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.     

Cockroaches on a treadmill: Aerobic running

Five species of cockroach were tested on a miniature treadmill at three velocities as O₂ consumption () was measured: Gromphadorhina chopardi, Blaberus discoidalis, Eublaberus posticus, Byrsotria fumagata and Periplaneta americana. All cockroaches showed a classical aerobic response to running: increased rapidly from a resting rate to a steady-state on-response varied from under 30 s to 3 min. Recovery after exercise was rapid as well; $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ off-response varied from under 30 s to 6 min. These times are faster or similar to mammalian values. varied directly with velocity as in running mammals, birds and reptiles. during steady-state running was only 4–12 times higher than at rest. Running is energetically much less costly per unit time than flying, but the cost of transport per unit distance is much more expensive for pedestrians. The minimal cost of transport (M_run), the lowest necessary to transport a given mass a specific distance, is high in cockroaches due to their small size. The new data suggest that insects may be less economical than comparable sized vertebrates.     

An allosteric pore model for sugar transport in human erythrocytes

Glucose transport in human erythrocytes is characterized by a marked asymmetry in the $\text{V}$ and $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for entry and for exit. In addition, they show a high $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ and a high $\text{V}$ for equilibrium exchange but low $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for infinite cis and for infinite trans exit and entry. An allosteric pore model has been proposed to account for these characteristics. In this model, substrate-induced conformational changes destabilize the interfaces between protein subunits (the pore gates).Pores doubly occupied from inside destabilize the transport gates and result in high $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ and high $\text{V}$ transport parameters. This effect is less marked when pores are doubly occupied from outside and therefore transport asymmetry results.     

Asymmetric or symmetric? Cytosolic modulation of human erythrocyte hexose transfer

(1) The Michaelis-Menten parameters for hexose transfer in erythroctes, erythrocyte ghosts and inside-out vesicles at 20°C were determined using the light scattering method of Sen and Widdas ((1962) J. Physiol. 160, 392–403). (2) The external $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for infinite-cis exit of d-glucose in cells and ghosts is $\text{3.6 ± 0.5}\text{mM}$. (3) Dilution of cellular solute (up to × 90 dilution) by lysing and resealing cells in varying volumes of lysate is without effect on the $\text{V}{}_{\mathrm{<mtext>m</mtext>}}$ for net d-glucose exit. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for net exit, however, falls from $\text{32.4 ± 3.7}\text{mM}$ in intact cells to $\text{12.9 ± 2.3}\text{mM}$ in ghosts. This effect is reversible. (4) Infinite-cis net d-glucose uptake measurements in cells and ghosts reveal the presence of a low $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$, high affinity internal site of $\text{5.9 ± 0.8}\text{mM}$. The $\text{V}{}_{\mathrm{<mtext>m</mtext>}}$ for net glucose entry increases from $\text{23.2 ± 3.7}\text{mmol/l per min}$ in intact cells to $\text{55.4 ± 6.3}\text{mmol/l per min}$ in ghosts. (5) The external $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for infinite-cisd-glucose exit in inside-out vesicles is $\text{6.8 ± 2.7}\text{mM}$. The kinetics of zero-transd-glucose exit from inside-out vesicles are changed markedly when cellular solute (obtained by lysis of intact cells) is applied to either surface of inside-out vesicles. When solute is present externally, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ and $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ for zero-trans exit are decreased by up to 10-fold. When solute is present at the interior of inside-out vesicles, $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ for zero-trans exit is reduced; $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for exit is unaffected. In the nominal absence of cell solute, transfer is symmetric in inside-out vesicles. The orientation of transporter in the bilayer is unaffected by the vesiculation procedure. (6) External application of cellular solute to ghosts reduces $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ for d-glucose exit but is without effect on the external $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for infinite-cis exit. (7) The inhibitory potency of cell lysate on hexose transfer is lost following dialysis indicating that the factors responsible for transfer modulation are low molecular weight species. (8) We consider the hexose transfer in human erythrocytes is intrinsically symmetric and that asymmetry of transfer is conferred by interaction of the system with low molecular weight cytosolic factors.     

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.     

Malate dehydrogenase. Kinetic studies with meso-tartrate and 2-keto-3-hydroxysuccinate, comparison of the mitochondrial and supernatant pig heart enzymes.

