Ultraviolet-induced mutations to asparagine independence in Jensen sarcoma cells in vitro

UV-irradiation induces an exponential increase in the frequency of mutation from asparagine requirement to asparagine non-requirement in Jensen sarcoma cells grown in vitro. The corrected mutation frequency increases from the spontaneous rate of 5.1·10^?6 per cell to 1248·10^?6 per cell with a dose of 180 erg/mm² of 254 nm UV A substantial increase was oberved even without correction for survivors, and no significant difference was observed in the UV sensitivity of asparagine-requiring and non-requiring Jensen clones. When Jensen cells were plated at low densities in a feeder layer of LMTK-cells inactivated by HAT medium, an increase in the cloning ability of the former was observed as compared to appropriate controls without the feeder layer, but the increase was constant over all doses of UV tested. Revertants are stable and possess measurable asparagine synthetase.It is concluded that UV is an extremely effective mutagen in this system.     

Ultraviolet light-induced responses of an mfd mutant of escherichia coli B/r having a slow rate of dimer excision

A mutant of Eschirichia coli B/r designated mfd has drastically reduced ability to exhibit “mutation frequency decline” (MFD) the irreversible loss of potential suppressor mutations which occurs when protein synthesis is briefly inhibited after irradiation with U.V. We have found that the initial rate of thymine dimer excision in the mfd mutant is only about one-third that of its mfd⁺ parent strain after a UV dose of 400 erg/mm². The yield of UV-induced Tyr⁺ revertants is 4–10 times higher in the mfd strain than in the mfd⁺ strain. This is comparable to the level of UV-mutability in the mfd⁺ strain in the presence of caffeine, an inhibitor of dimer excision. UV-mutability, prophage induction and Weigle reactivation of irradiated λ phage occur to a greater extent at low UV doses (10–50 erg/mm²) in the mfd strain compared to the mfd⁺ strain. We propose that the slow excision repair in the mfd mutant results in a shift in the induction threshold for these UV-inducible functions toward lower UV doses.     

Induction of forward mutations in mutationally defective yeast

Summary The 3 rev loci that reduce ultraviolet light (UV)-induced reversion in S. cerevisiae had a similar effect on forward mutation to auxotrophy induced by a single 400 erg/mm² UV dose: rev1-1, rev2-1 and rev3-1 reduced average frequencies of auxotrophs to 4%, 64% and 4% that in wild type and reduced frequencies of mutants at ade1 or ade2 to 19%, 88% and 2% wild type, respectively. The rev2-1 strain exhibited high frequencies of spontaneous mutation. It is suggested that rev1-1 and rev3-1 block steps in a general UV mutation mechanism controlling forward and reverse mutation throughout the genome. The small effect of rev2-1, compared to the effect of rev1-1 or rev3-1, is consistent with previously obtained data on UV reversion and could be due to a specificity for induced mutation involving only certain types of UV damage or, on the other hand, it may be related to mutator activity. Although rev caused varying degrees of sensitivity to ethylmethanesulfonate (EMS), there was little or no significant effect on mutation induced by a single 30 min. dose of 3% EMS. Auxotroph frequencies were 79%, 109% and 94% wild type, whild frequencies at ade1 or ade2 were 82%, 56% and 51% wild type in the respective strains. It is suggested that steps blocked by rev, although they may participate in repair of lethal EMS damage, do not themselves generate EMS-induced mutations.     

Ultraviolet Mutagenesis in Strains of <Emphasis Type="Italic">E. coli</Emphasis> deficient in DNA Polymerase

CERTAIN mutations in Escherichia coli which cause increased sensitivity to ultraviolet light (UV) drastically change the UV mutability of the sensitive strain. Strains lacking the ability to excise pyrimidine dimers, for example, exhibit greatly increased UV mutability, producing induced mutations at doses of UV far smaller than those required to induce mutations in wild type strains^{1, 2}. Mutants owing their UV sensitivity to reduced ability to perform genetic recombination, on the other hand, show reduced mutability in response to UV compared with the wild type and some (recA or exrA strains, for example) are stable to UV, producing no detectable induced mutations at any dose^3–5. Analysis of UV mutagenesis in such strains has led to the hypothesis that most UV-induced mutations in E. coli are errors in the recombinational repair of gaps in the daughter-strand which are located opposite unexcised pyrimidine dimers^{6, 7}.     

