Volume 44, Issue 5-6

By recording the temperature dependence of electroantennograms with Lepidoptera antennae optimum curves were obtained, and a hysteresis phenomenon was observed by passing tempera­ ture cycles. The latter also occur with single cell recording (electrosensillogram) in the receptor potentials as well as in the number of action potentials released per second. The results of the measurements are discussed on a molecular level, a scheme reflects the molecular processes which eventually are involved in the interaction of a signal molecule and receptor region.

Rabbit antisera raised against purified enoate reductase from Clostridium tyrobutyricum (DSM 1460) and horse-radish peroxidase-conjugated staphylococcal protein A or anti-rabbit immuno­ globulin G, respectively, were used to develop enzyme immunoassays. Sensitivity limits of the assay are about 250 pg antigen if the enzyme immunoassays are performed on membrane filters and examined visually, and 20 pg for tests in aqueous solution with spectrophotometrical evalua­tion. The procedures were applied for dot and Western blots as well as colony lifts. Immunologi­ cal distances between enoate reductases from different Clostridia were determined and the amounts of antigen present in bacterial crude extracts were estimated. In crude extracts of C. thermoaceticum a protein of approximately the size of the enoate reductase and its subunits from C. tyrobutyricum was immunologically detected. Gel filtration chromatography of identically pretreated crude extracts from C. thermoaceticum and C. tyrobuty­ricum produced immunological signals at similar molecular weights and revealed a lesser tendency of the presumed thermophilic enoatereductase from C. thermoaceticum to disintegrate into its subunits and fragments as compared to its mesophilic counterpart.

Bud exudates of some poplars are compared using GC-MS. P. candicans resembles P. balsami­fera. A sample of "Balm of Gilead" purchased in Oxford proved not to resemble its supposed source, P. candicans. Instead it was similar to P. nigra, from which it was presumably collected in Europe.

When opening in the morning flowers of Hibiscus mutabilis L. appear white or ivory. The flower colour changes to red by late afternoon due to the accumulation of the anthocyanin cyanidin-3-sambubioside. At the onset, and during the rapid phase of pigment accumulation, phenylalanine ammonia-lyase (PAL) activity in the petals increases rapidly to seven times its initial level and then decreases while the flower senesces. In excised petals, the PAL inhibitor L-α-aminooxy-β-phenylpropionic acid (AOPP) suppresses pigment formation and causes the accumu­lation of phenylalanine. Anthocyanin synthesis depends, therefore, on the de novo production of cinnamic acid.

Differentiated plants and suspension cultures of the taxonomic divisions Bryophyta, Pteridophyta, and Spermatophyta have been investigated as to their ability to detoxify heavy metals like Cd 2+ through the formation of (γ-Glu -Cys) n -Giy peptides, the phytochelatins. Over 200 individual plants have been checked and there was not a single exception observed. Only in the order Fabales several species mainly of the tribus Fabaceae form upon exposure to Cd 2+ ions peptides of the general structure (γ-Glu-Cys) n -β-Ala, the homo-phytochelatins. The existence of glutathione and homo-glutathione within a given species determines whether phytochelatins or their homo-derivatives are formed. The ability to form phytochelatins for metal homeostasis and metal detoxification is a principal feature of plant metabolism .

The cyanobacterium A nacystis nidulans was grown on nitrate or L-arginine as sole nitrogen source and in the presence of different divalent cation concentrations (1 mᴍ MgSO 4 and 0.1 mᴍ CaCl 2 or 0.1 mᴍ MgSO 4 and 0.05 mᴍ CaCl 2 ). The L-amino acid oxidase previously reported to be present in Anacystis nidulans (E . K. Pistorius and A. E. Gau, Biochim. Biophys. Acta 849, 203, 1986) was shown to be involved in L-arginine catabolism in cells grown with the lower divalentcation concentration. Under these conditions L-arginine was partly degraded via 2-ketoarginine and 4-guanidinobutyrate. On the other hand, at higher cation concentrations the ʟ-amino acid oxidase activity seem ed to be not sufficient to provide enough NH 4 + from ʟ-arginine for cell growth. Under those conditions photosystem II activity was initially reduced, and growth on ʟ-arginine could only start after photosystem II activity increased again and after arginase was induced. The arginase pathway was functional in A . nidulans grown on ʟ-arginine independently of the divalent cation concentration in the medium. A tentative scheme of the various functiona roles of the ʟ-amino acid oxidase protein in A . nidulans is given. This model combines the here presented and the previous results and suggests that the ʟ-amino acid oxidase is functional in photosynthetic and respiratory activities as well as in ʟ-arginine degradation in A . nidulans. All these activities of the ʟ-amino acid oxidase protein are greatly influenced by the divalent cation concentration in the growth medium.

