Kinetic alpha-deuterium isotope effects for enzymatic and nonenzymatic hydrolysis of nicotinamide-beta-riboside.

The kinetic α-secondary deuterium isotope effect, $\text{k}{}_{\mathrm{H}}\text{k}{}_{\mathrm{D}}$, for the pH-independent hydrolysis of nicotinamide riboside, yielding nicotinamide and ribose, in water at 25 ° is 1.14, establishing that this reaction proceeds with unimolecular substrate decomposition to yield a carboxonium ion, or related species, in the rate-determining step. Surprisingly, the corresponding isotope effect for the base-catalyzed decomposition of the same substrate is 1.12, a value indicating considerable sp² character at the Cl′ position in the transition state for this reaction. A similar result, $\text{k}{}_{\mathrm{H}}\text{k}{}_{\mathrm{D}}\text{= 1.15}$, was obtained for base-catalyzed hydrolysis of NAD⁺. The kinetic alpha deuterium isotope effect for the pig brain NAD glycohydrolasecatalyzed hydrolysis of nicotinamide riboside is 1.08. This value suggests that CN bond cleavage to form an intermediate carboxonium ion, or structurally related species, is at least partially rate-determining. In contrast, the corresponding value for the hydrolysis of this substrate catalyzed by Escherichia coli nicotinamide ribonucleotide glycohydrolase is very near unity, a result consistent with several interpretations including a rate-determining enzyme isomerization reaction.     

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.     

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.     

Alteration of the conductance of Na+ channels in the nodal membrane of frog nerve by holding potential and tetrodotoxin

(1) Na⁺ currents and Na⁺-current fluctuations were measured in myelinated frog nerve fibres at 15°C during 7.7 ms depolarizations to $\text{V = 40, 60}\text{and}\text{80}\text{mV}$. (2) The conductance γ of a single Na⁺ channel and the number $\text{N}{}_0$ of channels per node were calculated from ensemble average values of the mean Na⁺ current and the variance of Na⁺-current fluctuations. (3) For a hyperpolarizing holding potential of $\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{= ?28}\text{mV}$ the mean values of the channel conductance and number were $\text{γ = 9.8}\text{pS}$ and $\text{N}{}_0\text{= 74 000}$. (4) After changing the holding potential to the resting potential ($\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{= 0}$) the conductance γ increased by a factor of 1.37 whereas the number $\text{N}{}_0$ decreased by a factor of 0.60. (5) Addition of 8 nM tetrodotoxin at a holding potential of $\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{= ?28}\text{mV}$ increased γ by a factor of 1.55 and reduced $\text{N}{}_0$ by a factor of 0.25. (6) The increase of the channel conductance at reduced channel numbers suggests negative cooperativity between Na⁺ channels in the nodal membrane.     

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.     

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.     

Mechanistic studies of epoxide hydrolase utilizing a continuous spectophotometric assay

A precise continuous photometric assay has been devised and utilized for mechanistic studies of chicken and rat liver microsomal epoxide hydrolase (EH). The assay is based on monitoring the hydration of p-nitrostyrene oxide (PNSO) at 310 nm. Rat liver EH hydrates S-(+)- and R-(?)-PNSO differentially, the K_m and V values for the former being ca. four times those for the latter; in contrast, enantiomeric differences are negligible with chicken liver EH. With rat EH V increases slightly from pH 7 to 8 and then falls rapidly from pH 8 to 9.5; K_m remains constant from pH 7 to 8 and then increases steadily from pH 8 to 9.5. In 86 mol% D₂O the solvent isotope effect on V ($\text{H}{}_2\text{O}\text{D}{}_2\text{O}$) is 1.103 ± 0.015. Both rat and chicken EH show a 3% inverse isotope effect for the hydration of [7-²H]PNSO and a 4% normal isotope effect for the hydration of [8-²H₂]PNSO. These observations are discussed in terms of the possible participation of acid as well as base catalysis in the enzymatic mechanism.     

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.     

Effect of N-ethylmaleimide on leucine transport in the chang liver cell. II. Effect on the kinetics of Na+-independent transport

The Na⁺-independent leucine transport system is resolved into two components by their different affinity ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ about 44 μM and 8.0 mM) for leucine in the Chang liver cell. Treatment of the cells with $\text{N-}\text{ethylmaleimide}$ (1 mM) specifically stimulates the high-affinity component of the Na⁺-independent system by greatly increasing its $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ value, whereas the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ value of the low-affinity component is markedly lowered. The stimulatory effect of $\text{N-}\text{ethylmaleimide}$ on leucine transport is reduced by prior treatment of the cells with 2,4-dinitrophenol, but this phenomenon seems to be irrelevant to the ATP-depleting action of the uncoupler. The treatment with 2,4-dinitrophenol has been found not to be inhibitory on the subsequent Na⁺-independent leucine uptake itself. Treatment with dibucaine, a phospholipid-interacting drug, also reduces to varying degrees (depending on its concentration) the stimulatory effect of $\text{N-}\text{ethylmaleimide}$ on the subsequent leucine uptake, although pretreatment with dibucaine can stimulate the Na⁺-independent leucine uptake itself. We conclude that the stimulatory effect of $\text{N-}\text{ethylmaleimide}$ on leucine transport is not correlated with the energy level of cell, but involves the perturbation of the membrane bilayer structures.     

