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.     

The pH dependence of peptide hydrolysis by nitrocarboxypeptidase A

Hydrolysis of benzyloxycarbonyl-GlyGlyPhe by nitro(Tyr 248)carboxypeptidase A over the pH range 4.88–8.04 has been examined. The nitroenzyme retains appreciable activity near pH 6.5, and the limiting value of K_m is scarcely affected. The peptidase activity has a pH dependence characterized by the following parameters: pK_E1 of 6.37 ± 0.19 and pK_E2 of 6.60 ± 0.17 in $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$, and apparent pK of 5.59 ± 0.06 in K_cat. A spectroscopic pK of 6.75 ± 0.01, attributable to the nitro-Tyr 248 residue, has been determined. This correlates with the base-limb pK_E2 in the $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ profile, which appears to be shifted from a higher value, pK_E2 of 9.0, for the native enzyme. The single (acid-limb) pK which characterizes the k_cat profile of the native enzyme is also found to be perturbed to a lesser extent by nitration. A kinetically competent reverse protonation mechanism, based on chemical modification and crystallographic evidence for the enzyme, is described.     

The explicit analysis of consecutive pseudo-first-order reactions: Application to kinetics of thiolysis of dithiasuccinoyl (Dts) amino acids

An explicit set of general methods for the experimental determination of the rates k₁ and k₂ of consecutive pseudo-first-order reactions is described and discussed. These rely on the direct simultaneous analytical quantitation of the starting material, intermediate, and product of the reaction, and thus differ from present techniques based on measurement of coreactant consumption or coproduct appearance. The quantity $\text{k}{}^{\mathrm{<mtext>env</mtext>}}\text{=}\text{k}{}_1\text{k}{}_2\text{(k}{}_1\text{+ k}{}_2\text{)}$ is shown to define a good “envelope” approximation to product formation according to the simple law 100% [1 ? exp(?k^envt)]. The theory of envelopes is useful for comparing overall rates of reactions with widely differing values of $\text{κ =}\text{k}{}_2\text{k}{}_1$. The kinetic pattern of thiolysis of dithiasuccinoyl amino acids to carbamoyl disulfide intermediates to product free amino acids is analyzed and shown to agree quantitatively with theory.     

Presteady-state kinetic study of the elementary processes in the chymotrypsin-catalyzed hydrolysis of specific ester substrate. Rate-limiting association process due to the secondary binding.

Presteady-state kinetic studies of α-chymotrypsin-catalyzed hydrolysis of a specific chromophoric substrate, N-(2-furyl)acryloyl-l-tryptophan methyl ester, were performed by using a stopped-flow apparatus both under [E]₀ ? [S]₀ and [S]₀ ? [E]₀ conditions in the pH range of 5–9, at 25 °C. The results were accounted for in terms of the three-step mechanism involving enzyme-substrate complex (E · S) and acylated enzyme (ES′); no other intermediate was observed. This substrate was shown to react very efficiently, i.e., the maximum of the second-order acylation rate constant ($\text{k}{}_2\text{K}{}_{\mathrm{s}}\text{′}\text{)}{}_{\mathrm{<mtext>max</mtext>}}\text{= 4.2 × 10}{}^7\text{M}{}^{\mathrm{?1}}\text{s}{}^{\mathrm{?1}}$. The limiting values of K_s′ (dissociation constant of E · S), K₂ (acylation rate) and k₃ (deacylation rate) were obtained from the pH profiles of these parameters to be 0.6 ± 0.2 × 10^?5 m, 360 ± 15 s^?1 and 29.3 ± 0.8 s^?1, respectively. Likewise small values were observed for K_i of N-(2-furyl)-acryloyl-l-tryptophan and N-(2-furyl)acryloyl-d-tryptophan methyl ester and K_m of N-(2-furyl)acryloyl-l-tryptophan amide. The strong affinities observed may be due to intense interaction of β-(2-furyl)acryloyl group with a secondary binding site of the enzyme. This interaction led to a $\text{k}{}_{\mathrm{?1}}\text{k}{}_2$ value lower than unity, i.e., the rate-limiting process of the acylation was the association, even with the relatively low k₂ value of this methyl ester substrate, compared to those proposed for labile p-nitrophenyl esters.     

