Concentration Of Phenazine Methosulfate Research Articles

Oxidative stress antagonizes fluoroquinolone drug sensitivity via the SoxR-SUF Fe-S cluster homeostatic axis.

The level of antibiotic resistance exhibited by bacteria can vary as a function of environmental conditions. Here, we report that phenazine-methosulfate (PMS), a redox-cycling compound (RCC) enhances resistance to fluoroquinolone (FQ) norfloxacin. Genetic analysis showed that E. coli adapts to PMS stress by making Fe-S clusters with the SUF machinery instead of the ISC one. Based upon phenotypic analysis of soxR, acrA, and micF mutants, we showed that PMS antagonizes fluoroquinolone toxicity by SoxR-mediated up-regulation of the AcrAB drug efflux pump. Subsequently, we showed that despite the fact that SoxR could receive its cluster from either ISC or SUF, only SUF is able to sustain efficient SoxR maturation under exposure to prolonged PMS period or high PMS concentrations. This study furthers the idea that Fe-S cluster homeostasis acts as a sensor of environmental conditions, and because its broad influence on cell metabolism, modifies the antibiotic resistance profile of E. coli.

Repurposing the Electron Transfer Reactant Phenazine Methosulfate (PMS) for the Apoptotic Elimination of Malignant Melanoma Cells through Induction of Lethal Oxidative and Mitochondriotoxic Stress.

Redox-directed pharmacophores have shown potential for the apoptotic elimination of cancer cells through chemotherapeutic induction of oxidative stress. Phenazine methosulfate (PMS), a N-alkylphenazinium cation-based redox cycler, is used widely as an electron transfer reactant coupling NAD(P)H generation to the reduction of tetrazolium salts in biochemical cell viability assays. Here, we have explored feasibility of repurposing the redox cycler PMS as a superoxide generating chemotherapeutic for the pro-oxidant induction of cancer cell apoptosis. In a panel of malignant human melanoma cells (A375, G361, LOX), low micromolar concentrations of PMS (1–10 μM, 24 h) displayed pronounced apoptogenicity as detected by annexin V-ITC/propidium iodide flow cytometry, and PMS-induced cell death was suppressed by antioxidant (NAC) or pan-caspase inhibitor (zVAD-fmk) cotreatment. Gene expression array analysis in A375 melanoma cells (PMS, 10 µM; 6 h) revealed transcriptional upregulation of heat shock (HSPA6, HSPA1A), oxidative (HMOX1) and genotoxic (EGR1, GADD45A) stress responses, confirmed by immunoblot detection demonstrating upregulation of redox regulators (NRF2, HO-1, HSP70) and modulation of pro- (BAX, PUMA) and anti-apoptotic factors (Bcl-2, Mcl-1). PMS-induced oxidative stress and glutathione depletion preceded induction of apoptotic cell death. Furthermore, the mitochondrial origin of PMS-induced superoxide production was substantiated by MitoSOX-Red live cell fluorescence imaging, and PMS-induced mitochondriotoxicity (as evidenced by diminished transmembrane potential and oxygen consumption rate) was observable at early time points. After demonstrating NADPH-driven (SOD-suppressible) superoxide radical anion generation by PMS employing a chemical NBT reduction assay, PMS-induction of oxidative genotoxic stress was substantiated by quantitative Comet analysis that confirmed the introduction of formamido-pyrimidine DNA glycosylase (Fpg)-sensitive oxidative DNA lesions in A375 melanoma cells. Taken together, these data suggest feasibility of repurposing the biochemical reactant PMS as an experimental pro-oxidant targeting mitochondrial integrity and redox homeostasis for the apoptotic elimination of malignant melanoma cells.

