Interaction of ferredoxin-NADP+ oxidoreductase with triazine dyes. A rapid purification method by affinity chromatography.

The triazine dyes, Cibacron blue F3GA and Procion red HE3B inhibited diaphorase activity of ferredoxin-NADP+ reductase, in a competitive manner with respect to NADPH. The Ki values were 1.5 and 0.2 microM, respectively. Binding of the dyes to the flavoprotein, as measured by difference spectroscopy, indicated an apparent stoichiometry of 1 mol dye/mol reductase and was prevented by NADP+ or high ionic strength. Chemical modification of a lysine residue and a carboxyl group at the NADP(H) binding site of the enzyme prevented complex formation with Procion red. Procion red showed a higher affinity for ferredoxin-NADP+ reductase than Cibacron blue. The Kd values were 1.9 and 5 microM, respectively. Once covalently linked to a Sepharose matrix, the triazine compounds specifically bind the flavoprotein. The interaction is partially electrostatic and partially hydrophobic. The enzyme can be eluted by high concentrations of salt or low concentrations of the corresponding coenzyme. The use of this affinity column allows the rapid purification of ferredoxin-NADP+ oxidoreductase from spinach leaves with good yields.

Alkaloids as inhibitors of photophosphorylation in spinach chloroplasts.

A group of 12 alkaloids were tested as inhibitors of photophosphorylation in spinach chloroplasts. Ajmaline, a dihydroindole alkaloid, was found to be the strongest inhibitor of both cyclic and non-cyclic photophosphorylation. Low concentrations of ajmaline also inhibited the dark and light ATPases, and the coupled electron flow from water to ferricyanide, measured either as ferrocyanide formed or as oxygen evolved, but not the uncoupled electron transport or the pH rise of illuminated unbuffered suspensions of chloroplasts. Higher concentrations of ajmaline stimulated, instead of inhibiting, photosynthetic electron transport or oxygen evolution and decreased the pH rise, thus behaving as an uncoupler, such as ammonia. Photophosphorylation was partially inhibited by 100 microM dihydrosanguinarine, 100 microM dihydrochelerythrine (benzophenanthridine alkaloids); 500 microM O,O'-dimethylmagnoflorine, 500 microM N-methylcorydine (aporphine alkaloids) and 1 mM julocrotine. They also inhibited coupled oxygen evolution and only partially (dihydrosanguinarine and dihydrochelerythrine) or not at all (the other alkaloids) uncoupled oxygen evolution. Spegazzinine (dihydroindole alkaloid), magnoflorine, N-methylisocorydine, coryneine (aporphine alkaloids), candicine and ribalinium chloride were without effect on photophosphorylation at 500 microM.

Spegazzinine, a new inhibitor of mitochondrial oxidative phosphorylation.

The effects of spegazzinine, a dihydroindole alkaloid, on mitochondrial oxidative phosphorylation were studied. Spegazzinine inhibited coupled respiration and phosphorylation in rat liver mitochondria. The I50 was 120 microM. Uncouplers released the inhibition of coupled respiration. Arsenate-stimulated mitochondrial respiration was partially inhibited by spegazzinine. The stimulation of mitochondrial respiration by Ca2+ and the proton ejection associated with the ATP-dependent Ca2+ uptake were not affected by the alkaloid. Oxidative phosphorylation and the Pi-ATP exchange reaction of phosphorylating beef heart submitochondrial particles were strongly inhibited by spegazzinine (I50, 50 microM) while the ATP-dependent reactions, reduction of NAD+ by succinate and the pyridine nucleotides transhydrogenase were less sensitive (I50, 125 microM). Oxygen uptake by submitochondrial particles was not affected. The 2,4-dinitrophenol-stimulated ATPase activity of rat liver mitochondria was not affected by 300 microM spegazzinine, a concentration of alkaloid that completely inhibited phosphorylation. However, higher concentrations of spegazzinine did partially inhibit it. The ATPase activities of submitochondrial particles, insoluble and soluble ATPases were also partially inhibited by high concentrations of spegazzinine. The inhibitory properties of spegazzinine on energy transfer reactions are compared with those of oligomycin, aurovertin and dicyclohexylcarbodiimide. It is concluded that spegazzinine effects are very similar to the effects of aurovertin and that its site of action may be the same or near the site of aurovertin.

