AMP photophosphorylation and binding by chloroplasts

Stoichiometric amounts of chloroplast thylakoids photophosphorylate free AMP to tightly bound ADP. Free ADP is a poor competitor for this AMP photoreaction, which saturates below 16 micronAMP. The inhibitor, diadenosine pentaphosphate, abolishes AMP photophosphorylation, and inhibits dark ADP binding. Taken together, these data imply that this photoreaction involves the high affinity nucleotide binding site(s) of chloroplast coupling factor CF1, and that little mixing with free nucleotides occurs.     

ADP binding and ATP synthesis by reconstituted H-ATPase from chloroplasts

The H⁺-ATPase from chloroplasts, CF₀F₁, was isolated, purified and reconstituted into asolectin liposomes. The enzyme was brought either into the oxidized state or into the reduced state, and the rate of ATP synthesis was measured after energisation of the proteoliposomes with an acid—base transition ΔpH (pH_in = 5.0, pH_out = 8.5) and a K⁺/valinomycin diffusion potential, Δφ (K⁺_in = 0.6 mM, K⁺_out = 60 mM). A rate of 250 s⁻¹ was observed with the reduced enzyme (85 s⁻¹ in the absence of Δφ). A rate of 50 s⁻¹ was observed with the oxidized enzyme under the same conditions (15 s⁻¹ in the absence of Δφ). The reconstituted enzyme contained 2 ATP_bound per CF₀F₁ and 1 ADP_bound per CF₀F₁. Upon energisation the enzyme was activated and 0.9 ADP per CF₀F₁, was released. Binding of ADP to the active reduced enzyme was observed under different conditions. In the absence of phosphate the rate constant for ADP binding was 10⁵ M⁻¹·s⁻¹ under energized and de-energized conditions. In the presence of phosphate the rate of ADP binding drastically increased under energized conditions, and strongly decreased under de-energized conditions.     

Ribonucleic acid synthesis in chloroplasts

Chloroplasts isolated from young spinach leaves incorporate [(3)H]uridine into RNA. This incorporation shows an absolute requirement for light and does not occur in lysed chloroplasts. Fractionation by polyacrylamide-gel electrophoresis of the RNA synthesized in vitro reveals a major discrete product of molecular weight 2.7x10(6) and two minor products of molecular weight 1.2x10(6) and 0.47x10(6). These discrete products are super-imposed on a background of polydisperse RNA. The incorporation of (32)P(i) into chloroplast rRNA species (mol.wt. 1.05x10(6) and 0.56x10(6)) in excised spinach leaves proceeds after a distinct lag period compared with the incorporation into cytoplasmic rRNA species (mol.wt. 1.34x10(6) and 0.7x10(6)). Incorporation of (32)P(i) into chloroplast RNA species of molecular weight 2.7x10(6), 1.2x10(6), 0.65x10(6) and 0.47x10(6) proceeds without such a time-lag. The kinetics of labelling of the individual RNA components is consistent with the rapidly labelled RNA species of molecular weight 1.2x10(6) and 0.65x10(6) being precursors to the more slowly labelled rRNA species of molecular weight 1.05x10(6) and 0.56x10(6) respectively.     

Amino acid incorporation by ribosomes and polyribosomes from wheat chloroplasts

Sucrose-gradient and analytical ultracentrifugation showed that chloroplast polyribosomes from 4-day-old seedlings had mono-, di-, tri-, tetra- and traces of penta-ribosomes, in contrast with those from 7-day-old seedlings in which only the mono-, di- and traces of tri-ribosomes were present. Without Mg(2+) the polyribosomes dissociated into ribosomal subunits. The rate of l-[U-(14)C]phenylalanine incorporation was threefold greater for preparations from 4- than from 7-day-old seedlings. Incorporation by the latter was stimulated by polyuridylic acid. The rates of incorporation were similar whether the reaction mixture contained chloroplast or wheat-germ transfer RNA and amino acid synthetases purified on methylated albumin-on-kieselguhr and Sephadex G-75 columns respectively. The cofactor requirement was the same as for isolated intact chloroplasts. Osmotic rupture of chloroplasts with and without Triton X-100 revealed the presence of free and bound ribosomes. Free single ribosomes isolated by osmotic shrinkage or prepared by pancreatic ribonuclease digestion of chloroplast polyribosomes had negligible incorporation activity. This activity was increased by washing or by polyuridylic acid, but was still only a fraction of that given by polyribosomes. A comparison of incorporation activity of chloroplast polyribosomes with those from the surrounding cytoplasm showed the former to be 20 times more active.     

