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.     

Partial purification of d-glucose-6-phosphate dehydrogenase from bakers' yeast by affinity partitioning using polymer-bound triazine dyes

d-Glucose-6-phosphate dehydrogenase (d-glucose-6-phosphate:NADP⁺ 1-oxidoreductase EC 1.1.1.49) has been purified from bakers' yeast by liquid-liquid extraction using phase-restricted triazine dyes (Procion Yellow HE-3G, Procion Olive MX-3G, Procion Navy MX-RB and Cibacron Blue F3G-A). This method was combined with fractional precipitation with poly(ethylene) glycol) and batchwise treatment with DEAE-cellulose. This rapid procedure gave an enzyme preparation with a specific activity of 0.92 kat per kg protein within 5 h. The affinity extraction step can easily be scaled up and the good recovery of ligand-poly(ethylene glycol) should make the process useful for larger amounts of enzyme. The technical possibilities are discussed.     

Partial purification of d-glucose-6-phosphate dehydrogenase from bakers' yeast by affinity partitioning using polymer-bound triazine dyes

d-Glucose-6-phosphate dehydrogenase (d-glucose-6-phosphate:NADP⁺ 1-oxidoreductase EC 1.1.1.49) has been purified from bakers' yeast by liquid-liquid extraction using phase-restricted triazine dyes (Procion Yellow HE-3G, Procion Olive MX-3G, Procion Navy MX-RB and Cibacron Blue F3G-A). This method was combined with fractional precipitation with poly(ethylene) glycol) and batchwise treatment with DEAE-cellulose. This rapid procedure gave an enzyme preparation with a specific activity of 0.92 kat per kg protein within 5 h. The affinity extraction step can easily be scaled up and the good recovery of ligand-poly(ethylene glycol) should make the process useful for larger amounts of enzyme. The technical possibilities are discussed.     

Affinity and hydrophobic chromatography of Escherichia coli aspartate transcarbamoylase

Chromatography of aspartate transcarbamoylase from Escherichia coli on agarose-immobilized dyes and alkyl-agaroses of differing carbon length were investigated. The bacterial aspartate transcarbamoylase was bound by Procoin red HE3B-agarose and Cibacron blue F3GA-agarose nearly completely under the conditions chosen relative to other agarose-coupled dyes. The aspartate transcarbamoylase holoenzyme was eluted from the Procion red HE3B-agarose slightly later than from the Cibacron blue F3GA-agarose during salt gradient elution. The catalytic trimer of the enzyme as well as its regulatory dimer were eluted by a lower salt concentration from both dye-agarose gels than the concentration required to elute the haloenzyme. The interaction of the catalytic trimer with the Procion red HE3B-agarose and Cibacron blue F3GA-agarose gels may be a determinant in the holoenzyme being retained on these resins. Of those alkyl-agaroses tested, the ethyl-, propyl- and hexyl-agarose gels bound the majority of aspartate transcarbamoylase activity. Chromatography of aspartate transcarbamoylase on ethyl-agarose found it to be eluted by a low salt concentration. A purification scheme for relatively small amounts of aspartate transcarbamoylase utilizing Procion red HE3B-agarose and ethyl-agarose is presented. This purification scheme is particularly useful for mutant versions of aspartate transcarbamoylase which cannot be purified by literature procedures.     

A microcell for the temperature-jump technique

Pregnancy-specific β₁²-glycoprotein was purified to 99% purity by sequential chromatography on carboxymethyl-cellulose (to remove other pregnancy-specific β₁-glycoprotein variants), Cibacron blue 3G-A-Matrex Gel, Procion turquoise MX-G-Matrex Gel, Procion red H-8BN-Matrex Gel, and rabbit anti-normal human male serum-Matrex Gel. The purification was rapid, large-scale, and gave a yield of pregnancy-specific β₁-glycoprotein of 35.1%.     

Interaction of Cibacron Blue F3G-A and Procion Red HE-3B with sheep liver 5,10-methylenetetrahydrofolate reductase

Cibacron Blue F3G-A, a probe used to monitor nucleotide binding domains in enzymes, inhibited sheep liver 5,10-methylenetetrahydrofolate reductase competitively with respect to 5-methyltetrahydrofolate and NADPH. TheK _i values obtained by kinetic methods and theK _d value for the binding of the dye to the enzyme estimated by protein fluorescence quenching were in the range 0.9–1.2 μM. Another triazine dye, Procion Red HE-3B interacted with the enzyme in an essentially similar manner to that observed with Cibacron Blue F3G-A. These results as well as the interaction of the dye with the enzyme monitored by difference spectroscopy and intrinsic protein fluorescence quenching methods indicated that the dye was probably interacting at the active site of the enzyme by binding at a hydrophobic region.     