On the mechanism of the reaction of tris(hydroxymethyl)aminomethane with activated carbonyl compounds: A model for the serine proteinases

The pH dependence of the reaction of tris(hydroxymethyl)aminomethane (Tris) with the activated carbonyl compound 4-trans-benzylidene-2-phenyloxazolin-5-one (I) is given by the equation $\text{k′}{}_2\text{=}\text{k}{}_{\mathrm{<mtext>b</mtext>}}\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{(K}{}_{\mathrm{<mtext>a</mtext>}}\text{+ [}\text{H}{}^{\mathrm{+}}\text{])}\text{+}\text{k}{}_{\mathrm{<mtext>a</mtext>}}\text{[}\text{OH}{}^{\mathrm{?}}\text{]K}{}_{\mathrm{<mtext>a</mtext>}}\text{(K}{}_{\mathrm{<mtext>a</mtext>}}\text{+ [}\text{H}{}^{\mathrm{+}}\text{])}$, where K_a is the dissociation constant of TrisH⁺. Spectrophotometric experiments show that the Tris ester of α-benzamido-trans-cinnamic acid is formed quantitatively over a range of pH values, regardless of the relative contribution of k_b and k_a terms to k′₂. Hence, both terms refer to alcoholysis. While the mechanism of the reaction is not determined unequivocally in the present work, the magnitude of the k_b term, together with its dependence on the basic form of Tris, suggests that ester formation is occurring by nucleophilic attack of a Tris hydroxyl group on the carbonyl carbon of the oxazolinone, with intramolecular catalysis by the Tris amino group. The rate enhancement due to this group is at least 10² and possibly of the order 10⁶. This system is compared with other model systems for the acylation step of catalysis by serine esterases and proteinases.     

Mechanism of action of thrombin on fibrinogen. The reaction of thrombin with fibrinogen-like peptides containing 11, 14, and 16 residues

The following peptides were synthesized by classical methods in solution: Ac-Gly-Gly- Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-NHCH₃ (A), Ac-Ala-Glu-Gly-Gly-Gly-Val- Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-NHCH₃ (B), and Ac-Phe-Leu-Ala-Glu-Gly-Gly- Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-NHCH₃ (C). The rates of hydrolysis of the Arg-Gly bond of these three peptides by thrombin were measured, and the values of $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ were found to be 0.05 × 10^?7 (A), 0.02 × 10^?7 (B), and 1.6 × 10^?7 (C) [(NIH units/ liter)s]^?1. The value of$\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ for peptide C is less than 1% of that for fibrinogen [although the value of k_cat itself, for peptide C (but not for A or B), is comparable to that for fibrinogen]. These results indicate that phenylanine and leucine at positions P₉ and P₈, respectively, play a key role in the reaction of thrombin with fibrinogen. The data also show that factors outside of the 16 residues of peptide C are important in determining the rate of hydrolysis of fibrogen by thrombin.     

Isolation and characterization of isocitrate lyase of castor endosperm

Isocitrate lyase (threo-D_S-isocitrate glyoxylate-lyase, EC 4.1.3.1) has been purified to homogeneity from castor endosperm. The enzyme is a tetrameric protein (molecular weight about 140,000; gel filtration) made up of apparently identical monomers (subunit molecular weight about 35,000; gel electrophoresis in the presence of sodium dodecyl sulfate). Thermal inactivation of purified enzyme at 40 and 45 °C shows a fast and a slow phase, each accounting for half of the intitial activity, consistent with the equation: , where A₀ and A_t are activities at time zero at t, and k₁ and k₂ are first-order rate constants for the fast and slow phases, respectively. The enzyme shows optimum activity at pH 7.2–7.3. Effect of [S]on enzyme activity at different pH values (6.0–7.5) suggests that the proton behaves formally as an “uncompetitive inhibitor.” A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pK_a equal to 6.9. Successive dialysis against EDTA and phosphate buffer, pH 7.0, at 0 °C gives an enzymatically inactive protein. This protein shows kinetics of thermal inactivation identical to the untreated (native) enzyme. Full activity is restored on adding Mg²⁺ (5.0 mm) to a solution of this protein. Addition of Ba²⁺ or Mn²⁺ brings about partial recovery. Other metal ions are not effective.     

