Dual mechanisms of ion absorption in relation to long distance transport in plants

The characteristics of ion transport to the shoots of young corn seedlings were studied with respect to the nature of the isotherm through a wide concentration range, the competitive influence of closely related ions upon the transport of a given ion, and the influence of the counter-ion. Both with respect to ³⁶Cl and ⁸⁶Rb transport, the characteristics of the process in every way resemble uptake by non-vacuolate root tips wherein the plasma membrane is the only membrane involved in absorption, and where system 1 — of the 2 systems which can be shown to participate in absorption by vacuolate tissue — is the only system operative. Net ion uptake by the roots per se was shown to display both the high affinity (system 1) and low affinity (system 2) mechanisms. It is concluded that the symplastic theory of ion movement to the xylem is valid, and that the contention that system 1 operates at the plasma membrane while system 2 functions at the tonoplast is strengthened.     

Cellular uptake of taurine by lactating porcine mammary tissue

Milk taurine plays a critical role in neonatal development. Taurine uptake in lactating sow mammary tissue has not been characterized previously. The kinetic properties, ion dependence and substrate specificity of taurine uptake were characterized in mammary tissue collected from lactating sows at slaughter. Tissue explants were incubated in an isosmotic physiologic buffer with [³H]taurine tracer to measure taurine uptake. Taurine uptake was dependent upon the presence of extracellular sodium and chloride ions, which is consistent with the co-transport of sodium and chloride with taurine. Uptake was not dependent upon ion exchange mechanisms or upon furosemide-sensitive ion co-transport. Taurine uptake was saturable and exhibited an apparent K_m of 20 μM and a V_max of 386 μmol/kg cell water/30 min. Substrate specificity studies indicated a strong interaction of β-amino acids with the taurine transport system. Taurine transport in lactating sow mammary tissue is therefore a high affinity, sodium-dependent mechanism specific for β-amino acids, and is analogous to sodium-dependent taurine uptake in other tissues. The high affinity and high specificity of the taurine uptake system allows for concentration of taurine within the mammary cell and is ultimately responsible for provision of taurine required for neonatal development.     

Catalytic mechanism of Escherichia coli ribonuclease III: kinetic and inhibitor evidence for the involvement of two magnesium ions in RNA phosphodiester hydrolysis

Escherichia coli ribonuclease III (RNase III; EC 3.1.24) is a double-stranded(ds)-RNA-specific endonuclease with key roles in diverse RNA maturation and decay pathways. E.coli RNase III is a member of a structurally distinct superfamily that includes Dicer, a central enzyme in the mechanism of RNA interference. E.coli RNase III requires a divalent metal ion for activity, with Mg²⁺ as the preferred species. However, neither the function(s) nor the number of metal ions involved in catalysis is known. To gain information on metal ion involvement in catalysis, the rate of cleavage of the model substrate R1.1 RNA was determined as a function of Mg²⁺ concentration. Single-turnover conditions were applied, wherein phosphodiester cleavage was the rate-limiting event. The measured Hill coefficient (n_H) is 2.0 ± 0.1, indicative of the involvement of two Mg²⁺ ions in phosphodiester hydrolysis. It is also shown that 2-hydroxy-4H-isoquinoline-1,3-dione—an inhibitor of ribonucleases that employ two divalent metal ions in their catalytic sites—inhibits E.coli RNase III cleavage of R1.1 RNA. The IC₅₀ for the compound is 14 μM for the Mg²⁺-supported reaction, and 8 μM for the Mn²⁺-supported reaction. The compound exhibits noncompetitive inhibitory kinetics, indicating that it does not perturb substrate binding. Neither the O-methylated version of the compound nor the unsubstituted imide inhibit substrate cleavage, which is consistent with a specific interaction of the N-hydroxyimide with two closely positioned divalent metal ions. A preliminary model is presented for functional roles of two divalent metal ions in the RNase III catalytic mechanism.     

