Method for Determination and Productivity of Sclerothionine in Sclerotinia Fungi

For determining sclerothionine (STH, S-2-hydroxyethylergothioneine), the method of P. C. Jocelyn which determined ergothioneine in blood was applied, and it was shown that STH was necessary to be degraded with 70% KOH for 1 hr at 100°C, in order to get a theoretical amount of trimethylamine. Trimethylamine produced was trapped by picric acid and spectrophotometrically measured as picrate at 410 mμ. By this method and using paper chromatography, STH in Sclerotinia culture could be determined successfully, and it was found that, among Sclerotinia fungi, a strain of Sclerotinia libertiana which can form sclerotium normally only produced STH, and other various strains of the same genus produced ergothioneine. The cultural condition for production of STH by the Sclerotinia libertiana strain was investigated. As a result, in the shaking liquid culture containing wheat bran, 1.0%; glucose, 1.0%; Polypepton, 0.6%; KH₂PO₄, 0.05%; MgCl₂, 0.05% and cystine-HCl, 0.003% as nutrient, the addition of methionine at a later period in about 0.01% concentration was found to stimulate the accumulation of STH in mycelium.     

Role of Acetyl CoA: Deacetylcephalosporin C Acetyltransferase in Cephalosporin C Biosynthesis by Cephalosporium acremonium

Existence of an acetyltransferase, which catalizes acetylation of deacetylcephalosporin C to cephalosporin C, was demonstrated for the first time in cell-free extracts of Cephalosporium acremonium. The pH optimum of the enzyme appeared to be 7.0 to 7.5 and the enzyme required essentially Mg²⁺ as a cofactor for its reaction. The activity of this enzyme was not observed in the cell-free extracts of deacetylcephalosporin C-producing mutants Nos. 20, 29, 36 and 40, but was recovered in a revertant obtained from the mutant No. 40. These results indicate that deacetylcephalosporin C accumulation by these mutants was due to the lack of the acetyltransferase and made it reasonable that the terminal reaction of cephalosporin C biosynthesis in Cephalosporium acremonium proceeded by the catalytic action of acetyltransferase.     

Ionophorous Properties of SY-1 (20-Deoxysalinomycin) in Rat Liver Mitochondri

SY-1 (20-deoxysalinomycin), a monocarboxylic polyether antibiotic closely related to salinomycin, caused a rapid release of previously accumulated alkali metal cations by valinomycin or monazomycin in rat liver mitochondria, and simultaneously reversed swelling of mitochondria.

With a strict specificity for substrate and cation, SY-1 exhibited a property of inhibiting mitochondrial functions such as substrate oxidation, oxidative phosphorylation and ATP hydrolysis induced by valinomycin or monazomycin, In comparative study with salinomycin, SY-1 was found to be more effective on the mitochondrial functions than salinomycin.

On the basis of the results so far obtained, the inhibitory effect of SY-1 on mitochondria is interpreted as a result of interaction with essential cations, especially with K⁺, in mitochondria.     

Purification and Properties of Three Forms of α-Glucosidase from Germinated Green Gram (Phaseolus vidissimus Ten.)

Three forms of α-glucosidase have been isolated from 5-day-old green gram (Phaseolus vidissimus Ten.) seedlings, by a procedure including fractionation with ammonium sulfate and polyethylene glycol 6000, DEAE-cellulose column chromatography, SP-Sephadex column chromatography, preparative gel electrofocusing and preparative disc gel electrophoresis. The α-glucosidases isolated were designated as α-glucosidase I, α-glucosidase II–1 and α-glucosidase II–2. They were homogeneous on polyacrylamide disc gel electrophoresis. Their molecular weights were 145,000, 105,000 and 65,000, respectively. The three enzymes hydrolyzed maltose, maltotriose, phenyl α-maltoside, amylose and soluble starch liberating glucose, but did not act on sucrose. Their enzymes hydrolyzed phenyl α-maltoside into glucose and phenyl α-glucoside. They hydrolyzed amylose liberating α-glucose. Maltotriose was the main α-glucosyltransfer product formed from maltose by the three α-glucosidases.     