Ultrasonic Imaging of Endothelial CD81 Expression Using CD81-Targeted Contrast Agents in In Vitro and In Vivo Studies

This study is designed to investigate the feasibility for molecular imaging of endothelial CD81 expression in vitro and in vivo using the CD81-targeted ultrasound contrast agents (UCA). In the in vitro study, murine bEnd.3 cells were stimulated with phenazine methosulfate (PMS), an oxidative stress inducer. Changes in CD81 expression after stimulation were confirmed by Western blotting, tracked by using the targeted UCA and further imaged under ultrasound imaging system with 5 MHz transmit frequency. In the in vivo study, expression of endothelial CD81 proteins in murine carotid artery vessels was studied using high-frequency ultrasound system with 40 MHz transmit frequency. Our results showed that endothelial CD81 expression was gradually up-regulated with the increase of PMS concentration. Correspondingly, the accumulation of targeted UCA was gradually improved and could be inhibited significantly upon addition of free anti-CD81 antibodies. The mean video intensity (grey-level) of stimulated cells and vessels from backscatter of the CD81-targeted UCA was 17.2 (interquartile range [IQR] 15.4–19.8) and 27.2 (IQR 22.4–29.8), significantly greater than that of non-stimulated cells with 9.0 (IQR 8.6–10.8) (p < 0.01) and non-stimulated vessels with 11.3 (IQR 10.4–13.2) (p < 0.01), respectively. In conclusion, CD81-targeted UCA allows noninvasive assessment of the expression levels of CD81 on the vascular endothelium and may provide potential insights into early atherosclerotic plaque detection and treatment monitoring.

PMS: Photosystem I electron donor or fluorescence quencher

Light energy harvested by the pigments in Photosystem I (PSI) is used for charge separation in the reaction center (RC), after which the positive charge resides on a special chlorophyll dimer called P700. In studies on the PSI trapping kinetics, P700+ is usually chemically reduced to re-open the RCs. So far, the information available about the reduction rate and possible chlorophyll fluorescence quenching effects of these reducing agents is limited. This information is indispensible to estimate the fraction of open RCs under known experimental conditions. Moreover, it would be important to understand if these reagents have a chlorophyll fluorescence quenching effects to avoid the introduction of exogenous singlet excitation quenching in the measurements. In this study, we investigated the effect of the commonly used reducing agent phenazine methosulfate (PMS) on the RC and fluorescence emission of higher plant PSI–LHCI. We measured the P700+ reduction rate for different PMS concentrations, and show that we can give a reliable estimation on the fraction of closed RCs based on these rates. The data show that PMS is quenching chlorophyll fluorescence emission. Finally, we determined that the fluorescence quantum yield of PSI with closed RCs is 4% higher than if the RCs are open.

LOX-1 augments oxLDL uptake by lysoPC-stimulated murine macrophages but is not required for oxLDL clearance from plasma

Oxidized LDL (oxLDL) promotes lipid accumulation as well as growth and survival signaling in macrophages. OxLDL uptake is mainly due to scavenger receptors SR-AI/II and CD36. However, other scavenger receptors such as lectin-like oxLDL receptor-1 (LOX-1) may also play a role. We used mice with targeted inactivation of the LOX-1 gene to define the role of this receptor in the uptake of oxLDL and in activation of survival pathways. There was no difference in uptake or degradation of 125I-oxLDL in unstimulated macrophages from wild-type and LOX-1 knockout mice and no difference in the rate of clearance of oxLDL from plasma in vivo. However, when expression of LOX-1 was induced with lysophosphatidylcholine, oxLDL uptake and degradation increased 2-fold in wild-type macrophages but did not change in LOX-1 knockout macrophages. Macrophages lacking LOX-1 showed the same stimulation of PKB phosphorylation and enhancement of survival by oxLDL as wild-type cells. These data show that LOX-1 does not alter the uptake of oxLDL in unstimulated macrophages and is not essential for the pro-survival effect of oxLDL in these cells. However, LOX-1 expression is highly inducible by lysophosphatidylcholine and pro-inflammatory cytokines, and if that occurred in macrophages within atheromas, LOX-1 could substantially increase oxLDL uptake by lesion macrophages.