Molecular Design of Bisphosphonate-Modified Proteins for Efficient Bone Targeting In Vivo

To establish a rational molecular design for bisphosphonate (BP)-modified proteins for efficient bone targeting, a pharmacokinetic study was performed using a series of alendronate (ALN), a nitrogen-containing BP, modified proteins with various molecular weights and varying degrees of modification. Four proteins with different molecular weight—yeast glutathione reductase (GR; MW: 112,000 Da), bovine serum albumin (BSA; MW: 67,000 Da), recombinant human superoxide dismutase (SOD; MW: 32,000 Da), and chicken egg white lysozyme (LZM; MW: 14,000 Da)—were modified with ALN to obtain ALN-modified proteins. Pharmacokinetic analysis of the tissue distribution of the ALN-modified and unmodified proteins was performed after radiolabeling them with indium-111 (¹¹¹In) by using a bifunctional chelating agent. Calculation of tissue uptake clearances revealed that the bone uptake clearances of ¹¹¹In-ALN-modified proteins were proportional to the degree of ALN modification. ¹¹¹In-GR-ALN and BSA-ALN, the two high-molecular-weight proteins, efficiently accumulated in bones, regardless of the degree of ALN modification. Approximately 36 and 34% of the dose, respectively, was calculated to be delivered to the bones. In contrast, the maximum amounts taken up by bone were 18 and 13% of the dose for ¹¹¹In-SOD-ALN(32) and LZM-ALN(9), respectively, because of their high renal clearance. ¹¹¹In-SOD modified with both polyethylene glycol (PEG) and ALN (¹¹¹In-PEG-SOD-ALN) was efficiently delivered to the bone. Approximately 36% of the dose was estimated to be delivered to the bones. In an experimental bone metastasis mouse model, treatment with PEG-SOD-ALN significantly reduced the number of tumor cells in the bone of the mice. These results indicate that the combination of PEG and ALN modification is a promising approach for efficient bone targeting of proteins with a high total-body clearance.     

Simulation study of nutrient uptake by plants from soilless cultures as affected by salinity buildup and transpiration

Soilless plant growth systems are widely used as a means to save irrigation water and to reduce groundwater contamination. While nutrient concentrations in the growth medium are depleted due to uptake by the plants, salinity and toxic substances accumulate due to transpiration. A theoretical model is suggested, to simulate nutrient uptake by plants grown in soilless cultures with recycled solutions. The model accounts for salinity accumulation with time and plant growth, and its effects on uptake of the different nutrients by means of interaction with Na and Cl ions. The sink term occurs due to uptake by a growing root system. Influx as a function of the ion concentration is according to Michaelis–Menten active mechanisms for K⁺, NO₃ ^–-N, NH₄ ⁺-N, PO₄-P, Ca²⁺, Mg²⁺ and SO₄ ^2-, whose influx parameters are affected by Na and Cl^–, but not with time (age). Sodium influx is passive above a critical concentration. Sum of cations–anions concentrations is balanced by Cl^– to maintain electro-neutrality of the growth solution. Salinity (by means of Na concentration) suppresses root and leaf growth, which further effect uptake and transpiration. The model accounts for instantaneous transpiration losses, during daytime only and its effect on uptake of nutrients and plant development due to salt accumulation. The model was tested against NO₃ ^– and K⁺ uptake by plants associated with cumulative transpiration and with different NaCl salinity levels. Deviations from observed K⁺ uptake should be attributed to the salinity tolerance of the plants. In a study with data obtained from published literature, the model indicated that nutrient depletion and salinity buildup might be completely different with fully grown-up plants (that do not grow) and plants that grow with time. Depletion of different nutrients are according to their initial concentration and plant uptake rate, but also affected by their interactions with Na and Cl ions.     

Calcium uptake by excised maize roots and interactions with alkali cations

Ca²⁺ uptake was studied in short-term experiments using 5-day-old excised maize roots. This tissue readily absorbs Ca²⁺, and inhibition by dinitrophenol and low temperature shows that the process is metabolically mediated. The uptake of Ca²⁺, like that of other cations, is influenced by the counter ion, the pH and concentration of the ambient solution, and the presence of other cations. The rate of uptake from various salts decreases in the following order: NO₃⁻ > Cl⁻ = Br⁻ > SO₄²⁻. K⁺ and H⁺ greatly interfere with Ca²⁺ absorption, while Li⁺ and Na⁺ have only slight effects.     