Sulphydryl groups in photosynthetic energy conservation. I. Light-dependent inhibition of photophosphorylation by the sulphydryl reagent 2-2'dithio bis-(5-nitropyridine).

1. The sulphydryl reagent 2-2'dithio bis-(5-nitropyridine) (DTNP) inhibited photophosphorylation when the chloroplasts were preincubated with the reagent in the light. A maximum inhibition of about 50% was obtained in the presence of pyocyanine and MgCl 2 at 0.3 mumol DTNP per mg chlorophyll and was completed in about 40 s of preillumination. 2. Dithioerythritol, ADP plus Pi (or arsenate) and uncouplers prevented the inhibition when present during the preillumination while phloridzin, Dio-9 and discarine B were ineffective. Low concentrations of ADP or ATP afforded partial protection but other nucleotides had no effect. 3. DTNP inhibited the coupled electron transport rate to the basal level and had no effect on the uncoupled electron transport. The stimulation of proton uptake and inhibition of electron transport by ATP was prevented by DTNP. 4. The trypsin-activated but not the light- and dithioerythritol-triggered ATPase was inhibited by light preincubation of chloroplasts with DTNP. 5. Reversal of DTNP inhibition of photophosphorylation was obtained by a second preillumination in the presence of thiol groups. 6. More DTNP reacted with chloroplasts in the light than in the dark. Two mol of thione were formed in the light per mol of DTNP disappeared. 7. The results suggested that DTNP inhibition is related to the oxidation by DTNP of chloroplast vicinal dithiols probably exposed by a light-induced conformational change.

Sulphydryl groups in photosynthetic energy conservation. V. Localization of the new disulfide bridges formed by o-iodosobenzoate in coupling factor of spinach chloroplasts.

1. Chemical modification by o-iodosobenzoate of soluble chloroplast coupling factor 1 (CF1) during heat activation resulted in inhibition of its Ca-ATPase activity and in the formation of two new intrapeptide disulfide bridges as suggested by: (a) the disappearance of three out of four accessible thiol groups, two from gamma and one from a beta subunit as a consequence of CF1 modification by o-iodosobenzoate; (b) the total free sulphydryl groups of CF1 were reduced from 8 to 4 after modification of CF1 by o-iodosobenzoate. Two groups disappeared from beta and two from gamma subunits; (c) a second heating step of CF1 in the presence of 10 mM dithioerythritol reversed the inhibition of the ATPase and reduced both the newly formed disulfide bridges and those present in native CF1. 2. Modification of chloroplasts in the light with o-iodosobenzoate resulted in the inhibition of photophosphorylation and ATPase. CF1 isolated and purified from these chloroplasts had its Ca-ATPase activity inhibited and two new disulfide bridges. The total number of free sulphydryl groups was reduced from 8 to 4 and three accessible groups disappeared from beta and gamma subunits.

Functional arginine residues and carboxyl groups in the adenosine triphosphatase of the thermophilic bacterium PS-3.

Treatment of purified ATPase of the thermophilic bacterium PS-3 with the arginine reagent phenylglyoxal or with Woodward's reagent K, gave complete inactivation of the enzyme. The inactivation rates followed apparent first-order kinetics. The apparent order of reaction with respect to inhibitor concentrations gave values near to 1 with both reagents, suggesting that inactivation was a consequence of modifying one arginine or carboxyl group per active site. ADP and ATP strongly protected the thermophilic ATPase against both reagents. GDP and IDP protected less, whilst CTP did not protect. Experiments in which the incorporation of [14C]phenylglyoxal into the enzyme was measured show that extrapolation of incorporation to 100% inactivation of the enzyme gives 8-9 mol [14C]phenylglyoxal per mol ATPase, whilst ADP or ATP prevent modification of about one arginine per mol.