Some Properties of Excision-defective Recombination-deficient Mutants of Escherichia coli K-12

Strains of Escherichia coli that carry the mutation uvrA6 show no measurable excision of pyrimidine dimers and are easily killed by ultraviolet (UV) light, whereas strains that carry recA13 are defective in genetic recombination and are also UV-sensitive. An Hfr strain carrying uvrA6 was crossed with an F⁻ strain carrying recA13. Among the recombinants identified, one carrying uvrA recA proved to be of exceptional sensitivity to UV light. It is estimated from the UV dose (0.2 erg/mm² at 253.7 nm) required to reduce the number of colony-forming cells by one natural logarithm that about 1.3 pyrimidine dimers were formed in a genome of 5 × 10⁶ base pairs for each lethal event. This double mutant is 40 times more UV-sensitive than the excision-defective strain carrying uvrA6. The replication of one pyrimidine dimer is generally a lethal event in strains carrying recA13. Spontaneous breakdown and UV-induced breakdown of the deoxyribonucleic acid (DNA) of cells of the various genotypes were estimated by growing the cells in medium containing ³H-thymidine and measuring both acid-precipitable and acid-soluble radioactivity. The UV-induced degradation in strains with recA13 did not require the uvr⁺ genes and hence appears to depend upon a mechanism other than dimer excision. The greater level of survival after irradiation in Rec⁺ as compared to Rec⁻ bacteria may be due to a recovery mechanism involving the reconstruction of the bacterial chromosome through genetic exchanges which occur between the newly replicated sister duplexes and which effectively circumvent the damaged bases remaining in the DNA.     

Characteristics of β-adrenergic-agonist binding to rat adipocyte membranes: Evidence that (±)-[3H]hydroxybenzylisoproterenol interacts selectively with the adipocyte β-adrenergic receptors

The binding characteristics of the β-adrenergic agonist $\text{(±)-[}{}^3\text{H]hydroxybenzylisoproterenol}$ to rat adipocyte membranes were studied. Binding was rapid, reaching equilibrium within 10 min at 37°C (second order rate constant k₁=1.37·10⁷·M^?1·min^?1). Dissociation of specific binding by 0.5 mM (?)-isoproterenol suggested dissociation from two different sites with respective dissociation rate constants k₂ of 0.106·min^?1 and 0.011·min^?1.[³H]Hydroxybenzylisoproterenol binding was saturable (B_max=690±107 fmol/mg protein), yielding curvilinear Scatchard plots. Computer modeling of these data were consistent with the existence of two classes of [³H]hydroxybenzylisoproterenol binding sites, one having high affinity (K_D=3.5±0.7 nM) but low binding capacity (10% of the total sites) and one haveing low affinity (K_D=101±20 nM) but high binding capacity (90% of the sites). Adrenergic ligands competed with [³H]hydroxybenzylisoproterenol binding with the following order of potency=(?)-propranolol>(?)-isoproterenol>(?)-norepinephrine≈ (?)-epinephrine>>(+)-isoproterenol=(+)-propranolo, which is consistent with binding to β₁-adrenergic receptors. Competition curves of [³H]hydroxybenzylisoproterenol binding by the β-agonist (?)-isoproterenol were shallow and modeled to two affinity states of binding, whereas, competition curves by β-antagonist (?)-propranolol were steeper with Hill number near to one. Gpp[NH]p severely reduced [³H]hydroxybenzyl-isoproterenol binding, an effect which apparently resulted from the reduction of the number of both the high and low affinity sites. In membranes which had been previously exposed to (?)-isoproterenol, then number of [³H]hydroxybenzylisoproterenol binding sites was reduced by 50%, an effect which apparently resulted from the loss of part of both the high and low affinity state binding sites. Finally, the ability of (?)-isoproterenol to stimulate adenylate cyclase correlate closely with the ability of (?)-isoproterenol to displace [³H]hydroxybenzylisoproterenol binding. Comparison of these findings with the binding characteristics of the β-antagonist [³H]dihydroalprenolol to rat adipocyte membranes, led to conclude that [³H]hydroxybenzylisoproterenol can be successfully used to label the β-adrenergic receptors of rat fat cells and suggests that it might be a better ligand than [³H]dihydroalprenolol in these cells.     

