The site of inhibition of photosynthetic electron transfer by amphotericin B.

The inhibitory effect of the polyene antibiotic, amphotericin B, on photosynthetic electron transfer has been investigated. Treatment of chloroplasts with the inhibitor results in the release of plastocyanin from its site in the chloroplast membrane. This release is accompanied by a shift in the pH curve for ferricyanide photoreduction from water, which is similar to that observed when chloroplasts are treated by sonication or passage through a French press. Delayed light emission from photosystem 2 is not destroyed by amphotericin B treatment, indicating that photosystem 2 is not damaged. Amphotericin B does not inhibit photoreduction of ferricyanide from water by chloroplast preparations which are deficient in plastocyanin, such as maize bundle-sheath chloroplast fragments, Euglena chloroplasts, or maize mesophyll chloroplasts passed through a French press. Chloroplasts treated with amphotericin B are not able to photooxidize plastocyanin. This result demonstrates that little structural damage occurs to the membrane during treatment with the antibiotic as a capacity to photooxidize plastocyanin is observed only in damaged chloroplast membranes.     

Effects of metallic silver island films on resonance energy transfer between N,N'-(dipropyl)-tetramethyl- indocarbocyanine (Cy3)- and N,N'-(dipropyl)-tetramethyl- indodicarbocyanine (Cy5)-labeled DNA

Resonance energy transfer (RET) is typically limited to distances below 60 A, which can be too short for some biomedical assays. We examined a new method for increasing the RET distances by placing donor- and acceptor-labeled DNA oligomers between two slides coated with metallic silver particles. A N,N'-(dipropyl)-tetramethylindocarbocyanine donor and a N,N'-(dipropyl)-tetramethylindodicarbocyanine acceptor were covalently bound to opposite 5' ends of complementary 23 base pair DNA oligomers. The transfer efficiency was 25% in the absence of silver particles or if only one slide was silvered, and it increased to an average value near 64% between two silvered slides. The average value of the Forster distance increased from 58 to 77 A. The energy transfer data were analyzed with a model assuming two populations of donor-acceptor pairs: unaffected and affected by silver island films. In an affected fraction of about 28%, the apparent energy transfer efficiency is near 87% and the Forster distance increases to 119 A. These results suggest the use of metallic silver particles to increase the distances over which RET occurs in biomedical and biotechnology assays.     

Zinc depletion efficiently inhibits pancreatic cancer cell growth by increasing the ratio of antiproliferative/proliferative genes

We investigated the ability of the zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) to reduce pancreatic cancer cell viability. TPEN was much more efficient to inhibit pancreatic adenocarcinoma cell growth than a panel of anti-cancer drugs, including 5-fluorouracil, irinotecan, cisplatin, edelfosine, trichostatin A, mitomycin C, and gemcitabine, the gold standard chemotherapeutic agent for pancreatic cancer. Moreover, TPEN showed a dose- and time-dependent anti-proliferative effect significantly higher on pancreatic cancer cells than on normal primary fibroblasts. This effect may be explained by a significantly higher zinc depletion by TPEN in pancreatic cancer cells as compared to fibroblasts. Cell viability reduction by TPEN was associated to both G1-phase cell cycle arrest and apoptosis, and to the increased ratio of the expression level of cyclin-Cdk inhibitor versus cyclin genes and apoptotic versus anti-apoptotic genes. Finally, we show that apoptotic cell death induced by TPEN involved mitochondrial injury and caspase 3 and caspase 8 activation. In this study, we suggest that zinc depletion may be an efficient strategy in the treatment of pancreatic cancer because of its reduced antiproliferative effect on normal cells.     

Evidence for cyanide and mercury inactivation of endogenous plastocyanin

Cyanide and mercury treatment of chloroplast membranes inactivates plastocyanin as shown by the inability of the extracted plastocyanin to restore electron transport in a bioassay on chloroplasts depleted of their endogenous plastocyanin by digitonin treatment. The extraction procedure did remore the enzyme from cyanide and mercury treated chloroplasts as shown by sodium dodecyl sulfate polyacrylamide electrophoresis of the extracts. This procedure normally shows a plastocyanin band at 11,000 dalton molecular weight and the band was present in extracts from control and cyanide or mercury treated membranes.     