Chloride accumulation by mung bean root tips: a low affinity active transport system at the plasmalemma

Net uptake of Cl⁻ into root tips of mung bean (Phaseolus aureus) increases steadily with increasing external concentrations from 1 to 60 mm. Membrane potentials were measured to determine the equilibrium concentration of Cl⁻ in the tissue which could be due to diffusion. This concentration was readily exceeded in both the relatively nonvacuolate tips (0 to 1 mm) and the vacuolate, mature upper sectons (1 to 11 mm) of the roots. The activity coefficient of both cytoplasmic and vacuolar Cl⁻, measured with Cl⁻ sensitive microelectrodes, was approximately the same as that of a pure KCl solution of the same concentration. It is concluded that the “second mechanism” of ion uptake involves a large increase in the rate of active transport at the plasmalemma as the external concentration is increased above 1 mm.     

High-throughput isotopic analysis of RNA microarrays to quantify microbial resource use

Most microorganisms remain uncultivated, and typically their ecological roles must be inferred from diversity and genomic studies. To directly measure functional roles of uncultivated microbes, we developed Chip-stable isotope probing (SIP), a high-sensitivity, high-throughput SIP method performed on a phylogenetic microarray (chip). This approach consists of microbial community incubations with isotopically labeled substrates, hybridization of the extracted community rRNA to a microarray and measurement of isotope incorporation—and therefore substrate use—by secondary ion mass spectrometer imaging (NanoSIMS). Laboratory experiments demonstrated that Chip-SIP can detect isotopic enrichment of 0.5 atom % ¹³C and 0.1 atom % ¹⁵N, thus permitting experiments with short incubation times and low substrate concentrations. We applied Chip-SIP analysis to a natural estuarine community and quantified amino acid, nucleic acid or fatty acid incorporation by 81 distinct microbial taxa, thus demonstrating that resource partitioning occurs with relatively simple organic substrates. The Chip-SIP approach expands the repertoire of stable isotope-enabled methods available to microbial ecologists and provides a means to test genomics-generated hypotheses about biogeochemical function in any natural environment.     

Variations in Sulfhydryl, Disulfide, and Protein Content during Synchronous and Asynchronous Growth of HeLa Cells

The cellular contents of protein-bound and nonprotein sulfhydry (—SH) and disulfide (—SS—) groups were measured in both asynchronous and synchronous HeLa S3 cultures. About 90% of these groups are associated with proteins, the majority in the —SH form. The content of protein-bound groups, and hence the total content of —SH and —SS— groups (28 × 10^-15 moles/cell, or 1.1 × 10^-6 moles/g protein on average), changes in parallel with the protein content (which varies between 2 and 4 × 10^-10 g/cell) as asynchronous populations pass from the lag through the exponential to the stationary phase of growth. The concentration of nonprotein —SH groups, in contrast, increases 10-fold during lag phase and decreases in stationary phase; it follows the protein concentration closely during the exponential phase, at a level of about 2.8 × 10^-15 moles/cell. In synchronous cultures the protein content doubles during the cell cycle, possibly in an exponential fashion. The total —SH and —SS— content also doubles, but the rate of increase appears to fluctuate. The concentrations of the protein-bound groups show 2- to 3-fold fluctuations per unit protein: protein-bound —SH groups and mixed —SS— linkages rise to maxima while protein-bound —SS— groups fall to a minimum at the G₁/S transition, and fluctuations in these groups occur again during G₂. In addition, the protein-bound —SH concentration falls continuously during the S phase. The nonprotein —SH concentration undergoes the largest (relative) fluctuations, dropping from 4 × 10^-15moles/cell in early G₁ to about 0.4 × 10^-15 moles/cell (of standard protein content) at the end of G₁, and then rising to 30 times this value by the end of S.     

Comparative studies of three methods for measuring pepsin activity

Comparison has been made of a simple method originated by Absolon and modified in our laboratories for assay of proteolytic activity using RISA (radioactive iodinated serum albumin—Abbott Laboratories), with the commonly used photometric methods of Anson and Kunitz. In this method, pepsin was incubated with an albumin substrate containing RISA, followed by precipitation of the undigested substrate with trichloroacetic acid and measurement of radioactive digestion products in the supernatant fluid. The I¹³¹—albumin bond was shown in the present studies to be altered only by the proteolytic activity, and not by the incubation procedures at various values of pH. Any free iodine present originally in the RISA was removed by a single passage through a resin column (amberlite IRA-400-C1). Pepsin was shown to be most stable in solution at a pH of 5.5. Activity of pepsin was shown to be maximal when it was incubated with albumin at a pH of 2.5. Pepsin activity was shown to be altered in the presence of various electrolytes. Pepsin activity measured by the RISA and Anson methods as a function of concentration or of time of incubation indicated that these two methods are in good agreement and are equally sensitive. Consistently smaller standard errors were obtained by the RISA method of pepsin assay than were obtained with either of the other methods.     

