Studies on the mechanism of chloride action on photosynthetic water oxidation

In this paper, we describe experiments which were designed to probe the mechanism through which Cl^? anions exert their influence on electron transport on the oxidizing side of Photosystem II (PS II). We asked whether photosynthetically active Mn was released from and reinserted into the water-splitting enzyme upon Cl^? removal and subsequent repletion, and obtained evidence suggesting that it was not. To locate the site of the Cl^?-dependent lesion, we counted the number of electrons that were still available in Cl^?-free chloroplasts for rapid reduction of P-680⁺ following a flash, and compared our results with other, previously characterized methods of inhibition. Using both delayed and prompt fluorescence as measures of the lifetime of P-680⁺, we found that Cl^?-depleted thylakoids could deliver two electrons to the oxidized PS II reaction center. This is interpreted as indicating that two oxidizing equivalents can be generated and transiently stored by PS II after Cl^? removal. Two alternative schemes which describe the functional location of electron carriers in this portion of the electron transport chain are proposed to account for our data. An experiment designed to distinguish between them is discussed. We also investigated the stability of oxidants produced by the Cl^?-depleted PS II. The apparently contradictory results obtained by prompt fluorescence and luminescence measurements are tentatively resolved by postulating the existence of two pathways through which closed reaction centers reopen, only one of which proceeds via a luminescence-producing recombination mechanism. It is suggested that deactivation of the PS-II oxidizing equivalents through both pathways is accelerated by Cl^? removal.     

Evidence for a one-electron mechanism of 2-aminofluorene oxidation by prostaglandin H synthase and horseradish peroxidase

Previous studies have shown that the primary arylamine carcinogen 2-aminofluorene (2-AF) is oxidized by the prostaglandin H synthase peroxidase to mutagenic and electrophilic products capable of covalent binding to macromolecules. The present study was designed to identify the potential reactive intermediate(s) responsible for binding, and to characterize further the metabolic intermediates in 2-AF peroxidation. Both prostaglandin H synthase and horseradish peroxidase, with H2O2, oxidize 2-AF to azofluorene, 2-aminodifluorenylamine (2-ADFA), 2-nitrofluorene, polymeric and nonorganic-extractable material. Both enzymes show greater activity at pH 5.0 than at pH 7.0. In the presence of either 2-t-butyl-4-methoxyphenol or 2,6-dimethylphenol, arylamine/phenol adducts were formed in high yield, with the nitrogen of either 2-AF or 2-ADFA coupled to the para position of the phenol (loss of -OCH3 with 2-t-butyl-4-methoxyphenol). These structures were confirmed by mass spectrometry and NMR spectroscopy. Acid hydrolysis of N-hydroxy-2-AF to yield the nitrenium ion, in the presence of a phenol, also results in adduct formation, but only at times greater than 2 h and in very limited yield. The peroxidase-catalyzed adduct formation, however is rapid (less than 2 min) and extensive. These and other data support a one-electron pathway for 2-AF peroxidation, with a free radical or a free radical-derived product responsible for binding to protein and DNA. An N-hydroxy intermediate may therefore not be obligatory in the enzymatic activation of 2-AF to a mutagenic product.     

Studies on the mechanism of action of 6-mercaptopurine. Interaction with copper and xanthine oxidase.

Interaction between 6-mercaptopurine, Cu2+ and the enzyme xanthine oxidase (EC 1.2.3.2.) was examined. Whereas Cu2+ was found to inhibit the enzyme, 6-mercaptopurine could protect as well as reverse the enzyme inhibition produced by the metal ion. The formation of a complex between 6-mercaptopurine and Cu2+ seems to be responsible for the observed effect. Job's [(1928) Ann. Chem. 9, 113] method has shown the composition of the complex to be 1:1. The apparent stability constant (log K value), as determined by Subhrama Rao & Raghav Rao's [(1955) J. Sci. Chem. Ind. Res. 143, 278], method is found to be 6.74. It is suggested that the formation of a stable complex between 6-mercaptopurine molecules and Cu2+ may be an additional mechanism of action of 6-mercaptopurine, particularly with reference to its anti-inflammatory properties.     

Phthalazine PDE4 inhibitors. Part 2: the synthesis and biological evaluation of 6-methoxy-1,4-disubstituted derivatives

This communication describes the synthesis and in vitro evaluation of a novel and potent series of phosphodiesterase type IV (PDE4) inhibitors. The compounds described present substituents in position 4 of the phthalazine ring to replace the commonly observed cyclopentyloxy moiety of rolipram analogues. Preliminary evidences of reduced side effects compared to standards and improved pharmacokinetic properties for selected derivatives are also reported.     

Studies on the mechanism of action of oxygen-labile haemolysins.

The sensitivities of the binding step and the lytic step of haemolysis by pneumolysin to the action of various inhibitors and to variations in the assay conditions were studied. Binding was inhibited by HgCl2 and N-ethylmaleimide. Lysis by previously fixed lysin was insensitive to HgCl2 and only slightly sensitive to N-ethylmaleimide. Binding of pneumolysin was independent of ionic strength. Binding of pneumolysin and streptolysin O decreased above pH 8-0 and 8-4 respectively. These results suggest that binding requires a non-ionized unsubstituted sulphydryl group. Incubation of erythrocytes with NaF caused inhibition of pneumolysin, indicating that some metabolic function of the cell may be involved in lysis. The action of streptolysin O was not affected by NaF.     

Studies on the mechanism of action of the aromatase inhibitor, 4-hydroxyandrostenedione

[1 beta-3H], [1 alpha,2 alpha-3H] and [1 beta,2 beta-3H] 4-Hydroxyandrostenedione (4-OH-A) were synthesized to study the mechanism of inhibition of aromatase by 4-OH-A. Incubations of [1 beta-3H] and [1 beta,2 beta-3H] 4-OH-A with placental microsomes in the presence of NADPH showed very little loss of tritium, with aromatization of 4-OH-A ranging from 0.3 to 0.6 percent. No loss of tritium was observed in the absence of NADPH. The extent of covalent binding of 4-OH-A to microsomal proteins was higher with incubations in the absence of NADPH than with those in the presence of NADPH. These results are discussed in light of what has been proposed for the mechanism of androgen aromatization.     

Studies on the mechanism of the antifungal action of benzoate.

A method is described for the determination of the pH of intracellular water based on the distribution of [14C]benzoate (0.01 mM) between intra- and extra-cellular water. Benzoate at higher concentrations (2-10mM) enters the yeast cell in the undissociated form, and its neutralization within the cell can cause a shift of the pH of the intracellular water by more than 1 pH unit. Benzoate causes an accumulation of the two hexose monophosphates of yeast glucose fermentation and a decrease in intermediates beyond phosphofructokinase, suggesting inhibition at this stage. Benzoate also causes a concomitant fall in [ATP]. Phosphofructokinase is inhibited to a greater extent than hexokinase at acid pH. There is a relationship between intracellular pH, phosphofructokinase inhibition and CO2 production, suggesting that the antifungal action of benzoate is caused by an accumulation of benzoate at low external pH, which lowers the intracellular pH into the range where phosphofructokinase is sensitive. The subsequent inhibition of glycolysis causes a fall in [ATP] and thus restricts growth.