Organophosphorus compounds as active site-directed inhibitors of elastase

Four ethyl $\text{p}$-nitrophenyl alkylphosphonates were studied for the inhibition of elastase. A pH-dependence study using the assay substrate BOC-Ala-ONp or the phosphonate inhibitors showed the participation of an ionizing group with an apparent pK_a of 6.9 and a decrease of reaction or inhibition at higher pH. Out of the four compounds investigated ethyl $\text{p}$-nitrophenyl pentylphosphonate was found to be the best inhibitor of elastase as judged from the value of $\text{k}{}_2\text{K}{}_{\mathrm{<mtext>I</mtext>}}$. This value, which is the measure of inhibitory capacity, is the highest reported so far for the inhibition of elastase.     

Amino acid transport by the helicoidal colon of the new-born pig

The proximal colon of the new-born pig maintains a stable short-circuit current which is partly dependent upon the presence of methionine. This interaction between methionine and short circuit current shows Michaelis- Menten knetics with a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 0.24 mM and a $\text{V}$ of 27 μA·cm^?2. The net flux of methionine to the serosal surface of proximal colons also shows a hyperbolic relation to the external concentration of methionine ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{0.38}\text{mM}$; $\text{V 10.4}\text{nmol}\text{·}\text{cm}{}^{\mathrm{?2}}\text{·}\text{min}{}^{\mathrm{?1}}$). The proximal colon concentrates methionine within its epithelium giving a mucosal to medium ratio of $\text{11.2 ± 1.9}$ (90 min incubation in 1 mM methionine).The ability of the colon to transport methionine across and concentrate methionine within its mucosa is maintained for at least 24 h after birth. Colonic transport of amino acids could be physiologically important in the pig, where the immediate post-natal transfer of immune globulins has been shown to cause a temporary inhibition of normal intestinal function.     

Neutral amino acid transport in Leishmania promastigotes

Neutral amino acid transport was investigated in Leishmania promastigotes. Proline and alanine transport occur against their concentration gradient although there is a very rapid (40% at 30 min) conversion of proline to alanine. Uptake of these amino acids occurs by a sodium-independent route which is completely eliminated by addition of CCCP or KCN. $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for proline and alanine are 80 μM and 63 μM with $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ values of 6.4 and 7.2 nmol/min per mg dry weight, respectively. Countertransport of proline, alanine and phenylalanine was measured by loading the cells with a variety of neutral amino acids and proline analogs, followed by CCCP addition. The effect of aminooxyacetic acid, an inhibitor of alanine aminotransferase (EC 2.6.1.2), on proline and alanine countertransport was also examined. The results obtained are consistent with the presence of at least two systems for neutral amino acid transport in Leishmania promastigotes.     