Sulfhydryl groups in photosynthetic energy conservation. VI. Subunit distribution of sulfhydryl groups and disulfide bonds in chloroplast coupling factor and ATPase activity.

The subunit distribution of sulfhydryl groups and disulfide bonds of spinach chloroplasts coupling factor I has been determined. Native coupling factor I with a latent ATPase activity has eight sulfhydryl groups distributed 4 : 2 : 0 : 0 : 2 in the alpha, beta, gamma, delta and epsilon subunits, respectively. Heat treatment of coupling factor I, in addition to the activation of its ATPase activity, induces a dithiol-disulfide interchange between the gamma and the alpha subunit, changing the sulfhydryl groups' distribution to 2 : 2 : 2 : 0 : 2. Reduction of disulfide bonds of coupling factor I by dithioerythritol during heat treatment gives a subunit distribution of 4 : 4 : 4 : 0 : 2, suggesting that native coupling factor I has three disulfide bonds, two in the gamma subunit and one in one of the beta subunits. The results suggest an asymmetric redox state of some of the subunits of coupling factor I and an asymmetric positioning of some of them in the molecular structure of coupling factor I.

Sulphydryl groups in photosynthetic energy conservation. IV. Inhibition of the ATPase of chloroplast coupling factor 1 by sulphydryl reagents.

1. O-Iodosobenzoate and 2,2'-dithio bis-(5-nitropyridine) inhibited by about fifty per cent the ATPase activity of heat-activated chloroplast coupling factor 1 only when present during the heating but were without effect when added before or after the activation. Reversion of this inhibition was only obtained by a second heat treatment with 10 mM dithioerythritol. 2. The inhibition of the Ca2+-ATPase of coupling factor 1 by o-iodosobenzoate or 2,2'-dithio bis-(5-nitropyridine) was not additive with similar inhibitions obtained with the alkylating reagents iodoacetamide and N-ethylmaleimide. 3. The heat-activated ATPase of o-iodosobenzoate-treated coupling factor 1 had a higher Km for ATP, without modification of V. The modified enzyme was desensitized against the allosteric inhibitor ADP.

Structure and characterization of the gene encoding the ferredoxin-NADP reductase-binding protein from Zea mays L.

A genomic clone encoding ferredoxin-NADP reductase binding protein (BP) from Zea mays L. was sequenced and characterized. The promoter region (692 bp) shows several motives resembling those involved in enhancement, tissue-specific expression and light regulation in plants, besides the typical TATA and CAAT boxes. The coding sequence is interrupted by two introns. The deduced amino acid (aa) sequence corresponds to 22.85 kDa for the precursor polypeptide, including a transit peptide of 64 aa and a mature protein of 148 aa.

Protein phosphorylation in purple photosynthetic bacteria.

Endogenous protein phosphorylation was shown in both in vitro and in vivo experiments in R. rubrum and in other purple photosynthetic bacteria. Among the substrates of this protein kinase activity the apoproteins of the light harvesting complex were tentatively identified. Phosphoamino acid analysis revealed the presence of phosphoserine, phosphothreonine and phosphotyrosine in R. rubrum. A tyrosine kinase was partially purified in the same bacteria.

Isolation and expression of a barley beta-1,3-glucanase isoenzyme II gene.

A beta-1,3-glucanase gene from Hordeum vulgare was isolated by a PCR strategy, cloned and subsequently sequenced. The amplified sequence contained the entire coding region of the isoenzyme II, which is interrupted by a 165 bp intron at 73 bp downstream the starting codon. This intron contains all the elements required for the processing mechanism in monocots: a high A + U content, the appropriate splice sites in the 5' and 3' ends and four typical YUNAN consensus sequences. Transient transformation of wheat protoplasts with the complete beta-1,3-glucanase gene under the control of maize polyubiquitin promoter revealed that the intron sequence was spliced out. The gene was also expressed at high levels, probably due to an enhancer-like sequence found near the 3' end of the intron.