Enzyme Chemistry of C-P Compound Synthesis in Rumen Protozoa

瘤胃原生动物C-P化合物合成的酶化学

• 批准号: 60470141
• 负责人: HORIGUCHI Masaaki
• 金额: $ 3.84万
• 依托单位: TOHOKU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1985
• 资助国家: 日本
• 起止时间: 1985 至 1986
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-60470141/
• 关键词: C-P compound; Phosphonopyruvate; Rumen protozoa; Tetrahymena; 酵素化学; ホスホエノールピルビン酸ホスホムターゼ

1. Some properties and chromatographic behavior of the enzyme which catalyzes formation of 3-phosphonopyruvate (PnPY) in rumen protozoa were studied in an attempt to reveal the role of rumen protozoa in the characteristic metabolism of ruminants.2. In view of the difficulty of detection and quantitation of free PnPY by HPLC, PnPY-2,4-dinitrophenylhydrazone was separated on a reversed phase column and was detected at 377nm. 0.1 nmol of PnPY could be quantitated.3. A preliminary research with log phase cells of Tetrahymena, which is a close relative of rumen protozoa, demonstrated that the enzyme localized in the soluble fraction of the cells and required Mg ion and heat stable cofactors. The pH- activity profile of the crude enzyme was bell-shaped and the activity was maximal at about pH8.4. Mixed protozoa were collected from the contents of sheep rumen, and the cells were fractionated according to the procedures for Tetrahymena. The PnPY-forming enzyme localized in a soluble fraction of the cells, but the activity was very low compared to that in Tetrahymena. On gel permeation chromatography, the enzyme activity in the soluble fraction increased and exceeded the activity in Tetrahymena. The activity was maximal at about pH7.2 and the molecular weight was estimated at about 70k dalton.5. Results of ion-exchange chromatography revealed that the enzymes of both rumen protozoa and Tetrahymena were basic proteins.6. Natural occurrence of PnPY has now been established, and all the results were consistent with the biosynthetic mechanism of PnPY through an intramolecular rearrangement of phosphoenolphruvate. However, the final proof for this mechanism of PnPY synthesis must await isolation of the new enzyme, phosphoenolphruvate phosphomutase.

1.研究了瘤胃原虫催化3-磷酸丙酮酸（PnPY）生成酶的部分性质和色谱行为，旨在揭示瘤胃原虫在反刍动物特征代谢中的作用.针对游离PnPY难以用高效液相色谱法检测和定量的问题，采用反相柱分离PnPY-2，4-二硝基苯腙，检测波长为377nm。0.1 nmol的PnPY可以定量.对瘤胃原生动物近缘种四膜虫对数生长期细胞的初步研究表明，该酶定位于细胞的可溶性部分，需要镁离子和热稳定辅因子。该粗酶的pH-活性曲线呈钟形，在pH8.4左右活性最大。从绵羊瘤胃内容物中收集混合原虫，按照四膜虫的方法分离细胞。PnPY形成酶定位于细胞的可溶性部分中，但活性与四膜虫相比非常低。在凝胶渗透色谱上，可溶性组分中的酶活性增加并超过四膜虫中的酶活性。该酶在pH7.2左右活性最高，分子量约为70k道尔顿.离子交换层析结果表明，瘤胃原虫和四膜虫的酶均为碱性蛋白. PnPY的天然存在现已被确立，所有的结果与PnPY通过磷酸烯醇丙酮酸的分子内重排的生物合成机制一致。然而，这种机制的PNPY合成的最终证据必须等待分离的新酶，磷酸烯醇丙酮酸磷酸变位酶。

项目成果

T.Takada;M.Horiguchi: "Synthesis of 3-Phosphonopyruvate in Cell-Free Preparations of Tetrahymena." Abstr. General Meeting of Agric.Chem.Soc.Jpn. ( 1987 ) p.365.

T.Takada；M.Horiguchi：“四膜虫无细胞制剂中 3-磷酸丙酮酸的合成”。

TAKADA,Tatsuyuki;HORIGUCHI,Masaaki: XII Conference on Chemistry and Biochemistry of Natural C-P Compound.Shiga University,Otsu,November 14,1986.

TAKADA，Tatsuyuki；HORIGUCHI，Masaaki：第十二届天然 C-P 化合物化学和生物化学会议。滋贺大学，大津，1986 年 11 月 14 日。

HORIGUCHI,Masaaki;TAKADA,Tatsuyuki: XI Conference on Chemistry and Biochemistry of Natural C-P Compounds.Korea University,Seoul,Korea,November 1,1985.

HORIGUCHI,Masaaki;TAKADA,Tatsuyuki：第十一届天然 C-P 化合物化学和生物化学会议。高丽大学，首尔，韩国，1985 年 11 月 1 日。

高田達之,鈴木正弘,及川克徳,堀口雅昭: 第78回日本畜産学会大会,1987年.

Tatsuyuki Takada、Masahiro Suzuki、Katsunori Oikawa、Masaaki Horiguchi：日本动物科学学会第 78 届年会，1987 年。

T.Takada;M.Suzuki;K.Oikawa;M.Horiguchi: "Synthesis of 3-Phosphonopyruvate in Cell-Free Preparations of Rumen Protozoa and Purification of "C-P Mutase"." 78th General Meeting, Jpn.Soc.Zootechn.Sci.,April 2, 1987.

T.Takada;M.Suzuki;K.Oikawa;M.Horiguchi：“瘤胃原生动物无细胞制剂中 3-磷酸丙酮酸的合成以及“C-P 变位酶”的纯化。”