New synthetic substrates of mammalian nucleotide excision repair system

DNA probes for the studies of damaged strand excision during the nucleotide excision repair (NER) have been designed using the novel non-nucleosidic phosphoramidite reagents that contain N-[6-(9-antracenylcarbamoyl)hexanoyl]-3-amino-1,2-propandiol (nAnt) and N-[6-(5(6)-fluoresceinylcarbamoyl)hexanoyl]-3-amino-1,2-propandiol (nFlu) moieties. New lesion-imitating adducts being inserted into DNA show good substrate properties in NER process. Modified extended linear nFlu– and nAntr–DNA are suitable for estimation of specific excision activity catalysed with mammalian whole-cell extracts. The following substrate activity range was revealed for the model 137-bp linear double-stranded DNA: nAnt–DNA ≈ nFlu–DNA > Chol–DNA (Chol–DNA—legitimate NER substrate that contains non-nucleoside fragment bearing cholesterol residue). In vitro assay shows that modified DNA can be a useful tool to study NER activity in whole-cell extracts. The developed approach should be of general use for the incorporation of NER-sensitive distortions into model DNAs. The new synthetic extended linear DNA containing bulky non-nucleoside modifications will be useful for NER mechanism study and for applications.     

Carrier-Mediated Uptake of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid in Vacuoles Isolated from Catharanthus roseus Cells

The uptake of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), the conjugated form of the ethylene precursor, into vacuoles isolated from Catharanthus roseus cells has been studied by silicone layer floatation filtering. The transport across the tonoplast of MACC is stimulated fourfold by 5 millimolar MgATP, has a K_m of about 2 millimolar, an optimum pH around 7, and an optimum temperature at 30°C. Several effectors known to inhibit ATPase (N,N′-dicyclohexylcarbodiimide) and to collapse the transtonoplastic H⁺ electrochemical gradient (carbonylcyanide m-chlorophenylhydrazone, gramicidin, and benzylamine) all reduced MACC uptake. Abolishing the membrane potential with SCN⁻ and valinomycin also greatly inhibited MACC transport. Our data demonstrate that MACC accumulates in the vacuole against a concentration gradient by means of a proton motive force generated by a tonoplastic ATPase. The involvement of a protein carrier is suggested by the strong inhibition of uptake by compounds known to block SH—, OH—, and NH₂— groups. MACC uptake is antagonized competitively by malonyl-d-tryptophan, indicating that the carrier also accepts malonyl-d-amino acids. Neither the moities of these compounds taken separately [1-aminocyclopropane-1-carboxylic acid, malonate, d-tryptophan or d-phenylalanine] nor malate act as inhibitors of MACC transport. The absence of inhibition of malate uptake by MACC suggests that MACC and malate are taken up by two different carriers. We propose that the carrier identified here plays an important physiological role in withdrawing from the cytosol MACC and malonyl-d-amino acids generated under stress conditions.     

The uptake and incorporation of leucine and cystine by the mycelial and yeastlike phases of Blastomyces dermatitidis

Uptake and incorporation of L-leucine-C¹⁴ and L-cystine-S³⁵ was studied in the mycelial [MP] and yeastlike [YP] phases of the dimorphic fungal pathogen,Blastomyces dermatitidis. Both amino acids entered the cells of the two morphological forms ofB. dermatitidis by a permease-like system at low external concentrations of substrate. At high substrate levels, the amino acids entered the cells by a simple diffusion-like process in addition to the permease-like system. Michaelis-Menten constants [K_m] for L-leucine was found to be 1.1×10^–5 M and 4.4×10^–5 M for the MP and YP phases, respectively. The K_m for L-cystine was found to be 1.0×10^–5 M for the MP and 0.5×10^–5 M for the YP. A requirement for energy supplied by metabolic activity was demonstrated by the inhibition of uptake and incorporation of the amino acids by cells incubated with either 2,4-dinitrophenol or sodium azide. Amino acid uptake was broadly tolerant of hydrogen ion concentration, but definite optima were demonstrated at pH 7.0 to 7.5.     

