Metastable secondary structures in ribosomal RNA molecular hysteresis in the acid-base titration of Escherichia coli ribosomal RNA

The metastable conformational states which underlie the hysteresis displayed by Escherichia coli ribosomal RNA in its pH titration in the acid range have been analyzed in terms of acid-stable RNA secondary structures. Sedimentation measurements show that the phenomenon is intramolecular, so that analysis of the hysteresis loops can, in principle, reveal details of molecular architecture. Hysteresis cycles obtained spectrophotometrically and potentiometrically were compared for RNA in solutions of different ionic strengths and ionic compositions. The effect is much smaller at lower ionic strength and disappears in the absence of magnesium ions. The curve followed upon addition of acid appears to reflect the equilibrium state of the system at each pH value. On the “base branch” of the loop, a slow absorbance change (complete in hours) was observed after the pH was raised by addition of a portion of base. This slow process is attributed to the annealing of “mismatched” multihelical regions of the ribosomal RNA. Certain regions, however, remain in metastable configurations for days and it is these long-lived non-equilibrium structures that underlie the hysteresis. Titration at 35 °C gave hysteresis loops of the same size and shape as at 20 °C; indeed, we found that the metastabilities are not removed even at 80 °C. Ultraviolet light absorbance difference spectra at 80 °C between solutions at the same pH, but on different branches of the cycle, give insight into the nature of the metastable conformation(s).Our experimental observations lead us to propose that the hysteresis is due to the formation at acidic pH of double-helical structures involving protonated guanine and adenine base pairs. The G.G pairs seem especially important to account for the very high thermal stability, as well as for the fact that the structures formed at a given pH value as acid is added dissociate only at higher pH values when the solution is titrated with base. Titrations of transfer RNA, along with literature data on 16 S rRNA primary structure, imply that the metastable regions in rRNA may consist of perhaps 10 to 15 base pairs.     

Protonated polynucleotides structures - 23. The acid-base hysteresis of poly(dG).poly(dC).

The large hysteresis observed during the acid-base titration of poly(dG). poly (dC) was studied by CD and potentiometric scanning curves. Intermediate scanning loops as well as the equilibrium and metastable branches of the hysteresis loop have been determined. The potentiometric titrations showed, however, that the various complexes were not discrete entities, but were linked in "polycomplexes" as had been already suggested. This prevented a thermodynamic study of the system. The acid-base titration was further investigated as a function of ionic strength and temperature. The pK's showed considerably lower ionic strength dependence than observed for polyribonucleotide complexes. The thermal transitions permitted to establish the relative stabilities of the various complexes between pH 2.5 and pH 12.0.     

Studies on the assembly of a spherical plant virus. II. The mechanism of protein aggregation and virus swelling.

Potentiometric and spectrophotometric titrations have been undertaken to understand the mechanisms of control of the pH-dependent swelling of cowpea chlorotic mottle virus and of the formation of capsid by the viral protein. The results show a striking difference in the proton release by the virion and by the protein. Both the swelling of the virus and the capsid formation are accompanied by an anomalous proton release which shows a well-defined hysteresis in the pH range where these phenomena take place. The swelling is not accompanied by any major change of the u.v. absorption, but only by a small decrease (2% at 260 nm). Magnesium ions, which have a marked effect on the swelling, have no effect on the protein titration, but abolish the hysteresis and suppress the small decrease in absorbance associated with the swelling.The data suggest that two carboxyl groups with a pK near neutrality are responsible for controlling the swelling, and also probably control the capsid assembly. The effect of magnesium ions is presumably on the nucleic acid configuration and (or) the RNA-protein salt links.The existence of hysteresis, both in the formation of capsids from protein and in the swelling of the virus, is an interesting feature, showing the existence of a pathway through metastable states.     

Induction of mitochondrial contraction and concomitant inhibition of succinate oxidation by magnesium ions.

