Enzyme Chemistry of C-P Compound Synthesis in Rumen Protozoa

1. 研究了瘤胃原生动物中催化3-磷酸丙酮酸（PnPY）形成的酶的一些性质和色谱行为，试图揭示瘤胃原生动物在反刍动物特征代谢中的作用。考虑到高效液相色谱法检测和定量游离PnPY的难度，采用反相柱分离PnPY-2,4-二硝基苯腙，在377nm处检测，0.1 nmol的PnPY可定量。对瘤胃原生动物近亲四膜虫的对数相细胞的初步研究表明，该酶定位于细胞的可溶部分，需要Mg离子和热稳定的辅因子。粗酶的pH-活性曲线呈钟形，在pH8.4左右活性最高。从绵羊瘤胃内容物中采集混合原生动物，按四膜虫分离方法对细胞进行分离。pnpy形成酶定位于细胞的可溶部分，但与四膜虫相比活性很低。在凝胶渗透层析上，可溶性部分的酶活性增加并超过了四膜虫的酶活性。在pH7.2时活性最大，分子量约为70k道尔顿。离子交换色谱分析结果表明，瘤胃原生动物和四膜虫的酶均为碱性蛋白。PnPY的自然存在已被证实，所有结果都与PnPY通过磷酸烯醇酸酯分子内重排的生物合成机制相一致。然而，这种PnPY合成机制的最终证据必须等待新酶磷酸烯醇酸磷酸酶的分离。

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.