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Tumor sensitivity assays have been widely reported, and we have been performed SDI (succinic dehydrogenase inhibition) test using MTT.In this paper we have studied this test fundamentally by the use of a new synthesized tetrazolium salt, XTT.The assay is performed with 96-well microplates.In the assay using XTT, addition of appropriate concentration of PMS (phenazine methosulfate) to XTT results in a marked increase in optical density and continues to increase with reaction time, which means XTT is less toxic to cells than MTT. Therefore, we have studied from time to time the possibility of evaluation with a little number of cells comparing with MTT assay.Tumor cells used in this experiment are Sarcoma 180, Ehrlich carcinoma, NUGC4 and SW13 as human cell lines.In result, when we incubate tumor cell over 20-h with XTT, we are able to evaluate sufficiently with a little number of cells, and we studied I.I.(Inhibition Index) of each drug (MMC, ADM, CDDP). XTT assay is easy to be influenced by pH and requires the use of medium contained buffer.Formazan formed from XTT is soluble in water, so that XTT assay does not require DMSO (dimethyl sulf oxide), but the problem is the measure of colored drugs for example ADM etc.It is thought that XTT is a useful tetrazolium salt and the assay is simple and able to evaluate with a little number of cells.

An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT

A new tetrazolium salt XTT, sodium 3′-[1-[(phenylamino)-carbony]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate, was evaluated for use in a colorimetric assay for cell viability and proliferation by normal activated T cells and several cytokine dependent cell lines. Cleavage of XTT by dehydrogenase enzymes of metabolically active cells yields a highly colored formazan product which is water soluble. This feature obviates the need for formazan crystal solubilization prior to absorbance measurements, as required when using other tetrazolium salts such as MTT. Bioreduction of XTT by all the murine cells examined was not particularly efficient, but could be potentiated by addition of electron coupling agents such as phenazine methosulfate (PMS) or menadione (MEN). Optimal concentrations of PMS or MEN were determined for the metabolism of XTT by the T cell lines HT-2 and 11.6, NFS-60 a myeloid leukemia, MC/9 a mast cell line and mitogen activated splenic T cells. When used in combination with PMS, each of these cells generated higher formazan absorbance values with XTT than were observed with MTT. Thus the use of XTT in colorimetric proliferation assays offer significant advantages over MTT, resulting from reduced assay time and sample handling, while offering equivalent sensitivity.

Oxidation-Induced Changes in Microrheologic Properties of the Red Blood Cell Membrane

It has been hypothesized that some of the irreversible microrheologic abnormalities of sickle red blood cell (RBC) membranes could result from autoxidative perturbation. To model this possibility, we used micromechanical manipulation to examine the static extensional rigidity and inelastic or plastic behavior of normal RBCs exposed to phenazine methosulfate (PMS), an agent that generates superoxide from within the cell. In response to this stress, RBC membranes became stiff as evidenced by increasing extensional rigidity. At 50 mumol/L PMS they were as stiff as the membranes of most dense, dehydrated sickle RBCs; and at 25 mumols/L PMS the membranes were similar to somewhat less dense sickle RBCs. When examined for inelastic behavior, RBCs exposed to PMS even at 10 mumols/L showed hysteresis in loading and unloading phases of the curve relating aspiration length to suction pressure, and they developed membrane bumps that persisted after RBC release from the pipette. Examination of single cells in both isotonic and hypotonic buffers showed that the effect of PMS on RBC microheology is not mediated by cellular dehydration. Independent confirmation of the membrane stiffening effect of PMS was obtained by ektacytometric analysis of resealed RBC ghosts, with sickle-like increases in membrane rigidity observed between 50 and 100 mumol/L PMS. The rigidity of these ghosts was partially ameliorated by exposure to a thiol reductant. In terms of biochemical abnormalities, treated RBCs became significantly different from control RBCs at 25 mumol/L PMS, at which point they just began to enter the sickle range for amounts of membrane thiol oxidation and membrane-associated heme. The sickle average was achieved at 50 mumol/L PMS (for thiol oxidation) to 100 mumol/L PMS (for membrane heme). Thus, micromolar concentrations of PMS induce abnormalities of membrane microrheology that closely mimic those of unmanipulated sickle RBCs while reproducing similar degrees of oxidative biochemical change. We conclude that membrane protein oxidation could explain existence of an irreversible component to the abnormal rheology of the sickle membrane.