The addition of 1 Him MgCl₂ to partially swollen rat liver mitochondria respiring in an isoosmotic sucrose-sodium phosphate or sodium acetate medium containing 1 mm EDTA and 15 mm succinate (pH 7.2) initiates contraction, inhibits respiration, and alters the ultrastructural configuration of the inner membrane-cristae-matrix continuum. The maximal extent of contraction (A₅₂₀ increase) attainable with MgCl₂ in the phosphate medium is about 80% of the maximal contraction induced by 2,4-dinitrophenol but exceeds the maximal contraction induced with ADP by about 10%. The extent of mitochondrial contraction and the inhibition of succinate oxidation are dependent on MgCl₂ concentration. MgCl₂ at 1000 μm immediately inhibits about 70% of the succinate oxidation and initiates maximal extent of contraction concomitant with a distinct configurational change in ultrastructure which appears to differ from that initiated by either ADP or dinitrophenol. MgCl₂ at 150 μm does not inhibit the rate of succinate oxidation but it does initiate about 75% of the maximal contraction (A₅₂₀ increase) attained with 1000 μm MgCl₂. The rate of mitochondrial contraction is dependent on both the phosphate and MgCl₂ concentration. CaCl₂, in contrast to MgCl₂, immediately stimulates succinate oxidation and after a sharp contraction spike of short duration initiates additional expansion of the inner membrane-cristae-matrix continuum. The contraction spike occurs in a reaction system containing either phosphate or acetate. The results are consistent with the notion that Mg²⁺ and Ca²⁺ may modulate mitochondrial volume and exert control over certain oxidative processes.     

Protonated polynucleotide structures

The circular dichroism spectra of poly(dG). poly(dC) have been studied as a function of pH in 0.15 M NaCl solution. Acid titration to pH 2.5 showed two transitions, one around pH 5, the second below pH 3. These transitions are disproportionation reactions and are not due to the dissociation of the complexes. Alkaline back titration to neutrality showed only one step above pH 7, without appearance of the intermediate form. The hysteresis loop observed gave rise to a metastable (probably protonated) form at pH 7 which reverted to the neutral form upon heating or alkali treatment. In order to obtain again the metastable hysteretic form, a whole titration cycle to pH 2.5 had to be performed.Abbreviation CD circular dichroism Dedicated to Prof. A Janke (Vienna) on his 85th birthday.Part 13 is ref. [1].     

Hydrodynamic properties of 5.8S rRNA,salt and temperature effects

Sedimentation coefficients and apparent molecular masses of 5.8S rRNA from rat liver and yeast (Saccharomyces cerevisiae) depend considerably on the ionic strength and the kind of ions in solution. At 20°C the sedimentation coefficient of 5.8S rRNA in 10 mm sodium cacodylate, pH 7.0, amounts to 5.1 ± 0.2 S. By addition of NaCl up to 1.1 m the data increase reversibly to 6.1 ± 0.2 S (rat liver) or 5.4 ± 0.1 S (yeast) without significant changes of the molar mass (52 000 ± 2000) g/mol. Similar effects but with different extent were obtained using KCl or LiCl. These results can be explained by counterion effects on the conformation and changing of the water shell surrounding the RNA molecule. Short heat incubation (5 min at 65°C) and immediate cooling of rat liver 5.8S rRNA lead to dimer or oligomer formation. Its portions depend strongly on RNA concentration and are enhanced also with increasing NaCl concentration and incubation temperature as can be seen fro higher sedimentation coefficients and molecular masses as well as from additional bands in the electrophoretic pattern. At 20°C MgCl₂ provokes, in concentrations up to 1.5 mm, a reversible increase of sedimentation coefficients of rat liver 5.8S rRNA to 6.65 ± 0.1 S whereas the molecular mass remains unchanged indicating strong Mg⁺⁺ effects on conformation and/or water shell of the 5.8S rRNA. A further increase of sedimentation coefficients up to 8.2 ± 0.1 S combined with higher apparent molar masses up to 90 000 g/mol was observed in the presence of 30 to 50 mm MgCl₂. In this concentration range of Mg⁺⁺ the association constants of 5.8S rRNA dimerization increase from about $\text{10}{}^5\text{to}\text{3 × 10}{}^7\text{m}{}^{\mathrm{?1}}$. After removal of free Mg⁺⁺ by addition of EDTA the 5.8S rRNA dimers dissociate if no incubation step at higher temperature in involved. The Mg⁺⁺ induced 5.8S rRNA dimers differ in their stability from those formed by incubation at 65°C in the presence of higher concentrations of monovalent ions.     

