Studies on Phyto-peroxidase

Japanese-radish peroxidase c, a paraperoxidase exhibiting the optical absorption spectrum of low-spin nature, was found to transform to a high-spin state by removing a dissociable ligand of low molecular weight by the addition of the stoichiometric amount of p-chloro mercuribenzoate, as in the case of horseradish peroxidase I or wheat germ peroxidase 566. The reaction could be reversed by the addition of cysteine to remove p-chloromercuribenzoate. As this ligand would be possibly cyanide, the affinity of the high-spin form of the enzyme to sodium cyanide was determined, which was found to be much higher than that of Japanese-radish peroxidase a. The high-spin form of peroxidase c formed the usual Compound I by the addition of hydrogen peroxide, so that the peroxidatic reaction catalyzed by this enzyme should follow the common mechanism of plant peroxidases. However, Compound II was scarecely observed during the course of the stepwise reduction of Compound I by ascorbate, probably because of its more rapid conversion to the free enzyme.     

Studies on Phyto-peroxidase

Both crystalline Japanese-radish peroxidase a and c could catalyze the aerobic destruction of indole-3-acetic acid in the presence of a small amount of hydrogen peroxide as an inducer. The pH dependences of the enzymic activities of peroxidase a and c were different from each other. The optimum pH of peroxidase a was found at 3.6, while that of peroxidase c lied in a broad range over 3 to 4.8. In lower concentrations of both peroxidases at pH 3.8 and 6.0, the major product of the reaction was identical with that presented by Ray with using an Omphalia enzyme, which would probably be methyldioxindole, while, in higher concentrations of both peroxidases at pH 3.8, the major product was found to be indole-aldehyde.     

Studies on Phyto-peroxidase

Peroxidase c was isolated and purified from Japanese-radish roots by means of a chromatographic technique with carboxymethyl cellulose. Two or more components exhibiting the absorption spectrum of peroxidase c were separated chromatographically, and the most basic component was crystallized from ammonium sulphate solution. The Reinheit Zahl and the purpurogallin number of the crystalline preparation were found to be 3.55 and 1100 respectively. The absorption maxima were found at 420 and 540 mμ, for the oxidized form and at 425 and 560 mμ for the reduced form. The crystalline preparation contained 1.57% protohematin as the prosthetic group, and then the minimum molecular weight of peroxidase c was found to be 41500.     

Studies on ATP

The experiments described in this paper serve as a contribution to the solution of the discrepancies which exist in the assay of ATP:thiamine diphosphate phosphotransferase activity (EC 2.7.4.15), presently in use as a tool for the diagnosis of Leigh's disease (SNE, subacute necrotizing encephalomyelopathy). The results obtained with this phosphotransferase assay can, in part, be explained by the presence of thiamine triphosphate (ThTP) in the preparation of thiamine diphosphate (ThDP) used as a substrate, by the inhibition by ATP of the ThTP phosphohydrolase activity, present in fractions of rat brain homogenates, and by the stimulation by ThDP of the ATPase activity. When [2-¹⁴C-thiazole]thiamine was used for the synthesis of [¹⁴C]ThTP in fractions of rat brain, it was found that after chromatographic separation of thiamine and its phosphates,¹⁴C radio-activity could be demonstrated in the ThTP fractions, even in the absence of an enzyme source. Probably a complex is formed between [¹⁴C]thiamine and a phosphate ester which behaves chromatographically as ThTP. It is concluded that the assay system for the measurement of ThTP synthesis in its present form is, in our hands, not suitable for diagnostic purposes.     

Studies on Streptococci

The population levels of bacteria in the contents and the walls of the gastrointestinal tract of gnotobiotic rats inoculated with lactic acid bacteria (streptococci, lactobacilli, and bifidobacteria) from humans and rats were determined. Lactobacilli as well as streptococci isolated from rats colonized in the digestive tracts of the gnotobiotic rats at a high population level, characteristically highest in the stomach. On the other hand, in the rats inoculated with human lactic acid bacteria, streptococci were dominant in the lower tract. The human lactobacilli or bifidobacteria did not colonize when the organisms in each genus were inoculated together with streptococci. However, when all three genera were inoculated together, lactobacilli and bifidobacteria colonized. Observations on the species of streptococci showed that the intestinal type of streptococci was found to colonize at a high population level, but the oral type was not. Strains of the same genus of lactic acid bacteria from humans and from rats showed different colonization patterns.     

Studies on Streptomycetes

Investigation was made on the mycological properties of a strain No. 45449 isolated from a sample of soil collected in Fukuchiyama. Since the antibiotics produced by the strain resembled hydroxymycin and paromomycin, the strain was compared with the hydroxymycin and paromomycin-producing strains, S. paucisporogenes and S. rimosus forma paromomycinus, and as a result the strain No. 45449 was found to be different from the latter two strains. Among known strains, S. flavogriseus resembles the present strain, but they are different morphologically and in the kind of the antibiotics they produce. Thus, as the strain No. 45449 was found to be a new strain, it was named S. pulveraceus nov. sp. The antibiotics produced by the present strain are physiologically basic substances active against Gram-positive and negative bacteria and acid fast bacteria, and they are considered to belong to the neomycin-kanamycin group.     

Studies on Cereals

Five carbonyl compounds were found in the vapour of cooked rice, of which acetaldehyde, and n-caproaldehyde were identified from their retention times in gas chromatography and by their infrared spectra. Propionaldehyde or acetone, methylethylketone, and n-valeraldehyde were tentatively identified. The mechanisms of the formation of these carbonyl compounds were discussed from the view-point of Strecker degradation and lipid oxidation.

When stored rice (40°C, two months) was cooked, the stale flavor (komai-shu) was clearly detected by sensory test. Direct gas chromatographic analysis of head space vapors over cooked rice showed three main peaks which corresponded to propionaldehyde or acetone, n-valeraldehyde and n-caproaldehyde. On the other hand, the content of linoleic and linolenic acids of the rice decreased during storage at 40°C. This means that the unsaturated fatty acids autoxidized during storage and gave rise to carbonyl compounds responsible for the stale flavor of cooked rice.     

Studies on Pyrethrosin

Pyrethazulene, an azulene produced by zinc-dust distillation of pyrethrosin, is not 2,4,8-trimethylazulene but a mixture of 1,4-dimethylazulene (major component) and other azulenes (minor components). 1,4-Dimethyl-6-ethylnaphthalene was obtained on Se-dehydrogenation of pyrethrosin.