Mutagenesis in cultured mammalian cells. II. Induction of gene mutations in Chinese hamster cells

Mutations controlling the resistance to 6-mercaptopurine (6-M) and the ability to multiply in a medium with a low concentration of glucose (“glucose-independent” mutants) were induced in cultured Chinese hamster cells by N-nitrosomethylurea (NMU), 5-bromodeoxyuridine (BUdR), UV and X-rays. The chemical agents were found to be very active in induction of mutations to 6-M resistance (NMU and BUdR) and mutations of “glucose independence” (NMU). These agents increase the yield of mutations as compared to the spontaneous mutation rate by about two orders of magnitude. The induced rate of 6-M-resistant mutations by X-rays was 2.0 ? 10⁻⁷ per viable cell per roentgen. BUdR approximately equally increases the cell's sensitivity to both inactivating and mutagenic action of X-rays. The maximum induction of mutations to 6-M resistance by UV was observed at 100 erg/mm². This dose leads to 1 16-fold increase of the mutation frequency as compared to the spontaneous rate. Further increase of the UV dose up to 200 erg/mm² resulted in a lower yield of mutations per dose unit. The highest yield of mutations to 6-M resistance induced by NMU, BUdR and X-rays was observed if cells were plated in selective medium several generations after the mutagenic treatment. The maximum yield of mutations to 6-M resistance induced by UV and of glucose-independence induced by NMU was recorded if cells were transferred to selective media immediately after treatment. The kinetics of expression of mutations and the decline of their number observed after prolonged incubation of treated cells in nonselective conditions are discussed.     

Pathways of ultraviolet mutability in Saccharomyces Cerevisiae. IV. The relation between canavanine toxicity and ultraviolet mutability to canavanine resistance.

Seven umr mutants of Saccharomyces cerevisiae which had reduced capacity for ultraviolet light (UV)-induced forward mutation from CAN1 to can1 were tested for sensitivity to L-canavanine relative to one wild-type UMR strain and one slightly UV-sensitive but phenotypically umr⁺ strain (mutant 306). Relative UV mutation resistance was estimated by dividing the UV fluence needed to yeild a particular induced mutation frequency by that needed to reach the same frequency in the genotypic wild-type strain. The umr5 and umr6 strains were especially sensitive to canavanine growth inhibition, while umr1 was no more sensitive than either wild type; umr2, umr3, umr4, a umr7, and α umr7 were equally sensitive to an intermediate degree. Incubation at 30°C of wildtype cells plated on canavanine-selective agar for increasingly longer times before UV irradiation resulted in decreasing UV mutation frequencies (reduced to 50% in 1.6 h). All umr strains tested in this way lost UV mutability faster than wild type, including mutant 306, umr1 (not sensitive to growth inhibition), and umr6 (very sensitive to growth inhibition). Cells were grown to stationary phase in YEDP growth medium and assayed for arginine and tryptophan transport into the cell. The umr6 strain, which had weak UV mutation resistance but high sensitivity to canavanine growth inhibition, transported arginine and tryptophan at essentially wild-type levels. The umr1 strain, however, which had moderate UV mutation resistance and normal canavanine toxicity, transported both amino acids at rates tenfold higher than wild type. The data suggest that increased canavanine toxicity does not necessarily lead to defective mutability at CAN1, and that mutational deficiency cannot result solely from increased canavanine toxicity. Although exposure to canavanine was shown to block mutation fixation and/or expression, it is suggested that the degree of growth inhibition is not strictly correlated with the degree of mutation resistance.     

Cooperative response of chemically excitable membrane. II. Two-state models and their limitations

Various types of two-state models, classified by the type of direct receptorionophore coupling, were formulated based on the previously presented generalized two-state model of cooperativity (Kijima &; Kijima, 1978) and their dose-response relationships were examined. Hill coefficient at the mid-point of dose-response curve n_H^o the measure of the cooperativity of curves, is restricted for partial agonists in any two-state models because n_H^o is expressed by the product of two terms, one of which decreases when the other increases. In the independent gating unit model in which the channel opens only when the independent gating units are all in the activated state, the restriction of n_H^o is the most stringent: it never exceeds 2. In 2 ÷ 1·39 even for full agonist. It appears to be incompatible with most of the cooperative responses observed on chemically excitable membrane. In the basic model or one protomer-one channel model, n_H^o never exceeds 2·0 when 〈p〉_∞, the maximum fraction of open-channel, is less than $\text{2}\text{3}$. In the cooperative gating unit model, n_H^o is the least restricted, which is less than 2·8 when 〈p〉_∞ ≤ 0·5, but if the number of gating units, N in a receptor is practically reasonably small (N ≤ 12), n_H^o ≤ 2·0 when 〈p〉_∞ ≤ 0·58. It is discussed whether or not several representative drug-receptive membranes can be accounted for by two-state models. Response of the insect sugar receptor is out of the above limitations of two-state models and can be accounted for by three-state model. The origin of cooperative interaction can be inferred by the shapes of dose-response curves. Cooperative dose-response curves of two dimensional lattices or oligomerc systems with large number of protomers weakly interacting by long range forces bend upward more markedly at lower region than the curves of strongly interacting oligomers, when curves with the same n_H^o are compared.     