Determination of Fatty Acid in Surfactin and Elucidation of the Total Structure of Surfactin

Several properties of ATPase bound to the inner membrane of a psychrophilic marine bacterium Vibrio sp. strain ABE-1 were examined. The membrane-bound ATPase had two optimal peaks of the activity at pH 5.8 and 7.3. The ATPase activity was strongly inhibited by N,N’- dicyclohexylcarbodiimide (DCCD) and NaN₃ at pH 5.8 and 8.0, and stimulated by MgCl₂ and CaCl₂ at pH 8.0. At pH 8.0, the enzyme hydrolyzed GTP and ITP as well as ATP but not AMP or p-nitrophenylphosphate. CTP, UTP, and ADP were poor substrates. These characteristics indicate that there is a F₀F₁-type ATPase in the inner membrane of this bacterium. In addition, the ATPase activity was also significantly inhibited by Na₃ Vo₄, suggesting the coexistence of a P-type ATPase as a minor constituent. The membrane-bound ATPase activity was maximum at 50°C, but the strong DCCD-sensitivity observed at 20°C was greatly reduced at this temperature.     

Unusual Isothermal Hysteresis in DNA i-Motif pH Transitions: A Study of the RAD17 Promoter Sequence

We have interrogated the isothermal folding behavior of the DNA i-motif of the human telomere, dC₁₉, and a high-stability i-motif-forming sequence in the promoter of the human DNA repair gene RAD17 using human physiological solution and temperature conditions. We developed a circular-dichroism-spectroscopy-based pH titration method that is followed by analysis of titration curves in the derivative domain and found that the observed pH-dependent folding behavior can be significantly different and, in some cases, multiphasic, with a dependence on how rapidly i-motif folding is induced. Interestingly, the human telomere sequence exhibits unusual isothermal hysteresis in which the unfolding process always occurs at a higher pH than the folding process. For the RAD17 i-motif, rapid folding by injection into a low-pH solution results in triphasic unfolding behavior that is completely diminished when samples are slowly folded in a stepwise manner via pH titration. Chemical footprinting of the RAD17 sequence and pH titrations of dT-substituted mutants of the RAD17 sequence were used to develop a model of RAD17 folding and unfolding. These results may provide valuable information pertinent to i-motif use in sensors and materials, as well as insight into the potential biological activity of i-motif-forming sequences under stepwise or instantaneous changes in pH.     

A pH-dependent conformational change in the coat protein subunits from potato virus X.

Both the circular dichroism and fluorescence spectra of the dissociated coat protein subunits from potato virus X changed substantially over the pH range 8 to 4, irreversible changes resulted below pH 4, with tyrosyl and tryptophanyl residues affected most. The titration curves show a pKa of about 5.6 and do not require cooperative interactions between the coat protein subunits, thus they are in marked contrast to titrations of tobacco mosaic virus A-protein. The spectra of the intact virus were little changed between pH 8 and 4 and suggested that the coat protein was locked into a conformation similar to that of the subunits in solution at pH 7. It is proposed that the pH induced conformational change is responsible for determining the acidic branch of the pH profile for reconstitution of potato virus X from its dissociated coat protein subunits and RNA.     

Interdependence between mitochondrial matrix volume and the extramitochondrial ratio of [ATP]/[ADP] [inorganic phosphate].

ATP or combinations of ATP with EDTA and EGTA can act as chelators to support succinate-driven, phosphate-requiring expansion of mitochondrial inner membrane-matrices. Contraction of these swollen mitochondria can be induced with antimycin, MgCl₂ and ADP. The magnitude of ADP-induced contraction of mitochondria, swollen in the presence of ATP, is dependent on [ADP] and may be altered by the extramitochondrial concentrations of both P_i and ATP. In fact, the extent of contraction (+ΔA₅₂₀) is a linear function of the thermodynamic parameter, ?ΔGp (free energy of hydrolysis of ATP), provided excessive concentrations of reactants are not present and the extents of matrix swelling are similar ($\text{e.g.}\text{ΔA}{}_{520}$ is about 0.250) before starting contraction with ADP.     

