Metabolism of Glutamate in Purple Nonsulfur Bacteria Participation of Vitamin B12

Purple nonsulfur bacteria, Rhodospirillum rubrum and Rhodopseudomonas spheroides were found to possess coenzyme B₁₂-dependent glutamate mutase activity. Cell-free extracts of these bacteria grown on Co²⁺-containing media catalyzed the conversion of glutamate to β-methylaspartate and further to mesaconate. The activity of the cell-free extracts of these organisms cultivated on Co²⁺-deficient media was markedly lower than that of the normal cells. Addition of coenzyme B₁₂ to the former reaction mixture enhanced the mesaconate formation via β-methylaspartate. These results indicate the involvement of coenzyme Independent glutamate mutase of these bacteria in the dissimilation of glutamate to acetyl-CoA and pyruvate through the following pathway.

glutamate→β→methylaspartate→mesaconate→citramalate→→acetyl-CoA, pyruvate On the other hand, a greater part of glutamate was converted to α-hydroxyglutarate and succinate with the cell-free extracts of these photosynthetic bacteria. This fact, taking account of the presence of propionyl-CoA carboxylase in these bacteria, implies the participation of coenzyme B₁₂-dependent (R)-methylmalonyl-CoA mutase in the formation of succinate via the following route.

glutamate→α-ketoglutarate→α-hydroxyglutarate→propionate→propionyl-CoA→(S)-methylmalonyl-CoA→(R)-methylmalonyl-CoA→succinyl-CoA     

Changes of Linoleic,Arachidonic and Eicosapentaenoic Acids in Rat Platelet,Aorta and Plasma Lipids after Changing from a Sardine Oil Diet to a Corn Oil Diet

When rats were fed on a diet containing sardine oil (SO) for 10 days, 20:5 (eicosapentaenoic acid) was incorporated into the platelet, aorta and plasma lipids. The 20:5/20:4 (arachidonic acid) ratios of phospholipids were higher in platelets than in aortas. When the diet was changed from SO to corn oil (CO), the levels of 20:5 in the plasma lipids and platelet phospholipids decreased with an increase of 20:4. Although the 20:5 levels of phospholipids in the aortas decreased, 20:4 did not increase during feeding with the CO diet for 4 days. The 20:5/20:4 ratios of the phosphatidylcholine in platelets and aortas rapidly changed from 5.0 and 1.1 to 2.4 and 0.6, respectively. The linoleic acid level of phosphatidylcholine in platelets and aortas increased rapidly by changing from the SO diet to the CO diet, but this fatty acid was not incorporated into phosphatidylinositol of the platelets and aortas.     

Study of Microflora in Malaysian Dried Fishes and Their Decontamination by Gamma-irradiation

The distribution of microorganisms in 10 samples of salted dried fish and the effects of irradiation of them were studied. The total aerobic bacteria in commercial dried fish were determined to be from 2 × 10⁴ to 3 × 10⁶ per gram. Mold counts were 1 × 10² to 7 × 10³ per gram with a lower amount of yeasts. In spoiled dried fish, total aerobic bacteria were determined to be 4× 10⁶ or 1 × 10⁷ per gram with a few yeasts. Coliforms were not isolated on MacConkey agar plates from any of the samples. The predominant bacteria occurring in spoiled dried fish were Pediococcus halophilus, Vibrio costicola and Planococcus sp. More than 50% of the molds consisted of the Aspergillus niger group, whereas lower amounts of the A. flavus, A. fumigatus and A. ochraceus groups, Penicillium chrysogenum series, etc. were also isolated from many samples of dried fish. All kinds of putrefactive microorganisms were radiation sensitive, and a dose of ca. 500 krad appears to be sufficient for extension of the shelf-life of dried fish from 2 to 4 times.     

Fungal Proteolytic Enzymes

Two kinds of proteolytic enzyme, tentatively named acid protease A and B which showed a single peak on electrophoresis individually, were isolated from the crude enzyme powder obtained from the broth filtrate cultured with Asper gillus niger var. macrosporus. Acid protease B is similar too the fungal acid protease previously reported, bccause the enzyme exhibits optimum activity on milk casein at about pH 2.6 and 55°C when the incubation was done at pH 2.6. Acid protease A is a new proteolytic enzyme, because the enzyme exhibits optimum activity on milk casein at about 2.0 and 70°C or 60°C when the incubation was done at pH 2.6 or 1.5 respectively.     