Localization of the reaction side of plastocyanin from immunological and kinetic studies with inside-out thylakoid vesicles

(1) The effect of four active antisera against plastocyanin on Photosystem I-driven electron transport and phosphorylation was investigated in spinach chloroplasts. Partial inhibition of electron transport and stimulation of plastocyanin-dependent phosphorylation were sometimes observed after adding amounts of antibodies which were in large excess and not related to the plastocyanin content of the chloroplasts. This indicates effects of the antibodies on the membrane. (2) The antibodies against plastocyanin neither directly nor indirectly agglutinated unbroken chloroplast membranes. (3) The plastocyanin content of right-side-out and inside-out thylakoid vesicles isolated by aqueous polymer two-phase partition from chloroplasts disrupted by Yeda press treatment was determined by quantitative rocket electroimmunodiffusion. Right-side-out vesicles retained about 25%, inside-out vesicles none of the original amount of plastocyanin. (4) The effect of externally added plastocyanin on the reduction of P-700 was studied by monitoring the absorbance changes at 703 nm after a long flash. In inside-out vesicles P-700 was reduced by the added plastocyanin but not in right-side-out vesicles and class II chloroplasts. These results provide strong evidence for a function of plastocyanin at the internal side of the thylakoid membrane.     

Mechanism of KCN inhibition of photosystem I.

Experiments with chloroplasts and purified spinach plastocyanin suggest a mechanism for KCN inhibition of Photosystem I. KCN inhibition can be bypassed by a detergent or reversed by replacement of the inactive plastocyanin. KCN bleaches and inactivates purified plastocyanin. KCN releases copper from chloroplast membranes and from purified plastocyanin. Cyanide does not bind to the apoprotein produced when plastocyanin is treated with KCN, and KCN-produced apoplastocyanin has a N-ethylmaleimide-reactive sulfhydryl group not found in holoplastocyanin. Apoplastocyanin is not active in restoring Photosystem I activity to plastocyanin-depleted membranes. Holoplastocyanin restores Photosystem I activities to plastocyanin-depleted membranes prepared from either control or KCN-treated chloroplasts to about the same extent. KCN-treated chloroplast membranes are found to have higher amounts of apoplastocyanin than do control chloroplast membranes. These results offer evidence that KCN removes the copper from plastocyanin in the chloroplast membrane, leaving the inactive apoplastocyanin which is unable to transfer electrons to Photosystem I.     

Nicotinamide adenine dinucleotide phosphate photoreduction from water by agranal chloroplasts isolated from bundle sheath cells of maize

Photoreduction of NADP from water in agranal chloroplasts isolated from the leaf bundle sheath cells of Zea mays (var. DS 606A) or Sorghum bicolor (var. Texas 610) was dependent upon addition of plastocyanin as well as ferredoxin. Activity was further increased by the addition of ferredoxin NADP-reductase. Saturation for plastocyanin was reached at about 6 micromolar. In contrast, grana-containing chloroplasts isolated from leaf mesophyll cells of these plants or from pea (Pisum sativum L.) leaves did not require either plastocyanin or ferredoxin NADP-reductase for NADP photoreduction from water, although with some preparations plastocyanin stimulated the activity.     

A combined procedure for preparation of plastocyanin, ferredoxin and CF1.

Homogeneous preparations of ferredoxin, plastocyanin, and chloroplast coupling factor (CF1) have been isolated from spinach by a combined procedure in which supernatants from preparation of chloroplasts are used for isolation of ferredoxin and the chloroplasts serve as the source of plastocyanin. The proteins were purified by DEAE-cellulose chromatography and gel filtration, after precipitation with acetone in the case of ferredoxin or release from membranes in the case of plastocyanin. The proteins obtained by this procedure are pure, as evidenced by absorption ratios (ferredoxin, A420/A276 = 0.47-0.48; plastocyanin, A278/A597 = 1.2) and by the fact that both proteins migrate as single bands on polyacrylamide gels in the presence of sodium dodecyl sulfate.     

Inhibition of photosynthetic electron transfer by amphotericin B

The polyene antibiotic amphotericin B inhibits photosynthetic electron transfer by Class II maize mesophyll chloroplasts, from water to FeCN, DCIP and diquat but not to plastocyanin. Photosystem 1 activity is also inhibited by amphotericin B, but ferredoxin-NADP reductase activity is not affected. The activity of all the photosynthetic electron transfer systems inhibited by amphotericin B can be restored by the addition of carrier amounts of plastocyanin. The results suggest that amphotericin B inhibits photosynthetic electron transfer by acting only at the plastocyanin site in the chain, and that the primary site of reduction of FeCN and DCIP from water by Class II chloroplasts lies on the reducing side of photosystem 1.     