Phormidium foveolarum was grown with nitrate or ammonia as nitrogen source. With ammonia present respiration increased by a factor of 4 due to increase of cytochrome c oxidase. The inhibition of respiration by cyanide was measured during the growth period, indicating that only one terminal oxidase is present in Phormidium foveolarum . Cytochrome c oxidase from ammonia-grown filaments was solubilized and purified on phenylsuperose and by anion exchange 100-fold com pared to the crude cell extract. The difference spectrum (oxidized minus reduced) shows absorption peaks at 417 nm, 514 -520 nm. 550 nm and 605 nm. These maxima, together with a low inhibition constant for cyanide (K i = 0.4 μᴍ) and a higher one for azide (k i = 1.75 mᴍ) are interpreted in terms of an αα 3 -type cytochrome c oxidase present containing noncovalently-bound cytochrome c.

The reversible hydrogenase of the cyanobacterium Anacystis nidulans was purified 250-fold by classical methods. Activity staining on gels obtained by native PAGE allowed to identify two bands. A ntibodies were raised against the electrophoretically homogeneous protein. The molecu­lar weight of the hydrogenase was determined by SD S-PAGE follow ed by Western blot analysis with these antibodies. Crude extracts from the unicellular Anacystis and from heterocysts and vegetative cells of Anabaena variabilis showed precipitation bands of 56 and 17 kd. The reversible hydrogenase appeared to be com posed o f a larger and a smaller subunit as the NiFeS-hydrogen-ases from other bacteria. The hydrogenase subunits, particularly the smaller on e, were subject to proteolysis. By application of the protein A-immuno-gold labeling technique the reversible hydro­ genase was shown to associate with the cytoplasmic membrane in Anacystis. When A nacystis was grown microaerobically, the increase in the specific activity of the reversible hydrogenase paral­leled the raise in the gold-labeling at the cell periphery as measured from the electron micro­ graphs. It is concluded that Anacystis has two different hydrogenases: the reversible or bidirec­tional hydrogenase which is located exclusively at the cytoplasmic membrane of the cells, and a thylakoid-bound enzyme which catalyzes only the uptake of H 2 .

Carbon monoxide dehydrogenase of anaerobic bacteria catalyzes the reversible conversion of CO to "CO 2 " . With the enzyme from Peptostreptococcus productus it is shown that CO 2 rather than HCO 3 - (H 2 CO 3 ) is the active species of "CO 2 " formed by this dehydrogenase.

Carbon isotope fractionation during autotrophic growth o f different bacteria which possess different autotrophic CO 2 fixation pathways has been studied. 13 C / 12 C -Ratios in the cell carbon of the following bacteria were determined (CO 2 fixation pathway suggested or proven in paren­theses): Alkaligenes eutrophus (reductive pentose phosphate cycle), Desulfobacterium autotrophicum and Acetobacterium woodii (reductive acetyl-CoA pathway), Desulfobacter hydrogenophilus and Thermoproteus neutrophilus (reductive citric acid cycle). The Δδ 13 C values, which indicate the per mille deviation of the 13 C content of cell carbon from that of the CO : used as the sole carbon source, range from - 10% ° (reductive citric acid cycle) over - 26% ° (reductive pentose phosphate cycle) to -36% ° (reductive acetyl-CoA pathway). Acetate formed via the acetyl-CoA pathway by the acetogenic Acetobacterium woodii showed a Δδ 13 C = -40% °. These data are discussed in view of the different CO 2 fixation reactions used by the bacteria and especially with regard to the isotopic composition of sedimentary carbon through time.