Oxidation-induced changes in microrheologic properties of the red blood cell membrane

It has been hypothesized that some of the irreversible microrheologic abnormalities of sickle red blood cell (RBC) membranes could result from autoxidative perturbation. To model this possibility, we used micromechanical manipulation to examine the static extensional rigidity and inelastic or plastic behavior of normal RBCs exposed to phenazine methosulfate (PMS), an agent that generates superoxide from within the cell. In response to this stress, RBC membranes became stiff as evidenced by increasing extensional rigidity. At 50 mumol/L PMS they were as stiff as the membranes of most dense, dehydrated sickle RBCs; and at 25 mumols/L PMS the membranes were similar to somewhat less dense sickle RBCs. When examined for inelastic behavior, RBCs exposed to PMS even at 10 mumols/L showed hysteresis in loading and unloading phases of the curve relating aspiration length to suction pressure, and they developed membrane bumps that persisted after RBC release from the pipette. Examination of single cells in both isotonic and hypotonic buffers showed that the effect of PMS on RBC microheology is not mediated by cellular dehydration. Independent confirmation of the membrane stiffening effect of PMS was obtained by ektacytometric analysis of resealed RBC ghosts, with sickle-like increases in membrane rigidity observed between 50 and 100 mumol/L PMS. The rigidity of these ghosts was partially ameliorated by exposure to a thiol reductant. In terms of biochemical abnormalities, treated RBCs became significantly different from control RBCs at 25 mumol/L PMS, at which point they just began to enter the sickle range for amounts of membrane thiol oxidation and membrane-associated heme. The sickle average was achieved at 50 mumol/L PMS (for thiol oxidation) to 100 mumol/L PMS (for membrane heme). Thus, micromolar concentrations of PMS induce abnormalities of membrane microrheology that closely mimic those of unmanipulated sickle RBCs while reproducing similar degrees of oxidative biochemical change. We conclude that membrane protein oxidation could explain existence of an irreversible component to the abnormal rheology of the sickle membrane.

The aromatization of cyclohexanecarboxyl-CoA to hippuric acid by guinea pig liver mitochondria: submitochondrial localization.

The conversion of cyclohexanecarboxyl-CoA to hippuric acid in submitochondrial fractions from guinea pig liver was studied using a gas chromatographic-mass spectrometric method employing selected ion monitoring. Comparison of the activities of the cyclohexanecarboxyl-CoA to hippuric acid converting system (CCoAHC-system) and marker enzymes in the various submitochondrial fractions showed that the CCoAHC-system is localized in the mitochondrial matrix. Partial separation of the inner and outer membranes has been accomplished by treating mitochondria with digitonin in isotonic medium and fractionating the treated mitochondria by differential centrifugation. A digitonin-protein ratio of 2.6 mg of digitonin/10 mg of protein must be used in order to release significant amounts of amine oxidase activity (outer membrane marker) from low speed mitochondrial pellets. This pellet still contained most of the glutamate dehydrogenase activity and was insignificantly contaminated with adenylate kinase. Moderate concentrations of phenazine methosulfate (PMS) greatly stimulated the activity of the CCoAHC-system, even in intact mitochondria (optimal concentration of PMS: 1 mM) whilst higher concentrations (greater than 1 mM) decreased the activity. The formation of hippuric acid in these mitochondrial preparations was linear with time for at least 40 min and linear with respect to protein concentration up to approximately 2.0 mg mitochondrial protein X ml-1.

