoketone $\text{9}$ and the 2β-bromo isomer $\text{10}$ precedes the formation of the ketol $\text{11}$ in which the true intermediate might be the 2β-isomer $\text{10}$. However, rearranged androstane derivatives, 3β-hydroxy-2-ones $\text{18}$ and $\text{20}$, were stereoselectively obtained by treatment of the bromoketones $\text{12}$ and $\text{15}$ with an excess amount of sodium hydroxide.     

A new species of Hemibrycon (Characiformes,Characidae) from the upper San Juan River drainage,Pacific versant,Colombia

Hemibrycon sanjuanensis, new species, is described from the upper San Juan River drainage, Pacific versant, Colombia. It is distinguished from Hemibrycon boquiae, Hemibrycon brevispini, Hemibrycon cairoense, Hemibrycon colombianus, Hemibrycon mikrostiktos, Hemibrycon metae, Hemibrycon palomae, Hemibrycon rafaelense and Hemibrycon tridens by the presence of a circular or oblong humeral spot that is located two scales posterior to the opercle (vs. 3–4 scales in Hemibrycon palomae, Hemibrycon rafaelense, Hemibrycon brevispini and Hemibrycon cairoense, and 0–1 scales, in Hemibrycon metae and Hemibrycon boquiae). It further differs from Hemibrycon colombianus in having a round or oblong humeral spot (vs. rectangular). It differs from Hemibrycon beni, Hemibrycon dariensis, Hemibrycon divisorensis, Hemibrycon helleri, Hemibrycon huambonicus, Hemibrycon inambari, Hemibrycon jabonero, Hemibrycon jelskii, Hemibrycon mikrostiktos, Hemibrycon polyodon, Hemibrycon quindos, Hemibrycon raqueliae, Hemibrycon santamartae, Hemibrycon surinamensis, Hemibrycon taeniurus, Hemibrycon tridens, and Hemibrycon yacopiae in having melanophores on the posterior margins of the scales along the sides of body (vs. lacking melanophores on margins of scales along entire length of the sides of body). The new species differs from all congeners mentioned above in having, among other features, six teeth in the outer premaxillary row arranged in a straight line (vs. five or fewer teeth not arranged in straight line except Hemibrycon cairoense with two to six teeth in the outer premaxillary row).     

Association of α2-macroglobulin-thrombin and α2-macroglobulin-plasmin complexes with isolated hepatocytes

¹²⁵I-labelled $\text{α}{}_2\text{-}\text{macroglobulin}$ complexed with thrombin or plasmin bound to hepatocytes in a concentration-and time-dependent manner. The apparent $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ values calculated from displacement experiments were 7.9 · 10^?8 M for $\text{α}{}_2\text{-}\text{macroglobulin-thrombin}$ and 8.5 · 10^?8 M for $\text{α}{}_2\text{-}\text{macroglobulin-plasmin}$. Association of these complexes was only partially reversible; after a 180 min incubation period, 50–60% of the bound radioactivity was internalized by the cells. $\text{α}{}_2\text{-}\text{Macroglobulin}$ itself bound also to hepatocytes, but the affinity of the $\text{α}{}_2\text{-}\text{macroglobulin}$ complexes was higher than that of the inhibitor alone, and $\text{α}{}_2\text{-}\text{macroglobulin}$ was not internalized, either. ¹²⁵I-labelled thrombin or plasmin bound to hepatocytes as well. These bindings were also concentration-dependent and could be decreased with an excess of unlabelled ligands. Binding rates and amounts of the bound proteinases were higher than those of their $\text{α}{}_2\text{-}\text{macroglobulin}$ complexes. The $\text{α}{}_2\text{-}\text{macroglobulin-thrombin}$ complex competed with the $\text{α}{}_2\text{-}\text{macroglobulin-plasmin}$ complex in binding to hepatocytes, whereas there was no competition between these complexes and the antithrombin III-thrombin complex. These results suggest that the binding sites of hepatocytes for $\text{α}{}_2\text{-}\text{macroglobulin-proteinase}$ and antithrombin III-proteinase complexes are different.     

