Rate Of NADP Research Articles

Functional Screening of Cytochrome P450 Activity and Uncoupling by Capillary Electrophoresis

Cytochrome P450s (CYPs) are functionally diverse monooxygenases responsible for oxidation of endogenous and xenobiotic compounds. The function of nonmammalian CYPs are largely unknown and tools for characterization limited. CYPs critical for xenobiotic metabolism are prone to catalytic cycle uncoupling resulting in reactive oxygen species (ROS) generation that is highly dependent on the specific CYP isoform and substrate interaction. This study describes the rapid assessment of the activity and coupling efficiency of CYPs using capillary electrophoresis with UV detection. The coupling efficiency of five zebrafish (Danio rerio) CYP1 isoforms with a series of fluorogenic substrate probes was determined by the rate of NADP(+) formation and compared with fluorescent product turnover rates. In most cases, NADP(+) formation significantly overestimated CYP1 catalytic activity for substrate O-dealkylation suggesting uncoupling. ROS production was confirmed by elevated hydrogen peroxide generation in poorly coupled reactions. Reactions with β-estradiol confirmed that CYP1A, 1C1, and 1C2 have greater catalytic activity and coupling efficiency; CYP1B1 and 1D1 had coupling efficiencies under 4%. This work highlights the wide disparity in uncoupling induced by unproductive substrate binding among different CYP isoforms.

Effect of Chlorophyll-Protein Complex I Deficiency on the Physiological Character of a Chlamydomonas reinhardtii Mutant

In the mutant CC-1047 of Chlamydomonas reinhardtii, LDS-PAGE showed that the chlorophyll-protein complex I (CPI) is almost absent. The mutant could not grow in a culture medium without organic carbon source while the wild type (WT) C. reinhardtii grew quickly. When an organic carbon source was added into the culture medium, the mutant grew almost as well as WT. The rate of photosystem 1 (PS1) electron transport (DCPIP→MV) and the rate of whole chain electron transport (H2O→MV) of chloroplasts of the CC-1047 mutant were both lower than those of WT. The photophosphorylation activity, photosynthetic O2 evolution rate, and rate of NADP+ photoreduction of CC-1047 were also much lower than the activities of WT. There were some differences in ATPase activity between the mutant and WT. Two different activation ways were used to activate the latent ATPase using methanol and dithiothreitol (DTT) as activation substrate. More methanol and DTT were required for the mutant than WT to obtain the maximum activity. Thus the photosynthetic apparatus could not operate normally when CPI was absent because of the abnormal PS1 electron transport. Meanwhile, the other adjacent complexes of the thylakoid membrane, for example, ATP synthase complex, were slightly affected.

The Cysteine-proximal Aspartates in the FX-binding Niche of Photosystem I: EFFECT OF ALANINE AND LYSINE REPLACEMENTS ON PHOTOAUTOTROPHIC GROWTH, ELECTRON TRANSFER RATES, SINGLE-TURNOVER FLASH EFFICIENCY, AND EPR SPECTRAL PROPERTIES

The FX electron acceptor in Photosystem I (PS I) is a highly electronegative (Em = -705 mV) interpolypeptide [4Fe-4S] cluster ligated by cysteines 556 and 565 on PsaB and cysteines 574 and 583 on PsaA in Synechocystis sp. PCC 6803. An aspartic acid is adjacent to each of these cysteines on PsaB and adjacent to the proline-proximal cysteine on PsaA. We investigated the effect of D566PsaB and D557PsaB on electron transfer through FX by changing each aspartate to the neutral alanine or to the positively charged lysine either singly (D566APsaB, D557APsaB, D566KPsaB, and D557KPsaB) or in pairs (D557APsaB/D566APsaB and D557KPsaB/D566APsaB). All mutants except for D557KPsaB/D566APsaB grew photoautotrophically, but the growth of D557KPsaB and D557APsaB/D566APsaB was impaired under low light. The doubling time was increased, and the chlorophyll content per cell was lower in D557KPsaB and D557APsaB/D566APsaB relative to the wild type and the other mutants. Nevertheless, the rates of NADP+ photoreduction in PS I complexes from all mutants were no less than 75% of that of the wild type. The kinetics of back-reaction of the electron acceptors on a single-turnover flash showed efficient electron transfer to the terminal acceptors FA and FB in PS I complexes from all mutants. The EPR spectrum of FX was identical to that in the wild type in all but the single and double D566APsaB mutants, where the high-field resonance was shifted downfield. We conclude that the impaired growth of some of the mutants is related to a reduced accumulation of PS I rather than to photosynthetic efficiency. The chemical nature and the charge of the amino acids adjacent to the cysteine ligands on PsaB do not appear to be significant factors in the efficiency of electron transfer through FX.