Affinity labeling of spinach ferredoxin-NADP+ oxidoreductase with periodate-oxidized NADP+

Periodate-oxidized NADP⁺ (dialdehyde-NADP⁺) inactivated soluble ferredoxin-NADP⁺ oxidoreductase and combined covalently to the enzyme. This inactivation was first order with respect to dialdehyde-NADP⁺ and followed saturation kinetics, indicating that the enzyme initially forms a reversible complex with the inactivator. NADP⁺ afforded complete protection against inactivation, while spinach ferredoxin was uneffective. In the presence of exogenous ferredoxin and illuminated thylakoids, the nucleotide analog functioned as a coenzyme for the reductase, although with rather lower efficiency than NADP⁺. It also acted as a competitive inhibitor with respect to NADPH in diaphorase activity. Incorporation of radioactivity from periodate-oxidized [³H]NADP⁺ gave a stoichiometry of 0.85 mol of reagent/mol of reductase, indicating that the modification of a single residue in the flavoprotein is responsible for the loss of enzymatic activity.     

Complex formation by ferredoxin-NADP+ reductase with ferredoxin or NADP+

Complex formation by ferredoxin-NADP⁺ reductase (NADPH:ferredoxin oxidoreductase, EC 1.6.99.4) with ferredoxin was measured by the independent methods based on the changes of circular dichroism, fluorescence intensity and the chromatographic behavior on a Sephadex G-75 column of the two proteins after mixing. Complex formation between the flavoprotein and NADP⁺ was also detected from the changes of various optical properties of the protein. These experiments suggested that the optical changes accompanying the complex formation were due to a change of the chromophore group in ferredoxin-NADP⁺ reductase, but not due to that of ferredoxin.     

Affinity and hydrophobic chromatography of Escherichia coli aspartate transcarbamoylase

Chromatography of aspartate transcarbamoylase from Escherichia coli on agarose-immobilized dyes and alkyl-agaroses of differing carbon length were investigated. The bacterial aspartate transcarbamoylase was bound by Procoin red HE3B-agarose and Cibacron blue F3GA-agarose nearly completely under the conditions chosen relative to other agarose-coupled dyes. The aspartate transcarbamoylase holoenzyme was eluted from the Procion red HE3B-agarose slightly later than from the Cibacron blue F3GA-agarose during salt gradient elution. The catalytic trimer of the enzyme as well as its regulatory dimer were eluted by a lower salt concentration from both dye-agarose gels than the concentration required to elute the holoenzyme. The interaction of the catalytic trimer with the Procion red HE3B-agarose and Cibacron blue F3GA-agarose gels may be a determinant in the holoenzyme being retained on these resins. Of those alkyl-agaroses tested, the ethyl-, propyl- and hexyl-agarose gels bound the majority of aspartate transcarbamoylase activity. Chromatography of aspartate transcarbamoylase on ethyl-agarose found it to be eluted by a low salt concentration. A purification scheme for relatively small amounts of aspartate transcarbamoylase utilizing Procion red HE3B-agarose and ethyl-agarose is presented. This purification scheme is particularly useful for mutant versions of aspartate transcarbamoylase which cannot be purified by literature procedures.     

Complex-forming properties of butanedione-modified ferredoxin-NADP+ reductase with NADP+ and ferredoxin.

The plastidic ferredoxin-NADP⁺ reductase from the xanthophycean alga Bumilleriopsis forms a stoichiometric 1:1 complex with ferredoxin and NADP⁺ which is demonstrated by difference spectra of both complexes. Butanedione modification of the flavoprotein results in loss of its enzymatic activities (transhydrogenase and diaphorase) concurrently with its capability to form a complex with NADP⁺, whereas the ferredoxin-binding site is practically not influenced by the modifying reagent and complex formation is still possible. It is assumed, therefore, that butanedione specifically reacts with the arginine residue of the protein involved in binding of pyridine nucleotides at the active site. Further, the data presented strongly support the previous proposal of different binding sites for ferredoxin and pyridine nucleotides at the reductase.     

The effect of ligand presaturation on the interaction of serum albumins with an immobilized Cibacron Blue 3G-A studied by affinity gel electrophoresis.

