Cooperativity and effects of ionic strength on the binding of sodium n-dodecyl sulphate to lysozyme

The binding of sodium n-dodecyl sulphate to lysozyme has been measured by equilibrium dialysis at 25°C and pH 3.2 over a range of ionic strengts from 0.0119 to 0.2119. Binding isotherms in the region corresponding to ionic binding between the surfactant anions and cationic amino acid residues on the protein have been interpreted in terms of the Hill equation and exhibit positive cooperativity with Hill coefficients in the region of 7–11. The Gibbs energies of binding have been calculated from the Hill binding constants and from the Wyman binding potentials. The stability of the surfactant-protein complexes is discussed in relation to the stability of surfactant micelles. Ionic binding of the surfactant is weakened and hydrophobic binding strengthened by increasing ionic strength.     

Phloem unloading and ‘sink strength’: The parallel between the site of attachment of Cuscuta and developing legume seeds

Sink regions play a central role in determining assimilate distribution patterns. Two factors are discussed which have a strong effect on the sink strength of a sink, viz. phloem unloading and turgor-sensitive transport. Sink strength may be defined as the capacity of phloem in the sink region to import assimilates from other parts of the plants and to release the imported substances into the sink apoplast.A stem parasitized by Cuscuta represents a very strong sink. A review is presented of data on enhanced phloem unloading, at the site of attachment of Custuta. Recent data on metabolically controlled sucrose and amino acid unloading into the seed coat apoplast of developing legume seeds show a remarkable parallel with phloem unloading in a parasitized Vicia faba stem. Data on turgor-sensitive sucrose and amino acid transport into developing seeds are presented, which throw new light on the pressure flow theory of phloem transport.     

The oxidation-reduction kinetics of the reaction of cytochrome c1 with non-physiological redox agents

The kinetics of the oxidation-reduction reactions of cytochrome c₁ with ascorbate, ferricyanide, triphenanthrolinecobalt(III) and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) have been examined using the stopped-flow technique. The reduction of ferricytochrome c₁ by ascorbic acid is investigated as a function of pH. It is shown that at neutral and alkaline pH the reduction of the protein is mainly performed by the doubly deprotonated form of ascorbate. From the ionic-strength-dependence studies of the reactions of cytochrome c₁ with ascorbate, ferricyanide and triphenanthrolinecobalt(III), it is demonstrated that the reaction rate is governed by electrostatic interactions. The second-order rate constants for the reaction of cytochrome c₁ with ascorbate, ferricyanide, TMPD and triphenanthrolinecobalt(III) are 1.4·10⁴, 3.2·10³, 3.8·10⁴ and 1.3·10⁸ M^?1·s^?1 (pH 7.0, I = 0, 10°C), respectively. Application of the Debye-Hückel theory to the the ionic-strength-dependence studies of these redox reactions of cytochrome c₁ yielded for ferrocytochrome c₁ and ferricytochrome c₁ a net charge of ?5 and ?4, respectively. The latter value is close to that of ?3 for the oxidized enzyme, calculated from the amino acid sequence of the protein. This implies that not a local charge on the surface of the protein, but the overall net charge of cytochrome c₁ governs the reaction rate with small redox molecules.     

Anion and ionic strength effects upon the oxidation of cytochrome c by cytochrome c oxidase

Citrate and other polyanion binding to ferricytochrome c partially blocks reduction by ascorbate, but at constant ionic strength the citrate-cytochrome c complex remains reducible; reduction by TMPD is unaffected. At a constant high ionic strength citrate inhibits the cytochrome c oxidase reaction competitively with respect to cytochrome c, indicating that ferrocytochrome c also binds citrate, and that the citrateferrocytochrome c complex is rejected by the binding site at high ionic strength. At lower ionic strengths, citrate and other polyanions change the kinetic pattern of ferrocytochrome c oxidation from first-order towards zero-order, indicating preferential binding of the ferric species, followed by its exclusion from the binding site. The turnover at low cytochrome c concentrations is diminished by citrate but not the K_m (apparent non-competitive inhibition) or the rate of cytochrome a reduction by bound cytochrome c. Small effects of anions are seen in direct measurements of binding to the primary site on the enzyme, and larger effects upon secondary site binding. It is concluded that anion-cytochrome c complexes may be catalytically competent but that the redox potentials and/or intramolecular behaviour of such complexes may be affected when enzyme-bound. Increasing ionic strength diminishes cytochrome c binding not only by decreasing the ‘association’ rate but also by increasing the ‘dissociation’ rate for bound cytochrome c converting the ‘primary’ (T) site at high salt concentrations into a site similar kinetically to the ‘secondary’ (L) site at low ionic strength. A finite K_m of 170 μM at very high ionic strength indicates a ratio of $\text{K}{}^{\mathrm{\infty }}{}_{\mathrm{<mtext>M</mtext>}}\text{K}{}^0{}_{\mathrm{<mtext>M</mtext>}}$ of about 5000. It is proposed that anions either modify the E¹₀ of cytochrome c bound at the primary (T) site or that they perturb an equilibrium between two forms of bound c in favour of a less active form.     