Molecular Weight Determination of Component Polypeptides of Glutenin after Fractionation by Gel Filtration

Reduced and cyanoethylated glutenin was fractionated into three fractions (F I, F II and F III) by gel filtration on Sephadex G–100 in 0.1 m acetic acid. The molecular weight determination was made with these three fractions by sedimentation equilibrium in 6.5 m guanidine hydrochloride containing 0.01 m acetic acid. The molecular weight obtained was 44,000 for F II, and 32,000 for F III. F I showed a distribution of molecular weight due to the aggregation. The average molecular weight of F I was 52,000, being 27,000 at the meniscus and 98,000 at the bottom. The estimation of molecular weight by SDS–PAGE* gave overestimated values for glutenin polypeptides, as was already reported for gliadin.     

Structural Studies on “Isosclerotan”, a New Glucan Isolated from Sclerotinia Fungus,by Physical,Chemical and Enzymatic Methods

“Isosclerotan”, a polysaccharide constituent extracted with a sodium hydroxide solution from sclerotia of Sclerotinia libertiana, could be purified by the successive precipitation with the followings; a mixture of copper sulfate and sodium hydroxide, ammonium sulfate, and ethyl alcohol. The preparation proved homogeneous by ultracentrifugal analysis. From sedimentation and viscosity measurements, the molecular weight of isosclerotan was calculated as 6.13 × 10⁶, andas 1.60 × 10⁵ after treatment with a dilute oxalic acid solution. Isosclerotan was scarecely soluble in cold water but soluble in hot water, yielding a highly viscous solution. It exhibited a low positive optical rotation, + 23.0° (in water), and infrared spectrum had a sharp absorption at 890~898 cm^?1, which indicated the prevalence of the β-glycosidic linkage in isosclerotan. Through degradation by acids and enzymes of isosclerotan, there were obtained various oligosaccharides containing β-1.3, β-1.4, and β-1.6 linkages. From results obtained by periodate oxidation and methylation, it is assumed that the polysaccharide involves the 1.3, 1.4, and 1.6 linkages in 47.7%, 16.6% and 35.7%, respectively, and a branching structure about 12.5%.     

Evaluation of Polyethylene Glycol as a Water-soluble Marker: Absorption and Excretion of 14C-labeled Polyethylene Glycol in Rats

The absorbability of polyethylene glycol (PEG), a water-soluble nutritional marker, from the gastrointestinal tract of rat was examined using the [¹⁴C]-labeled compound ([¹⁴C]PEG) having a molecular weight of 4000. Intravenously injected [¹⁴C]PEG was readily excreted and recovered almost completely in the urine and neither hepatic nor renal uptake of the PEG was observed. Intragastrically administered [¹⁴C]PEG was eliminated in the urine with an average recovery of only 0.43 ± 0.13% (Mean ± S.D., n= 10) of the dose over 24 hr. From the gel column chromatographic profile of the radioactivity excreted in the urine after an oral dose, [¹⁴C]PEG was suggested to be absorbed in two forms, as an original form and as a low molecular weight component. The latter component might be the degraded product of PEG in the gastrointestinal tract. From these results it was confirmed that PEG with a molecular weight of 4000 is a satisfactory marker because of its low absorbability.     

Two Forms of Glucoamylase from Mucor rouxianus 1. Purification and Crystallization

Mucor rouxianus produced two forms (isoenzymes) of glucoamylase which could be separated from each other by polyacrylamide gel electrophoresis or by chromatography on SP-Sephadex C-50, and they were designated glucoamylase I and glucoamylase II. Glucoamylases I and II were isolated in crystalline form, and were homogeneous in poly acrylamide gel electrophoresis and in ultracentrifugation, respectively. The sedimentation coefficient () molecular weight of glucoamylase I were 4.39 S and 59,000, and those of glucoamylase II were 4.29 S and 49,000, respectively.     

Purification and Characterization,of Rice Bran Lipase II

A species of rice bran lipase (lipase II) was purified by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-cellulose, Sephadex G–75 and CH-Sephadex C–50. Both polyacrylamide disc electrophoresis and ultracentrifugation demonstrated that the enzyme protein is homogeneous. The isoelectric point of the enzyme was 9.10 by ampholine electrophoresis. The sedimentation coefficient of the enzyme was evaluated to be 2.60 S, and the molecular weight to be 33,300 according to Archbald’s method. The enzyme showed the optimum pH between 7.5 and 8.0, and the optimum temperature at about 27°C. It was stable over the pH range from 5 to 9.5 and below 30°C. In substrate specificity, the enzyme exhibited a high specificity toward triglycerides having short-carbon chain fatty acids, although it was capable of hydrolyzing the ester bonds in the rice and olive oil.