Light scattering during the hysteresis effect in poly(A)·2poly(U)

Light-scattering studies on buffered aqueous solutions of the triple-stranded polyribonucleic acid poly(A)·2poly(U) were carried out at neutral pH and during titration. At pH 7.1 and 22°C, a sample of commercially available polymer in 0.005M phosphate buffer gave a Zimm plot which yielded values for the weight-average molecular weight, M _w, of 874,000 ± 1800 g/mol, a root-mean-square radius, ρ of 930 ± 22 Å, and a second viral coefficient of 0.51 ± 0.05 × 10 ^?3 cm³g^?1 mol. The light-scattering data were also analyzed by serval linear and nonlinear least-squares programs which were devised to determine the model (e.g., rod, coil, or zigzag) which could best describe the shape of the molecule. It was found that a rodlike model, perhaps with a few bends, was in best overall agreement with the data. The assumption that the molecule is a thin rod leads to a value for the linear density of 206 g mol^?1 Å^?1 and a translation of 3.3 Å per residue. These values are also in close agreement with those expected for a triple-stranded, thin, base-stacked molecule. During titration from neutral pH with 0.1M HCl, the observed apparent molecular weight slowly increased until at about pH 3.5 a sudden, large increase (about 30-fold) occurred. The root-mean-square radius, on the other hand, after an initial small decrease (of about 25%), also exhibited a large increase (about 4-fold). Upon back titration with 0.1M NaOH, the molecular parameters did not retrace the original path, but instead exhibited hysteresis—the M _w and ρ _z are both larger on the basic branch than on the acid branch at a corresponding pH. A plot of long ρ _z against log(M _w) during the interval in which the high-moelcular-weight form was present (below pH 3.5 on the acid branch, and on the basic branch) gave a straight line with a slope of ?. This suggests that the aggregates were composed of some tens of rather open radom coils, presumably of poly(A)·poly(A), and that the hysteresis may be caused under conditions by the metastability of the entangled coils.     

The random coil → β transition of copolymers of L-lysine and L-isoleucine: Potentiometric titration and circular dichroism studies

Copolymers of L -lysine and L -isoleucine [poly(L -Lys^f,L -Val^{1 ? f})] containing 4–15% isoleucine were investigated using potentiometric titration and circular dichroism (CD) spectroscopy. With increasing isoleucine content, β-sheet formation is favored over α-helix formation at high pH and room temperature. The fraction of β-sheet present, as a function of pH, calculated from titrations of poly(L -Lys^85.2,L -Ile^14.8), agreed well with data obtained from CD studies for the same copolymer. Thermodynamic parameters were determined from titrations using the method of Zimm and Rice; the partial free energy (ΔG°_{C → β}) at 25° for the coil-to-β-sheet transition for isoleucine was estimated to be ?515 cal/mol; from the temperature dependence of free energy, the partial entropy (ΔS°_cβ), and the partial free enthalpy (ΔH°_{c → β}) of the coil → β transition for isoleucine is estimated to be 2.6 e.u. and 260 cal/mol, respectively. The partial thermodynamic parameters obtained for lysine are in good agreement with literature values. It is concluded from these studies that isoleucine has a very high potential for a β-sheet formation.     

Ionic strength dependence of the hysteresis in the polyriboadenylate-polyribouridylate system.

The hysteresis observed in cyclic acid-base titrations of the three-standed polyribonucleotide helix poly (A)-2 POLY (U) strongly depends on ionic strength. For NaCl and at 25 degrees C, hysteresis occurs in the limited concentration range between 0.03 M and 1.0 M(NaCl). The transition points associated with the cyclic conversions between the triple helix and the poly (A)-poly (A) double helix and (free) poly (U) constitute a (pH ionic strength) phase diagram covering the ranges of stability and metastability of the hysteresis system. Variations with NaCl concentration of some hysteresis parameters can be quantitatively described in terms of polyelectrolyte theories based on the cylinder-cell model for rodlike polyions. The results of this analysis suggest that the metastability is predominantly due to dlectrostatic energy barriers preventing the equilibrium transition of the partially protonated triple helix above a critical pH value. Ultraviolet absorbance and potentiometric titration data of poly (A)in the acidic pH range can be analyzed in terms of two types of double-helical structures. Spectrophotometric titrations reveal isosbestic wavelengths for structural transitions of poly (A). "Time effects" commonly observed in poly (A) titrations are suggested to reflect helix transitions between the two acidic structures.     