Initial rate, product inhibition, and isotope rate kinetic studies of pig heart mitochondrial and supernatant malate dehydrogenases, acting upon the nonphysiological substrates, meso-tartrate and 2-keto-3-hydroxysuccinate, are reported. The measured spontaneous keto-enol equilibrium for 2-keto-3-hydroxysuccinate in 0.05 m Tris-acetate (pH 8.0) at 25 °C favors the enol form, dihydroxyfumarate, with an apparent equilibrium constant of 0.036. The enzyme-catalyzed reaction favors meso-tartrate with an apparent equilibrium constant of 1.25 × 10^?6, M^?1 at pH 8.0. The mechanism apparently remains ordered bi bi for both enzymes when these nonphysiological substrates are used, and the chemical-converting hydride transfer step becomes more rate limiting for both enzymes. This conclusion is supported by $\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{V}{}_{\mathrm{<mtext>D</mtext>}}$ and $\text{(}\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{K}{}_{\mathrm{<mtext>H</mtext>}}\text{)}\text{V}{}_{\mathrm{<mtext>D</mtext>}}\text{K}{}_{\mathrm{<mtext>D</mtext>}}$ values of 2.6 and 3.1, respectively, for the mitochondrial enzyme and 1.9 and 2.9, respectively, for the supernatant enzyme.     

Synthesis and characterization of a DNA probe complementary to rat liver 28S ribosomal RNA.

Conditions for the production of a complementary DNA sequence for use in studies of ribosomal RNA are described. $\text{E}$. $\text{coli}$ DNA polymerase I is used to transcribe highly purified 28S ribosomal RNA from rat liver. The reaction is sensitive to the tertiary structure of the rRNA template-primer. The complementary DNA hybridizes to its rRNA template with a $\text{R}{}_{\mathrm{o}}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 0.02. The hybrid formed between 28S ribosomal RNA and complementary DNA has a T_m of 73°C. The probe reacts with total rat nuclear RNA with a $\text{R}{}_{\mathrm{o}}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 1.0.     

The diffusion-concentration product of oxygen in lipid bilayers using the spin-label T1 method

A method is described to measure the oxygen diffusion-concentration product, $\text{D}{}_{\mathrm{<mtext>o</mtext>}}\text{[}\text{O}{}_2\text{]}$, at any locus that can be probed or labeled using nitroxide radicals. The method is based on the dependence of the spin-lattice relaxation time $\text{T}{}_1$ of the spin label on the bimolecular collision rate with oxygen. Strong Heisenberg exchange between spin label and oxygen contributes directly to $\text{T}{}_1$ of the spin label, while dipolar interactions are negligible. Both time-domain and continuous wave saturation methods for studying $\text{T}{}_1$ are considered. The method has been applied to phospholipid liposomes using fatty acid spin labels. A discontinuity in $\text{D}{}_{\mathrm{<mtext>o</mtext>}}\text{[}\text{O}{}_2\text{]}$ at the main phase transition was observed.     

Quenching of chlorophyll fluorescence and primary photochemistry in chloroplasts by dibromothymoquinone

The quenching action of dibromothymoquinone on fluorescence and on primary photochemistry was examined in chloroplasts at ?196 °C. Both the initial (F₀) and final (F_M) levels of fluorescence as well as the fluorescence of variable yield (F_v = F_M ? F₀) were quenched at ?196 °C to a degree which depended on the concentration of dibromothymoquinone added prior to freezing. The initial rate of photoreduction of C-550 at — 196 °C, which was assumed to be proportional to maximum yield for primary photochemistry, ?_Po, was also decreased in the presence of dibromothymoquinone. Simple theory predicts that the ratio $\text{F}{}_{\mathrm{<mtext>V</mtext>}}\text{F}{}_{\mathrm{<mtext>M</mtext>}}$ should equal ?_Po. Excellent agreement was found in a comparison of relative values of ?_Po with relative values of $\text{F}{}_{\mathrm{<mtext>V</mtext>}}\text{F}{}_{\mathrm{<mtext>M</mtext>}}$ at various degrees of quenching by dibromothymoquinone. These results are taken to indicate that F₀ and F_V are the same type of fluorescence, both emanating from the bulk chlorophyll of Photosystem II.Dibromothymoquinone appears to create quenching centers in the bulk chlorophyll of Photosystem II which compete with the reaction centers for excitation energy. The rate constant for the quenching of excitation energy by dibromothymoquinone is directly proportional to the concentration of the quencher. Rate constants for the de-excitation of excited chlorophyll molecules by fluorescence, k_F, by nonradiative decay processes, k_D, by photochemistry, k_P, and by the specific quenching of dibromothymoquinone, k_Q, were calculated assuming the absolute yield of fluorescence at F₀ to be either 0.02 or 0.05.     

pH kinetic studies of the N-demethylation of N,N-dimethylaniline catalyzed by chloroperoxidase