6-Phosphogluconate dehydrogenase: the mechanism of action investigated by a comparison of the enzyme from different species

The mechanism of action of 6-phosphogluconate dehydrogenase with the alternative substrate 2-deoxy 6-phosphogluconate was investigated using enzymes from sheep liver, human erythrocytes and Trypanosoma brucei. The three enzymes oxidize 2-deoxy 6-phosphogluconate, but only the sheep liver enzyme releases the intermediate 2-deoxy,3-keto 6-phosphogluconate. Kinetic comparison showed that an increase in the rate of NADP + reduction at high pH is due to increased release of the intermediate, rather than an increase in the overall reaction rate. 2-Deoxy,3-keto 6-phosphogluconate is decarboxylated by the erythrocyte and trypanosome enzymes but not the liver one in the absence of either NADPH or 6-phosphogluconate, which act as activators. The pH dependence of decarboxylation and the degree of activation suggest that 6-phosphogluconate is the activator which operates under normal assay conditions, while NADPH acts mainly by increasing the binding of the intermediate. The data suggest that the activity of 6PGDH is subjected to a two-way regulation: NADPH, which regulates the pentose phosphate pathway, inhibits the enzyme, while 6-phosphogluconate, levels of which rise when NADPH inhibition is removed, acts as an activator ensuring that 6-phosphogluconate is rapidly removed.

Photoreduction of NADP(+) in photosystem II of higher plants: requirement for manganese.

The effects of reversible managanese extraction on NADP(+) photoreduction were studied with higher plant subchloroplast preparations of photosystem II (PS II). Under anaerobic conditions, when the reaction centers (RCs) of PS II are "closed" (i.e. in the state [P680 Pheo] QĀ), and in the presence of ferredoxin-ferredoxin-NADP(+) reductase, NADP(+) reduction is observed at a rate of 0.8-1.1 μmol/mg x chlorophyII x h. After complete removal of manganese from PS II, the rate of NADP(+) reduction is reduced 40-5- fold. Upon the addition of Mn at a concentration of approx. 4 Mn atoms per reaction center, the NADP reduction is restored up to 85-90% of the initial value, When half of this amount of Mn is combined with about 40 times of the equivalent concentration of other divalent ions (Ca²⁺, Sr²⁺, Mg²⁺ etc) the reaction is also reactivated. Dinoseb (10⁻⁶M) an inhibitor of electron transfer in PS II prevents NADP(+) photoreduction. It is concluded that under conditions when the first quinone acceptor, Q(A), is in its reduced state (QĀ), electrons are transferred from reduced pheophytin (Pheo(-)) to NADP(+), indicating that PS II can reduce NADP(+) without the participation of PS I. On the basis of these and literature data, and alternate pathway for electron phototransfer in PS II reaction centers of higher plants is suggested. Some problems concerning the Z-scheme are discussed.

Photosynthetic metabolism in bundle sheath cells of the C 4 species Zea mays: Sources of ATP and NADPH and the contribution of photosystem II

Rates of ATP and NADPH consumption during photosynthesis by isolated strands of Zea mays bundle sheath cells were calculated from an analysis of intermediates labeled from assimilated 14C0 2. The NADPH generated during decarboxylation of added malate (via malic enzyme) was also calculated so that the component of total NADPH produced by photoreduction could be deduced. Adding ribose 5-phosphate, malate, and aspartate to cells provided with HCO 3 − substantially increased the rates of ATP and NADPH utilization by the reactions of the photosynthetic carbon reduction cycle. The rate of NADP + photoreduction was reduced by adding malate and aspartate. The ratio of ATP produced by photophosphorylation to NADP + photoreduced was highest (5 to 11) in systems provided with malate and aspartate. Only low rates of oxygen evolution were observed in the presence of HCO 3 − and ribose 5-phosphate; these rates were further reduced when malate and aspartate were added. Observed rates of oxygen evolution were in close agreement with the rates of NADP + photoreduction estimated by analysis of labeled intermediates. Simultaneous measurements of oxygen evolution and oxygen uptake, using isotopic oxygen, indicated that the rate of pseudocyclic electron transport was low. Oxygen evolution was light dependent and inhibited by NH 4C1 and 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Cells provided with malate and sufficient 3-(3,4-dichlorophenyl)-l,1-dimethylurea to completely inhibit NADP + photoreduction retained a substantial capacity for ATP synthesis. Reducing the oxygen concentration partially reversed 3-(3,4-dichlorophenyl)-l,l-dimethylurea inhibition. We propose that these treatments modify the poising of the cyclic electron transport system and that cyclic photophosphorylation is responsible for up to 80% of the ATP produced in these cells.

