Saburo Uchiyama

Intraspecies and interspecies variations in the substrate specificity of D-amino acid oxidase.

1. Substrate specificity of D-amino acid oxidase was examined in the kidney homogenates of the mouse and the six species of fishes. 2. The enzyme of the mouse did not show a significant intraspecies variation in the substrate specificity. The sex and age of the mouse did not affect the substrate specificity of the enzyme. 3. The degree of similarity in the substrate specificity of the enzymes was quantified as the variation index. The values of the variation index among the enzymes of the fishes paralleled their taxonomic relatedness.

Localization of fluorescent substances in the cellular slime moldDictyostelium discoideum cells during growth and development

The localization of fluorescent substance was observed microscopically in livingDictyostelium discoideum cells. The fluorescence was localized in the vacuoles of the vegetative cells. The fluorescent vacuoles were not observed in the dead cells. The fluorescent vacuoles in the cytoplasm were lost in starved cells which are able to form an aggregate and to differentiate. The fluorescent vacuoles were not lost but decreased slightly in the cytoplasm of full grown cells and of cells grown in liquid nutrient medium for an extended period of time (stationary phase cells). On a solid substratum, fluorescent vacuoles were also lost from the cells, where the vegetative cells aggregate and form a slug-shaped mass of cells. The whole slug showed homogeneous fluorescence. In a finally constructed fruiting body, the spore mass showed fluorescence. In a spore mass, the fluorescence was not observed in the spores but in the interspore space of the spore mass. It is suggested that vegetative cells secrete fluorescent substance into the inter-cellular space in the mass of cells during development.

Determination of the molecular weights of ribonuclease isozymes in a cell-free crude extract of Dictyostelium discoideum, by activity-staining of gels after SDS-PAGE

1. In a previous report we described three isozymes of intracellular ribonuclease in Dictyostelium discoideum, which were found in vegetative cells. Here we report that the molecular weights of the three isozymes from vegetative cells. 2. They are 14.3 kDa, 60 kDa and 80 kDa, as determined by activity-staining of gels after SDS-PAGE. 3. For renaturation of ribonucleolytic activity from D. discoideum cells after SDS-PAGE, fibrinogen-containing gels were used and gels were washed in aqueous isopropanol to remove detergent. Results of studies by this method suggest that each of these isozymes is composed of only a single polypeptide. 4. The effect of the buffer system on this technique is discussed.

An assay for ribonuclease activity, based on ultraviolet absorption of RNA hydrolysate, using phosphotungstic acid.

In the method for the determination of ribonuclease activity that depends on the ultraviolet absorption of the RNA hydrolysate, the uranium reagent (25% perchloric acid solution containing 0.75% uranyl acetate) is commonly used for the efficient precipitation of the unhydrolyzed RNA. However, this reagent is always contaminated by the presence of radioactive isotopes. Radioactive uranium is one of the substances used for atomic nuclear fuel and therefore, at least in Japan, the use of uranium compounds requires permission from the government. We tried to find another efficient and non-radioactive precipitant of RNA to replace the uranium reagent, and have developed a phosphotungsten reagent (25% perchloric acid solution containing 0.75% phosphotungstic acid plus 0.6% bovine serum albumin solution) which functions as efficiently as the uranium reagent in the precipitation of RNA. A cell-free crude extract of Dictyostelium discoideum was used as the source of ribonuclease.

Lumazine-like Fluorescence in a Mass of Spores of the Cellular Slime Mold, Dictyostelium discoideum

Using a fluorospectrophotometer, we examined the fluorescence of a crude preparation from the spore masses ofDictyostelium discoideum. Fluorescence emission spectra and excitation spectra suggested that the fluorescence of the crude preparation was a lumazine-like fluorescence rather than a pterin-like fluorescence. By using a microspectrophotometer, we observedin situ the fluorescence emission of a lumazine-like substance localized only in the spore mass of the fruiting body.

INTRACELLULAR RIBONUCLEASE ISOZYME ACTIVITY OF THE CELLULAR SLIME MOLD CELLS, DICTYOSTELIUM DISCOIDEUM AX2 AND DICTYOSTELIUM MUCOROIDES

The isozymes of ribonuclease were analyzed in cell‐free, crude extracts of cells by reversed activity staining of polyacrylamide gradient gels after electrophoresis. We previously reported three isozymes of the intracellular ribonuclease in Dictyostelium discoideum NC4, which were found in vegetative cells. In the present study, we examined the ribonucleolytic activity of an axenic strain of D. discoideum Ax2 cells which can grow with or without bacteria. The mobility of these isozymes (Dd I, II and III) was the same regardless of the difference in the feeding condition. We also found the three isozymes of D. mucoroides cells, Dm I, II and III. Dm I was different from Dd I in D. discoideum cells, while Dm II and Dm III were the same as Dd II and Dd III in terms of the mobility in polyacrylamide gradient gels during electrophoresis.