Reactions of antibodies against ferredoxin, ferredoxin-NADP+ reductase and plastocyanin with spinach chloroplasts

Purified antisera against ferredoxin, ferredoxin-NADP+ reductase and plastocyanin agglutinated osmotically shocked and washed spinach chloroplasts, prepared according to standard procedures. The monomeric antibody (immunoglobulin G fraction) of the reductase antiserum agglutinated chloroplasts specifically and directly, indicating that protruding structures (for example, the coupling factor) do not act as steric hindrances as has been suggested. With ferredoxin antiserum, the presence of a pentameric antibody (immunoglobulin M fraction) was obligatory to observe a positive agglutination reaction. Immunoglobulin G only inhibited ferredoxin-dependent reactions, like NADP+-photoreduction, but did not cause agglutination. Ferredoxin seems to be located in depressions of the membrane, possibly caused by a partial release of this protein in shocked chloroplasts. Similar results were obtained with purified immunoglobulins from a plastocyanin antiserum. Again the immunoglobulin G fraction inhibited electron transport reactions catalyzed by plastocyanin, whereas immunoglobulin M showed a positive agglutination, but had no influence on electron transport. It is concluded that ferredoxin, ferredoxin-NADP+ reductase and plastocyanin are peripheral electron transport components, located at the outer thylakoid membrane.     

Lateral distribution and diffusion of plastocyanin in chloroplast thylakoids

The lateral distribution of plastocyanin in the thylakoid lumen of spinach and pea chloroplasts was studied by combining immunocytochemical localization and kinetic measurements of P700+ reduction at high time resolution. In dark-adapted chloroplasts, the concentration of plastocyanin in the photosystem I containing stroma membranes exceeds that in photosystem II containing grana membranes by a factor of about two. Under these conditions, the reduction of P700+ with a halftime of 12 microseconds after a laser flash of saturating intensity indicates that to greater than 95% of total photosystem I a plastocyanin molecule is bound. An analysis of the labeling densities, the length of the different lumenal regions, and the total amounts of plastocyanin and P700 shows that most of the remaining presumable mobile plastocyanin is found in the granal lumen. This distribution of plastocyanin is consistent with a more negative surface charge density in the stromal than in the granal lumen. During illumination the concentration of plastocyanin in grana increases at the expense of that in stroma lamellae, indicating a light-driven diffusion from stroma to grana regions. Our observations provide evidence that a high concentration of plastocyanin in grana in the light favors the lateral electron transport from cytochrome b6/f complexes in appressed grana across the long distance to photosystem I in nonappressed stroma membranes.     

Reactions of plastocyanin and cytochrome 553 with Photosystem I of Scenedesmus

Chloroplast material active in photosynthetic electron transport has been isolated from Scenedesmus acutus (strain 270/3a). During homogenization, part of cytochrome 553 was solubilized, and part of it remained firmly bound to the membrane. A direct correlation between membrane cytochrome 553 and electron transport rates could not be found. Sonification removes plastocyanin, but leaves bound cytochrome 553 in the membrane. Photooxidation of the latter is dependent on added plastocyanin. In contrast to higher plant chloroplasts, added soluble cytochrome 553 was photooxidized by 707 nm light without plastocyanin present. Reduced plastocyanin or cytochrome 553 stimulated electron transport by Photosystem I when supplied together or separately. These reactions and cytochrome 553 photooxidation were not sensitive to preincubation of chloroplasts with KCN, indicating that both redox proteins can donate their electrons directly to the Photosystem I reaction center. Scenedesmus cytochrome 553 was about as active as plastocyanin from the same alga, whereas the corresponding protein from the alga Bumilleriopsis was without effect on electron transport rates.

It is suggested that besides the reaction sequence cytochrome 553 → plastocyanin → Photosystem I reaction center, a second pathway cytochrome 553 → Photosystem I reaction center may operate additionally.     