The effect of pH on the kinetic parameters for the chloroperoxidase-catalyzed N-demethylation of N,N-dimethylaniline supported by ethyl hydroperoxide was investigated from pH 3.0 to 7.0. Chloroperoxidase was found to be stable throughout the pH range studied. Initial rate conditions were determined throughout the pH range. The V_max for the demethylation reaction exhibited a pH optimum at approximately 4.5. The K_m for N,N-dimethylaniline increased with decreasing pH, while the K_m for ethyl hydroperoxide varied in a manner paralleling V_max. Comparison of the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{K}{}_{\mathrm{m}}$ values for N,N-dimethylaniline and ethyl hydroperoxide indicated that the interaction of N,N-dimethylaniline with chloroperoxidase compound I was rate-limiting below pH 4.5, while compound I formation was rate-limiting above pH 4.5. The log of the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{K}{}_{\mathrm{m}}$ for ethyl hydroperoxide was independent of pH, indicating that chloroperoxidase compound I formation is not affected by ionizations in this pH range. The plot of the log of the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{K}{}_{\mathrm{m}}$ for N,N-dimethylaniline versus pH indicated an ionization on compound I with a pK of approximately 6.8. The plot of the log of the V_max versus pH indicated an ionization on the compound I-N,N-dimethylaniline complex, with a pK of approximately 3.1. The results show that chloroperoxidase can demethylate both the protonated and neutral forms of N,N-dimethylaniline (pK approximately 5.0), suggesting that hydrophobic binding of the arylamine substrate is more important in catalysis than ionic bonding of the amine moiety. For optimal catalysis, a residue in the chloroperoxidase compound I-N,N-dimethylaniline complex with a pK of approximately 3.1 must be deprotonated, while a residue in compound I with a pK of approximately 6.8 must be protonated.     

Biochemical adaptation of mitochondria,muscle, and whole-animal respiration to endurance training

The experimental intervention of exercise training has been used to study mitochondrial biosynthesis, and the physiologic integration of subcellular, cellular, and whole-animal energetics. Gross mitochondrial composition was unchanged in rat muscle by a 10-week program of endurance treadmill running. The mitochondrial concentration of iron-sulfur clusters, cytochromes, flavoprotein, dehydrogenases, oxidases, and membrane protein and lipid, as well as the ratios of each component to the others, maintained constant proportions. The mitochondrial content of muscle, however, increased by approximately 100% as did absolute tissue oxidative capacity. The soluble portions of mitochondria maintained a constant total protein content and mass, relative to the membrane fraction, despite adaptive changes in the specific activities of some citric acid-cycle enzymes. Mitochondria from endurance-trained muscles generated normal transmembrane potentials, ADP/O ratios, and respiratory control ratios. Muscle oxidase activity was highly correlated (r = 0.92) with endurance capacity, which increased 403%. Whole-animal maximal O₂ consumption (), however, increased only 14% and was a relatively poor predictor of endurance. Thus, mitochondrial factors, rather than , must play an important role in dictating the limits of endurance activity. Conversely, was strongly related to the maximal intensity of work which could be attained aerobically (r = 0.82). Comparison of O₂ consumption at the mitochondrial, muscle, and whole-animal levels revealed that maximal muscle oxidase activity was not an absolute limitation to : It is concluded that other factors intervene to control the percentage of muscle O₂ consumption capacity which may be utilized during exercise.     

Lactose transport in Escherichia coli cells Dependence of kinetic parameters on the transmembrane electrical potential difference

We determine the kinetic parameters $\text{V}$ and $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ of lactose transport in Escherichia coli cells as a function of the electrical potential difference (Δψ) at pH 7.3 and $\text{Δ}\text{pH}\text{= 0}$. We report that transport occurs simultaneously via two components: a component which exhibits a high $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ (larger than 10 mM) and whose contribution is independent of Δψ, a component which exhibits a low $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ independent of Δψ (0.5 mM) but whose $\text{V}$ increases drastically with increasing Δψ. We associate these components of lactose transport with facilitated diffusion and active transport, respectively. We analyze the dependence upon Δψ of $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ and $\text{V}$ of the active transport component in terms of a mathematical kinetic model developed by Geck and Heinz (Geck, P. and Heinz, E. (1976) Biochim. Biophys. Acta 443, 49–63). We show that within the framework of this model, the analysis of our data indicates that active transport of lactose takes place with a H⁺/lactose stoichiometry greater than 1, and that the lac carrier in the absence of bound solutes (lactose and proton(s)) is electrically neutral. On the other hand, our data relative to facilitated diffusion tend to indicate that lactose transport via this mechanism is accompanied by a H⁺/lactose stoichiometry smaller than that of active transport. We discuss various implications which result from the existence of H⁺/lactose stoichiometry different for active transport and facilitated diffusion.     

