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The effects of 2-phenylisatogen and menadione on some mitochondrial reactions have been studied. The respiration with NAD+-linked substrates was stimulated, but respiration with succinate was not affected. The stimulation of respiration was inhibited by p-hydroxymercuribenzoate, but not by other respiratory chain inhibitors. Potassium cyanide and sodium sulphide stimulated NADH oxidation in the presence of 2-phenylisatogen or menadione. 2-Phenylisatogen was found to be converted to 2-phenylindolone. It is concluded that both 2-phenylisatogen and manadione are acting as electron acceptors in the NADH dehydrogenase region of the respiratory chain. Possible mechanisms for the reaction and for the stimulation observed in the presence of cyanide are discussed.

The isomeric prostaglandins, A and B, can be readily distinguished by differences in the mass spectra of their derivatives. The mass spectra of the PGA1- or PGA2-methyl ester (ME)-trimethyl silyl (TMS) ether derivatives have a prominent ion at [M − 71]+ or [M − C5H11]+ while those of the PGB1- or PGB2-ME-TMS derivatives have a predominant ion at [M − 99]+ or [M − C6H11O]+ in addition to that at [M − 71]+. Ions of similar origin characterize the spectra of the PGA1- or PGA2-TMS ether-TMS ester and PGB1- or PGB1-TMS-TMS derivatives, respectively. The fragmentation of other derivatives of PGA1, PGA2, PGB1 and PGB2 such as the ME-t-Bu-DMS (t-butyl-dimethylsilyl ether); ME-MO (methoxime)-TMS; ME-MO-Ac (acetate), and ME-Ac are also described comparatively. The composition of important ions was confirmed by deuterium labeling and/or high resolution mass spectroscopy, where appropriate. The potential advantages and limitations of the derivatives for quantitative analysis of prostaglandins by the specialized technique of multiple ion detection (MID) are described.

In a recent paper Sweetman et al. (1971) described the effects of a novel energy-transfer inhibitor, 2-phenylisatogen, on rat liver mitochondria. The effects of 2-phenylindolone, one of the reduction products of the isatogen, on energy-linked reactions in rat liver submitochondrial particles have now been investigated by A. P. G., A. J. S. and M. H. at the Sunderland Polytechnic. Since the results obtained showed a marked similarity to those obtained by K. J. C., C. R. L. and R. B. B. at Shell Research Ltd., who have been using [‘4C]N-ethylmaleimide to correlate inhibition of the partial reactions of oxidative phosphorylation with the binding of the thiol-blocking reagent to mitochondria1 membranes, we present them as a joint communication. Previous work (Sanadi et al., 1968; Foucher et al., 1969; Foucher & Gaudemer, 1971) suggested that thiol groups were involved in the reactions leading to ATP synthesis, but no attempt had been made to label isolated membrane components containing these thiol groups. Ox heart submitochondrial particles were used in the work on N-ethylmaleimide to avoid problems with the mercurial-sensitive phosphate-porter (Tyler, 1968 ; Fonyo & Bessman, 1968). The reactions studied with 2-phenylindolone and N-ethylmaleimide included NADH and succinate oxidation, energy-linked and non-energy-linked transhydrogenases, Mg’+-stirnulated adenosine-triphosphatase and the ATP-driven reduction of NAD+ by succinate. Without preincubation, lOnmol of 2-phenylindolone/mg of submitochondrialparticle protein inhibited the non-energy-linked transhydrogenase by 37.7 %, the ATPdriven transhydrogenase by 55.7 % and the aerobically driven reaction by 65.1 %. The same concentration inhibited the ATP-driven reduction of NAD+ by succinate by 72.5 %, but had no effect on the oxidation of succinate or NADH or on the Mg’+-stirnulated adenosine triphosphatase. The degree of inhibition of the two transhydrogenases was very similar to that seen with the thiol-blocking reagent N-ethylmaleimide. It was found that lOnmol of Nethylmaleimide/mg of submitochondrial-particle protein inhibited the non-energylinked reaction by 60.2 % and the ATP-driven reaction by 89.7 %. The N-ethylmaleimide was incubated with the particles for 14h at 2°C before measurement of the reaction. It was also found that lOnmol of inhibitor/mg of submitochondrial-particle protein failed to inhibit the Mg*+-stimulated adenosine triphosphatase during 2.5 h at 2°C; on the contrary, a variable stimulation of between 10 and 50% was observed. The ATP-driven reduction of NAD+ by succinate was inhibited by 35 % with lOnmol of N-ethylmaleimide/mg of submitochondrial-particle protein during 2.5 h at 2°C. However, the same concentration of N-ethylmaleimide inhibited NADH oxidation by 33% after lOmin at 2°C. It was therefore concluded that the inhibition of the ATPdriven reduction of NAD+ by succinate by N-ethylmaleimide was probably due to an interaction with the NADH dehydrogenase enzyme system present in the particles. The binding of [14C]N-ethylmaleimide was correlated with its inhibitory activity against the non-energy-linked and energy-linked transhydrogenases and with its stimulation of the Mg’+-stimulated adenosine triphosphatase (Fig. 1). The transhydrogenase reaction was chosen for this work since it does not involve the electrontransport-chain enzymes, some of which are sensitive to thiol-blocking reagents. After measurement of the reactions the submitochondrial particles were solubilized in a sodium dodecyl sulphate-dithiothreitol-EDTA solution and subjected to electrophoresis in polyacrylamide gels (Cattell et al., 1971). The distribution of radioactivity was then determined in each gel. The results obtained were surprising, since the single major