LIGHT-INDUCED REDUCTION AND OXIDATION OF PLASTOCYANIN BY CHLOROPLAST PREPARATIONS

In the light the intact chloroplasts of spinach reduce plastocyaninwith a reaction rate comparable to that of the usual HILL reaction,while no reduction nor oxidation of the copper protein is inducedby the chloroplasts in the dark. The dependency of the rateof photoreduction upon light intensity, pH and presence of variousreagents was similar to that of the HILL reaction with the usualHILL oxidants. The photoreduction of plastocyanin was acceleratedby the addition of complete phosphorylating system or ammoniumsulfate to the chloroplasts. Digitonin-treated chloroplast wasfound to be inactive in photoreducing plastocyanin but highlyactive in photo-oxidizing reduced plastocyanin. The rate ofphotooxidation was saturated at about 5, 000 lux, and showeda rather broad pH optimum around pH 8.0—8.5. The effectsof various poisons on the reaction rate were studied. When thedigitonin-treated chloroplasts were fractionated with ethanolinto a chlorophyll-bearing participate fraction and a solublefrac tion, the former was active in catalyzing the photooxidationof reduced plastocyanin, but not in photooxidizing reduced cytochromec.An ap preciable photooxidation of reduced cytochrome cwiththe ethanol-precipi tated fraction was obtained on additionof the soluble fraction, which was effectively replaced by plastocyanin.The properties of the reaction systems responsible for photooxidationof plastocyanin and cytochrome C were compared, and a possiblerole of plastocyanin in the photooxidatory process of the chloroplastswas suggested. ¹A part of the present investigation was supported by a researchgrant (GAMN 6208) from ROCKEFELLER Foundation. ²Present address; C.F. KETTERING Research Laboratory, YellowSprings, Ohio, U.S.A.     

Copper tolerance in Silene cucubalus

The uptake, translocation and subcellular distribution of copper as well as its effect on chloroplasts and plastocyanin synthesis were studied in a copper-sensitive and a copper-tolerant population of Silene cucubalus (L.) Wib. As a function of time, the copper concentration in roots of tolerant plants increased more slowly than that in roots of sensitive ones. Translocation to the shoot occurred more rapidly in tolerant plants than in sensitive ones. Although it was accumulated in leaf cells, copper was not accumulated in the chloroplasts of either sensitive or tolerant plants. Chlorophyll content was not affected by copper in tolerant plants, whereas sensitive plants became chlorotic. Plastocyanin synthesis was not enhanced as a result of high copper concentrations and no difference in plastocyanin content between tolerant and sensitive plants was detected. Measurements of copper in purified cell walls revealed that storage of the metal in cell-wall material does not play an important role in tolerance mechanism. Uptake characteristics, distribution and cytoplasmic detoxification of copper are discussed.     

Effect of surface potential on P-700 reduction in chloroplasts

The effect of salt addition on the rate of reduction of P-700 oxidized by flash illumination was analyzed. In broken chloroplasts, the rate of P-700 reduction was accelerated by salts of mono-, di- and trivalent cations, with the increasing effectiveness in this order, in the presence of various artificial electron donors or acceptors. The rate was not dependent on the concentration and the valence of anions. On the other hand, in Photosystem I-enriched subchloroplast particles, added KCl did not induce the acceleration of direct reduction of P-700 by reduced DCIP.At low KCl concentrations (below 10 mM), the rate of P-700 reduction was also accelerated by added KCl in sonicated chloroplasts to which purified plastocyanin was added. The curves of dependence of the reduction rate on plastocyanin concentration were not of the Michaelis-Menten type, but sigmoidal. The maximal of P-700 reduction was higher at higher salt concentrations and the half-maximal plastocyanin concentration for P-700 reduction became lower with increasing NaCl concentrations.In broken chloroplasts treated with 50 mM glutaraldehyde, the rate of P-700 reduction was not accelerated by added KCl.The Debye-Hückel theory and the Gouy-Chapman theory were applied to our data to analyze the electrostatic interaction between electron tranfer components on thylakoid membranes. It is suggested that the major factor determining the rate of P-700 reduction is the donation of electrons from plastocyanin to P-700. Most of the observed effect is probably due to the increase in the local concentration or accessibility of plastocyanin to the site of P-700 reduction which is expected when the negative surface potential rises when salt is added.     

Interaction between immobilized plastocyanin and cytochrome f and the photosystem I reaction center in a reconstituted system

The effects of redox conversions of plastocyanin copper chromophore on the formation of plastocyanin complexes with cytochrome f and the reaction center of photosystem I from pea chloroplasts were studied. In order to investigate the complex formation plastocyanin and cytochrome f were immobilized on Sephadex G-200. The cytochrome f and reaction center assembly takes place on the immobilized plastocyanin, which is necessary for cytochrome f photooxidation. It was found that in a reconstituted system the reduced plastocyanin forms more stable complexes with the proteins than the oxidized one, which is due to its lower pI value.     