Protein binding to two-dimensional hydrophobic binding-site lattices: adsorption hysteresis on immobilized butyl-residues

Long-lived metastable states involving multiple binding sites of a protein ligand with immobilized alkyl residues on a solid phase can be observed at high ionic strength between butyl agaroses (5.21 μ mol/ml packed gel) and phosphorylase b by perturbations enforcing either the on-reaction (adsorption) or the off-reaction (desorption). These apparent equilibrium states are suggested because the adsorption isotherms of phosphorylase b on butyl agaroses are not retraced by the desorption isotherms. In this first example of macromolecular adsorption hysteresis on immobilized alkyl residues, it can be shown that the irreversible entropy (Δ_iS) produced in an adsorption-desorption cycle lies between 6 (5 μ mol/ml packed gel) and 40 (21 μ mol/ml packed gel) J mol ¹ K⁻¹. For the latter gel the apparent standard entropy of adsorption (Δ_aS_i⁰′) is 160 J mol⁻¹ K⁻¹. The metastable state observed during adsorption is probably due to an energy barrier which must be overcome for the nucleation of protein binding on the matrix. Other metastable states may possibly be encountered during desorption when the adsorbed enzyme resists the breakage of hydrophobic interactions. In the transition from the adsorption branch to the desorption branch of the hysteresis loop, the apparent affinity of the enzyme-matrix interaction is enhanced. For the desorption branch, the apparent association constant of half-maximal saturation corresponds to K_d,0.5′ = 4.2 × 10⁹ ]m⁻¹ as compared to the respective constant of adsorption K_{a, 0.5}′ = 1.6 × 10⁵m⁻¹ (gel: 21μ mol/ml packed gel). Since the area of the hysteresis loops (see also Δ_iS) depends strongly on the density of butyl residues on the gel, it is concluded that the number of alkyl residues interacting with the protein molecule is crucial for the metastable states and hysteresis. It is unlikely that hysteresis is due to the pore structure of the agarose or to nearest neighbour interactions of ligand molecules. Since thermodynamic irreversibility and hysteresis may be encountered when macromolecules, such as proteins, are adsorbed to cell membranes or cell organelles: an analysis and understanding of these phenomena should be of general biological significance.     

Production and characterization of keratinase from<Emphasis Type="Italic">Paracoccus</Emphasis> sp. WJ-98

A bacterial strain WJ-98 found to produce active extracellular keratinase was isolated from the soil of a poultry factory. It was identified asParacoccus sp. based on its 16S rRNA sequence analysis, morphological and physiological characteristics. The optimal culture conditions for the production of keratinase byParacoccus sp. WJ-98 were investigated. The optimal medium composition for keratinase production was determined to be 1.0% keratin, 0.05% urea and NaCl, 0.03% K₂HPO₄, 0.04% KH₂PO₄, and 0.01% MgCl₂·6H₂O. Optimal initial pH and temperature for the production of keratinase were 7.5 and 37°C, respectively. The maximum keratinase production of 90 U/mL was reached after 84 h of cultivation under the optimal culturing conditions. The keratinase fromParacoccus sp. WJ-98 was partially purified from a culture broth by using ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, followed by gel filtration chromatography on Sephadex G-75. Optimum pH and temperature for the enzyme reaction were pH 6.8 and 50°C, respectively and the enzymes were stable in the pH range from 6.0 to 8.0 and below 50°C. The enzyme activity was significantly inhibited by EDTA, Zn²⁺ and Hg²⁺. Inquiry into the characteristics of keratinase production from these bacteria may yield useful agricultural feed processing applications.     

Calorimetric investigation of binding of NADH to pig muscle lactate dehydrogenase

Thermal titrations have been performed to study the enthalpy of binding (Δ H_b) of the reduced coenzyme, NADH, to the pig muscle isoenzyme (M₄) of lactate dehydrogenase (EC 1.1.1.27). It has been shown that at 25°C, pH 7.0, in 0.2 M phosphate buffer Δ H_b is ?32.5 ± 1.5 kcal per mole of enzyme. The calorimetric titration data can be well represented within the limits of experimental error by a theoretical binding curve calculated on the assumption of four independent and identical binding sites.     

Letter: Hysteresis of proton binding to tobacco mosaic virus protein associated with metastable polymerization.

Proton binding to tobacco mosaic virus protein at 20 °C has been found to exhibit a reproducible hysteresis which results from the metastability of high molecular weight helical, virus-like rods. In a titration from pH 4 or 5 to 7, the time for depolymerization of such rods, as measured by ultracentrifugation, decreases from days to minutes over a range of about a tenth of a pH unit, near pH 6·6 at 20 °C. Relative to the extent of proton binding in the depolymerized state at 4 °C, the magnitude of the hysteresis near pH 6·2 corresponds to more than 50% of the protons bound per subunit in the equilibrium polymerized state.     