The interaction of the immobilized triazine dye Cibacron Blue 3G-A with rat, rabbit, sheep, goat, bovine and human serum albumins was studied by affinity gel electrophoresis. Dissociation constants were estimated in each instance and showed human serum albumin to have a significantly higher affinity for the dye than did albumin from any other species. Pretreatment of the defatted proteins with bilirubin (3 mol of bilirubin/mol of protein) did not increase the dissociation constants of the serum albumins, whereas pretreatment with palmitate (7 mol of palmitate/mol of protein) increased the dissociation constant in all cases: 3-fold for human serum albumin, 15-fold for other serum albumins. Increasing the bilirubin/albumin ratio (to 7:1) did not affect the dissociation constant of the albumins studied. Decreasing the palmitate/albumin ratio decreased the dissociation constant for human serum albumin, but did not affect those of bovine and rat albumins. Altering the chain length of the presaturating fatty acid dramatically changed the dissociation constant of both human and bovine serum albumins. Butyrate, hexanoate, octanoate and decanoate did not significantly influence the dissociation constants of bovine and human serum albumins for Cibacron Blue, whereas laurate, myristate and palmitate greatly increased the dissociation constant. These data are discussed in relationship to the behaviour of albumins during dye--agarose column chromatography. In Addendum the effect of nucleotide presaturation on the interaction between Bacillus stearothermophilus 6-phosphogluconate dehydrogenase and the immobilized triazine dyes Cibacron Blue 3G-A and Procion Red HE-3B was examined, and the implications for dye--ligand chromatography are discussed.     

Affinity chromatography on immobilised triazine dyes. Studies on the interaction with multinucleotide-dependent enzymes

A systematic investigation into the interaction of several triazinyl dyes with two enzymes from purine metabolism, IMP dehydrogenase (IMP: NAD+ oxidoreductase, EC 1.2.1.14( and adenylosuccinate synthetase (IMP: L-aspartate ligase (GDP-forming), EC 6.3.4.4) has been conducted. Evidence from kinetic inhibition studies, enzyme inactivation with specific affinity labels and specific elution techniques from agarose-immobilised dyes indicate that triazine dyes such as Procion Blue H-B (Cibacron Blue F3G-A), Red HE-3B and Red H-3B are able to differentiate between the nucleotide-binding sites of these enzymes. This information has been exploited to design specific elution techniques for the purification of these enzymes by affinity chromatography.     

15-ketoprostaglandin delta13 reductase from human placenta: purification, kinetics, and inhibitor binding.

An NADH-dependent 15-ketoprostaglandin Δ¹³ reductase has been purified to near homogeneity from human placenta by a procedure which includes affinity chromatography on blue Sepharose. The enzyme utilizes as substrates 15-ketoprostaglandins of the E, F, A, and B series, and the reaction is experimentally irreversible. Molecular weight estimations on Sephadex G-100 and sodium dodecyl sulfate disc gel electrophoresis suggest that the enzyme is a dimer. The subunits appear to be similar in size if not identical and have a molecular weight of 35,000. The mechanism of the reaction of 15-ketoprostaglandin E₂ and NADH catalyzed by this enzyme has been investigated by steady-state kinetic methods. The 13,14-dihydro-15-ketoprostaglandin product is an inhibitor of the reaction, being competitive with respect to 15-ketoprostaglandin E₂ and noncompetitive with respect to NADH; NAD⁺ does not inhibit the reaction. NADPH and Cibacron blue 3G-A are “dead-end” inhibitors of the reaction; both act competitively with respect to NADH and noncompetitively with respect to 15-ketoprostaglandin E₂. These observations are consistent with a rapid equilibrium random mechanism with the formation of an unreactive enzyme · NADH · 13,14-dihydro-15-ketoprostaglandin E₂ complex. The interaction of NADPH and Cibacron blue 3G-A with the free enzyme was investigated further by fluorimetry. Both substances bind to the free enzyme and quench its fluorescence. This property was utilized to titrate the enzyme, and a value of 3.28 × 10^?11 mol of binding sites/mU of enzyme was obtained.     