Precipitation by polycation as capture step in purification of plasmid DNA from a clarified lysate

The demand for highly purified plasmids in gene therapy and plasmid-based vaccines requires large-scale production of pharmaceutical-grade plasmid. Large-scale purification of plasmid DNA from bacterial cell culture normally includes one or several chromatographic steps. Prechromatographic steps include precipitation with solvents, salts, and polymers combined with enzymatic degradation of nucleic acids. No method alone has so far been able to selectively capture plasmid DNA directly from a clarified alkaline lysate. We present a method for selective precipitation of plasmid DNA from a clarified alkaline lysate using polycation poly(N, N'-dimethyldiallylammonium) chloride (PDMDAAC). The specific interaction between the polycation and the plasmid DNA resulted in the formation of a stoichiometric insoluble complex. Efficient removal of contaminants such as RNA, by far the major contaminant in a clarified lysate, and proteins as well as 20-fold plasmid concentration has been obtained with about 80% recovery. The method utilizes a inexpensive, commercially available polymer and thus provides a capture step suitable for large-scale production.     

Effect of metalloproteins on the photochemical activity of chloroplasts treated with polyene antibiotics]

The effects of various metall-containing proteins (plastocyanin, plantacyanin, azurine and cytochromes of the f type) on the activity of photosystem I of chloroplasts, treated with polyene antibiotics, were studied. The inhibiting effect of the polyenes, surgumycin and philipin, was completely removed by an addition of copper-containing protein plastocyanin. No similar effect was exerted by other Cu-containing proteins--azurine and plantacyanin. The cytochromes of the f type isolated from the green algae chlorella, blue-green algae spiruline and aphanezomenone, having different electrophoretic properties, restored the activity of photosystem I of chloroplasts incubated with antibiotics in a different degree. Acid cytochrome f of chlorella restored the activity by 80--100%; less acid cytochrome f from spiruline-only by 50%. The least restoring effect was exerted by aphanezomenone cytochrome, which possesses some basic properties. The chloroplasts treatment with surgumycin did not affect the isolation of the terminal enzyme of the chloroplast electron-transporting chain of ferredoxin--NADP--reductase. Possible environment of plastocyanin in the chloroplast membrane and the mechanism of photosystem I restoration are discussed.     

Polylysine-enhanced effectiveness of plastocyanin in photosystem I

1. Chloroplast fragments from either Chlamydomonas reinhardi or spinach, which lack plastocyanin, or from Euglena gracilis depleted of cytochrome c552, require a large excess of exogenously added plastocyanin or cytochrome c552 to restore Photosystem I activity.2. In the presence of a small amount of polylysine, Photosystem I activity of chloroplast fragments is stimulated greatly by plastocyanin or cytochrome c552, and the reaction is saturated at a lower concentration of these proteins. Higher concentrations of polylysine inhibit Photosystem I activity; the inhibition is not reversed by plastocyanin or cytochrome c552.3. Salt protects chloroplast fragments from stimulation by polylysine plus plastocyanin or cytochrome c552, and also reverses this stimulation.4. The data suggest that polylysine, at low concentration, enhances binding of plastocyanin or cytochrome c552 to chloroplast membranes, thereby increasing the effective concentration at their site of function. The total inhibition of Photosystem I activity, independent of the presence of plastocyanin or cytochrome c552, at higher polylysine concentrations is similar probably to that observed previously in chloroplasts which retain their plastocyanin.     

Lipase catalysed synthesis of diacyl hydrazines: an indirect method for kinetic resolution of chiral acids

The acylation of hydrazine, to afford the N,N′-diacyl derivatives, was catalysed by a number of lipases. The rates of the first and second steps depended on the lipase and the type of solvent used. Water, up to 0.4 M, had no detrimental effect on the yield and complete conversion to the N,N′-diacyl derivative was accomplished with some lipases. The hydrazide of 2-(4-isobutylphenyl)propanoic acid (ibuprofen), prepared by non-enzymatic reaction of ibuprofen methyl ester with hydrazine, acted as a nucleophile towards several lipases that do not accept ibuprofen derivatives as the acyl donor, but the enantiomer differentiation was inefficient in most cases. The best result was obtained with Pseudomonas lipoprotein lipase on EP 100 which formed the (R) enantiomer of the product (N-octanoyl-N′-2-(4-isobutylphenyl)propanoylhydrazine) with an enantiomeric ratio E of 26.