The main chlorophyll a/b protein complex of the chloroplast thylakoid membrane is organized into two subpopulations; one inner which is tightly bound to the photosystem II core and one outer which is bound more loosely or peripherally. In this study, changes in the LHC II com position due to long-term light acclimation were analyzed and quantified in spinach thylakoids and isolated stroma lamellae vesicles. The results show that; photosystem II located in the appressed thylakoid regions (α-centres) which have a relatively large antenna size, contains both the inner and outer LHC II with a predominance of the latter (58-70%). At low light the antenna size o f the α-center becomes larger due to a specific increase of the outer pool o f LHC II. The antenna size of photosystem II in the stroma thylakoids (β-centres) is smaller and contains mainly or only the inner LHC II pool. In contrast to the α-centres the β-centres centres do not undergo adaptive changes in their size in response to long-term changes in the light intensities.

The antenna complex B 880 of Rp. marina has been isolated by applying ion-exchange chromatography on Whatman DE -52 resin and sucrose density centrifugation of LDAO-solubilized photosynthetic membranes. The antenna polypeptides B 880-α and B 880-β were pre­pared by organic solvent extraction of extensively dialyzed and freeze-dried B 880 antenna complex material or photosynthetic membranes. Gel filtration on Sephadex LH-60 and ion-exchange chromatography on Whatman DE -32 resin in the presence of organic solvents and an additional step on a C-8 reversed phase column yielded pure α-and β-apoproteins. Their complete primary structures have been elucidated using automated Edman degradation and carboxypeptidase diges­tion. According to quantitative Edman degradation the ratio of B 880-α and B 880-β has been determined as 1:1 in the isolated antenna complex as well as in the photosynthetic membrane. B 880-α of Rp. marina, presumably N -form ylated, consists of 52 amino acid residues and is 75, 56, 52 and 44% homologous to the corresponding core antenna polypeptides of Rs. rubrum, Rp. viridis, Rb. capsulatus and Rb. sphaeroides. In contrast, B 880-β (56 amino acid residues) is less homologous to the corresponding core β-antenna polypeptides of the same strains (57. 51. 41 and 42%). It shows an extended N-terminal domain as compared to the B 880-a polypeptide. Apart from the typical structural features of bacterial membrane-bound antenna polypeptides (three domain structure, His-residue in the hydrophobic stretch) the antenna polypeptides of Rp. marina are structurally related to polypeptides of core antenna complexes with strong near infra­red circular dichroism signals.

Cytochrome b-559. Spinach Thylakoids. PS II Particles. High and Low Potential. Interconvertible Forms Cytochrome b-559 has been purified from both spinach (Spinacia oleracea L.) thylakoids and photosystem II particles in order to compare some of its more polemic molecular properties. In the two cases, cytochrome 6-559 (which is initially present in its reduced high-potential form and in its oxidized low-potential form) is purified as a single species by preparative disc electrophoresis in Triton-containing polyacrylamide gel, the isolated protein exhibiting similar molecular mass and almost identical low midpoint redox potential and absorption spectra. Moreover, sodium dodecyl sulfate polyacrylamide gel electrophoresis of the purified cytochrom e b-559 reveals, again in both cases, only one polypeptide band of similar molecular mass. Integration o f the isolated low-potential form into liposomes partly restores the high-potential form. These data corroborate our previous results and indicate that there exists in thylakoids only a cytochrome b -559 molecular species tightly bound to PS II and that the two naturally occurring low-and high-potential couples are physiological and correspond to interconvertible states of the genuine hem e protein.

In the present study the effect of TBTQ on PS II and its mutual interaction with DCMU was analyzed by measurements of the oxygen yield oscillation pattern and of DCMU binding. It was found: 1)TBTQ in its reduced form is able to induce the reduction of D ox which gives rise to an accelerated decay of S 2 and S 3 of the wateroxidizing complex. 2) Triton X-100 treatment used for isolation of PS II membrane fragments does not significantly affect the lateral mobility of p lastoquinone within the membrane. TBTQ bound to the thylakoid membrane does not enhance the electron pool capacity in PS II membrane fragments. 3) Preincubation of thylakoids with TBTQ diminishes the blockage of O2-evolution by DCMU significantly. In correspondence with previous findings [18, 19] the effect strongly depends on the order of addition of TBTQ and DCMU . 4) Excitation with a single saturating flash causes enhanced DCMU binding in TBTQ pretreated samples leading to the inhibition of flash induced oxygen evolution. The rate of the latter process depends on the DCMU concentration. 5) In thylakoids pretreated in the d ark with TBTQ the oxygen yield of the 3rd flash slowly declines as a function o f dark incubation time at constant DCMU concentration. Based on the above mentioned findings it is inferred that a mutual interaction between TBTQ and DCMU takes place at the PS II acceptor side. Two alternative mechanisms are discussed: i) TBTQ tightly (covalently?) bound at the Q B -site (or very close to it) is modified in its function by DCMU via structural effects (allosteric type), or ii) there occurs a TBTQ /DCMU exchange that is fast in the light and slow in the dark.