Restoration by silicotungstic acid of DCMU-inhibited photoreactions in spinach chloroplasts

The restoration by silicotungstic acid of the reversible light-induced pH rise mediated by pyocyanine in EDTA-treated chloroplasts corresponds to an irreversible fixation of the acid. The proton uptake is linearly related to the amount of fixed acid (4 protons per molecule of acid) as long as the amount of silicotungstic acid does not exceed 200 nmoles/mg of chlorophyll.In the same conditions silicotungstic acid partly restores ferricyanide reduction and O₂ evolution in chloroplasts suspensions supplemented with DCMU. These photoreactions are observed only with chloroplasts and these chloroplasts must have an unimpaired water-splitting mechanism.Silicotungstic acid does not impair DCMU fixation on the specific sites. More likely in its presence the properties of the membrane change and ferricyanide can accept electrons from a part of the electron transport chain, between the Photosystem II reaction center and the block of the electron flow by DCMU.     

Relationship between inhibitor binding by chloroplasts and inhibition of photosynthetic electron transport

The binding of radioactively labelled atrazin, metribuzin and phenmedipham by broken chloroplasts was studied. From the double-reciprocal plots (bound vs. free inhibitors) a high affinity binding reaction is graphically isolated which is related to the inhibition of photosynthetic electron transport. It is concluded that the specific binding sites correspond to the electron carrier molecules which are attacked by the inhibitors. The relative concentration of specific binding sites is 1 per 300–500 chlorophyll molecules.The binding of the labelled substances is competitively inhibited by each of the indicated unlabelled substances, by DCMU and by several pyridazinone derivatives. These results suggest that triazines, triazinones, pyridazinones, biscarbamates and phenylureas interfere with the same electron carrier of the photosynthetic electron transport chain, according to the same molecular mechanism.     

Removal of Mn from spinach chloroplasts by sodium cyanide and the binding of Mn2+ to Mn-depleted chloroplasts.

Manganese and copper were released from spinach chloroplasts by NaCN-treatment, though iron was not affected. The Hill reaction activity was also inhibited by this treatment, but was partially recovered by the addition of either Mn2+ or Cu2+, but not of Fe3+. The interaction of Mn2+ with manganese-depleted chloroplasts by NaCN-treatment was studied using 54Mn2+. A Scatchard plot shows the high and low affinity binding sites of Mn2+ on NaCN-treated chloroplast membrane; high affinity binding being specific for NaCN-treated chloroplast with a binding constant, KH, of 1.9 X 10(5) M-1, and a maximum binding number, NH, of 0.0016 g-atom per mole of chlorophyll. The low binding site was also found on untreated chloroplasts; its binding constant, KL, being 1.2 X 10(4) M-1, and its maximum binding number, NL, of 0.0112 g-atom per mole oc chlorophyll at pH 8.2 NH was proportional to the degree of the removal of Mn by NaCN-treatment and was constant at pH 4--9. NL markedly increased at a high pH with a midpoint of pH 7.9 indicating the exposure of a new, similar binding site. Light illumination partially inhibited the binding of Mn2+. Within 1 min in the dark the binding reaction reached equilibrium in the absence of pyrophosphate, however, 20 min were required to transform into pyrophosphate-resistant form. The pH dependence of the binding of Mn2+ with pKa 7.2 and the ineffectiveness of p-chloromercuribenzoate suggest the possible ligand of Mn2+ is the imidazole nitrogen of the histidine residue.     

