Ikuo Igaue

Purification and Characterization of Membrane-Bound Inositol Phospholipid-Specific Phospholipase C from Suspension-Cultured Rice (Oryza sativa L.) Cells (Identification of a Regulatory Factor).

A membrane-bound inositol phospholipid-specific phospholipase C was solubilized from rice (Oryza sativa L.) microsomal membranes and purified to apparent homogeneity using a series of chromatographic separations. The apparent molecular mass of the enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 42,000 D, and the isoelectric point was 5.1. The optimum pH for the enzyme activity was approximately 6.5, and the enzyme was activated by both Ca2+ and Sr2+. The chemical and catalytic properties of the purified membrane-bound phospholipase C differed from those of the soluble enzyme reported previously (K. Yotsushima, K. Nakamura, T. Mitsui, I. Igaue [1992] Biosci Biotech Biochem 56: 1247–1251). In addition, we found a regulatory factor for the phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolyzing activity of phospholipase C from rice cells. The regulatory factor was dissociated from the catalytic subunit of phospholipase C during the purification. The regulatory factor was necessary to induce PIP2-hydrolyzing activity of both membrane-bound and -soluble phospholipase C; these purified enzymes had no activity alone. Because the plasma membranes isolated from rice cells could also act as a regulatory factor, the regulatory factor seems to be localized in the plasma membranes. Regulation of inositol phospholipid turnover in rice cells is discussed.

Structure and Function of the Golgi Complex in Rice Cells (II. Purification and Characterization of Golgi Membrane-Bound Nucleoside Diphosphatase)

Inosine diphosphatase bound to Golgi membranes was studied in rice (Oryza sativa L. cv Nipponkai) cells. The enzyme was solubilized with Triton X-100 from isolated rice Golgi membranes and was highly purified employing a series of chromatography steps in the presence of 20% glycerol and 0.1% Triton X-100. The apparent molecular mass of the enzyme was estimated by gel filtration column chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 200 and 55 kD, respectively. The isoelectric point of the enzyme was determined to be 7.5. The optimal pH for the enzyme activity was around 7 and the enzyme required Mg2+ for hydrolyzing activity. IDP, UDP, and GDP were effective substrate for the purified rice Golgi membrane-bound inosine diphosphatase, whereas activity with ADP, CDP, and thymidine 5[prime]-diphosphate was 10 to 20% of IDP. The Km values for IDP, UDP, and GDP were 0.48, 0.50, and 0.67 mM, respectively, and Vmax values were 1.85, 1.54, and 1.67 [mu]mol min-1 mg-1, respectively. These results indicate that the rice Golgi enzyme is a nucleoside diphosphatase that is specific for IDP, UDP, and GDP. Furthermore, this rice Golgi nucleoside diphosphatase stimulated the activity of glucan synthase I also localized in rice Golgi membranes. The results strongly support the view that this nucleoside diphosphatase is involved in regulation of [beta]-glucan synthesis in the plant Golgi complex.

Characterization and Solubilization of β-Glucan Synthases from Cultured Rice Cells

Two types of β-glucan synthases, GS-I and GS-II, were found in cultured rice cells (Oryza sativa L.). In glycerol density gradient centrifugation, GS-I activity peak co-migrated with a marker enzyme of the Golgi membrane, while GS-II co-migrated with the plasma membrane. Analysis of the reaction products of GS-I and GS-II, suggested that GS-I and GS-II were mainly β-1,4,- and β-1,3-glucosyltransferases, respectively. GS-I had a higher substrate affinity for UDP-glucose than GS-II, and needed divalent cations for its activity. Effects of nucleotides on the activity were also considerably different between GS-I and GS-II. GS-I was solubilized well with CHAPS and digitonin, and GS-II was solubilized effectively with sucrose monolaurate.

Some Physico-Chemical Properties of Non-Waxy Paddy Rice Starch in Niigata Prefecture:Part I. Relation of Properties of Starch to Eating and Cooking Qualities of Milled Rice

Fifteen samples of milled rice had protein contents ranging from 5.53 to 7.83% and amylose contents ranging from 17.6 to 22.1%. Eating and cooking qualities of cooked rice gave gross palatability indicies ranging from 100 to 61, cohesiveness values by balance method from 64 to 9, and starch-iodine color test values of milled rice from 0.297 to 0.574. Pasting characteristics of a 8% suspension of rice starch gave maximum viscosity values ranging from 550 to 380 Brabender Unit (B.U.), temperature at maximum viscosity from 82° to 92.5°C, break down values from 260 to 20B.U., and set back values from −0.5 to 60B.U. Good correlations were found between cooking and eating qualities of rice and rheological property of starch, and between rheological property and amylose content of starch, respectively.