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.     

Equilibrium constants for association of guanidinium and ammonium ions with oxyanions: The effect of changing basicity of the oxyanion

Using guanidinium and n-butylammonium cations (C⁺) as models for the positively charged side chains in arginine and lysine, we have determined the association constants with various oxyanions by potentiometric titration. For a dibasic acid, H₂A, three association complexes may exist: $\text{K}{}_{\mathrm{<mtext>1M</mtext>}}\text{=}\text{[CHA]}\text{[C}{}^{\mathrm{+}}\text{]}\text{[HA}{}^{\mathrm{?}}\text{]}$; $\text{K}{}_{\mathrm{<mtext>1D</mtext>}}\text{=}\text{[CA}{}^{\mathrm{?}}\text{]}\text{[C}{}^{\mathrm{+}}\text{]}\text{[A}{}^{\mathrm{2?}}\text{]}$; $\text{K}{}_{\mathrm{<mtext>2D</mtext>}}\text{=}\text{[C}{}_2\text{A]}\text{[C}{}^{\mathrm{+}}\text{]}\text{[CA}{}^{\mathrm{?}}\text{]}$. For guanidinium ion and phosphate, K_1M = 1.4, K_1D = 2.6, and K_2D = 5.1. The data for carboxylates indicate that the basicity of the oxyanion does not affect the association constant: acetate, pK_a = 4.8, K_1M = 0.37; formate, pK_a = 3.8, K_1M = 0.32; and chloroacetate, pK_a = 2.9, K_1M = 0.43, all with guanidinium ion. Association constants are also reported for carbonate, dimethylphosphinate, benzylphosphonate, and adenylate anions.     

Flow analysis in a biological system adopting the buffer capacitor concept.

The flow measurement of each component in each compartment is important in works on transport phenomena in a biological system. The method of flow measurement was studied adopting the capacitor concept derived from network thermodynamics.A biologically active component i in a compartment is defined as follows,

$\text{n}{}_{\mathrm{i}}\text{=n}{}_1\text{+n}{}_2\text{=c}{}_1\text{V+c}{}_2\text{V}$

where the total quantity ni consists of a measurable form n_i (free form, conc. c₁) and concealed form n₂ (conc, c₂). Capacitor for the species i of a compartment is defined as follows,

$\text{C=}\text{d}\text{n}{}_{\mathrm{i}}\text{d}\text{μ}{}_{\mathrm{i}}\text{=}\text{1+}\text{c}{}_2\text{c}{}_1\text{c}{}_1\text{dV}\text{dμ}{}_{\mathrm{i}}\text{+}\text{1+}\text{dc}{}_2\text{dc}{}_1\text{v}\text{dc}{}_1\text{dμ}{}_{\mathrm{i}}$

,

$\text{=Ac}{}_1\text{dV}\text{dμ}{}_{\mathrm{i}}\text{+BV}\text{d}\text{c}{}_1\text{d}\text{t}$

Thus flow of each component is expressed as,

$\text{J}{}_{\mathrm{i}}\text{=}\text{d}\text{n}{}_{\mathrm{i}}\text{d}{}_{\mathrm{i}}\text{=}\text{d}\text{n}{}_{\mathrm{i}}\text{dμ}{}_{\mathrm{i}}\text{dμ}\text{n}{}_{\mathrm{i}}\text{dt}\text{=C}\text{d}\text{μ}{}_{\mathrm{i}}\text{dt}$

,

$\text{=Ac}{}_1\text{d}\text{V}\text{dt}\text{+BV}\text{d}\text{c}{}_1\text{dt}$

Method of determination of capacitor coefficients A and B by titration experiment was also considered. For an experimental case, the capacitance for H⁺ of blood compartment was determined. The relationship between the capacitor concept and the buffer value of Van Slyke was discussed.     

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.     