Study on models of single populations: an expansion of the logistic and exponential equations

Using the adsorption theory of chemical kinetics, a new equation concerning the growth of single populations is presented:

$\text{d}\text{X}\text{d}\text{t}\text{=}\text{μ}{}_{\mathrm{c}}\text{X(1 ?)}\text{X}\text{X}{}_{\mathrm{m}}\text{1?}\text{X}\text{X}{}_{\mathrm{m}}{}^{\mathrm{\prime }}$

or in its integral form:

$\text{ln}\text{X}\text{X}{}_{\mathrm{o}}\text{?}\text{ln}\text{X}{}_{\mathrm{m}}\text{?X}\text{X}{}_{\mathrm{m}}\text{?X}{}_{\mathrm{o}}\text{+}\text{X}{}_{\mathrm{m}}\text{X}{}^{\mathrm{\prime }}{}_{\mathrm{m}}\text{X}{}_{\mathrm{m}}\text{?X}\text{X}{}_{\mathrm{m}}\text{?X}{}_{\mathrm{o}}\text{=μ}{}_{\mathrm{c}}\text{(t?t}{}_{\mathrm{o}}\text{)}$

This equation attempts to explain the relationship between population increment and limiting resources. It can be reduced to either the logistic or exponential equation under two extreme conditions. The new equation has three parameters, X_m, X′_m and μ_c, each of which has ecological significance. $\text{X}{}_{\mathrm{m}}\text{X′}{}_{\mathrm{m}}$ concerns the efficiency of nutrient utilization by an organism. Its value is between zero and one. With ratios approaching unity, the efficiency is high; lower ratios indicate that population increment is quickly restricted by limiting resources. μ_c, is a velocity parameter lying between μ_e, (exponential growth) and μ_L (logistic growth), and is dependent on the value of $\text{solX}{}_{\mathrm{m}}\text{X′}{}_{\mathrm{m}}$. From μ_c we can predict the time course of population incremental velocity ($\text{d}\text{X}\text{d}\text{t}$), and can observe that it is not symmetrical, unlike that derived from the logistic equation. At $\text{X}{}_{\mathrm{m}}\text{X′}{}_{\mathrm{m}}\text{= 1}$ the maximum velocity of the population increment predicted from the new equation is twice that of the logistic equation.Population growth in nature seems to support the new equation rather than the logistic equation, and it can be successfully fitted by means of a least square method.     

Preferential solvation of methylmercurated calf thymus deoxyribonucleic acid.

The changes in polymer-solvent interactions that occur when native calf thymus DNA is dialyzed against Na₂SO₄ solutions of a given ionic strength and buffer concentration but of varying concentrations in methylmercuric hydroxide have been investigated with the help of solution density measurements at 25 °C and pH 6.8–7.0. From measurements executed under equilibrium dialysis conditions at the three salt levels 5 mm, 0.05 m, and 0.5 m Na₂SO₄ (m refers to molality) and in the presence of 5 mm cacodylic acid buffer, the density increments $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ for native calf thymus DNA were determined as a function of CH₃HgOH concentration. $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ was found not to vary with organomercurial concentration, irrespective of the concentration of supporting electrolyte, until a certain CH₃HgOH concentration level has been reached, viz., , beyond which $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ increases strongly with increasing concentration of CH₃HgOH. As is shown by optical melting, $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ becomes a function of organomercurial concentration the moment DNA undergoes denaturation brought about by the complexing of CH₃HgOH with the various N-binding sites of the base residues in the DNA double helix.Polymer-solvent interactions, expressed in terms of preferential water interactions (“net hydration”) and preferential salt interactions (“salt solvation”), were derived from the $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ data in combination with data obtained on the preferential interaction of CH₃HgOH with denatured DNA and data on the partial specific volumes of all major solution components, gathered from density measurements on solutions with fixed concentrations of diffusible components. Evidence is presented which shows that denaturation in general decreases the net hydration while salt becomes preferentially associated with the polyelectrolyte. This process is further amplified by the interaction of CH₃HgOH with denatured DNA: Methylmercurated DNA alters the redistribution of diffusible components at dialysis equilibrium to such an extent that in a formal sense large amounts of water are rejected from the immediate vicinity of the polymer. The molecular implications of these findings are explored. The results are further discussed in the light of previous findings where the methylmercury-induced denaturation of DNA had been studied with the help of buoyant density measurements in a Cs₂SO₄ density gradient and by velocity-sedimentation in a variety of sulfate media.     