Phenazine methosulfate stimulation of ouabain-sensitive Rb+ uptake by HeLa cells: effects of respiratory inhibitors, anaerobiosis, and ascorbate.

phenazine methosulfate (PMS) stimulates ouabain-sensitive Rb+ uptake by HeLa cells. This stimulation cannot be attributed to the effect of the dye on the intracellular Na+ or ATP content. Respiratory inhibitors, such as 5 mM NaCN and 5 microM rotenone, and anaerobic conditions enhance the stimulation of Rb+ uptake by PMS. Cellular respiration is stimulated, but lactate production is reduced in the presence of PMS, irrespective of the presence of respiratory inhibitors. Cellular NADH is oxidized markedly on addition of PMS plus inhibitors, but it is not affected by addition of the inhibitors only. In the presence of a high concentration of PMS, PMS-stimulated ouabain-sensitive Rb+ uptake is inhibited by addition of ascorbate. From these results it is concluded that Na+K-pump activity is closely related to the cellular redox state.

Destruction of leishmania mexicana amazonensis amastigotes within macrophages in culture by phenazine methosulfate and other electron carriers

Exposure of macrophages infected with Leishmania mexicana amazonensis to phenazine methosulfate (PMS) resulted in rapid damage and disappearance of the intracellular amastigotes without obvious ill effects to the host cells. The reduction of the percent infection was related to the concentration of PMS and to the duration of the pulse. Most Leishmania disappeared within 2 h of a 2-h pulse with 10 muM of the drug. In contrast, pretreatment of the macrophages with PMS followed by removal of the drug before infection did not result in disappearance of the parasites. The pH of the PMS medium markedly influenced the disappearance of Leishmania: maximum effect was observed at pH 8.0, while the effect was negligible at pH 6.3. The pH effect may be related to pseudobase formation by the PMS cation. Dose-response curves for PMS were similar for resident, elicited, or activated macrophages. Observations by time-lapse cinemicrography documented the explosion-like fragmentation of the amastigotes within 1-2 h of exposure of infected macrophages to the drug. Parasite-derived granules and vacuoles were seen to scatter within the parasitophorous vacuoles. This early damage to the parasites was confirmed by transmission electron microscopic observations. Infected macrophages incubated with PMS displayed detectable vacuolar fluorescence, indicating that PMS or a metabolite of PMS had access to the vacuoles. A series of other electron carriers, including phenyl methanes, phenazines, oxazines, a xanthene, and a naphthoquinone, given continuously for 18 h, also induced the disappearance of the Leishmania. The most potent was crystal violet, active at 70 nM. The presence of apolar substituents enhanced activity and this is probably related to increased permeation of the dyes. Finally, PMS, as well as other electron carriers examined, also reduced the growth of Leishmania promastigotes in culture. The results are compatible with a direct effect of the drugs on the intracellular amastigotes, involving only a permissive participation of the macrophages. We propose that the diverse agents destroy the amastigotes by redox-cycling generation of active oxygen metabolites at or near the parasites. Alternatively, the effect of the drugs could be mediated by toxic free radical reduction species of the drugs or by interference with electron flow or with the intermediary metabolism of Leishmania.

Evidence for the involvement of superoxide anion in dopamine-beta-hydroxylase system.

SummaryDopamine-β-hydroxylase activity was markedly activated by a NADH-phenazine methosulfate-O2 model system known to generate superoxide anion (O2-) in the absence of ascorbate. This activation of the enzyme activity was shown to be dependent on the concentration of phenazine methosulfate. When superoxide dismutase was added along with ascorbate to the system, DBH activity was markedly inhibited. Furthermore, the addition of the superoxide scavenger, nitro blue tetrazolium, to the enzyme either in the presence of ascorbate or NADH-phenazine methosulfate, resulted in the inhibition of DBH activity. These data suggest that O2- is essential for the enzyme activity and that the role of ascorbate is probable in the generation of O2-.