Structure and function of the Q B -protein of a metribuzin resistant mutant of Chlamydomonas reinhardii were analyzed. The amino acid residue Leu-275 of the wild type protein is changed to Phe as was determined by RNA -sequence analysis. This mutation caused a 20-fold and 5-fold resistance to metribuzin and DCMU , respectively. No resistance to atrazine was observed. The kinetics of the electron transport from Q A to O B was similar to that of the wild type (t 1/2 = 0.4 ms).

The conformations and electronic properties of several urea/amide, uracil, s-triazine and methylthiopyrimidine inhibitors of photosystem II were determined using molecular mechanics (CHARMM , SYBYL) or quantum semi-empirical (MNDO , AM1) methods. Spectroscopic measurements of the in vitro hydrogen bonding capacity of these com pounds were also carried out. The validity of the calculated values was estimated from experimental measurements. The conformational and electronic properties of the inhibitors studied are then discussed in terms of their mechanism of action on the D1 site and its modification by triazine resistance.

Azido-ioxynil (3,5-diiodo-2-azido-4-hydroxy-benzonitrile) is a potent photosystem II inhibitor (pI 50 -value 7.38) and as effective as the parent compound ioxynil itself. [ 125 I]azido-ioxynil exhibits specific binding to isolated thylakoids with a binding constant K b = 7.14. Upon UV -illumination it binds covalently to thylakoids or photosystem II particles. It labels predominantly the 32 kDa D-1 photosystem II reaction center protein . A 41 kDa protein is only tagged in trace amounts. After proteolytic treatment of labeled D -1 protein with Staphylococcus aureaus V8 -protease two major and two minor fragments are obtained. Automated gas phase sequencing of a 7 kDa cleavage peptide revealed that Val 249 is the primary target of azido-ioxynil binding. Compared to urea type herbicides, this places the ioxynil binding site in a different environment of the D -1 photosystem II protein.

A characteristic 23.5 kD a degradation intermediate of the 32 kDa photosystem II reaction center protein is produced upon illumination in UV , visible and far red light. We suggest a similar degradation pathway is employed in these three spectral regions, even though the light can enter the system through different photo receptors.

The to pography of chloroplast cytochromes f and b 6 was probed with proteases carboxypeptidase A (CpA ), trypsin, and Staph, aureus V8. The cytochrome and its proteolytic products were detected by heme stain and, in most experiments, by immunoreaction. In thylakoids, the only protease that significantly affected the intactness of cytochrome f was CpA that caused a small (ΔM r = -1 - 2000) decrease in the apparent molecular weight. In SDS -treated thylakoids, both trypsin and V8 degraded cytochrome f. The inferred topography of cytochrom f , with the COOH -terminus on the stromal (n) side, one membrane-spanning a-helix n ear the COOH-terminus, and most of the Cyt f mass on the lumen (p) side, is consistent with that previously inferred by others. Cytochrome b 6 was not sensitive to CpA . but was more sensitive to trypsin and V8 protease than cytochrome f , cytochrome b-559, or the 17 kDa OEC extrinsic protein. Trypsin caused a small decrease in size of cytochrome b 6 , which was observed using whole protein antibody as a single smaller band (ΔM r ≈ 2000) or two smaller discrete bands (ΔM r = -1000 and 2500, respectively) which, unlike the untreated protein , did not react with antibody generated to a peptide mimicking A sp-5 -Gln-14 near the NH 2 -terminus. These shorten ed tryptic fragments were attributed to cleavage after R-10 and K-23 n ear the NH 2 -terminus, implying an orientation with the NH 2 -terminus on the stromal side of the membrane. The sensitivity of cytochrome b 6 toward this trypsin cleavage was increased if the membranes were first incubated with CpA , showing that the NH 2 -terminal region of cytochrome b 6 is masked by the COOH -terminal domain of one or more thylakoid proteins. Under conditions where degradation of cytochrome b-559, cytochrome f, or the 17 kDa OEC extrinsic protein on the lumen side of the membrane was small ( < 10-20% ). V8 protease degraded 80-90% of cytochrome b 6 . The main polypeptide fragment generated by V8 protease had an apparent molecular weight M r = 16,000, reacted with antibody to a peptide mimicking Ala-146-Asp-155, and was more pronounced in membranes treated with SDS. The fragment could then result from cleavage at two sites, Glu-74 and /or Glu-166. It is concluded that the loop of cytochrome b 6 protruding from the lumen side of the membrane between helices III and IV , and possibly that between helices I and II as w ell, contains a single Glu residue conferring a sensitivity to V8 protease greater than that of the lumen-side mass of cytochrome f which has many more Glu residues. This implies a lumen-skeletal structure in which one or two loops of cytochrome b 6 on the lumen side are more exposed to the aqueous phase than cytochrome f.

