Oxidase-peroxidase enzymes of Datura innoxia. Oxidation of formylphenylacetic acid ethyl ester.

An enzyme system from Datura innoxia roots oxidizing formylphenylacetic acid ethyl ester was purified 38-fold by conventional methods such as (NH4)2SO4 fractionation, negative adsorption on alumina Cy gel and chromatography on DEAE-cellulose. The purified enzyme was shown to catalyse the stoicheiometric oxidation of formylphenylacetic acid ethyl ester to benzoylformic acid ethyl ester and formic acid, utilizing molecular O2. Substrate analogues such as phenylacetaldehyde and phenylpyruvate were oxidized at a very low rate, and formylphenylacetonitrile was an inhilating agents, cyanide, thiol compounds and ascorbic acid. This enzyme was identical with an oxidase-peroxidase isoenzyme. Another oxidase-peroxidase isoenzyme which separated on DEAE-chromatography also showed formylphenylacetic acid ethyl ester oxidase activity, albeit to a lesser extent. The properties of the two isoenzymes of the oxidase were compared and shown to differ in their oxidation and peroxidation properties. The oxidation of formylphenylacetic acid ethyl ester was also catalysed by horseradish peroxidase. The Datura isoenzymes exhibited typical haemoprotein spectra. The oxidation of formylphenylacetic acid ethyl ester was different from other peroxidase-catalysed reactions in not being activated by either Mn2+ or monophenols. The oxidation was inhibited by several mono- and poly-phenols and by catalase. A reaction mechanism for the oxidation is proposed.     

An enzyme degrading reduced nicotinamide-adenine dinucleotide in Proteus vulgaris.

Cell-free extracts of a strain of Proteus vulgaris degrade NADH to reduced nicotinamide riboside, adenosine and two molecules of phosphate. The system is weakly active in fresh cell extracts, but activity is increased about 10-fold on rapid heating to 70-100 degrees C. On returning to room temperature, the activity returns rapidly to its initial low value but can be re-activated by again heating to 70-100 degrees C. Reversible activation can also be effected by extremes of pH or by teatment with 8M-urea. Activation appears to be due to reversible changes in conformation of the protein of the enzyme rather than to combination of the enzyme with a heat-labile inhibitor. The active form can be stabilized by addition of PPi. The system, which also possesses 5'-nucleotidase activity not separable from the NADH pyrophosphatase, requires Co2+ (0.4mM) for maximum activity. Although activated at relatively high temperatures, it is not enzymically active until cooled to 50-60 degrees C. It may be purified by affinity chromatography (with NAD+ as ligand) to an activity over 400 times that of the crude cell extract, and yields only one major band on polyacrylamide-gel electrophoresis.     

Oxidation of reduced nicotinamide-adenine dinucleotide by the malate—Aspartate shuttle in Ehrlich ascites tumour cells

The capability of ascites tumour mitochondria to oxidize externally formed NADH has been investigated in intact cells. Lactate has been used as the source of reducing equivalents and the oxidation of this substrate to pyruvate has been estimated. Ascites cells, under conditions of endogenous metabolism, are able to produce pyruvate upon addition of lactate. This effect is prevented by aminooxyacetate, an inhibitor of glutamate—oxalacetate transaminase (EC 2.6.1.1). Half-maximal inhibition by aminooxyacetate is attained at a concentration of approx. 30 μM. Oxidation of lactate is also sensitive to inhibitors of mitochondrial electron and energy transfer and it is enhanced by -oxoglutarate plus aspartate. These data demonstrate that reducing equivalents can be transported across the mitochondrial membrane of intact Ehrlich ascites tumour cells by the malate—aspartate shuttle.     

Induced transcription-dependent synthesis of mitochondrial reduced nicotinamide-adenine dinucleotide dehydrogenase in Drosophila.

The 23S rRNA of Agrobacterium tumefaciens contains at least two nicks which result in the formation of RNA components with mol.wts. of 0.52 X 10(6) and 0.48 X 10(6). Thus under the usual conditions of extraction and analysis, no 23S rRNA was recovered from the bacterium. The experiments show that 23S rRNA is synthesized as a continuous chain, in which one or two nicks are formed almost immediately near the ends of the molecule and an additional nick in the middle at a later time.     

Some rate constants for the phenazine methsulphate-catalysed oxidation of reduced nicotinamide-adenine dinucleotide

Kinetic measurements have been made on the phenazine methosulphate-catalysed oxidation of NADH(2) and the effects of cyanide, EDTA, Fe(2+), catalase and an unidentified mitochondrial factor on this reaction are examined. Chemical equations are formulated and values of the rate constants are determined. The data suggest the existence of an unreactive form of the reduced phenazine methosulphate; the stability and possible nature of such a compound are discussed.     

Embryogenesis in suspension cultures of Datura innoxia Mill.

In vitro plant regeneration via embryogenesis was obtained in suspension cultures of Datura innoxia Mill. Embryogenesis was induced in suspension cultures raised from callus of androgenetic origin, using LS liquid medium supplemented with 0.22 mg/l 2,4-D. The total number of embryos formed was variable over time in culture. Embryos differentiated and matured in the liquid medium itself as also evidenced by histological observation. Embryos germinated to form plantlets on semisolid MS medium without growth substances. The regenerated plants had haploid number of chromosomes.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - Kn Kinetin - LS Linsmaier and Skoog (1965) - MS Murashige and Skoog (1962)