FLASH PHOTOLYSIS‐ELECTRON SPIN RESONANCE STUDIES OF THE INTERACTION OF PHENAZINE METHOSULFATE WITH REACTION‐CENTER PREPARATIONS FROM THE R‐26 MUTANT OF RHODOPSEUDOMONAS SPHEROIDES*

Abstract— Using the technique of flash photolysis‐electron spin resonance, we have shown, by means of a kinetic analysis, that phenazine methosulfate (PMS) interacts with reaction‐center preparations from the blue‐green mutant R26 of Rhodopseudomonas spheroides. At intermediate concentrations of PMS, biphasic decay kinetics of the P870+ ESR signal are observed demonstrating that the PMS radical interacts with reaction centers by a specific binding mechanism. With PMS bound to reaction centers, the P870+ ESR signal decays in ˜ 1 ms; whereas, in unbound reaction centers the decay is ˜ 120 ms. A model is proposed involving the interaction of PMS on the donor side of P870.

Effects of phenazine methosulfate on glucose metabolism in rat adipose tissue

Segments of epididymal fat-pad tissue of rats were incubated with glucose labeled uniformly with 14C and in carbons 1 and 6 and with tritium in positions 3, 5, and 6. The effect of phenazine methosulfate (PMS) on incorporation of glucose carbon and tritium into metabolic products and on the formation and utilization of cytoplasmic and mitochondrial reducing equivalents was determined. PMS concentrations below 5 μ m have little effect on glucose metabolism. PMS between 5 and 100 μ m stimulates the uptake and oxidation of glucose but is without effect on its incorporation into fatty acids and glycerol. Higher concentrations of PMS inhibit glucose oxidation and lipogenesis. The lactate-pyruvate ratio in tissue incubated with glucose ranged from 3 to 10. With PMS the ratio was decreased to less than 0.5. At a PMS concentration of 50 μ m, glucose uptake was increased in tissue of fed rats by 30–50% and CO 2 production by 100–200%, The contribution of the pentose cycle to glucose metabolism increased from 15–20% to 40–50%, and oxidation via the Krebs cycle increased by 50–300%. In tissue of starved-refed rats with a very high rate of lipogenesis, the stimulation of the pentose cycle and Krebs cycle oxidation was less pronounced. A balance of reducing equivalents was calculated. In the absence of PMS the formation of cytoplasmic reducing equivalents is equal or in a small excess over that needed for lipogenesis and lactate production. In the presence of PMS a large excess is generated. The generation and utilization of ATP was calculated. Without PMS, ATP generation is in excess over that needed for known biosynthetic requirements. The excess is of the order of 20 μmoles/gram/hour for tissue of fed rats, and in tissue of fasted-refed rats, of the order of 40–50 μmoles/gram/hour. PMS increases oxidation via the Krebs cycle in tissue of normal rats but it is likely that the excess oxidation is not coupled to phosphorylation. When tissue was incubated with [5-T] glucose virtually the sole product was labeled water. This label is useful in measuring glucose uptake or turnover in vivo and in vitro by determination of the yield of labeled water. We conclude that lipogenesis is not affected by an increase in cellular pyruvate concentration or the NAD +/NADH and NADP +/NADPH ratios in the cytoplasm, or by a large decrease in the concentration of free reduced pyridine nucleotides.