Stigmatellin causes spectral changes of both hemes of cytochrome b or b6 in cytochrome bc1/b6f-complexes. It also affects the redox potentials of all three hemes, including cytochrome c l and f, in addition to the dramatic rise of the redox potential it exerts on the Rieske FeS-center. We conclude that stigmatellin changes the overall conformation of the complexes.

Thylakoids and photosystem I (PS I) preparations from Euglena gracilis and from the cyanobacterium Anacystis nidulans were analyzed for their prenylquinone content. Both organisms contained 5 '-monohydroxyphylloquinone as the dominant quinone of PS I, and not phylloquinone as other organisms. A stoichiometry of about two 5 '-monohydroxyphylloquinone per P700 (the primary electron donor of PS I) was determined for A nacystis. However, both the PS I preparation and the thylakoids from Euglena contained only one 5 '-monohydroxyphylloquinone per P700. Phylloquinone and an unidentified naphthoquinone were present in trace amounts.

Rates of photophosphorylation were measured at constant saturating phosphate concentration , varying ADP concentration , and varying light intensity. As the transmembrane proton gradient is decreased by phosphorylation to different extents depending on the concentration of ADP . rates of ATP formation obtained at the different ADP concentrations were plotted versus the actual steady state ΔpH (in the absence of ΔΨ) during the course of the reaction . ΔpH was monitored by the calibrated 9-aminoacridine fluorescence technique. In secondary plots phosphorylation as function of ADP concentration at different constant ΔpH values were obtained . The results indicate Michaelis-Menten kinetics. The true K m for ADP is virtually unaffected by ΔpH whereas V max (at ADP saturation ) strongly depends on ΔpH . The results are discussed in the framework of a simple enzyme kinetic model which considers the intrathylakoidal proton (at constant external pH ) as a third substrate for ATP formation. The model is capable o f explaining the reported results as well as a variety of important results from the literature.

The amino acid residues of spinach CF 1 subunit delta are identified which are accessible and thus exposed within the quaternary structure of the ATP-synthase complex on the thylakoid membrane. Two types of antibodies in the monospecific polyclonal antiserum 306 against CF 1 , delta, described in the previous publication [Z. Naturforsch. 44c, 153 - 160 (1989)], were separated by virtue of their different affinity to thylakoid membranes and used for specific analysis of the products of proteolytic digestion of delta in situ. Polypeptide delta in situ, i.e. within the CF 0 C F 1 , complex on the membrane, is not susceptible to digestion by aminopeptidase M and trypsin, but is shortened by about 1 kDa by carboxypeptidase Y and digested at residues Glu 173 and Glu 179 by the Staphylococcus aureus protease V8 . The epitope on delta reacting with the agglutinating antibodies from serum 306 is lost after these proteolytical treatments and there fore situated on residues Met 180 -Val 187 . Since trypsin destroys this epitope only after prolonged incubation and with at least 50 μg trypsin/mg Chl, residue Lys 169 of delta probably is inaccessible in situ. We conclude that the C -terminal am phipathic α-helix of spinach CF 1 subunit delta is exposed on the thylakoid membrane, with the hydrophilic face directed to the outside, and th at CF 1 delta starts to be shielded within the quaternary structure of the CF 0 CF 1 complex between Glu 173 and Lys 169 . The hydrophobic face of the c-terminal helix may be part of the binding surface towards CF 0 . Antibodies from serum 306 inhibit the PMS mediated cyclic photophosphorylation by reacting with C -terminal residues of δ.