The covariance of relatives derived from a random mating population.

Attention is drawn to errors common in the derivation of forms for the genotypic covariance of noninbred relatives from a Hardy-Weinberg population of diploids. A synthesis of Fisher's least-squares method of partitioning the genotypic variance and Malécot's probability method of expressing kinship, yields a general form. For one locus, the form is $\text{(P}{}_{\mathrm{ss}}\text{+ P}{}_{\mathrm{sd}}\text{+ P}{}_{\mathrm{ds}}\text{+ P}{}_{\mathrm{dd}}\text{)}\text{1}\text{2}\text{σ}{}_{\mathrm{a}}{}^2\text{+ (P}{}_{\mathrm{ss}}\text{P}{}_{\mathrm{dd}}\text{+ P}{}_{\mathrm{sd}}\text{P}{}_{\mathrm{ds}}\text{) σa}{}_{\mathrm{d}}{}^2$, where σ_a² is the additive genetic variance, α_d² is the variance of dominance deviations, p_ij is the probability that parental gamete i is identical by descent to parental gamete j, i = s, d indexes the parents of one relative, and j = s, d indexes those of the other. The form provides a framework for obtaining the covariance of relatives from an equilibrium population with linkage.     

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.     

The determination of positive and negative co-operativity with allosteric enzymes and the interpretation of sigmoid curves and non-linear double reciprocal plots for the MWC and KNF models

(i) It is proved that only four independent constants can ever be obtained by extrapolation procedures applied to non-hyperbolic steady-state or binding data, (ii) Analysis of the algebraic graphs $\text{y}\text{x}$, $\text{(1/y)}\text{(1/x)}$, $\text{y}\text{(}\text{y}\text{x}\text{)}$ and ($\text{x}\text{y}\text{)/x}$ is shown to require a knowledge of the sign of six curve shape determinants. In each case, the sign is a necessary and sufficient condition for a specific curve shape feature, (iii) The precise graphical effect of positive and negative co-operativity then requires the definition of two reference curves, the osculating hyperbola at zero substrate concentration, OH(0), and the osculating hyperbola at infinite substrate concentration OH(∞). These are better first order approximations than the Hill equation, (iv) Rules for determining unambiguously the sign of initial, final and overall co-operativity coefficients by inspection of non-hyperbolic binding curves are then possible, (v) These rules require that saturation data for:

$\text{y=}\text{∑}\text{i=1}\text{n}\text{a}{}_{\mathrm{i}}\text{x}{}^{\mathrm{i}}\text{∑}\text{i=0}\text{n}\text{β}{}_{\mathrm{i}}\text{x}{}^{\mathrm{i}}$

be fitted by computer for low concentrations to the hyperbola:

$\text{OH}\text{(o)=}\text{(}\text{-a}{}_1{}^2\text{ψ}{}_{11}{}^{20}\text{)x}\text{[(}\text{-a}{}_1\text{β}{}_0\text{ψ}{}_{11}{}^{20}\text{)+x]}$

while regression of high substrate concentration data is to:

$\text{OH}\text{(∞)=}\text{(}\text{a}{}_{\mathrm{n}}\text{β}{}_{\mathrm{n}}\text{)x}\text{[(}\text{φ}{}_{\mathrm{n,n-1}}\text{a}{}_{\mathrm{n}}\text{β}{}_{\mathrm{n}}\text{)+x]}$

. Comparisons of the best fit pseudo-kinetic constants then gives the type of co-operativity present in an unambiguous way with no assumptions as to molecular mechanism, (vi) These rules are then applied to the MWC and KNF allosteric models of ligand binding and the constraints necessary for specific curve shape effects are given, (vii) The graphical expression of positive or negative final co-operativity depends only on events at high substrate concentration but overall and initial co-operativities produce specific geometric effects depending upon the difference between behaviour of saturation data at both extremes of concentration, (viii) This apparent anomaly is explained by a discussion of the relationships between the osculating hyperbolae, the theoretical parent hyperbola and the Hill plot asymptotes.     

