Sakuzo Fukui

Structure of rhodotorucine A, a novel lipopeptide, inducing mating tube formation in Rhodosporidiumtoruloides

Abstract Rhodotorucine A is a peptidyl factor which induces mating tube formation in Rhodosporidium toruloides . The amino acid sequence of the factor was determined by Edman degradation and enzymatic hydrolysis. Rhodotorucine A was shown to contain a lipophilic amino acid, S-farnesyl cysteine, at C-terminus by proton magnetic resonance, mass spectrometry and chemical synthesis. We proposed the following structure for rhodotorucine A . H-Tyr-Pro-Glu-Ile-Ser-Trp-Thr-Arg-Asn-Gly-Cys(S-farnesyl)-OH

Requirements of chemical structure for hormonal activity of lipopeptidyl factors inducing sexual differentiation in vegetative cells of heterobasidiomycetous yeasts

Biological activities of both peptidyl and isoprenyl constituents of yeast sex hormones (lipopeptides), rhodotorucine A and farnesyl tremerogen A -10, were examined. The chemically synthesized simple peptides (peptidyl constituents) and farnesyl cystein (isoprenyl one) did not exhibit any biological activities, such as hormonal and hormone-inhibitory (or -accelerative) activities. The mixture of both constituents also had no activity. Then, it is assumed that the heterobasidiomycetous yeasts ( Rhodosporidium toruloides and Tremella mesenterica ) recognize the total structure of lipopeptidyl molecule for the biological response, sexual differentiation, and the term “Pattern Recognition” is proposed for the mode of this recognition.

Isolation, solubilization and reaggregation of outer membrane of Escherichia coli

Abstract Cytoplasmic (inner) and outer membranes of Escherichia coli K-12 were isolated with fair separation from each other, and their chemical, biological and morphological properties were compared. The outer membrane isolated was composed of protein, phospholipid and lipopolysaccharide as major high molecular weight components in a ratio of 100:82:34 (by wt), and was solubilized in 1% sodium dodecyl sulfate without any sediments. In polyacrylamide disc gel electrophorsis with the sodium dodecyl sulfate-solubilized outer membrane, six proteins were found to be major. Removal of sodium dodecyl sulfate from the sodium dodecyl sulfate-solubilized outer membrane by dialysis induced a self-assembly to form a membrane structure which has similar properties in chemical composition, density and morphology to those of the original outer membrane.

Binding of rhodotorucine A, a lipopeptidyl mating hormone, to a cells of Rhodosporidium toruloides for induction of sexual differentiation.

Rhodotorucine A , a lipopeptidyl mating hormone, selectively bound to the cells of Rhodosporidium toruloides M-1057, mating type a , having a large number of hormone-binding site, approximately 5 × 10 7 molecules per cell. The binding is temperature-independent. The bound hormone is sensitive to pronase, while the binding site is resistant to the protease. The cells, which were preincubated with the hormone at 25°C for 10 min (10-min hormone-preincubation), completely lost the mating tube-forming ability by pronase treatment, while the cells of 15-min hormone-preincubation remained the ability after the treatment. Thus, it is concluded that the establishment of intracellular information inducing sexual differentiation depends on the term of preincubation, such as 15 min, with the hormone at 25°C.

Preparation of artificial vesicles having an L-alanine uptake activity which requires NADH as energy source

The vesicles (proteoliposomes) were artificially prepared by sonic oscillation from hydrophobic protein, which was isolated from cytoplasmic membrane fraction of Bacillus subtilis W-23, and phospholipids or lipids, which were isolated from different organims; phospholipids from hen-egg yolk and soybean, and lipids from B . subtilis W-23. In the vesicles, remarkable activity of L-alanine uptake was demonstrated in the presence of NADH or ascorbate-phenazine methosulfate as an energy source. The uptake activity was temperature-dependent and inhibited by respiration inhibitor (KCN) and uncoupler (ClCCP). Replacement experiment was also performed.

Crystalline DPNH Oxidase from Lactobacillus plantarum No.11

plantarum No.11 was remarkably enhanced when the organism was grown in a medium containing D-gluconate or D-ribose as a carbon source in place of glucose. During growth in such a medium, the ability of oxidative fermentation on D-ribose was also induced. From the studies on the absorption spectrum of the cells, the yellow pigment was identified as a flavin compound which could be converted into reduced form by the addition of D-ribose as a hydrogen donor2). The reduced flavin compound was readily reoxidized by aeration. Flavin in these cells was found to be present mostly as FAD in a bound form and its concentration was estimated to be

An electron microscopic study on the mating tube formation in the heterobasidiomycete Tremella mesenterica

Ultrastructure of the mating tube formed in yeast haplont of the heterobasidiomycete Tremella mesenterica was studied by electron microscopy. Cell wall of the mating tube emerged as evagination of the inner layers, rupturing outer layers of the mother cell wall. Comparison with budding cells suggested that the tube emergence place at bud scar and the process of tube emergence was the same as that of bud emergence. Electron transparent vesicles of 0.1 μm diameter were scattered in the cytoplasm of the mating tube. Nucleus-associated organelle was located at one side of the nuclear envelope which extended towards the mating tube. A few microtubules were detected in the mating tube, but their association with a nucleus was not clear. The cytoplasmic structure of the mating tube was discussed in comparison with that of hyphae of the filamentous fungi.

Application of photochemical reactions of bis-azido compounds to preparation of an enzyme-polymer film

Abstract β-Glucosidase (EC 3.2.1.21) was immobilized in fibroin film by using a photo-crosslinking agent, 4,4′-diazidostilbene-2,2′-disodium sulfonate. Crosslinking and immobilization reactions proceeded by light irradiation for 20 min in air. The immobilized enzyme showed approximately 50% of its native activity with an apparent Michaelis constant of 3.1 m m . The Michaelis constant of the native enzyme was 2.3 m m . Some properties of the immobilized and native enzymes were compared.

Purification and properties of glucose 1-phosphate binding protein in Agrobacterium tumefaciens

Abstract The presence of two kinds of glucose 1-phosphate-binding protein in sonic extract of sucrose-grown cells of Agrobacterium tumefaciens was demonstrated by DEAE-cellulose column chromatography. Both of them showed high specificity for glucose 1-phosphate ( Glc -1-P ) and were purified as homogeneous proteins in polyacrylamide disc gel electrophoresis. The properties of the two binding proteins (I and II) are as follows: ratio of A 280 nm /A 260 nm , 1.82 and 1.70; molecular weight, 35 000 and 42 000; K D for Glc -1-P , 8 · 10−7 M and 1.3 · 10−6 M; substrate binding ( Glc -1-P mole/protein mole), 0.84 and 0.93; optimal pH, 8.0–8.4 (narrow range) and 5.4–8.4 (wide range). The binding protein II was exclusively released from cells by osmotic shock, and the restoration of Glc -1-P uptake activity of shocked cells occurred on addition of the binding protein II. On the other hand, by treatment with the binding protein I, the isolated cell envelope which was prepared from sonic extract of resting cells gained Glc -1-P- binding activity, while by treatment with the binding protein II the envelope gained no activity.