Factors affecting the efficacy of methyl bromide fumigation to control Liriomyza trifolii in imported chrysanthemum cuttings

Laboratory investigations were made into the effect of cultivar type, prior cold storage, fumigation temperature and methyl bromide concentration, on the efficacy of a fumigation treatment to control Liriomyza trifolii in chrysanthemum cuttings. The tests related to the standard quarantine treatment used in the UK to control Spodoptera littoralis on imported chrysanthemum cuttings: cold storage for 2 days at 1 – 2°C followed by methyl bromide fumigation at 15°C with a concentration time product (CTP) of 54 g h/m³. L. trifolii larvae, within detached leaves, and 1 – 2 and 2 – 3 day old pupae, were treated. Methyl bromide concentrations of 6·75 or 13·5 g/m³ were used to achieve a range of CTPs and thus obtain accurate dose-response lines and estimates of the LD99 and LD99·9 for each insect stage. Fumigation temperatures were 8, 11 or 15°C. Efficacy of the standard treatments differed between the three cultivars tested, but the LD99 for larvae remained below 54 g h/m³. Decreasing fumigation temperature to 11°C or less increased LD99 values for larvae and pupae and substantially increased variability. There is therefore little scope for using fumigation temperatures of less than 15°C for quarantine purposes. Omitting the cold storage treatment prior to fumigation did not significantly affect efficacy of fumigation. Reducing the methyl bromide concentration from 13·5 to 6·75 g/m³ did not significantly affect the LD99 for larvae but significantly reduced LD99s for pupae.     

Effect of glucose starvation on the melting points of DNA following UV irradiation ofEscherichia coli 15 T−U−his−

The effect of glucose starvation during the pre-irradiation period on the UV resistance and on the DNA melting points was investigated. The radio-resistance ofEscherichia coli 15 T^?U^? his ^? increases markedly with the length of starvation. The DNA melting point of an exponentially growing culture is decreased by a dose of 500 erg/mm² by 3 C. The same dose does not affect the melting point of DNA in a prestarved culture.     

ATP-dependent deoxyribonuclease in Bacillus subtilis and a mutant deficient in this activity

ATP-dependent DNAse activity was measured in rec⁺ and several rec strains of B. subtilis 168. One of the strains (marker recE₅) was found to lack this activity. The enzyme from the wild type was partially purified and some of its properties were determined. The pH optimum is 9.5. Activity is higher at 50° but inactivation occurs on standing at this temperature. The enzyme requires Mg²⁺ (10^?2M) or Mn²⁺ (2·10^?4M). ATP is an absolute requirement and the only other nucleoside triphosphate that can partially replace it is dATP. Lack of activity in the mutant does not seem to be due to the presence of an inhibitor. Results so far do not allow us to conclude as to whether or not the mutant produces an altered enzyme.     

Photosynthesis, Photorespiration and Respiration of Detached Spruce Twigs as Influenced by Oxygen Concentration and Light Intensity