Light-induced rapid absorption changes during photosynthesis. VII. Some reactions in sonicated chloroplasts

1. Spinach chloroplasts subjected to sonication show light-induced absorption changes at 700 mμ characteristic of the photooxidation of the chlorophyll component P700. The appearance of P700 absorption changes probably resulted from the release of plastocyanin thus interrupting the electron flow between pigment systems 1 and 2. The general features of the absorption-change transients are similar to those observed previously with digitonin-treated chloroplasts. The addition of 2 mM ascorbate or 10 μM 3-(3,4-dichlorophenyl)-1, 1-dimethylurea had practically no effect on either the magnitude or the dark decay of the transient absorption change. 2. Phenazine methosulfate (PMS) (in the presence or in the absence of ascorbate) reduction appeared to be coupled to P700 photooxidation, as shown by the corresponding transients at 430 and 388 mμ. The absorbance changes at these two wavelengths indicate that the amount of PMS photoreduced was equivalent to that of P700 photooxidized. Higher PMS concentrations accelerate the dark decay of the P700 signal. When PMS alone is present, anaerobiosis caused the dark decay to become more rapid than in the presence of ascorbate. 3. Unlike PMS, other redox agents such as 2,6-dichlorophenolindophenol, N, N, N′, N′-tetramethyl- p-phenylenediamine or diaminodurol in the presence of excess ascorbate, did not noticeably affect the kinetics of the dark decay at 430 or 703 mμ, suggesting that these reduced species are not efficiently coupled to photooxidized P700. 4. The onset and decay rates of the P700 transient in the presence of PMS and excess ascorbate was insensitive to temperature between 25° and o°. However, when the chloroplast sample was frozen at temperatures ranging from −5° to −196°, all reactions ceased. When the frozen (−196°) sample was brought back to the room temperature, the reaction was restored completely. Fresh broken chloroplasts behave similarly. Digitonin-treated chloroplasts persisted down to about −25° but with diminishing magnitude and slower decay.

SITES OF PHOTOSYNTHETIC ELECTRON-TRANSPORT SYSTEMS COUPLING PHOSPHORYLATION WITH CHROMATOPHORES FROM RHODOSPIRILLUM RUBRUM.

Abstract 1. 1. With chromatophores from Rhodospirillum rubrum , phosphorylations coupled with photosynthetic electron-transport systems have been investigated. 2. 2. Under illumination with white light at a fixed high intensity, the addition of a strictly limited concentration of phenazine methosulfate was required to induce photophosphorylation at a maximal rate. The maximal rate of photophosphorylation was always much faster when induced by phenazine methosulfate than when induced by the other reagents tested such as ascorbate or succinate. The ratio of maximal rate of the phenazine methosulfate-induced to ascorbate-induced photophosphorylation varied from batch to batch of chromatophore preparations with experimental values from 2 to 5. 3. 3. The phenazine methosulfate-induced, and ascorbate-induced photophosphorylations were inactivated in a similar manner by illumination of chromatophores with ultraviolet irradiation and by additions of the following inhibitors: Na 2 SO 4 , quinacrine hydrochloride, 2,4-dinitrophenol, dicumarol EDTA and phenylmercuric acetate. Chromatophore suspensions, if resonicated or heated, lost the activity of ascorbate-induced photophosphorylation somewhat more rapidly than that of phenazine methosulfate-induced photophosphorylation. In the presence of an appropriate concentration of o -phenanthroline, the ascorbate-induced and succinate-induced photophosphorylations were inactivated completely, but the phenazine methosulfate-induced photophosphorylation was not affected. With the chromatophores treated with an appropriate concentration of Triton X-100, the ascorbate-induced and succinate-induced photophosphorylations were inactivated completely, whereas the phenazine methosulfate-induced photophosphorylation was inactivated only partially. 4. 4. The photooxidations of cytochrome c 2 , and cytochrome c were stimulated in rate by the addition of ADP. The stimulation by ADP was not significantly influenced in the presence or absence of P i . The photooxidation was stimulated by 2,4-dinitrophenol in the absence but not in the presence of ADP. The concentraton of 2,4-dinitrophenol required for the stimulation was nearly identical with that required for the inhibition of photophosphorylation. 5. 5. ATP was synthesized from ADP and P i , coupled with the photooxidations of cytochrome c 2 , and cytochrome c . The values of the P/O ratio obtained were less than one. 6. 6. Based on these findings, the site for phosphorylation coupled with the photosynthetic electron-transport systems are suggested.