Model of singlet oxygen scavenging by α-tocopherol in biomembranes

Quenching of singlet molecular oxygen (${}^1\text{Δ}{}_{\mathrm{g}}\text{O}{}_2$) by α-tocopherol (I) involves the hydroxy function of the chromanol ring of I. In phosphatidylcholine (PC) uni- and multilamellar vesicles this structural element of I is localized at the interface polar headgroup/hydrophobic core. A dielectric constant of ? ～ 25 was determined for this special region of the PC bilayer. The ratio k_Q/k_R of rate constants of quenching processes (k_Q) and irreversible reactions (k_R) of I with ${}^1\text{Δ}{}_{\mathrm{g}}\text{O}{}_2$ increases with decreasing polarity of the solvent. In ethanolic solutions where ? = 25.5, k_Q/k_R is about 40. Extrapolation of these results to phospholipid bilayers suggests that at the nearness of the ester carbonyl oxygen of the PC fatty acid moieties, α-tocopherol can deactivate approximately 40 ${}^1\text{Δ}{}_{\mathrm{g}}\text{O}{}_2$ molecules before being destroyed. It is concluded that in vivo, one may expect to find a higher k_Q/k_R ratio if the chromanol ring of I hides within the more hydrophobic interiors of the membrane surface peptides.     

Hymenolepis diminuta: The non-saturable component of methionine uptake

Lussier P. E., Podesta R. B. and Mettrick D. F. 1982. Hymenolepis diminuta: the non-saturable component of methionine uptake. International Journal for Parasitoiogy12: 265–270. The concentration dependence of in vitro unidirectional methionine influx by Hymenolepis diminuta was analysed by the relation: $\text{J =}\text{(J}{}_{\mathrm{<mtext>m</mtext>}}\text{C}{}_{\mathrm{<mtext>b</mtext>}}\text{)}\text{(K}{}_{\mathrm{t}}\text{+ C}{}_{\mathrm{<mtext>b</mtext>}}\text{)}\text{+ K}{}_{\mathrm{d}}\text{(C}{}_{\mathrm{<mtext>b</mtext>}}\text{)}$, where J_m is the maximum uptake rate, K_t is the the apparent affinity constant and C_b is the medium substrate concentration. The linear component was separated using an asymptotic least squares curve fitting procedure and the resulting constant, K_d, is thought to be an apparent permeability coefficient. K_d may be a reflection of a simple diffusive component, a second mediated component or a combination of a passive and mediated influx. The low Q₁₀ value of the K_d's for methionine uptake (Q₁₀ = 1.31) indicated that this component is probably a reflection of diffusion within the membrane. However, the decrease in the K_d component in the presence of leucine and glycine, implies that there is also a small, second, mediated component in addition to the diffusive component. K_d derived from the asymptotic portion of the concentration-flux relation was compared with the residual flux of methionine after near complete inhibition of the mediated component with leucine and glycine. The K_d component was found to be pH-sensitive, increasing as the pH decreased and was not affected by external sodium. Results indicate that the mediated component of methionine influx was accelerated by increasing external Na⁺ and H⁺ concentrations.     

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.     

Energization of sulfate transport in yeast

Sulfate uptake by Saccharomyces cerevisiae is stimulated about 12-fold by preincubation of cells with 1% d-glucose or 1% ethanol. The $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ remains unchanged (0.34–0.38 mM), the $\text{J}{}_{\mathrm{<mtext>mar</mtext>}}$ increase from 18–20 to 195–230 and 170–185 nmol/min per g dry wt., respectively, after glucose and ethanol preincubation. The stimulation involves protein synthesis (it is suppressed by cycloheximide), has a half-time of 18 min and requires mitochondrial respiration (no or low effect in respiration-deficient mutants and those lacking ADP-ATP transport in mitochondria, as well as after anaerobic preincubation of the wild-type strain, and in low-phosphate cells). The presence of NH₄⁺ and some amino acids (e.g., leucine, aspartate, cysteine and methionine) depressed the stimulation while that of cationic amino acids (typically arginine and lysine) and of K⁺ increased it by 50–80%. The stimulated (i.e., newly synthesized) transport system was degraded with a half-life of about 10 min.     