Evidence for cooperative hydrogen ligand binding to sperm whale metmyoglobin

Potentiometric titrations of sperm whale metmyoglobin from pH 10 to 3 shows the well-known exposure of groups between pH 4.5 and 4. However the reverse titration, at low protein concentration, results in the regeneration of its ionic property in the form of a reversible hysteresis, which was obtained by titrating the same solution twice. The Soret intensity band indicates reversibility for about 94%. Apomyoglobin shows an acid-base reversible titration from pH 9.5 to 3. Since the analysis by electrostatic interaction models do not adequately describe the overall ionic equilibria of metmyoglobin, a cooperative model was introduced. The model exactly reproduces the titration curve, indicating that 6 groups ionize with a cooperative coefficient of 9. The cooperative model explains the anomalous behaviour toward hydrogen ions and the incomplete spectral reversibility of sperm whale metmyoglobin as a molecular mechanism with physiological significance.     

Thermal denaturation of nucleohistones--effects of formaldehyde reaction

Thermal denaturation of native or partially dehistonized nucleohistones shows two melting bands at 66 and 81° in 2.5 × 10^?4 M EDTA, pH 8.0. These correspond to the melting of DNA segments bound by the less basic and the more basic half-molecules of histones, respectively. These two melting bands combine into a broad melting band from around 70 to 85° when these nucleohistones are pre-treated with formaldehyde. A formaldehyde reaction which fixes histones on DNA by covalent bonds account for the effect. Formaldehyde fixation also increases the melting temperature of some free DNA segments from around 42 to around 55°. This is interpreted as a result of closed or rigid boundaries between free DNA and formaldehyde-reacted histone-bound DNA segments. MgCl₂ dissociates histones from DNA more effectively and leaves longer free DNA segments than does NaCl. Thermal denaturation of a formaldehyde-reacted nucleoprotein thus provides an effective tool for comparing the relative size of free DNA regions on nucleoproteins. The effect of reversible binding of ligands on helix-coil transition of DNA is descussed and found not adequate for thermal denaturation of nucleohistones.     

A requirement for chelation in obtaining functional chloroplasts of sunflower and wheat.

In studying conditions for obtaining photosynthetically functional chloroplasts from mesophyll protoplasts of sunflower and wheat, a strong requirement for chelation was found. The concentration of chelator, either EDTA or pyrophosphate (PP_i), required for maximum activation depended on the pH, the concentration of orthophosphate (P_i) in the assay, and the chelator used. Studies with EDTA indicate that including the chelator in the isolation, resuspension, and assay media, in the absence of divalent cations, was most effective. Increased concentration of EDTA from 1 to 10 mm broadened the pH response curve for photosynthesis, inasmuch as a higher concentration of chelator was required for activation of photosynthesis at lower pH.Either EDTA, PP_i, or citrate could activate photosynthesis of sunflower chloroplasts isolated and assayed at pH 8.4. At pH 7.6, PP_i and EDTA were equally effective at low P_i concentrations but PP_i was particularly effective in shortening the induction period at high concentrations of P_i (2.5 mm) in the assay medium. Including 1 mm 3-phosphoglycerate in the assay medium with or without P_i could not replace the need for chelation. However, 3-phosphoglycerate + EDTA in the assay medium with 0.5 mm P_i, pH 7.6, gave a short induction period and rates of photosynthesis similar to those with 10 mm PP_i. The results suggest that PP_i can have a dual effect at the lower pH through chelation and inhibition of the phosphate transporter.Photosynthesis by sunflower chloroplasts isolated and assayed at pH 8.4 with 0.2 mm EDTA (+ 0.5 mm P_i in the assays) was severely inhibited by 2 mM CaCl₂, MgCl₂, or MnCl₂. Wheat chloroplasts isolated and assayed at pH 8.4 without chelation, and assayed with 0.2 mm P_i, had low rates of photosynthesis (25 μmol O₂ evolved mg^?1 chlorophyll h^?1) which were strongly inhibited by 2 to 4 mm MgCl₂, MnCl₂, or CaCl₂. With inclusion of EDTA and P_i at optimum levels, isolated chloroplasts of sunflower and wheat have high rates of photosynthesis and PP_i or divalent cations are not of benefit.