Peculiarities of the Na+/d-glucose cotransport system in necturus renal tubules

The effects of d-glucose addition to a glucose-free luminal perfusate were investigated in the proximal tubule of Necturus kidney, by electrophysiological techniques. The main findings are: (1) In the presence of sodium, d-glucose produces $\text{10.5}\text{mV}\text{± 1.1}$ (S.E.) depolarization. (2) Phlorizin reduces the magnitude of this response to $\text{2.1 ± 0.1}\text{mV}$. (3) The glucose-evoked depolarization, $\text{ΔV}{}_{\mathrm{<mtext>G</mtext>}}$, does not alter the intracellular K⁺ activity nor is it affected by peritubular addition of ouabain. (4) Isosmotic reduction of Na⁺ concentration in luminal perfusate from 95 to 2 mmol/l (choline or Li⁺ substituting for Na⁺) does not change the magnitude of $\text{ΔV}{}_{\mathrm{<mtext>G</mtext>}}$; complete removal of sodium from the lumen lowers the value of $\text{ΔV}{}_{\mathrm{<mtext>G</mtext>}}$ ($\text{3.2 ± 0.2}\text{mV}$) but the response is not abolished. This observation suggests that the d-glucose carrier of renal tubules in Necturus is poorly specific with regard to the cotransported cation species.     

Anomerization of glucose 6-phosphate: catalysis by phosphoglucose isomerase.

The anomerase (1-epimerase) activity of phosphoglucose isomerase (d-glucose 6-phosphate ketol-isomerase EC 5.3.1.9) has been studied. The pH-V_max profile, assayed by two different methods, shows a dependence on two ionizable groups in the enzyme with pK values of 7.0 and 9.3 at 0 °C. Additionally, an unusual reversal of the basic leg of the normal profile to yield a large increase in V_max is observed above pH 9.5. Deuterium solvent isotope effects of are observed for isomerase and anomerase activities respectively. An anomerase mechanism similar to noncatalyzed anomerization is postulated with a discussion of the catalytic groups involved.     

The one-electron transfer redox potentials of free radicals. I. The oxygen/superoxide system

The method of determination of Redox potentials of radicals, using the pulse radiolysis technique, is outlined. The method is based on the determination of equilibrium constants of electron transfer reactions between the radicals and appropriate acceptors. The limitations of this technique are discussed.The redox potentials of several quinones-semiquinones are calculated, as well as the standard redox potential of the peroxy radical. and the redox oxidation properties of the peroxy radical in various systems and pH are discussed. The value determined for the redox potentials of $\text{O}{}_2\text{O}{}_2{}^{\mathrm{?}}$ is higher by more than 0.2 V than earlier estimates, which has important implications on the possible role of O₂^? in biological processes of O₂ fixation.     

Pseudoperoxidase activity of mushroom tyrosinase

Under anaerobic conditions, ethyl hydroperoxide functions as a two-electron acceptor in the tyrosinase-catalyzed oxidation of 4-tert-butylcatechol to 4-tert-butyl-o-benzoquinone, apparently by the following mechanism:

$\text{T?[Cu(II)]}{}_2\text{+ TBC = T?[Cu(I)]}{}_2\text{+ TB?}\text{o?}\text{BQ + 2H}{}^{\mathrm{+}}$

$\text{T?[Cu(I)]}{}_2\text{+ EtOOH + 2H}{}^{\mathrm{+}}\text{= T?[Cu(II)]}{}_2\text{+ EtOH +H}{}^2\text{O}$

This is a direct demonstration of the pseudoperoxidase activity of tyrosinase. Ethyl hydroperoxide failed to oxidize either oxy- or deoxyhemocyanin.     

Electrophoretic measurements about the relation between transition voltage and ζ-potential of biological membranes

The effects of inorganic cations, $\text{n-}\text{hexanol}$, saccharose and ²H₂O on the electrophoretic mobility and ζ-potential of membrane vesicles from nerve myelin were measured and the results compared with the corresponding effects of the same reagents on the transition voltage, $\text{V}{}_{\mathrm{<mtext>Tr</mtext>}}$, of the nerve axon membrane. Different cation concentrations and ²H₂O affect both potentials, the ζ-potential and $\text{V}{}_{\mathrm{<mtext>Tr</mtext>}}$, in a parallel way. Saccharose and $\text{n-}\text{hexanol}$, however, shift $\text{V}{}_{\mathrm{<mtext>Tr</mtext>}}$ but leave the electrophoretic mobility of the myelin vesicles unchanged. These results suggest that $\text{V}{}_{\mathrm{<mtext>Tr</mtext>}}$ shifts are not necessarily linked to changes in the membrane surface charge density but may also be caused by an interaction between the reagent and non-polar groups of the membrane interior.