The effects of oxygen concentration and light intensity on the rates of apparent photosynthesis, true photosynthesis, photorespiration and dark respiration of detached spruce twigs were determined by means of an infra-red carbon dioxide analyzer (IRCA). A closed circuit system IRCA was filled with either 1 per cent of oxygen in nitrogen, air (21 % O₂) or pure oxygen (100 % O₂). Two light intensities 30 × 10³ erg · cm ^?2· s^?1 and 120 × 10³ erg · cm^?2· s^?1 were applied. It has been found that the inhibitory effect of high concentration of oxygen on the apparent photosynthesis was mainly a result of a stimulation of the rate of CO₂ production in light (photorespiration). In the atmosphere of 100 % O₂, photorespiration accounts for 66–80 per cent of total CO₂ uptake (true photosynthesis). Owing to a strong acceleration of photorespiration by high oxygen concentrations, the rate of true photosynthesis calculated as the sum of apparent photosynthesis and photorespiration was by several times less inhibited by oxygen than the rate of apparent photosynthesis. The rates of dark respiration were essentially unaffected by the oxygen concentrations used in the experiments. An increase in the intensity of light from 30 × 10³ erg · cm^?3· s^?1 to 120 · 10³ erg · cm^?2· s^?1 enhanced the rate of photorespiration in the atmospheres of 21 and 100 % oxygen but not in 1 % O₂. The rate of apparent photosynthesis, however, was little affected by light intensity in an atmosphere of 1 % oxygen.     

In vitro carotenogenesis by wild type and mutants of Phycomyces blakesleeanus

Cell extracts from shake cultures of the wild type and six mutant strains of Phycomyces converted [2-¹⁴C] MVA into carotenes, squalene and prenyl phosphates. Oxygen was required for the desaturation of phytoene. When compared with the wild type, cells extracts of carB and carR mutants are much less effective in phytoene dehydrogenation and lycopene cyclization, respectively. This confirms previous conclusions about the biochemical functions of the carB and carR genes, which were based on genetic and in vivo studies. CarA strain mutants accumulate, in vivo, much less β-carotene than the wild type. This correlates with a 10-fold decrease in carotenogenesis in vitro. The addition of retinol to incubations of cell extracts of the wild type and C2 strains stimulated β-carotene formation. Both carB and carR mutants show enhanced total carotenogenic activities in vitro and the carS mutant shows a higher β-carotene-synthesizing activity than the wild type. It is suggested that the feed-back regulatory mechanism known to control this pathway operates at the level of enzyme synthesis.     

[3H]norepinephrine binding and lipolysis by isolated fat cells

[³H]norepinephrine was shown to bind to specific sites on isolated fat cells. A Scatchard plot of norepinephrine binding showed two apparent K_a of 1.9 · 10⁶ and 1.2 · 10⁵ LM^?. 1.4 · 10^?4 M Norepinephrine covalently-linked to agarose beads reduced [³H]norepinephrine binding by over 50%. Several structurally related drugs were compared as inhibitors of [³H]norepinephrine binding and as stimulators of lipolysis in preparations of similarly prepared cells. Dose-response curves for norepinephrine, epinephrine and isoproterenol showed the affinities for binding inhibition and for stimulation of lipolysis to be in the same range of 6 · 10^?7-2 · 10^?6 M. Dopamine and dopa were potent inhibitors of [³H]norepinephrine binding at 8.5 · 10^?7 M and 2.0 · 10^?6 M respectively, but did not stimulate lipolysis even at 10^?4 M. Propranolol, a β-adrenergic antagonist, had no effect on [³H]norepinephrine binding at 10^?4 M but completely inhibited catecholamine-stimulated lipolysis at 10^?5 M. Phentolamine, an α-adrenergic antagonist, did not inhibit binding or catecholamine-stimulated lipolysis at 10^?4 M. Ephedrine, metaraminol, phenylephrine and normetanephrine were also ineffective both as [³H]norepinephrine binding inhibitors and as stimulators of lipolysis. The results suggested the catechol ring of catecholamines is more important than the ethanolamine side chain as a requirement for binding, while both an intact catechol moiety and ethanolamine function appear necessary for physiological effect.     

Interactions between gnathiid isopods,cleaner fish and other fishes on Lizard Island,Great Barrier Reef