Properties of Crystalline 3β-Hydroxysteroid Oxidase of Brevibacterium sterolicum

3β-Hydroxysteroid oxidase (3β-hydroxysteroid: oxygen oxidoreductase, EC 1.1.3.6.) from the culture supernatant of Brevibacterium sterolicum ATCC 21387 has a molecular weight of 32,500 and an isoelectric point of 8.9. The enzyme contained 258 amino acid residues and the composition revealed a distinctive feature of a relatively high amount of proline and the absence of alanine and tryptophan. The crystalline enzyme exhibited an absorption spectrum characteristic of a flavoprotein with absorption maxima at 280, 390, and 470 nm with a shoulder at 490 nm. Anaerobic addition of dehydro-epi-androsterone as well as sodium dithionite to the enzyme produced a disappearance of the peaks at 390 and 470 nm. The flavin moiety of the enzyme was isolated and identified as flavin adenine dinucleotide, 1 mole of which was found per mole of protein. The enzyme is sulfhydryl dependent and was inactivated by silver and mercury compounds. Analysis of the enzyme protein by atomic absorption spectrophotometry failed to detect any significant quantity of heavy metals.

Various 3β-hydroxysteroids were oxidized and the relative rates of the oxidation were cholesterol, 100; dehydro-epi-androsterone, 41; pregnenolone, 22; and β-sitosterol, 20. The oxidation product of cholesterol by the enzyme was crystallized and identified as 4-cholesten-3-one by melting point, elementary analysis, optical rotation, UV, IR and NMR spectra. The oxidation of cholesterol proceeded as follows:

The enzyme would be used for some analytical and preparative purposes in the field of steroid chemistry, e.g., microdetermination of cholesterol in serum.     

Isolation and Crystallization of Extracellular 3β-Hydroxysteroid Oxidase of Brevibacterium sterolicum nov. sp.

Among about 500 strains tested, a newly isolated soil bacterium, Brevibacterium sterolicum nov. sp. KY 3463 (ATCC 21387) showed the highest potency in production of 3β-hydroxysteroid oxidase in the culture fluid.

The 3β-hydroxysteroid oxidase was purified from the culture filtrate by a procedure involving ammonium sulfate fractionation, DEAE-cellulose and hydroxyapatite column chromatographies and Sephadex G–75 gel filtration. Crystals of the enzyme were obtained from solutions of the purified preparation by the addition of ammonium sulfate. The crystals appeared as fine rods, with a bright yellow color.

The enzyme is homogeneous by disc gel electrophoresis and ultracentrifugation. Sedimentation velocity yields a value of . It exhibits a typical flavoprotein spectrum of absorption maxima at 280, 390, and 470 mμ.     

Purification,Crystallization and Properties of Tyrosine Phenol Lyase from Erwinia herbicola

Crystalline tyrosine phenol lyase was prepared from the cell extract of Erwinia herbicola grown in a medium supplemented with l-tyrosine. The crystalline enzyme was homogeneous by the criteria of ultracentrifugation and acrylamide gel electrophoresis. The molecular weight was determined to be approximately 259,000. The crystalline enzyme catalyzed the conversion of l-tyrosine into phenol, pyruvate and ammonia, in the presence of added pyridoxal phosphate. The enzyme also catalyzed pyruvate formation from d-tyrosine, S-methyl-l-cysteine, 3, 4-dihydroxyphenyl-l-alanine, l- and d-serine, and l- and d-cysteine, but at lower rates than from l-tyrosine. l-Phenyl-alanine, l-alanine, phenol and pyrocatechol inhibited pyruvate formation from l-tyrosine.

Crystalline tyrosine phenol lyase from Erwinia herbicola is inactive in the absence of added pyridoxal phosphate. Binding of pyridoxal phosphate to the apoenzyme is accompanied by pronounced increase in absorbance at 340 and 425 mμ. The amount of pyridoxal phosphate bound to the apoenzyme was determined by equilibrium dialysis to be 2 moles per mole of enzyme. Addition of the substrate, l-tyrosine, or the competitive inhibitors, l-alanine and l-phenyl-alanine, to the holoenzyme causes appearance of a new absorption peak near 500 mμ which disappears as the substrate is decomposed but remains unchanged in the presence of the inhibitor.     

Impaired Neural Differentiation of Induced Pluripotent Stem Cells Generated from a Mouse Model of Sandhoff Disease

Sandhoff disease (SD) is a glycosphingolipid storage disease that arises from mutations in the Hexb gene and the resultant deficiency in β-hexosaminidase activity. This deficiency results in aberrant lysosomal accumulation of the ganglioside GM2 and related glycolipids, and progressive deterioration of the central nervous system. Dysfunctional glycolipid storage causes severe neurodegeneration through a poorly understood pathogenic mechanism. Induced pluripotent stem cell (iPSC) technology offers new opportunities for both elucidation of the pathogenesis of diseases and the development of stem cell-based therapies. Here, we report the generation of disease-specific iPSCs from a mouse model of SD. These mouse model-derived iPSCs (SD-iPSCs) exhibited pluripotent stem cell properties and significant accumulation of GM2 ganglioside. In lineage-directed differentiation studies using the stromal cell-derived inducing activity method, SD-iPSCs showed an impaired ability to differentiate into early stage neural precursors. Moreover, fewer neurons differentiated from neural precursors in SD-iPSCs than in the case of the wild type. Recovery of the Hexb gene in SD-iPSCs improved this impairment of neuronal differentiation. These results provide new insights as to understanding the complex pathogenic mechanisms of SD.