Abstract— A quantitative radiometric assay utilising incorporation of 14C from labelled precursors as a measure of melanin formation by human brain in vitro is described. The assay was validated by comparison with various criteria of melanin formation. Catecholamines, DOPA and 5HT were precursors for brain melanin formation. Melanin formation was detected in all brain regions studied and was highest in substantia nigra and striatum. The assay was used to evaluate various hypotheses of brain melanin formation. No evidence for enzymic activity was found and it is concluded that brain melanin formation may be a largely non‐enzymic process.

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The mechanisms of toxicity to isolated rat hepatocytes of two structurally related naphthoquinones have been studied. Both 5-OH-1,4-naphthoquinone (5-OH-1,4-NQ; juglone) and 2-OH-1,4-naphthoquinone (2-OH-1,4-NQ; lawsone) caused a concentration-dependent cytotoxicity to hepatocytes which was preceded by a depletion of intracellular glutathione. 5-OH-1,4-NQ caused a depletion of intracellular glutathione when incubated either at 4 degrees C or 37 degrees C whereas 2-OH-1,4-NQ caused a depletion of intracellular glutathione when the hepatocytes were incubated at 37 degrees C but not at 4 degrees C. 5-OH-1,4-NQ but not 2-OH-1,4-NQ reacted with glutathione in buffered solution. These results suggested that the depletion of intracellular glutathione by 2-OH-1,4-NQ is enzyme mediated whereas in the case of 5-OH-1,4-NQ the direct chemical reaction with gluathione may be largely responsible for the depletion. A critical role for depletion of protein thiols in menadione-induced cytotoxicity has been proposed. In agreement with earlier work, menadione caused a decrease in protein sulphydryls prior to cell death, however, at cytotoxic concentrations of both 2-OH-1,4-NQ and 5-OH-1,4-NQ this decrease only accompanied rather than preceeded cell death. The mechanism of toxicity of 5-OH-1,4-NQ is similar to that of other naphthoquinones and involves formation of its corresponding naphthosemiquinone, active oxygen species and redox cycling as it stimulated a disproportionate increase in both microsomal NADPH oxidation and oxygen consumption.(ABSTRACT TRUNCATED AT 250 WORDS)

SUMMARY : An enzyme which splits high molecular weight polymetaphosphate (PMP) to orthophosphate has been extracted from Corynebacterium xerosis, partially purified and some of its properties studied. A method for the preparation of bacterial P M P labelled with 32P is described. The enzyme hydrolyses bacterial or synthetic P M P at the same rate. No formation of short-chain phosphate polymers was detected during the reaction and the enzyme did not attack metaphosphates smaller than hexametaphosphate. It does not transfer phosphate from P M P to ADP to form ATP, nor does it synthesize PMP from orthophosphate (Pi) in the presence of ATP or other nucleotides. All the metal ions tested, including Mg++, inhibited the enzyme. Sodium ethylenediaminetetraacetate (EDTA ; 0.2 mM) stimulated the rate of reaction ; higher concentrations inhibited. The significance of polymetaphosphate and enzymes which split it in micro-organisms is briefly discussed.Enzymes which split polymetaphosphate (PMP) to orthophosphate (Pi) occur in extracts of a variety of micro-organisms including bacteria, yeasts and moulds (Mann, 1944; Kitasato, 1928). Most of the earlier observations were, however, made with polymetaphosphatases which split metaphosphates of low molecular weight, e.g. trimetaphosphate, tetrametaphosphate and hexametaphosphate. More recently Malmgren (1952) studied the breakdown of very high molecular weight P M P preparations by extracts from Aspergillus niger, Penicillium expansum and some other organisms. By measuring the decrease in viscosity and the amount of orthophosphate formation for 2 days at pH 5.3 he concluded that low molecular weight compounds such as tetrametaphosphate and pentametaphosphate were the major end-products. More recently, Kornberg, Kornberg & Simms (1956) prepared an enzyme from Escherichia coli which could synthesize P M P from adenosine triphosphate (ATP); an unidentified primer was also necessary to initiate the reaction. Kornberg (1957) showed that this enzyme also catalysed the reverse reaction, i.e. synthesis of ATP from P M P and adenosine diphosphate (ADP). A similar enzyme has also been slightly purified from Corynebacterium diphtheriae (Kornberg, 1957). The aim of the present investigation was to purify the polymetaphosphatase (PMP-ase) from C. xerosis and to study some of its properties ; a relatively simple assay system has been devised. We were also interested in finding out whether this enzyme catalyses the transfer of orthophosphate (Pi) from P M P to a suitable substrate such as ADP, besides breaking P M P to Pi, and whether P M P could directly phosphorylate other compounds, such as glucose, without the intermediation of the ADP-ATP system.