Ligand-dependent reactivity of (Na+ + K+)-ATPase with showdomycin

Showdomycin inhibited pig brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ with pseudo first-order kinetics. The rate of inhibition by showdomycin was examined in the presence of 16 combinations of four ligands, i.e., Na⁺, K⁺, Mg²⁺ and ATP, and was found to depend on the ligands added. Combinations of ligands were divided into five groups in terms of the magnitude of the rate constant; in the order of decreasing rate constants these were: (1)$\text{Na}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}$, (2) $\text{Mg}{}^{\mathrm{2+}}$, $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{K}{}^{\mathrm{+}}$, $\text{K}{}^{\mathrm{+}}$ and none, (3) $\text{Na}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{,}\text{Na}{}^{\mathrm{+}}$, $\text{K}{}^{\mathrm{+}}\text{+}\text{Na}{}^{\mathrm{+}}$ and $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}$, (4) $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{ATP}\text{,}\text{K}{}^{\mathrm{+}}\text{+}\text{ATP}$ and $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}\text{, (5)}\text{K}{}^{\mathrm{+}}\text{+}\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}$ and ATP. The highest rate was obtained in the presence of Na⁺, Mg²⁺ and ATP. The apparent concentrations of Na⁺, Mg²⁺ and ATP for half-maximum stimulation of inhibition ($\text{K}{}_{0.5}{}^{\mathrm{<mtext>s</mtext>}}$) were 3 mM, 0.13 mM and 4μM, respectively. The rate was unchanged upon further increase in Na⁺ concentration from 140 to 1000 mM. The rates of inhibition could be explained on the basis of the enzyme forms present, including E₁, E₂, ES, E₁-P and E₂-P, i.e., E₂ has higher reactivity with showdomycin than E₁, while E₂-P has almost the same reactivity as E₁-P. We conclude that the reaction of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ proceeds via at least four kinds of enzyme form (E₁, E₂, E₁ · nucleotide and EP), which all have different conformations.     

Influence of ionic strength upon the proton inventory for the water-catalyzed hydrolysis of N-acetylbenzotriazole

Proton inventory investigations of the hydrolysis N-acetylbenzotriazole at pH 3.0 (or the equivalent point on the pD rate profile) have been conducted at two different temperatures and at ionic strengths ranging from 0 to 3.0 M. The solvent deuterium isotope effects and proton inventories are remarkably similar over this wide range of conditions. The proton inventories suggest a cyclic transition state involving four protons contributing to the solvent deuterium isotope effect for the water-catalyzed hydrolysis. The hydrolysis data are described by the equation $\text{k}{}_{\mathrm{n}}\text{= k}{}_{\mathrm{<mtext>o</mtext>}}\text{(1 ? n + nπ}{}_{\mathrm{<mtext>a</mtext>}}{}^1\text{)}{}^4$ with $\text{π}{}_{\mathrm{<mtext>a</mtext>}}{}^1\text{～ 0.74}$, where k_o is the observed first-order rate constant in protium oxide, n is the atom fraction of deuterium in the solvent, k_n is the rate constant in a protium oxide-deuterium oxide mixture, and $\text{π}{}_{\mathrm{<mtext>a</mtext>}}{}^1$ is the isotopic fractionation factor.     

Localized coupling in oxidative phosphorylation by mitochondria from Jerusalem artichoke (Helianthus tuberosus)

Delocalized chemiosmotic coupling of oxidative phosphorylation requires that a single-value correlation exists between the extent of and the kinetic parameters of respiration and ATP synthesis. This expectation was tested experimentally in nigericin-treated plant mitochondria in single combined experiments, in which simultaneously respiration (in State 3 and in State 4) was measured polarographically, FΔψ (which under these conditions was equivalent to ) was evaluated potentiometrically from the uptake of tetraphenylphosphonium⁺ and the rate of phosphorylation was estimated from the transient depolarization of mitochondria during State 4-State 3-State 4 transitions. The steady-state rates of the different biochemical reactions were progressively inhibited by specific inhibitors active with different modalities on various steps of the energy-transducing process: succinate respiration was inhibited competitively with malonate or noncompetitively with antimycin A, or by limiting the rate of transport into the mitochondria of the respiratory substrate with phenylsuccinate; was dissipated by uncoupling with increasing concentrations of valinomycin; ADP phosphorylation was limited with oligomycin. The results indicate generally that when the rate of respiratory electron flow is decreased, a parallel inhibition of the rate of phosphorylation is also observed, while very limited effects can be detected on the extent of . This behavior is in marked contrast to the effect of uncoupling where the decreased rate of ATP synthesis is clearly due to energy limitation. Extending previous observations in bacterial photosynthesis and in respiration by animal mitochondria and submitochondrial particles the results indicate, therefore, that respiration tightly controls the rate of ATP synthesis, with a mechanism largely independent of . These data cannot be reconciled with a delocalized chemiosmotic coupling model.     