Role of β-carotene in the reaction centres of Photosystems I and II of spinach chloroplasts prepared in non-polar solvents

Spinach chloroplasts have been prepared nonaqueously using non-polar solvents ( n-hexane, CCl 4, n-heptane) and the β-carotene content extracted in a controlled manner. This procedure is reproducible and does not result in large structural or spectral changes of the chloroplasts. The organisation of the chlorophyll-proteins is unaltered, as fragmentation with digitonin results in the appearance of the same fractions as found previously for aqueously-prepared chloroplasts, including the pink zone containing cytochromes f and b 6 in the ratio 1:2. The chloroplasts possess both Photosystem I activity ( P-700 photo-bleaching, and NADP + photoreduction) and Photosystem II activity (parabenzoquinone reduction with Mn 2+ as electron donor, and chlorophyll fluorescence induction). Use of moderate intensity red illumination has allowed a study of the role of β-carotene in photochemistry separate from its roles in energy transfer and photoprotection. Removal of the fraction of β-carotene closely associated with the Photo-system I reaction centre caused the rate of NADP + photoreduction to fall to a low, but significantly non-zero level. Thus, in the complete absence of β-carotene, photochemistry can still be observed, however the specific association of β-carotene with the reaction centre is required for maximal rates. We propose that β-carotene bound at the reaction centre decreases the rate of transfer of excitation energy away from the reaction centre, and increases the rate of photochemistry. It is possible that this occurs via formation of an exciplex between ground state β-carotene and chlorophyll in the first excited state.

Herbicidal interference with the photochemical activities of chloroplasts ( in vivo and in vitro) of certain crop and weed species

The independent modes of action of diuron and atrazine on the photochemical activities of chloroplasts ( In vivo and in vitro) from the leaves of crop plants Pisum sativum and Pennisetum typhoides and the weeds Amaranthus viridis and Cyperus rotundus were investigated. Hill reaction activity (DCPIP photoreduction) of in vivo chloroplasts (chloroplasts isolated from herbicide-sprayed plants) was unaffected by treatment at sublethal or intermediate levels of diuron or atrazine while that of in vitro chloroplasts (chloroplasts incubated in the required herbicidal concentration) was severely inhibited. The ferricyanide catalyzed noncyclic photophosphorylation was markedly reduced in both the in vivo and in vitro chloroplast systems. N-Methyl phenozonium sulfate (PMS)-mediated cyclic photophosphorylation was inhibited in the in vivo system while a pronounced enhancement of activity was noticed in the in vitro chloroplasts. The rate of NADP + photoreduction was severely inhibited in the in vitro chloroplasts. The unaffected in the in vivo system. The herbicidal effects on the photoreactions of isolated chloroplasts were compared with chloroplasts isolated from herbicide-sprayed plants.

Characterization of Glutamate Dehydrogenase from the Ammonia-oxidizing Chemoautotroph Nitrosomonas europaea

Abstract Glutamate dehydrogenase, purified 160-fold by ammonium sulfate precipitation and chromatography on diethylaminoethyl cellulose and Bio-Gel P-300, was characterized as a catalyst of both nicotinamide adenine dinucleotide phosphate reduction and NADPH oxidation at the optimal pH of each reaction (8.7 and 7.7, respectively). In the pH range 7.5 to 8, the rate of enzyme-catalyzed NADPH oxidation is 10 to 15 times greater than the rate of NADP+ reduction. The enzyme is specific for NADPH (Km, 4.9 x 10-5 m), α-keto-glutarate (Km, 4.3 mm), NADP+ (Km, 7.9 x 10-6 m), and glutamate (Km, 6.7 mm). The Km for NH4Cl is 16 mm. The oxidation of NADPH was 50% inhibited by 0.5 m NaCl or NH4Cl, 20 mm d-glutamate, 2 x 10-4 m NADH, 10-3 m NADP+, 4.4 x 10-4 m NADPH, and 5 µm p-hydroxymercuribenzoate. The reduction of NADP+ was 50% inhibited by 1 mm NH4Cl, 20 mm d-glutamate or β-alanine, 4 x 10-4 m NADH, and 3 x 10-5 m NADPH. Enzyme activity was not affected by 2 x 10-4 m adenosine or guanosine triphosphate. It appears that in Nitrosomonas the enzyme catalyzes the reaction primarily in the direction of glutamate formation and has a high enough activity to account for all cellular amino nitrogen.