Studies on Q-Enzyme of Rice Plant

Purification of Q-enzyme prepared from the extract of rice seed at the milky stage employing precipitation by lead acetate, fractionation by ammonium sulfate and chromatography on hydroxylapatite was carried out. The highly purified preparation of Q-enzyme was nearly homogeneous in electrophoresis, chromatography and solubility, and was nearly free from amylase, maltase, isoamylase, phosphorylase and phosphatase activities. Some of the physico-chemical and enzymic properties of the preparation were also investigated.

Fungal Enzymes Active in Degrading Gentiobiose Part II

(1) ハイドロール中の苦味成分として知られるゲンチオビオースの酵素的分解を目的として,多数の糸状菌からゲンチオビオースを唯一の炭素源とする合成培地によく生育し,かつゲンチオビオース分解能の高い菌の選択を行ない,Aspergitlus japonicus SAITOと同定された1株を分離した.本菌は麩固形培地によく生育し,かつ強力にβ-グルコシダーゼ(ゲンチオビアーゼ)を生産する. (2) タンニン酸沈澱法,硫安分画,DEAE-セファデックスクロマトグラフィー,セファデックスG-75ゲル濾過,デンプンブロック電気泳動によってβ-グルコシダーゼを収量20%,比活性増加100倍のタンパク的にほぼ単一と考えられる精製標品を得た.電気泳動における易動度-1.26×10-5cm2・V-1・sec-1(pH6.0, μ0.1),沈降分析におけるS20,w5.5(pH6.0, μ0.1,υ0.75として)であった. (3) 最適pH4～4.5,最適温度55～60°C, pH 2～6(40°C,24時間)で安定であり,また50～55°C (pH 4.5,15分)以上で失活が起こる. (4) ゲンチオビオース,セロビオース,ラミナリビナース,サリシンおよびρ-ニトロフェニルβ-グルコシドのいずれに対しても作用し,それぞれに対するKmは1.4×10-3,1.5×10-3, 2.3×10-3,5.4×10-3および1.35×10-3Mで,互に大きな差異は見出せなかった.

Studies on the Eating Quality of White Milled Non Waxy Rice Produced in Niigata Prefecture (Part7)

The eating quality of various rices were tested. Rices of superior quality (sticky, offflavor, transparent color) are Norin No.1, Koshihikari, Koshiziwase, Yatikogane, Koshisakae, and those of poor quality (less sticky, less off-flavor, white color) are Norin No.43, Nihonkai, Yoneyama, Shin No.7, Norin No.41, Hokuriku No.52, San-in No.52, Mihonishikiand Tangin.Results of statistical analysis of the samples barvested in 1958 and 1959 showed that a highlysignificant negative correlation of -0.921 existed between palatability and iodine-starchblue value of the residual cooking liquid. The tests showed a highly significant negativecorrelation of -0.899 between the palatability and total solids in the residual cooking liquidalthough there were exceptions.Regression lines between cohesiveness of cooked rice and iodine-starch blue value of theresidual cooking liquid were obtained. The line of the 1958 sample showed Y=124.29-209.19X, and that of the 1959 sample showed Y=103.27-194.28X.Standard errors for both regressionlines were very small.

Studies on Q-Enzyme of Rice Plant. Part VIII

(1) 水稲ウルチ,モチ両種の乳熟期種実から抽出したQ-酵素,ホスホリラーゼ,アミラーゼ,イソアミラーゼ,マルターゼについて酵素活性を測定し,両種間に有意の差の認められないことを示した. (2) ウルチ種(とわだ),モチ種(こがねもち)の胚乳,稈,葉鞘デンプンの成熟中における理化学的性質について検討した.両種胚乳デンプンの分画について,アミロース含量,ヨード呈色度,平均末端鎖長,極限粘度を測定した結果は,とわだでは乳熟期においてやや差を認めたが以後完熟期まで大きな変化はなく,こがねもちは各熟期を通じてほとんど差は認められなかった.両種間においてもアミロースの有無を除いて大した差異は見出せなかった. 稈,葉鞘のデンプン分画は胚乳に比較してかなり差があったが,両種間に差はなく,ともにウルチ型であった. (3) 以上の結果からウルチ,モチ両種の胚乳デンプン構造の生成に関して考察を行なった. 終始御鞭撻を載いた新潟大学農学部倉沢文夫教授,九州大学農学部山藤教授に厚く御礼申し上げます. また有益な御教示をいただいた九州大学農学部永松土巳教授,林武助教授,農技研生理遺伝部明峰英夫氏,新潟大学農学部木戸三夫教授,界面活性剤を御分与いただいたライオン油脂株式会社研究部,実験に協力された中村直道,田沢俊介の両君に謝意を表します.