Influence of root diameter on the penetration of seminal roots into a compacted subsoil

A field experiment was conducted to evaluate the influence of root diameter on the ability of roots of eight plant species to penetrate a compacted subsoil below a tilled layer. The soil was a fine sandy loam red-brown earth with a soil strength of about 3.0 MPa (at water content of 0.13 kg kg^-1, corresponding to 0.81 plastic limit) at the base of a tilled layer. Relative root diameter (RRD), which was calculated as the ratio of the mean diameters of roots of plants grown in compacted soil to the mean diameters of those from uncompacted soil, was used to compare the sensitivity of roots to thicken under mechanical stress.Diameters of root tips of plants grown in soil with a compacted layer were consistently larger than those from uncompacted soil. Tap-rooted species generally had bigger diameters and RRDs than fibrous-rooted species. A higher proportion of thicker roots penetrated the strong layer at the interface than thinner roots. There were differences between plant species in the extent to which root diameter increased in response to the compaction. The roots which had larger RRD also tended to have higher penetration percentage.The results suggest that the size of a root has a significant influence on its ability to penetrate strong soil layers. It is suggested that this could be related to the effects which root diameter may have on root growth pressure and on the mode of soil deformation during penetration.     

Year-class strength regulation in plaice (Pleuronectes platessa L.) on the Swedish west coast

Year-class strength of plaice (Pleuronectes platessa) has been studied over periods of 10 and 35 years, respectively, in two shallow sandy areas on the Swedish west coast. In one area, Gullmar Bay (N 58 ° 19 – W 11 ° 33), 0-group plaice were sampled quantitatively with a drop trap at 0–0.7 m depth and densities between 0.2 and 3.8 ind./m² were recorded in early summer. In the other area, Laholm Bay (N 56 ° 30 – W 12 ° 55), 0-group plaice were sampled semi-quantitatively in August with young-fish trawl at 1.5 m depths. Densities between 0.001 and 0.28 ind./m² were assessed.The effects of temperature and wind (in the winter and early spring) and predation (in early summer), on the recruitment of 0-group plaice were investigated. In Gullmar Bay high recruitment occurred after severe winters and in years when on-shore winds dominated during spring. No such correlations were found in Laholm Bay. In Gullmar Bay a significant inverse relationship was found between the density of O-group plaice in early summer and the biomass of brown shrimp (Crangon crangon) and the shore crab (Carcinus maenas), the main predators in the nursery area.The importance of physical and biological factors regulating recruitment in plaice are discussed.     

Studies on the yield and gel strength of agar from Gracilaria domingensis Sonder ex Kuetzing (Gracilariales,Rhodophyta) following the addition of calcium

Studies were carried out on the seasonal variation in yield and gel strength of agar from Gacilaria domingensis with and without the addition of calcium chloride. Extraction was done with and without treatment with 1% hydrochloric acid. The results showed an increase in yield and gel strength when an alkaline solution of calcium was used, but the gel strength was low. For commercial use, Gracilaria domingensis should be mixed with better quality Gracilaria species because of its low gel strength.     

Ionic strength effects on cytochrome aa3 kinetics

1. The occurrence of an optimal ionic strength for the steady-state activity of isolated cytochrome aa₃ can be attributed to two opposite effects: upon lowering of the ionic strength the affinity between cytochrome c and cytochrome aa₃ increases, whereas in the lower ionic strength region the formation of a less active cytochrome c-aa₃ complex limits the ferrocytochrome c association to the low affinity site.2. At low ionic strength, the reduction of cytochrome c-aa₃ complex by ferrocytochrome c₁ proceeds via non-complex-bound cytochrome c. Under these conditions the positively charged cytochrome c provides the electron transfer between the negatively charged cytochromes c₁ and aa₃.3. Polylysine is found to stimulate the release of tightly bound cytochrome c from the cytochrome c-aa₃ complex. This property points to the existence of negative cooperativity between the two binding sites. We suggest that the stimulation is not restricted to polylysine, but also occurs with cytochrome c.4. Dissociation rates of both high and low affinity sites on cytochrome aa₃ were determined indirectly. The dissociation constants, calculated on the basis of pre-steady-state reaction rates at an ionic strength of 8.8 mM, were estimated to be 0.6 nM and 20 μM for the high and low affinity site, respectively.     

The reaction between cytochrome C1 and cytochrome C

The kinetics of electron transfer between the isolated enzymes of cytochrome c₁ and cytochrome c have been investigated using the stopped-flow technique. The reaction between ferrocytochrome c₁ and ferricytochrome c is fast; the second-order rate constant (k₁) is 3.0 · 10⁷ M^?1 · s^?1 at low ionic strength (I = 223 mM, 10°C). The value of this rate constant decreases to 1.8 · 10⁵ M^?1 · s^?1 upon increasing the ionic strength to 1.13 M. The ionic strength dependence of the electron transfer between cytochrome c₁ and cytochrome c implies the involvement of electrostatic interactions in the reaction between both cytochromes. In addition to a general influence of ionic strength, specific anion effects are found for phosphate, chloride and morpholinosulphonate. These anions appear to inhibit the reaction between cytochrome c₁ and cytochrome c by binding of these anions to the cytochrome c molecule. Such a phenomenon is not observed for cacodylate. At an ionic strength of 1.02 M, the second-order rate constants for the reaction between ferrocytochrome c₁ and ferricytochrome c and the reverse reaction are k₁ = 2.4 · 10⁵ M^?1 · s^?1 and k_?1 = 3.3 · 10⁵ M^?1 · s^?1, respectively (450 mM potassium phosphate, pH 7.0, 1% Tween 20, 10°C). The ‘equilibrium’ constant calculated from the rate constants (0.73) is equal to the constant determined from equilibrium studies. Moreover, it is shown that at this ionic strength, the concentrations of intermediary complexes are very low and that the value of the equilibrium constant is independent of ionic strength. These data can be fitted into the following simple reaction scheme: cytochrome c²⁺₁ + cytochrome c³⁺ai cytochrome c³⁺₁ + cytochrome c²⁺.