Fatty acid synthesis by spinach chloroplasts III. Relationship between fatty acid synthesis and photophosphorylation

The modes of actions of photosynthetic inhibitors on photosynthesisand fatty acid synthesis were examined. DCMU, an electron transport inhibitor, inhibited fatty acidsynthesis and photophosphorylation to the same extent, suggestingdependence of fatty acid synthesis on photosynthesis. The samewas also the case with FCCP, a photophosphorylation uncoupler.In contrast, NH₄Cl and phlorizin at concentrations completelysuppressing ATP formation, only partially inhibited the fattyacid synthesis. These facts suggest that a certain level ofhigh-energy intermediate (state) is responsible for the lightenhancement of fatty acid synthesis. This idea is further supportedby the fact that the partial inhibition of fatty acid synthesisby NH₄Cl was relieved by addition of DCCD at low concentrationssuppressing the ATP formation but not completely destroyingthe high energy intermediate. The lag period in the initial period of fatty acid synthesiswas shortened by preillumination of chloroplasts, even in theabsence of ADP. This indicates that the light dependent fattyacid synthesis is closely associated with the high-energy intermediate(state), but not directly with ATP formation by photophosphorylation. ¹ Present address: Radioisotope Centre, University of Tokyo,Yayoi, Bunkyo, Tokyo 113, Japan. (Received August 26, 1974; )     

Inhibition of ATPase from chloroplasts by a hydroxamic acid from the gramineae

-DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one), a hydroxamic acid from the Gramineae involved in the resistance of cereals to aphids, inhibits     

Selective binding of cholesterol by recombinant fatty acid binding proteins

The sterol binding specificity of rat recombinant liver fatty acid binding protein (L-FABP) and intestinal fatty acid binding protein (I-FABP) was characterized with [3H]cholesterol and a fluorescent sterol analog dehydroergosterol. Ligand binding analysis, fluorescence spectroscopy, and activation of microsomal acyl-CoA:cholesterol acyltransferase activity showed that L-FABP-bound sterols. 1) Lipidex-1000 assay showed a dissociation constant Kd = 0.78 +/- 0.18 microM and stoichiometry of 0.47 +/- 0.16 mol/mol for [3H]cholesterol binding to L-PABP. 2) With [3H]cholesterol/phosphatidylcholine liposomes, the cholesterol binding parameters for L-FABP were Kd = 1.53 +/- 0.28 microM and stoichiometry 0.83 +/- 0.07 mol/mol. 3) L-FABP interaction with dehydroergosterol altered the fluorescence intensity and polarization of dehydroergosterol. Dehydroergosterol bound to L-FABP with Kd = 0.37 microM and a stoichiometry of 0.83 mol/mol. 4) Cholesterol and dehydroergosterol decreased L-FABP tyrosine lifetime. Dehydroergosterol binding produced sensitized emission of bound dehydroergosterol with longer lifetime.5) L-FABP bound two cis-parinaric acid molecules/molecule of protein. Cholesterol displaced one of these bound cis-parinaric acids. 6) L-FABP enhanced acyl-CoA:cholesterol acyltransferase in a concentration-dependent manner. In contrast, these assays indicated that I-FABP did not bind sterols. Thus, L-FABP appears able to bind 1 mol of cholesterol/mol of L-FABP, the L-FABP sterol binding site is equivalent to one of the two fatty acid binding sites, and L-FABP stimulates acyl-CoA:cholesterol acyltransferase by transfer of cholesterol.     

The 2-hydroxylation of trans-cinnamic acid by chloroplasts from Melilotus alba Desr

Chloroplasts isolated from sweetclover leaves contain an enzyme which converts trans-[3-¹⁴C]cinnamic acid to 2-hydroxy-trans-[3-¹⁴C]cinnamic (o-coumaric) acid. The identity of the product has been verified by recrystallization with unlabeled o-coumaric acid to constant specific activity, and by gas-liquid cochromatography of unlabeled o-coumaric acid and the radioactive product.The enzyme has an optimum of pH 7.0 and its activity can be enhanced ~ 4-fold by adding 4 mm glucose-6-phosphate to the reaction mixture. Light can replace glucose-6-phosphate, presumably as a source of reducing power required for the hydroxylation system. It was found that approximately 50% of the hydroxylase activity is bound to the lamellar membranes, from which it can be released by sonication.     