Determination of the kinetic constants of fructose transport and phosphorylation in the perfused rat liver

The kinetics of fructose uptake was determined in perfused rat liver during steady-state fructose elimination. On the basis of the corresponding values of fructose concentration in the affluent and in the effluent medium, and the fructose and ATP concentration in biopsies, the kinetics of membrane transport and intracellular phosphorylation in the intact organ was calculated according to a model system. Carrier-mediated fructose transport has a high $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ (67 mM) and $\text{V}$ (30 μmoles · min^?1 ·g^?1). The calculated kinetic constants of the intracellular phosphorylation were compared with values obtained with an acid-treated rat liver high speed supernatant (values given in parentheses). $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ with fructose 1.0 mM (0.7 mM), $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ with ATP 0.54 mM (0.37 mM), $\text{V}$ 10.3 μmoles · min^?1 · g^?1 (10.1 μmoles · min^?1 · g^?1, calculated on the basis of the highest measured rate of fructose uptake correcting the ATP concentration to saturating values). The kinetics of fructose uptake reveals that at Physiological fructose concentrations the membrane transport limits the rate of fructose uptake, thus protecting the liver from severe depletion of adenine nucleotides.     

Double dependence of organic acid active transport in proximal tubules of surviving frog kidney on sodium ions I. Influence of sodium ions in bath medium on the uptake and run out of fluorescein and uranin

With the aid of direct microfluorimetric determination of marker organic anions (fluorescein and uranin) accumulated in the proximal tubules the influence of Na⁺ in the bath medium on the active transport of these anions was studied. Kinetic analysis of the rate dependence of organic acid active transport into tubules on their concentration in the bath medium with a constant Na⁺ concentration permitted to define values of apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ and $\text{V}$ for uranin and fluorescein transport in the medium with different Na⁺ content. It was shown that a decrease of Na⁺ concentration in the medium increases $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ and lowers the $\text{V/K}{}_{\mathrm{<mtext>m</mtext>}}$ ratio with uncharged $\text{V}$. By varying the Na⁺ concentration in the medium with a constant concentration of the marker anion the and values for fluorescein and uranin transport were determined. A value for fluorescein in twice as much that for uranin. The $\text{1/K}{}_{\mathrm{<mtext>m</mtext>}}$ value for uranin transport is a linear function of Na⁺ concentration, while for fluorescein transport it is a quadratic one. Therefore it is concluded that two Na⁺ from the medium participate in active transfer of one fluorescein anion whereas only one Na⁺ from the medium is required for active transfer of one uranin anion. The run out of fluorescein from tubules preloaded with this acid is sharply reinforced by the Na⁺ omission from the medium. Thus, active transport of organic acids in proximal tubules of frog kidney is Na⁺-dependent, and Na⁺ from the medium is likely to participate directly in formation of a transport complex. When Na⁺ is absent in the medium a carrier fulfils a facilitated diffusion only.     

Involvement of the proton electrochemical gradient in genetic transformation in Escherichia coli

Plasmid pIY2 DNA which encodes for ampicillin-resistance was used to study the energetics of Ca⁺⁺-induced transformation in Escherichia coli. When cells are exposed to DNA in the presence of carbonylcyanide-m-chlorophenylhydrazone or 2,4-dinitrophenol, two protonophores that collapse the proton electrochemical gradient across the cell membrane ($\text{Δ}\text{μ}{}_{\mathrm{H}}\text{+}$), transformation to ampicillin-resistance is drastically reduced with little or no effect on viability. Furthermore, when the components of $\text{Δ}\text{μ}{}_{\mathrm{H}}\text{+}$ are altered by varying ambient pH or by performing transformation in the presence of valinomycin or nigericin, the efficiency of transformation is directly correlated with the magnitude of the membrane potential and changes in the pH gradient have no significant effect. It is concluded that $\text{Δ}\text{μ}{}_{\mathrm{H}}\text{+}$, more specifically the membrane potential, plays a critical role in Ca⁺⁺-induced transformation.     

Elasnin,a new human granulocyte elastase inhibitor produced by a strain of Streptomyces

Elasnin, a new human granulocyte elastase inhibitor, has been isolated from $\text{Streptomyces}\text{noboritoensis}$ KM-2753. Elasnin is a neutral, lipophilic colorless and viscous oil ($\text{n}{}_{\mathrm{D}}{}^{17}\text{=1.4983}$, $\text{[α]}{}_{\mathrm{D}}{}^{18}\text{?0.9°}$, λ_max^EtOH 291 nm (ε, 7760)). The molecular formula was C₂₄H₄₀O₄ (M.W.: 392) as determined by its elemental analysis and mass spectrum. Elasnin inhibits markedly human granulocyte elastase, but is almost ineffective for pancreatic elastase, trypsin, chymotrypsin, thermolysin and papain.     

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. pH dependence and solvent isotope effects.

The anomerization of α-d-glucose 6-phosphate has been examined using a spectrophotometric coupled enzyme assay. The pH-rate profile for spontaneous d-glucose 6-phosphate anomerization reveals that the d-glucose 6-phosphate dianion is the species giving rise to the much higher rate of d-glucose 6-phosphate anomerization over that of d-glucose. A deuterium solvent isotope effect of is consistent with the postulated intramolecular general-base catalysis by the phosphate.