Affinity purification and characterization of 2,4-dichlorophenol hydroxylase from Pseudomonas cepacia

2,4-Dichlorophenol hydroxylase, a flavoprotein monooxygenase from Pseudomonas cepacia grown on 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, was purified to homogeneity by a single-step affinity chromatography on 2,4-DCP-Sepharose CL-4B. The enzyme was eluted from the affinity matrix with the substrate 2,4-dichlorophenol. The enzyme has a molecular weight of 275,000 consisting of four identical subunits of molecular weight 69,000 and requires exogenous addition of FAD for its complete catalytic activity. The enzyme required an external electron donor NADPH for hydroxylation of 2,4-dichlorophenol to 3,5-dichlorocatechol. NADPH was preferred over NADH. The enzyme had Km value of 14 microM for 2,4-dichlorophenol, and 100 microM for NADPH. The enzyme activity was significantly inhibited by heavy metal ions like Hg2+ and Zn2+ and showed marked inhibition with thiol reagents. Trichlorophenols inhibited the enzyme competitively. The hydroxylase activity decreased as a function of increasing concentrations of Cibacron blue and Procion red dyes. The apoenzyme prepared showed complete loss of FAD when monitored spectrophotometrically and had no enzymatic activity. The inactive apoenzyme was reconstituted with exogenous FAD which completely restored the enzyme activity.     

Isolation and characterization of an acyl-coenzyme A carboxylase from an erythromycin-producing Streptomyces erythreus

Streptomyces erythreus produces erythromycin presumably from methylmalonyl-coenzyme A, (CoA) which might be generated by carboxylation of propionyl-CoA. A biotin-containing enzyme which carboxylates acetyl-CoA, propionyl-CoA, and butyryl-CoA was purified to near homogeneity from S. erythreus using DEAE-cellulose, affinity chromatography on monomeric avidin-Sepharose, and blue Sepharose. The enzyme carboxylates propionyl-CoA (100%) with a K_m of 0.09 mm and V of 0.86μmol/mg/min, acetyl-CoA (16%) with a K_m of 0.17 mm and V of 0.08μmol/mg/min, and butyryl-CoA (7.7%) with a K_m of 0.67 mm and V of 0.044 μmol/mg/min. The native enzyme has a molecular weight of 537,000 and consists of two types of subunits with molecular weights of 67,000 and 61,000, respectively, indicating an octameric α₄β₄ type of structure. Biotin is associated with the large subunit (α). The enzyme has a pH optimum between 7.5 and 7.8. It is stimulated (three- to fourfold) by K⁺, Rb⁺ and Cs⁺ but not by Na⁺ or Li⁺ and is inhibited by high concentrations of NH₄⁺ and C1^?. Neither citrate nor free CoA stimulated the enzyme. The enzyme was shown to be stereospecific and generated onlyS-methylmalonyl-CoA from the carboxylation of propionyl-CoA. The present case appears to be the first enzyme possibly involved in erythromycin production to be isolated in homogeneous form.     

Photoacclimation in the Red Alga Porphyridium cruentum: Changes in Photosynthetic Enzymes, Electron Carriers, and Light-Saturated Rate of Photosynthesis as a Function of Irradiance and Spectral Quality

Acclimation of the photosynthetic apparatus to changes in the light environment was studied in the unicellular red alga Porphyridium cruentum (American Type Culture Collection No. 50161). Absolute or relative amounts of four photosynthetic enzymes and electron carriers were measured, and the data were compared with earlier observations on light-harvesting components (F.X. Cunningham, Jr., R.J. Dennenberg, L. Mustárdy, P.A. Jursinic, E. Gantt [1989] Plant Physiol 91: 1179-1187; F.X. Cunningham, Jr., R.J. Dennenberg, P.A. Jursinic, E. Gantt [1990] Plant Physiol 93: 888-895) and with measurements of photosynthetic capacity. P_max, the light-saturated rate of photosynthesis on a chlorophyll (Chl) basis, increased more than 4-fold with increase in growth irradiance from 6 to 280 μeinsteins·m⁻²·s⁻¹. Amounts of ferredoxin-NADP⁺ reductase, ribulose-1,5-bisphosphate carboxylase, and cytochrome f increased in parallel with P_max, whereas numbers of the light-harvesting complexes (photosystem [PS] I, PSII, and phycobilisomes) changed little, and ATP synthase increased 7-fold relative to Chl. The calculated minimal turnover time for PSII under the highest irradiance, 5 ms, was thus about 4-fold faster than that calculated for cultures grown under the lowest irradiance (19 ms). A change in the spectral composition of the growth light (irradiance kept constant at 15 μeinsteins·m⁻²·s⁻¹) from green (absorbed predominantly by the phycobilisome antenna of PSII) to red (absorbed primarily by the Chl antenna of PSI) had little effect on the amounts of ribulose-1,5-bisphosphate carboxylase, ATP synthase, and phycobilisomes on a Chl, protein, or thylakoid area basis. However, the number of PSI centers declined by 40%, cytochrome f increased by 40%, and both PSII and ferredoxin-NADP⁺ reductase increased approximately 3-fold on a thylakoid area basis. The substantial increase in ferredoxin-NADP⁺ reductase under PSI light is inconsistent with a PSI-mediated reduction of NADP as the sole function of this enzyme. Our results demonstrate a high degree of plasticity in content and composition of thylakoid membranes of P. cruentum.     