Hydroxylation of p-Coumaric acid by illuminated chloroplasts. The role of superoxide.

1. Chloroplasts isolated from leaves of spinach-beet (Beta vulgaris L. ssp. vulgaris) do not catalyse the hydroxylation of p-coumaric acid in the dark unless a reductant (such as ascorbate, NADH or NADPH) is added. Superoxide dismutase has no effect on this reaction. 2. Illuminated chloroplasts catalyse the hydroxylation in the absence of added reductant. This reaction is completely inhibited by superoxide dismutase, but catalase has little effect. 3. Both hydroxylation in the light and hydroxylation in the dark in the presence of reductants are inhibited by diethyldithiocarbamate, EDTA, cyanide and 2-mercaptoethanol. 4. It is proposed that O-2- generated by illuminated chloroplasts is involved in the provision of a reductant to the enzyme phenolase.     

Insights into binding of fatty acids by fatty acid binding proteins

Members of the phylogenetically related intracellular lipid binding protein (iLBP) are characterized by a highly conserved tertiary structure, but reveal distinct binding preferences with regard to ligand structure and conformation, when binding is assessed by the Lipidex method (removal of unbound ligand by hydrophobic polymer) or by isothermal titration calorimetry, a true equilibrium method. Subfamily proteins bind retinoids, subfamily II proteins bind bulky ligands, examples are intestinal bile acid binding protein (I-BABP) and liver fatty acid binding protein (L-FABP) which binds 2 ligand molecules, preferably monounsaturated and n-3 fatty acids. Subfamily III intestinal fatty acid binding protein (I-FABP) binds fatty acid in a bent conformation. The fatty acid bound by subfamily IV FABPs has a U-shaped conformation; here heart (H-) FABP preferably binds n-6, brain (B-) FABP n-3 fatty acids. The ADIFAB-method is a fluorescent test for fatty acid in equilibrium with iLBP and reveals some correlation of binding affinity to fatty acid solubility in the aqueous phase; these data are often at variance with those obtained by the other methods. Thus, in this review published binding data are critically discussed, taking into account on the one hand binding increments calculated for fatty acid double bonds on the basis of the solubility hypothesis, on the other hand the interpretation of calorimetric data on the basis of crystallographic and solution structures of iLBPs.     

Glycolate metabolism in algal chloroplasts: inhibition by salicylhydroxamic acid (SHAM)

Unicellular green algae such as Chlamydomonas and Dunaliella excrete small amounts of glycolate during active photosynthesis. This phenomenon has been explained by the fact that these algae do not have leaf-type peroxisomes and glycolate oxidase; instead, they have a limited capacity to metabolise glycolate in their mitochondria by a membrane-associated glycolate dehydrogenase. Salicylhydroxamic acid (SHAM), an inhibitor of alternative oxidase in plant and algal mitochondria, stimulates glycolate excretion by the algae or their isolated chloroplasts 5-fold. In the presence of SHAM, cells of Chlamydomonas or Dunaliella grown with high-CO₂ (5% CO₂ in air, v/v) or adapted with air levels of CO₂ excreted glycolate at a rate of about 14 µmol glycolate mg⁻¹ Chl h⁻¹. Aminooxyacetate (AOA), an inhibitor of aminotransferases, also increases glycolate excretion by the algal cells or chloroplasts but at a lower rate (about 50%) than SHAM. The algal, light dependent, SHAM-sensitive glycolate oxidizing system in the chloroplasts appears to be the primary site for glycolate oxidation, and it is different and more active then the minor mitochondrial glycolate dehydrogenase.     

Specific insulin binding to chloroplasts isolated from Acetabularia mediterranea]

Kinetics of insulin fixation to chloroplasts, exponential dissociation of bound hormone, competition between radioactive and native insulins for binding and for release indicate that chloroplasts possess insulin receptors.