Characterization of cytochrome P-450SCC-containing liposomes

Purified cytochrome $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ from bovine adrenocortical mitochondria was incorporated into liposomes by the cholate-dilution method utilizing either dialysis or Sephadex gel filtration. Among synthetic phospholipids tested, dioleoylglycerophosphocholine showed the best stability during the incorporation of $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ into liposomes. A maximum amount of heme was incorporated into liposomes at a molar ratio of phospholipid to the cytochrome of approx. 200. When $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ was incorporated into the dioleoylglycerophosphocholine liposomes by the cholate-filtration method, the $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}\text{-}\text{containing liposomes}$ showed two major populations on the elution pattern of the Sepharose 4B gel filtration, and were seen at a diameter of 200–600 Å and its aggregated forms. When the cytochrome was incorporated into dioleoylglycerophosphocholine liposomes or cholesterol-free adrenocortical mitochondrial liposomes, $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ was less stable than $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ in aqueous solution. Cholesterol or adrenodoxin markedly stabilized the liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$. Liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ required cholesterol for its optimum reduction with adrenodoxin, adrenodoxin reductase, and NADPH in the presence of CO. About 70% of the total heme in the dioleoylglycerophosphocholine liposomes was reduced by the enzymatic reduction in the presence of cholesterol, indicating that 70% of the total molecules are exposed to the surface of the outer monolayer. In order to see the location of the heme in membrane, the dioleoylglycerophosphocholine-liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ was subjected to $\text{p-}\text{chloromercuriphenyl sulfonic acid}$ treatment. This reagent destroyed the liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$. These results suggest that the heme is located in the proximity of the $\text{p-}\text{chloromercuriphenyl sulfonic acid}$ reacting sites which are exposed to the surface, or located on the vincinity of polar heads of the membrane.     

Transfer of N, N'-diacetylchitobiose from dolichyl diphosphate into a gray matter membrane glycoprotein

Gray matter and white matter membranes catalyze the transfer of label from UDP-N-acetyl-[${}^{14}\text{C}$] glucosamine into N-acetyl[${}^{14}\text{C}$]glucosaminyl-pyrophosphoryl-dolichol, N,N′-diacetyl [${}^{14}\text{C}$]chitobiosyl-pyrophosphoryl-dolichol, and N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein. Gel filtration of the Pronase digests of gray matter N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein reveals two N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide fractions. One fraction (A) contains approximately eight glycose units. All of the radioactivity is at nonreducing termini and can be released by treatment with an exo-β-N-acetylglucosaminidase. A smaller N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide (B) is recovered in the elution volume expected for an asparaginyl disaccharide. Structural studies show that the labeled saccharide unit in glycopeptide B is N,N′-diacetyl[${}^{14}\text{C}$]chitobiose. The linkage between the ${}^{14}\text{C}$-labeled disaccharide and the polypeptide has the properties of an N-glycosidic attachment to asparagine. Only the larger N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide (A) is found in Pronase digests of white matter membrane N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein after incubation with UDP-N-acetyl[${}^{14}\text{C}$]glucosamine. When gray matter membranes are incubated with UDP-N-acetyl[${}^{14}\text{C}$]glucosamine in the presence of tunicamycin or UMP, the labeling of glycolipid and the asparaginyl disaccharide is inhibited. UMP and tunicamycin have no effect on the transfer of N-acetyl[${}^{14}\text{C}$]glucosamine to external acceptor sites of the larger glycopeptide (A). The transfer of N,N′-diacetyl[${}^{14}\text{C}$]-chitobiose from carrier lipid to protein is observed when extensively washed membranes containing endogenous, prelabeled ${}^{14}\text{C}$-labeled glycolipids are incubated in the presence or absence of unlabeled GDP-mannose. UMP treatment of the prelabeled membranes selectively discharged over 80% of the label from N-acetyl[${}^{14}\text{C}$]glucosaminyl-pyrophosphoryl-dolichol, but had no effect on the transfer of the ${}^{14}\text{C}$-labeled disaccharide to protein. All of these results are concordant with transfer of N,N′-diacetylchitobiose from dolichyl diphosphate to gray matter glycoprotein. The major membrane glycoprotein labeled by the lipid-mediated [${}^{14}\text{C}$]disaccharide transfer reaction has an apparent molecular weight of 24,000. Tunicamycin prevents the enzymatic labeling of the gray matter glycoprotein having an apparent molecular weight of 24,000.     

