Satoru Akune

DNA synthesis and activities of pyrimidine-synthesizing enzymes in the silk gland of Bombyx mori

Abstract Synthesis of DNA in the silk gland of Bombyx mori was investigated by studying incorporation of DNA precursors and determining activities of related enzymes in the developmental stage from the fourth apolysis to the middle fifth instar. Uptake of 32 Pi (for 10 hr) into pyrimidine nucleotides of DNA was higher than that into purine nucleotides of DNA. 3 H-Thymidine and 14 C-orotic acid were found to be active precursors for DNA and, seemingly, the incorporation of the former was more active than the latter. Activities of thymidine kinase and thymidylate kinase exhibited their highest levels on day 4 of the instar, and aspartate transcarbamylase showed a peak on the fifth day. Administration of ecdysterone (10 or 20 μg) into a larva resulted in decreased incorporation (by 30 or 60 per cent, respectively) of 3 H-thymidine into DNA fraction from intracellular pools. Uptake of 32 Pi into the fractions of the gland fell by 50 per cent after administration of the hormone (10 μg). A study of daily changes of incorporation of 3 H-thymidine into the DNA fraction demonstrated that its maximum rate was on the fourth day, in accordance with the appearance of peaks of the above-mentioned enzymes. The implications of these observations are discussed in relation to the known features of development of the insect during the period.

Correlation of nucleolytic activities with the stage of growth of the silkworm

Abstract From the tissues of the silkworm several nucleolytic enzymes were detected. Among these, acid ribonuclease (RNase) and alkaline nuclease were dominant. The optimal pH values for these are 5·3 and 10·3, respectively. An acid deoxyribonuclease (DNase), optimally active at pH 5, was also found in the tissues of the insect. Changes of activity were investigated of the acid RNase and the alkaline nuclease through the whole life cycle of the silkworm. The former remains very active during each step of development. By contrast, the latter exists at higher levels only during the feeding period, and then drops abruptly to an almost undetectable level at the initiation of spinning. The acid DNase activity showed a peak 3 days after pupation. The acid RNase and DNase showed a wide distribution in the tissues of the silkworm, whereas the alkaline nuclease is restricted to the gut juice and to the gut.

Purification and properties of acid ribonucleases from posterior silk gland of silkworm

Abstract The posterior silk glands of domestic silkworm larvae, Bombyx mori, contain acid ribonuclease (RNase) activity. This was purified and separated into two components by fractionation with ammonium sulphate followed by chromatography on carboxymethyl (CM-) cellulose. Each of the two components was separately purified further by gel-filtration through a column of Sephadex G-100. These two components showed pH optima of activity at about 5·5. Paper chromatographic analysis of the products of digestion of RNA by the two components showed that each of them can split all of the internucleotide linkages in RNA to produce ribonucleoside 3′-phosphates via 2′,3′-cyclic phosphates as intermediates. The two components also appeared similar to each other with respect to the properties such as the effect of ions on activity, and relative affinity for sRNA and high-molecular-weight RNA. These facts contrast with the case of the tissues of mammalian species which in general have both acid and alkaline RNases and so may indicate that the RNA metabolism in the posterior silk gland of this insect is different in some way from that in ordinary mammalian tissues. However, biological significance of the coexistence of the two acid RNases of almost similar properties is not known at present.

Studies on the Metabolism of Azuki Seed Protein:Part V. Purification of Azuki Protease

In order to investigate the role of a protease in protein metabolism during the germination of azuki seeds, the purification of the portease was carried out by employing fractionation with ammonium sulfate, dialysis against distilled water, and elution chromatography on a column of DEAE-cellulose. The enzyme preparation thus obtained was homogeneous in ultracentrifugal patterns.

Nutritional and Pathological Studies of Aseptic Rearing Silkworm (Part 1)

容易に飼育できる無菌動物の一つとして家蚕の無菌飼育を試み, 脱脂米糠, 桑葉粉末などを主体とした人工飼料を調製した。脱脂米糠は希硫酸溶液或いはメタノールなどで抽出処理をおこなうことにより食下性が改善されることを知った。また希硫酸処理糠は脱脂米糠より飼料としてすぐれているものと推測された。

Mode of Action of a Silkworm Nuclease and an Analysis of Oligonucleotide

(1) Oligonucleotideの塩基順列決定法として,家蚕消化液ヌクレアーゼの使用を考え,次のモデル実験を行なった.まず,RNase-IによるRNA分解物より,penta-からheptanucleotideを調製し,各々をモデルの基質として,このヌクレアーゼを作用させた,続いて再分解物は,7M尿素DEAE-セルロースカラムにより分離し,脱塩後,アルカリ分解,ペーパークロマトグラフィーにより同定した.この結果,どのoligonucleotideの再分解物も,7M尿素DEAE-セルロースカラム(pH7.5)で,燐酸基による陰電荷順に6つのピークに分かれた.(-1)価に相当するピークはNpNであり,(-2)価のピークは少量のpNを含むが,大部分はNpNpNであり,いずれも5末端に由来するfragmentである.そして,これらを重ね合せることにより,5末端から3個までのヌクレオチド順列を確実に決めることができる.一方,(-5)価,(-6)価のピークはそれぞれ3末端から生じたpNpNpとpNpNpNpであり,やはり同様に3末端から3個までのヌクレオチドを決定することができる.また,(-3)価はpNpN,(-4)価はpNpとpNpNpNであり,このうちpNp以外は,元の基質の的部鎖に由来するものであり,アルカリ分解するか,このまま分解せずに分析することにより内部の塩基順列の確認,あるいは高級oligonucleotideの分析に役立つ.そして本法は単にoligonucleotideの分析だけでなく,5末端に燐酸基を持たない高分子の核酸であれば,5末端から3個までのヌクレオチド順列決定が可能である. (2)この酵素はpentanucleotideを,最初次のように分解した NpNpNpNpNp_??_NpN+pNpNpNp_??_NpNpN+pNpNp