1. Small particles from Escherichia coli catalyse an ATP-dependent reduction of NADP+ by NADH. 2. The reaction was stimulated by Mg2+ and only ATP and ITP could serve as energy donors. 3. Studies of the stoicheiometry of the reaction indicate that 1–2mol of ATP are utilized/mol of NADH produced. 4. The reaction is inhibited by uncoupling agents such as 2,4-dinitrophenol (200μm) and tetrachlorotrifluorobenzimidazole (2μm) but oligomycin does not inhibit. 5. The reaction is inhibited by relatively high concentrations (200μm) of piericidin A.

A 4-year-old boy ingested between 1 and 2 Gm. of Dilantin and was hospitalized with marked ataxia, tremor, nystagmus, and hyperreflexia. The highest serum level of Dilantin was 72 μg per milliliter. The signs subsided as the serum level of the drug declined. The time required for the serum level to halve itself declined from 60 hours to 26 hours. At the high level of serum Dilantin concentration the patient had hyperglycemia with relative hypoinsulinemia. The daily excretion of parahydroxydilantin (p-HPPH), the major metabolite of Dilantin, was 115 mg. In addition, a dihydrodiol metabolite has been identified in the urine.

1. A method for the preparation of small particles from Escherichia coli is described. 2. These particles catalyse the ATP-driven reduction of NAD+ by succinate. 3. ATP utilized/NADH produced ratios below 2.0 were measured and a stoicheiometry of 1 molecule of ATP utilized per molecule of NADH produced is proposed. 4. The reaction is not inhibited by 2,4-dinitrophenol or by oligomycin but is inhibited by other uncouplers such as pentabromophenol and dicoumarol. 5. The specificity of the energy source, the specificity of the electron acceptor, the effects of pH, Mg2+, Pi and temperature have been studied.

Conversion of prostaglandin E(PGE) into the methyl ester, 15-trimethylsilyl ether of either PGA or PGB, makes possible the estimation of PGE in the sub-nanogram range, using vapor-phase analysis. PGE methyl ester can be efficiently converted at sub-nanogram levels to the TMS derivative of PGA by treatment with N,O,- bis -trimethylsilylacetamide in pyridine. The well-known, base-catalysed dehydration and rearrangement of PGE to PGB can similarly be achieved using sub-nanogram levels of prostaglandin. The methyl ester, trimethylsilyl ethers of PGA or PGB are shown to possess excellent properties for vapor-phase analysis, presenting minimal difficulties due to adsorption or thermal degradation, and have mass spectra characterized by only one or two predominant ions, facilitating their quantification into the sub-nanogram range, using mass spectrometry. Quantitative determination, with improved sensitivity into the sub-nanogram range of the derivative of PGB, has also been achieved using the electron capture detector. The same system can be applied to the estimation of PGA in the low nanogram range. These derivatives and analytical methods have the potential to provide quantitative estimation, with excellent sensitivity and specificity, of 9-keto-prostaglandins at low levels in biological samples.

The effects of alphaxalone, a new intravenous anaesthetic, on mitochondrial reactions are described. At low concentrations (20–100 μM) the compound was found to be an inhibitor of the oxidation of NAD + -linked substrates in rabbit brain mitochondria. It was concluded that the site or action was at, or near, the rotenone-sensitive site on the NADH dehydrogenase enzyme system. When succinate was substrates, and when higher concentrations (300 μM) were used, alphaxalone was found to release respiratory control, stimulate state 4 respiration, relieve oligomycin-inhibited state 3 respiration and stimulate the mitochondrial ATPase. It was concluded that this second effect was due to an uncoupling action.

ALTHOUGH the reactions of aromatic ‘disulphoxides’ have been studied by Smiles and his co-workers1 and by Vinkler and Klivenyi1 among others, the structures of these compounds remained uncertain until their infra-red spectra were examined by Cymerman and Willis3. The latter work confirmed earlier evidence that the compounds hitherto known as disulphoxides should be assigned the thiolsulphonate structure (I) rather than the disulphoxide structure (II).

Peracid oxidation of wool or bovine plasma albumin greatly increases the apparent thiol contents of the proteins as estimated by reaction with organomercuric halides. This apparent anomaly results principally from reaction of the mercurials with disulphide monoxide (thiolsulphinate) groups present (as S-monoxycystyl residues) in the protein. SS-Dioxycystyl residues may also react under some conditions. The mercurial reagents similarly combine with cystine (�)-S-monoxide.