Photoconversion and regeneration of active protochlorophyll(ide) in mutants defective in the regulation of chlorophyll synthesis

Seedlings carrying mutations in regulatory genes for protochlorophyll(ide) synthesis accumulate protochlorophyll(ide) in darkness in amounts exceeding the wildtype level. Thus, +/tig-d¹² and $\text{tig-b}{}^{24}\text{tig-b}{}^{24}$accumulate 2-fold, $\text{tig-o}{}^{34}\text{tig-o}{}^{34}$ 5- to 6-fold, and $\text{tig-d}{}^{12}\text{tig-d}{}^{12}$ 15-fold more protochlorophyll(ide) than the wild type.The amount of photoconvertible protochlorophyll(ide) accumulated in darkness is the same in all genotypes, despite the large differences in total protochlorophyll(ide) content, indicating a constant number of photoconversion sites.When briefly illuminated leaves are returned to darkness, regeneration of active protochlorophyll(ide) from the pool of inactive protochlorophyll(ide) takes place in wild-type and mutant leaves. Compared to the wild type, the rate of protochlorophyll(ide) activation during 4- and 10-min dark periods is higher in +/tig-d¹², $\text{tig-b}{}^{24}\text{tig-b}{}^{24}$, and $\text{tig-o}{}^{34}\text{tig-o}{}^{34}$, but lower in $\text{tig-d}{}^{12}\text{tig-d}{}^{12}$.There was no indication that the accumulation of protochlorophyll(ide) influences the conversion sites of the protochlorophyll(ide) holochrome, as the kinetics of photoconversion of initially active protochlorophyll(ide) in leaves with the genotypes +/+, +/tig-o³⁴, and $\text{tig-o}{}^{34}\text{tig-o}{}^{34}$ are similar or identical.     

Electronic structure of established and potential oxidizing agents in biological systems

The electronic structure of 19 established and potential biological oxidants has been studied by semiempirical all-valence-electron quantum-chemical methods. Electronic ground and excited states of O₂, HO₂, HO, H₂O₂, H₃O, H₄O₂ and their (radical) ions have been investigated in order to get information on the geometry, vertical ionization potentials, vertical electron affinities and low-lying electronic excited states. The actual aim has been (i) to arrange the studied species according to their oxidizing power as given by gas-phase electron affinity.

$\text{9·HO·OH}{}_2\text{O}{}^1{}_2\text{>(}{}^1\text{?}{}^{\mathrm{+}}{}_{\mathrm{g}}\text{).·OH>O}{}^1{}_2\text{(}{}^1\text{δ}{}^{\mathrm{+}}{}_{\mathrm{g}}\text{) >HO}{}^1{}_2\text{(}{}^2\text{A′)>O}{}^1{}_2\text{(}{}^2\text{A′)>O}{}_2\text{(}{}^3\text{?}{}^{\mathrm{-}}{}_{\mathrm{g}}\text{>HO·}{}_2\text{)}$

and (ii) to contribute to the thermodynamics of early changes of the O₂ molecule

$\text{O}{}_2\text{+e→O}{}^{\mathrm{?}}{}_2\text{·;O}{}^{\mathrm{?}}{}_2\text{·+H}{}^{\mathrm{+}}\text{→HO·}{}_2$

. Moreover, it has been found theoretically that the hydrated form of the hydroxyl radical (·HO.OH₂) should be a relatively stable species with very high electron affinity (2·4 eV, INDO method). This circumstance and the theoretically predicted, extraordinarily low-lying, excited doublet state of the peroxyl radical (about 6000 cm^?1) could be of biological significance.