Properties of chloroplast membrane-bound ferredoxin--NADP + reductase during cold hardening of wheat. No indication of qualitative membrane changes during cold hardening

Kinetic parameters of the chloroplastbound ferredoxin-NADP⁺ reductase from two varieties of wheat (Triticum aestivum), hardy Kharkov 22 MC (winter wheat) and less hardy Rescue (spring wheat), were followed during induction of frost hardiness as a means of examining possible changes in chloroplast membranes during hardening. No changes were found in the Michaelis constants for NADPH and 2,6-dichlorophenol indophenol, inhibition constants for p-chloromercuriphenylsulfonate, and activation energy values of the enzyme in either variety. The data suggest that no qualitative changes occurred in the properties of wheat chloroplast membranes related to ferredoxin-NADP⁺ reductase during cold hardening.     

Functional sulfhydryl groups of ferredoxin-NADP+ oxidoreductase

Chemical modification of membrane-bound ferredoxin-NADP⁺ oxidoreductase with oxidants of vicinal dithiols caused inactivation of NADP⁺ photoreduction, with no effect on the diaphorase activity. Inactivation was partially prevented by ferredoxin and reversed by dithioerythritol. N-Ethylmaleimide inhibited both activities, even though with a different kinetic pattern. Inactivation of NADP⁺ reduction by either N-ethylmaleimide or o-iodosobenzoate was greater in the light than in the dark. The results suggest the existence of essential sulfhydryl groups related with the ferredoxin site, in addition to those described in the soluble flavorprotein. The role of SH residues in the activity and regulation of membrane bound reductase is discussed.     

Effects of Light on the Membrane Potential of Protoplasts from Samanea saman Pulvini : Involvement of K Channels and the H -ATPase

Rhythmic light-sensitive movements of the leaflets of Samanea saman depend upon ion fluxes across the plasma membrane of extensor and flexor cells in opposing regions of the leaf-movement organ (pulvinus). We have isolated protoplasts from the extensor and flexor regions of S. saman pulvini and have examined the effects of brief 30-second exposures to white, blue, or red light on the relative membrane potential using the fluorescent dye, 3,3′-dipropylthiadicarbocyanine iodide. White and blue light induced transient membrane hyperpolarization of both extensor and flexor protoplasts; red light had no effect. Following white or blue light-induced hyperpolarization, the addition of 200 millimolar K⁺ resulted in a rapid depolarization of extensor, but not of flexor protoplasts. In contrast, addition of K⁺ following red light or in darkness resulted in a rapid depolarization of flexor, but not of extensor protoplasts. In both flexor and extensor protoplasts, depolarization was completely inhibited by tetraethylammonium, implicating channel-mediated movement of K⁺ ions. These results suggest that K⁺ channels are closed in extensor plasma membranes and open in flexor plasma membranes in darkness and that white and blue light, but not red light, close the channels in flexor plasma membranes and open them in extensor plasma membranes. Vanadate treatment inhibited hyperpolarization in response to blue or white light, but did not affect K⁺ -induced depolarization. This suggests that white or blue light-induced hyperpolarization results from activation of the H⁺ -ATPase, but this hyperpolarization is not the sole factor controlling the opening of K⁺ channels.     

Immunodetection of the Ferredoxin-NADP Oxidoreductase-Binding Protein Complex in Thylakoids of Different Higher Plant Species

Monospecific polyclonal antibodies against thylakoid ferredoxin-NADP⁺ oxidoreductase and its binding protein from Spinacia oleracea were used to detect the presence of these proteins in different higher plants, including C₃, C₄, and Crassulacean acid metabolism species. A remarkable conservation of antigenic determinants in all the species analyzed was demonstrated for both the reductase and its binding protein. The association of these polypeptides in a complex was detected by immunoprecipitation.