The composition-dependence of the extent of reaction between a multivalent acceptor and a bivalent ligand: Implications of self-interaction of the ligand in precipitin effects

Theory is presented relating to the reversible interaction of an f-valent acceptor, A, with a bivalent ligand, B, which leads to the formation of a series of complexes comprising networks of alternating A and B molecules. An explicit expression is derived for the overall extent of reaction in terms of the total molar concentrations of reactants $\text{(}\text{m}{}_{\mathrm{A}}$ and $\text{m}{}_{\mathrm{B}}\text{)}$, the valency of the acceptor and the site-binding constant, k, governing the equilibria. It is shown by differentiation of this expression holding $\text{m}{}_{\mathrm{A}}$ (or $\text{m}{}_{\mathrm{B}}$) fixed that relations are available for the independent evaluation of f and k from a combination of precipitin and radioimmunoassay experiments. Moreover, it is established that dilution with solvent ($\text{m}{}_{\mathrm{A}}\text{/}\text{m}{}_{\mathrm{B}}$ fixed) cannot lead to the appearance of a precipitate with this type of crosslinking system. The latter observation forms the background for the development of theory pertaining to the joint operation of ligand dimerization, 2B?B₂, and crosslinking of the multivalent acceptor with bivalent B₂. The theoretical examination of this system is developed in terms of site-probability functions and involves the delineation of unique solutions for the extent of crosslinking reaction aided by the definition of the extent of binding in defined limits. It is shown with the use of numerical examples that the system involving self-associating ligand may result in the appearance of a precipitate on dilution with solvent and the conditions for the operation of this phenomenon are elucidated. It is noted that other types of ligand self-interaction may lead to similar effects in crosslinking systems, and the general principles emerging from this study are discussed in terms of systems in which antibody ligands are known to be involved in association reactions or are suspected to be so involved on the basis of precipitation effects observed on dilution with solvent.     

A theory of dynamic and steady responses in chemoreception

A theoretical model which can account for both the dynamic and steady responses is proposed based on the occupation theory. The reaction scheme used is;

Here, S and A are stimulus chemicals and receptor sites unbound, respectively. The binding of S to A leads to an active complex (SA)_active, which is successively transformed into an inactive complex (SA)_active. The response is assumed to be proportional to number of (SA)_active. When a stimulating solution is applied instantaneously at t = 0, the solution to the set of differential equations based on the above scheme is obtained as follows;

where p and C stand for the fraction of (SA)_active to the total number of receptor sites and stimulus concentration, respectively, and α_i, and ω_i (i = 1, 2) are numerical parameters depending on the rate constants and on C. The steady response is expressed as the third term in the above equation, which indicates that the response accords with the Beidler taste equation. Mathematical analysis of the above scheme shows that the dynamic response appears when k₁C > k_?2, and the calculated results for the dynamic response agree approximately with the Hill equation. The Hill coefficient lays within 1·00 and 0·79 and reaches unity with increasing $\text{k}{}_{\mathrm{?1}}\text{k}{}_2$, which implies that the dynamic response under this condition satisfies the Beidler taste equation. For the case of gradual application of stimuli, i.e. the experimental condition, the time course of p is simulated with use of an analogue computer rather than with a numerical solution to the above equation. The results indicate that the dynamic response diminishes with decreasing the application speed of stimulus solution. The present theory accounts consistently for various experimental data observed in the chemoreceptor systems.     

Comparative studies on human carboxypeptidases B and N

A series of dicarboxylic acid bi-product analogs of lysine and arginine have been tested as competitive inhibitors of human pancreatic carboxypeptidase B and human plasma carboxypeptidase N. The most effective derivative was guanidinoethylmercaptosuccinic acid with K_is of 0.5 and 1.0 × 10^?6m for Carboxypeptidases B and N, respectively. Values for the all-carbon guanidinopropylsuccinic acid were similar. In addition the kinetic parameters, K_m and $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$, have been determined for the hydrolysis of benzoyl-alanyl-lysine and benzoylalanyl-arginine by human Carboxypeptidases B and N. These substrates have been proposed for use in improved spectrophotometric assays. An enhanced affinity of these substrates versus benzoyl-glycyl-lysine or benzoyl-glycyl-arginine indicates a significant participation of the penultimate amino acid in catalysis of substrate.