A chloroplast type of fructose-1,6 -bisphosphatase, a central regulatory enzyme of photosynthetic carbon metabolism, has been partially purified from Chlamydomonas reinhardtii. Unlike its counterpart from spinach chloroplasts, the algal FBPase showed a strict requirement for a dithiol reductant irrespective of Mg 2+ concentration. The enzymes from the two sources resembled each other immunologically, in subunit molecular mass and response to pH. In the presence of dithiothreitol, the pH optimum for both the algal and spinach enzymes shifted from 8.5 to a more physiological value of 8.0 as the Mg 2+ concentration was increased from 1 to 16 mᴍ . At 1 mᴍ Mg 2+ , a concentration estimated to be close to physiological, the Chlamydomonas FBPase was active only in the presence of reduced thioredoxin and was most active with Chlamydomonas thioredoxin f. Under these conditions, the enzyme showed a pH optimum of 8.0. The data suggest that the Chlamydomonas enzyme resembles its spinach counterpart in most respects, but it has a stricter requirement for reduction and less strict reductant specificity. A comparison of the prop­erties of the FBPases from Chlamydomonas and spinach will be helpful for elucidating the mechanism of the reductive activation of this enzyme.

Sulfate uptake into the unicellular green alga Chlorella fusca Shihira et Krauss strain 211-8 b was described as a pH-, temperature-and energy-dependent process. Transport followed triphasic kinetics with K m -values of 1.3 x 10 -5 , 3.9 x 10 -5 and 2.8 x 10 -4 M . The Hill plot showed coeffi­cients of 1.0 for each uptake isotherm. Sulfate uptake was substantially inhibited by chromate, selenate, sulfite, thiosulfate and tetrathionate. Cysteine and other mercaptides diminished sulfate transport, whereas methionine was not inhibitory. Uptake was sensitive towards inhibitors of photosynthesis and respiration and towards SH-group reagents. Sulfate uptake could be strongly enhanced by polyvalent cations, apparently via reduction of the surface potential. Sulfate uptake in S-deprived algae was markedly different from constitutive sulfate uptake. Time kinetics was not linear but showed an initial rapid phase. Uptake was neither multiphasic nor conformed to simple Michaelis-Menten kinetics. Sulfate uptake was hardly affected by pH and temperature in the suboptimal range. The Arrhenius plot was biphasic with a discontinuity at 38 °C.

Higher plant leaf protein was investigated for the enzyme activity catalyzing a thioredoxin-dependent reduction of 3'-phosphoadenylylsulphate (PAPS) to sulphite. The enzyme became detectable when heterologous thioredoxin from Escherichia coli was used substituting for the hitherto unidentified plant thioredoxin. The enzyme's cross-reactivity with heterologous thioredo­ xin enabled the partial purification and brief characterization. The molecular weight of the en­ zyme as estimated by HPLC size exclusion and gel filtration was 68-72 k. The protein reduced PAPS only when thioredoxin was present as cosubstrate. The function of this enzyme in the assimilation of inorganic sulphate by higher plants is discussed in comparison to the function of the respective enzymes from Escherichia coli and Saccharom yces cerevisiae.

Processes of 35 S-labelled sulfite uptake and efflux by oat protoplasts and their chloroplasts are very rapid: during the first minutes (or perhaps even seconds) after sulfite supply a state of equilibrium is established between the protoplasts and their chloroplasts and the incubation medium. Bicarbonate concentrations ranging from 1 to 5 mᴍ in the medium had no significant effect on sulfite uptake. At pH 7.0 sulfite uptake is much greater than at pH 7.6. Light of low intensity (9 W m -2 ) had no significant effect on this uptake. The 35 S concentration in chloroplasts was always much higher than in the whole protoplasts. One hour's incubation at sulfite concentra­ tions of 0.1 and 0.5 mᴍ in the medium, concentrations slightly stimulating or inhibiting photosyn­ thesis, results in sulfite concentrations of 0.004 to 0.230 mᴍ in chloroplasts. At a sulfite concentration photosynthesis, these values are 0.120 to 0.551 and 2.045 to 3.531, respectively.