Two categories of c/c ratios for higher plants

¹³C/¹²C ratios have been determined for plant tissue from 104 species representing 60 families. Higher plants fall into two categories, those with low δ_PDBI¹³C values (—24 to —34‰) and those with high δ ¹³C values (—6 to —19‰). Algae have δ ¹³C values of —12 to —23‰. Photosynthetic fractionation leading to such values is discussed.     

Effects of membrane potential and surface potential on the kinetics of solute transport

A theoretical study has been made of the influence of the transmembrane potential difference and the surface potential of living cells on the kinetics of carried-mediated solute transport. It is assumed that the form of the free energy barrier within the membrane may be approximated by one dominant symmetrical peak, and that the electrical field is constant. Both single-ion transport kinetics and cotransport of an ion with a neutral solute are dealt with. Provided that the surface potential and the transmembrane potential are constant, the concentration dependence of the uptake rate is given by the Michaelis-Menten equation. The kinetic parameters, the maximal rate of uptake and the K_m, depend on both the surface potential and the membrane potential in a rather complex way. It is shown that the intuitive notion, that the maximal rate of cation uptake will increase when the cell membrane is hyperpolarized, is wrong in its generallity. Both an increase or a decrease may occur, depending on the characteristics of the transport system involved. If the magnitude of the membrane potential and the surface potential depends on the substrate concentration, marked deviations from Michaelis-Menten kinetics may come to the fore. This may result in either apparent positive or apparent negative homotrope cooperative effects. Enhancement of the uptake rate of the substrate ion may occur on adding another cation, despite the fact that the membrane will become depolarized. The same type of complex transport kinetics as found for Rb⁺ and Na⁺ uptake in yeast cells can be simulated by using a single-site transport model and including effects of the membrane potential.     

Importance in catalysis of a magnesium ion with very low affinity for a hammerhead ribozyme

Available evidence suggests that Mg²⁺ ions are involved in reactions catalyzed by hammerhead ribozymes. However, the activity in the presence of exclusively monovalent ions led us to question whether divalent metal ions really function as catalysts when they are present. We investigated ribozyme activity in the presence of high levels of Mg²⁺ ions and the effects of Li⁺ ions in promoting ribozyme activity. We found that catalytic activity increased linearly with increasing concentrations of Mg²⁺ ions and did not reach a plateau value even at 1 M Mg²⁺ ions. Furthermore, this dependence on Mg²⁺ ions was observed in the presence of a high concentration of Li⁺ ions. These results indicate that the Mg²⁺ ion is a very effective cofactor but that the affinity of the ribozyme for a specific Mg²⁺ ion is very low. Moreover, cleavage by the ribozyme in the presence of both Li⁺ and Mg²⁺ ions was more effective than expected, suggesting the existence of a new reaction pathway—a cooperative pathway—in the presence of these multiple ions, and the possibility that a Mg²⁺ ion with weak affinity for the ribozyme is likely to function in structural support and/or act as a catalyst.     

Mechanistic model of nutrient uptake explains dichotomy between marine oligotrophic and copiotrophic bacteria

Marine bacterial diversity is immense and believed to be driven in part by trade-offs in metabolic strategies. Here we consider heterotrophs that rely on organic carbon as an energy source and present a molecular-level model of cell metabolism that explains the dichotomy between copiotrophs—which dominate in carbon-rich environments—and oligotrophs—which dominate in carbon-poor environments—as the consequence of trade-offs between nutrient transport systems. While prototypical copiotrophs, like Vibrios, possess numerous phosphotransferase systems (PTS), prototypical oligotrophs, such as SAR11, lack PTS and rely on ATP-binding cassette (ABC) transporters, which use binding proteins. We develop models of both transport systems and use them in proteome allocation problems to predict the optimal nutrient uptake and metabolic strategy as a function of carbon availability. We derive a Michaelis–Menten approximation of ABC transport, analytically demonstrating how the half-saturation concentration is a function of binding protein abundance. We predict that oligotrophs can attain nanomolar half-saturation concentrations using binding proteins with only micromolar dissociation constants and while closely matching transport and metabolic capacities. However, our model predicts that this requires large periplasms and that the slow diffusion of the binding proteins limits uptake. Thus, binding proteins are critical for oligotrophic survival yet severely constrain growth rates. We propose that this trade-off fundamentally shaped the divergent evolution of oligotrophs and copiotrophs.     