The rate of emergence of micropredatory gnathiid isopods from the benthos, the proportion of emerging gnathiids potentially eaten by Labroides dimidiatus, and the volume of blood that gnathiids potentially remove from fishes (using gnathiid gut volume) were determined. The abundance (mean ±s.e .) of emerging gnathiids was 41·7 ± 6·9 m^?2 day^?1 and 4552 ± 2632 reef^?1 day^?1 (reefs 91–125 m²). The abundance of emerging gnathiids per fish on the reef was 4·9 ± 0·8 day^?1; but excluding the rarely infested pomacentrid fishes, it was 20·9 ± 3·8 day^?1. The abundance of emerging gnathiids per patch reef was 66 ± 17% of the number of gnathiids that all adult L. dimidiatus per reef eat daily while engaged in cleaning behaviour. If all infesting gnathiids subsequently fed on fish blood, their total gut volume per reef area would be 17·4 ± 5·6 mm³ m^?2 day^?1; and per fish on the reefs, it would be 2·3 ± 0·5 mm^?3 fish^?1 day^?1 and 10·3 ± 3·1 mm³ fish^?1 day^?1 (excluding pomacentrids). The total gut volume of gnathiids infesting caged (137 mm standard length, L_S) and removed from wild (100–150 mm L_S) Hemigymnus melapterus by L. dimidiatus was 26·4 ± 24·6 mm³ day^?1 and 53·0 ± 9·6 mm³ day^?1, respectively. Using H. melapterus (137 mm L_S, 83 g) as a model, gnathiids had the potential to remove, 0·07, 0·32, 0·82 and 1·63% of the total blood volume per day of each fish, excluding pomacentrids, caged H. melapterus and wild H. melapterus, respectively. In contrast, emerging gnathiids had the potential of removing 155% of the total blood volume of Acanthochromis polyacanthus (10·7 mm L_S, 0·038 g) juveniles. That L. dimidiatus eat more gnathiids per reef daily than were sampled with emergence traps suggests that cleaner fishes are an important source of mortality for gnathiids. Although the proportion of the total blood volume of fishes potentially removed by blood‐feeding gnathiids on a daily basis appeared to be low for fishes weighing 83 g, the cumulative effects of repeated infections on the health of such fish remains unknown; attacks on small juvenile fishes, may result in possibly lethal levels of blood loss.     

Algae,bacteria and detritus as food for the harpacticoid copepod, Heteropsyllus pseudonunni Coull and Palmer

The harpacticoid copepod, Heteropsyllus pseudonunni Coull and Palmer, when fed Juncus roemerianus Scheele (needlerush) detritus produced significantly more eggs than when fed the alga Dunaliella sp. Five food rations and a starved control were used: A, algal concentration ≈ 10⁴ cells · ml^?1; LA, algal concentration 10³ cells · ml^?1; D, detritus concentration 0.25 g · ml^?1; LD, detritus concentration 0.025 g · ml^?1; LDLA, a mixture of the LD and LA rations. Geometric mean total egg production values were: LDLA 132.5; LD 132.6; D 120.5; A 65.8; LA 5.0. The LA results were significantly less (P = 0.05; Student-Newman-Keuls' test) than all others, while the A results were significantly less than the LDLA, LD and D results. The LDLA, LD and D results were not statistically different. Similar trends were obtained when mean brood sizes and mean number of broods were compared. In a second feeding experiment, using Juncus detritus, bacteria on sand grains, and algae (Nitzschia sp.) on sand grains, Heteropsyllus pseudonunni produced significantly more eggs (P = 0.05; SNK test) when fed detritus (166.0) or bacteria (132.2) than algae (58.9). The detritus and bacteria results were not significantly different.     

Factors influencing hydroxylamine inactivation of photosynthetic water oxidation

The kinetics of Mn release during NH₂OH inactivation of the water oxidizing reaction is largely insensitive to the S-state present during addition of NH₂OH. This appears to reflect reduction by NH₂OH of higher S-states to a common more reduced state (S₀ or S_?1) which alone is susceptible to NH₂OH inactivation. Sequences of saturating flashes with dark intervals in the range 0.2–5 s^?1 effectively prevent NH₂OH inactivation and the associated liberation of manganese. This light-induced protection disappears rapidly when the dark interval is longer than about 5 s. Under continuous illumination, protection against NH₂OH inactivation is maximally effective at intensities in the range 10³–10⁴ erg · cm^?2 · s^?1. This behavior differs from that of NH₂OH-induced Mn release, which is strongly inhibited at all intensities greater than 10³ erg · cm^?2 · s^?1. This indicates that two distinct processes are responsible for inactivation of water oxidation at high and low intensities. Higher S-states appear to be immune to the reaction by which NH₂OH liberates manganese, although the overall process of water oxidation is inactivated by NH₂OH in the presence of intense light. The light-induced protection phenomenon is abolished by 50 μM DCMU, but not by high concentrations of carbonyl cyanide m-chlorophenylhydrazone, which accelerates inactivation reactions of the water-splitting enzyme, Y (an ADRY reagent). The latter compound accelerates both inactivation of water oxidation and manganese extraction in the dark.