The fragmentation of KMB (4-(methylthio)-2-oxobutyric acid) as an indicator reaction for the production of strong oxidants is catalyzed by diesel soot particles in the light. This reaction operates also in the dark and can be strongly stimulated by the addition of sulfite. The cooperative activity of diesel soot particles (DP) and sulfite also bleaches the carotenoid crocin. This molecule represents a model for both the groups of plant pigments as well as polyene structures embedded as flexible backbones in cellular membranes. Enhanced chemiluminescence generated by linolenic acid in the presence of either ter/-butylhydroperoxide or hydrogenperoxide is detected in the presence of both DP and HSO 3 - . Luminescence originates from singlet oxygen or chemically excited molecules such as triplet ketones and is thus a valuable indicator for peroxidation of lipids involved in membrane damage. All reactions were inhibited by SOD (superoxide dismutase) whereas catalase had no effect.

Effects of Pi deficiency on photosynthesis ot isolated spinach chloroplasts were examined. The following observations were made: (1) Chloroplasts isolated in Pi-free media evolved oxygen in the light in the absence of added Pi until acid-extractable P i in the chloroplasts had decreased to 1 to 2.5 m M . This P i was unavailable for photophosphorylation as shown by the inability of the chloroplasts to respond by oxygen evolution to the addition of PGA. In the state of P i- deficiency, stromal ATP to A DP ratios were in the light close to or below ratios observed in the dark. In the presence of 2 mᴍ PGA, addition of 20 μm Pi was insufficient to increase ATP to ADP ratios, but sufficient for appreciable oxygen evolution. (2) More Pi was available for oxygen evolution of phosphate-deficient chloroplasts at low levels of C0 2 than at high levels. This was due mainly to the suppression of oxygenation of RuBP by high C0 2 levels which prevented formation of phosphoglycolate and the subsequent release of Pi into the chloroplast stroma. (3) More oxygen was produced by phosphate-deficient chloroplasts at a low light intensity than at a high light intensity. This was due to increased availability of endogenous Pi under low light and to photoinhibition of the chloroplasts by high light. The main product of photosynthesis of phosphate-deficient chloroplasts in the presence of a high bicarbonate concentration was starch, and the main soluble product was PGA. (4) After phosphate-deficient chloroplasts had ceased to evolve oxygen in the light, they be­ came photosensitive. Part of the loss of the capacity for oxygen evolution is attributed to leakage of PGA, but the main reason for loss of function is photoinactivation of electron transport. Both photosystems of the electron transport chain were damaged by light. (5) Protection against photoinactivation was provided by coupled electron transport. Photo­ inactivation of phosphate-deficient chloroplasts was less extensive in the presence of low C0 2 concentrations which permitted oxygenation of RuBP than at high CO 2 concentrations. Electron transport to C0 2 and other physiological electron acceptors and to the herbicide methylviologen was also protective. However, electron transport to oxygen in the Mehler reaction failed to provide appreciable protection against high light intensities, because oxygen reduction is slow and reactive oxygen species produced in the light contribute to photoinactivation.

Plant The effect of chemical modification of histidyl residues using diethyl pyrocarbonate (DEP) on plant mitochondrial electron transport was studied. Mitochondrial membranes from potato tubers were isolated and electron flow from NADH to oxygen, NADH to ferricyanide and ascorbate to oxygen were monitored in pro», nee and/or absence of DEP. Measurements were made at various concentrations of DEP and at different pHs either by using an oxygen electrode or spectro-photometrically. The results show that DEP inhibits flow of electrons from NADH to oxygen, however partial electron transport from NADH to ferricyanide and ascorbate to oxygen was unaffected. Maximum inhibition was observed at pH 6.5. The time course of the DEP action revealed a biphasic nature of inhibition. Effects on the levels of reduction of cytochromes b and c by DEP during electron transport indicated that histidyl residues may be present before or at cytochrome b , which are being modified.