Sayoko Mihara

Metabolic roles of inorganic polyphosphates in chlorella cells

1. 1. A new type of inorganic polyphosphate (poly-Pi) was detected in Chlorella cells and named Poly-Pi “D”. It is extractable with 2 N KOH at 37° together with Poly-Pi “C” which has been reported earlier. Upon neutralization of the extract with perchloric acid, Poly-Pi “C” is co-precipitated with perchloric acid while Poly-Pi “D” remains dissolved. 2. 2. Using uniformly 32P-labeled Chlorella cells which are subjected to various environmental conditions, the metabolic role of Poly-P1 “D”, as well as those referred to earlier as Poly-Pis “A”, “B” and “C” were investigated. 3. 3. On incubating the 32P-labeled algal cells in a phosphate-free medium under photosynthetic conditions, ribonucleic acid increased and Poly-Pi “D” decreased. When the 32P-labeled algal cells were subcultured in the normal “cold” medium under photosynthetic conditions, a large amount of external phosphate but only a small amount of endogenous 32P was incorporated into the ribonucleic acid fraction. In the meantime, the 32P in Poly-Pis “C” and “A” decreased considerably while that in Poly-Pis “B” and “D” decreased slightly or remained constant. It has been reported that deoxyribonucleic acid and phosphoprotein increased with contamitant decrease of Poly-P1s “A”, “B” and “C” in a phosphate-free medium under photosynthetic condition, and that 32P in the fractions of deoxyribonucleic acid and protein continued to increase when the 32P-labeled algal cells were subcultured in the normal “cold” medium1. It was therefore, inferred that, under photosynthetic conditions, Poly-Pis “C” and “A” serve as intermediates in the transfer of phosphate to the phosphorus compounds synthesized such as deoxyribonucleic acid and phosphoprotein, while Poly-Pis “B” and “D” function as reservoirs of phosphate which are utilized only in the absence of exogenous phosphate source. 4. 4. In the dark the 32P in Poly-Pi “A” continued to increase slowly both in the phosphate-free medium and in the normal cold medium, while no significant change was observed in the levels of 32P or total phosphate of the other phosphorus compounds. This suggests that Poly-Pi “A” accepts phosphate—independently of light— from some intracellular phosphate source.

The role of sulfur in the cell division of chlorella

Summary1.The role of sulfur in the process of cell division of Chlorella was studied using the technique of synchronous culture. When the “dark cells” (smaller and strongly photosynthesizing cells), which had been grown in a normal nutrient medium, were further cultured in an S-deficient medium under photosynthesizing conditions, the cells grew up to some extent, showing about two-fold increase of DNA-content followed by the division of nucleus into two. At this stage, however, the cells fell into a stalemate, being unable to complete cellular division.2.When such cells were transferred to a medium containing potassium sulfate only, some synthesis of DNA and further division of nuclei occurred. Under non-photosynthesizing conditions, the process of cellular division ensued, giving rise to a formation of small daughter cells. Under photosynthesizing conditions, on the other hand, the cells increased appreciably in size, without, however, being able to perform cellular division. The process of cellular division thus halted could be resumed when the cells were further supplied with nitrate and sulfate under photosynthesizing condition.3.Based on these observations, it was concluded that sulfur plays, in cooperation with some nitrogenous substance(s), an essential role in the process of DNA-formation and nuclear division as well as in the process of cellular division.

Role of sulfur in the cell division of Chlorella, studied by the technique of synchronous culture

1. 1. Chlorella was grown synchronously under controlled supply of S- and N-sources so as to make the processes of nuclear and cellular division proceed stepwise, and by using 35S as a tracer the fates of S-compounds in various fractions of cell material (extracts with 70% ethanol, with 10% trichloroacetic acid, and the residue) were followed. 2. 2. In the cells grown in the normal medium, the S-content (in %) of the ethanol- and TCA-extracts decreased in the growing stage and increased markedly at the ripening stage. 3. 3. The ethanol extract contained an unknown S-compound and a non-S-containing peptide-like substance, the latter substance appearing only at certain stages of cell ripening. When chromatographed on paper the TCA extract gave only 1 radioactive (but ninhydrin-negative) spot, which, on hydrolysis with HCl, turned ninhydrin-positive owing to the liberation of several amino acids including cyst(e)ine. 4. 4. In the cells, whose nuclear and cellular division were controlled by limited supply of S- and N-sources, the quantity of the S-containing substance in the TCA-extract increased markedly before nuclear division, and the unknown non-S-containing substance in the ethanol extract appeared only before and concomitantly with, the occurrence of cellular division.

Sulfur-containing peptide-nucleotide complex isolated from Chlorella and yeast cells

Abstract 1. 1. From the cells of a green alga (Chlorella) and yeasts (Saccharomyces formosensis and S. cerevisiae), a new sulfur-containing peptide-nucleotide complex was obtained by extracting the cells with cold trichloroacetic acid (10%). The complex was revealed to contain cyst(e)ine as the major sulfur component in the peptide moiety, and adenine and uracil as the major bases in the nucleotide moiety. 2. 2. In the field of zone electrophoresis, the complex as a whole moved, unlike free nucleotides and polynucleotides, toward the cathode at pH 3.6 in the case of Chlorella and at pH 3.6–5.0 in the case of yeast. At pH 5.0, the complex obtained from Chlorella split into a number of components, the majority of which moved toward the anode. The unsplit complex obtained at pH 3.6 gave, on hydrolysis followed by paper chromatography, several unidentified components, besides adenine, uracil, and a number of amino acids. Some of these unidentified components gave a red color with ninhydrin reagent, and they were assumed to be the factors making the complex assume a positive charge as a whole. 3. 3. The complex obtained at pH 3.6 from Chlorella was also split into a number of subunits when it was subjected to an anion-exchange chromatography (Dowex 1). The complex has, thus, a property of being readily disintegrated by changes of pH or other conditions into its components of different degrees of complexity.