SLC25A23 augments mitochondrial Ca2+ uptake,interacts with MCU,and induces oxidative stress–mediated cell death

Emerging findings suggest that two lineages of mitochondrial Ca²⁺ uptake participate during active and resting states: 1) the major eukaryotic membrane potential–dependent mitochondrial Ca²⁺ uniporter and 2) the evolutionarily conserved exchangers and solute carriers, which are also involved in ion transport. Although the influx of Ca²⁺ across the inner mitochondrial membrane maintains metabolic functions and cell death signal transduction, the mechanisms that regulate mitochondrial Ca²⁺ accumulation are unclear. Solute carriers—solute carrier 25A23 (SLC25A23), SLC25A24, and SLC25A25—represent a family of EF-hand–containing mitochondrial proteins that transport Mg-ATP/Pi across the inner membrane. RNA interference–mediated knockdown of SLC25A23 but not SLC25A24 and SLC25A25 decreases mitochondrial Ca²⁺ uptake and reduces cytosolic Ca²⁺ clearance after histamine stimulation. Ectopic expression of SLC25A23 EF-hand–domain mutants exhibits a dominant-negative phenotype of reduced mitochondrial Ca²⁺ uptake. In addition, SLC25A23 interacts with mitochondrial Ca²⁺ uniporter (MCU; CCDC109A) and MICU1 (CBARA1) while also increasing I_MCU. In addition, SLC25A23 knockdown lowers basal mROS accumulation, attenuates oxidant-induced ATP decline, and reduces cell death. Further, reconstitution with short hairpin RNA–insensitive SLC25A23 cDNA restores mitochondrial Ca²⁺ uptake and superoxide production. These findings indicate that SLC25A23 plays an important role in mitochondrial matrix Ca²⁺ influx.     

Uptake of Some Quaternary Ammonium Ions by Human Erythrocytes

In many biophysical studies on erythrocytes some quaternary ammonium ions are used as replacements for Na⁺ and K⁺ of the physiological solutions. The object of this work was to study the possible uptake of quaternary ammonium ions by erythrocytes. Uptake of C¹⁴–choline chloride and C¹⁴–tetramethylammonium chloride by human erythrocytes was proved. It was shown that the compounds were neither incorporated into phospholipids of the cell nor converted to any other metabolites. Studies of uptake as a function of time, at several external concentrations of choline and tetramethylammonium, showed that within the first 4 hours uptake was a linear function of time regardless of the external concentration of the quaternary ammonium ions. The effects of various external concentrations of choline and tetramethylammonium ions on the rate of uptake by the cells were studied. The results showed the presence of two distinct mechanisms for the uptake of choline: one, a facilitated uptake mechanism which becomes saturated at low external concentrations of the ion; the other, a simple diffusion mechanism in which the rate of uptake is proportional to concentration. For the facilitated part of the uptake the external choline concentration at which half-maximum rate was obtained was found to be 0.02 mm. Although the kinetic studies with tetramethylammonium ion were not as extensive as those with choline, they did suggest the presence of similar mechanisms for the uptake of both ions. Tetramethylammonium and tetraethylammonium ions were shown to be competitive inhibitors of the facilitated choline uptake.     

Photoreaction of manganese catalyst in photosynthetic oxygen evolution

The formation of active O₂ evolving centers following addition of Mn²⁺ to Mn deficient Anacystis nidulans cells yielded an estimate of 6 to 12 Mn atoms associated with each O₂ evolving reaction center. Restoration of activity upon addition of Mn ions is affected in 3 ways: (1) Stimulation of the uptake of exogenous Mn into the cells—this uptake occurs in darkness, but is enhanced 5 to 10 fold by light; a high concentration of DCMU (1 × 10⁻⁵m) decreases this light enhanced influx no more than 50 to 75%; (2) Photoreactivation of the O₂ evolving centers, after excess Mn has been accumulated in the cells essentially no increase in Hill activity is observed unless the cells are illuminated. This photoreactivation is fully inhibited by 10⁻⁶m DCMU and partially by benzoquinone. The Q₁₀ of photoreactivation proper is close to 1; (3) Photoinhibition of the activation—photoreactivation occurs most effectively in weak intensities (< one-fiftieth photosynthetic saturation in normal cells). Apparently at higher intensities an inhibitory photoprocess is overriding. This inhibition proved reversible. The photoreactivation leads to new stable O₂ evolving centers as evidenced by an increase in the rate at saturating intensity, quantum yield, and the O₂ gush.