光/暗对高粱叶片磷酸烯醇式丙酮酸羧化酶基因表达的调节

高粱幼苗黄化叶片经照光转绿后,其PEP-Case活性提高4～15倍,mRNA含量提高了1.03倍,并测定出PEPCase mRNA的分子量为3.4kb。以等量的总RNA及mRNA进行体外翻译,发现转绿后PEPCase专一性翻译活性提高了51％～53％。这表明光照可以在转录水平上调节PEP-Case的基因表达。     

Glyphosine Inhibits Maize Leaf Phosphoenolpyruvate Carboxylase

Glyphosine [N, N-bis-(phosphonomethyl) glycine] inhibited maizeleaf P-enolpyruvate carboxylase competitively with respect toP-enolpyruvate. The inhibition was dependent on glyphosine concentrationand pH. Glycine, but not glucose-6-phosphate, protected theenzyme from the effect of glyphosine. A related compound, glyphosate[N-(phosphonomethyl) glycine], produced little or no inhibition.P-enolpyruvate carboxylase could be one of the targets of glyphosineaction, causing growth inhibition as reported (Croft, S. M.,C. J. Arntzen, L. N. Vanderhoef and C. S. Zettinger (1974) Biochim.Biophys. Acta 335: 211-217). (Received July 10, 1986; Accepted December 4, 1986)     

转玉米pepc基因水稻的某些光合特性

测定转玉米pepc基因水稻和未转化粳稻品种Kitaake在分蘖初期、分蘖盛期、拔节期、始穗期、齐穗期、成熟期和剑叶不同生长时期的光合特性动态变化的结果表明:转玉米pepc基因水稻的磷酸烯醇式丙酮酸羧化酶(PEPCase)活性和净光合速率(Pn)在不同时期各不相同,相对于Kitaake而言,均有不同程度的提高;剑叶完全展开时的PEPCase活性和Pn提高最明显.     

Structure and Regulation of Phosphoenolpyruvate Carboxylase from Sorghum Leaves

Phosphoenolpyruvate carboxylase (ortho-phosphate: oxaloacetate carboxylase, EC 4.11.31, PEPCase), an enzyme widely occurringin bacteria, algae and plants, is an importantcarboxylating enzyme serving a variety of func-tions ranging from photosynthetic carbon dioxidefixation to nitrogen assimilation (Latzko andKelly 1983, O'Leary 1982). It is a key regula-tory enzyme in both C_4 and CAM photosyn-thesis. In C_4 plants, PEPCase is localized inthe mesophyll-cell cytoplasm and catalyzesthe conversion of PEP and bicarbonate to     

神经系统特异表达基因调控机理的研究进展

神经系统特异性基因正确的时空表达受细胞内外信号的调控,信号传导途径最终的靶位点是能结合特异转录因子的DNA序列.目前发现的决定神经系统基因特异性表达的顺式作用元件既有增强子,也有沉默子.它们可以特异性地增强基因在神经系统的表达,或特异性抑制基因在非神经系统的表达. 顺式元件要发挥这些作用,依赖于与其结合的反式因子,而这些反式因子又能与其他蛋白质或DNA序列发生互动, 通过协调作用,共同决定基因的时空表达顺序.     

Modification of Maize Leaf Phosphoenolpyruvate Carboxylase with Fluorescein Isothiocyanate

Fluorescein isothiocyanate inactivates phosphoenolpyruvate carboxylasefrom maize leaves, presumably by reacting with lysyl groups.The reaction appears to involve at least two groups of lysineson the enzyme. The more rapid reaction is with groups whichare protected by the substratemagnesium phosphoenolpyruvateand thus probably are located in the active site. In addition,fluorescein isothiocyanate apparently binds more slowly at asite which desensitizes the enzyme to activation by glucose-6-phosphate. Using the fluorescence of the complex of fluorescein isothiocyanatewith phosphoenolpyruvate carboxylase it was shown that bothmagnesium phosphoenolpyruvate and glucoses-6-phosphate causechanges in the conformation of the enzyme and influence thebinding of fluorescein isothiocyanate as well. Light scattering measurements showed that fluorescein isothiocyanateinduced disaggregation of the enzyme, while glucose-6-phosphatecaused aggregation, although less when fluorescein isothiocyanatewas present. ¹Supported in part by National Science Foundation grant no.DMB 88-12484.     

deltaC Values in Maize Leaf Correlate with Phosphoenolpyruvate Carboxylase Levels

Values of δ¹³C and levels of phosphoenolpyruvate carboxylase and ribulose 1,5-bisphosphate carboxylase/oxygenase were analyzed in segments from the fourth leaf of young maize (Zea mays L.) plants. The δ¹³C values became significantly more negative from the base to the tip of the leaves. Phosphoenolpyruvate carboxylase levels and ribulose bisphosphate carboxylase levels both increased from the base to the tip. The principal effect of phosphoenolpyruvate carboxylase levels or δ¹³C should arise through its effect on the carboxylation/diffusion balance in the mesophyll. In this case, δ¹³C values should become more negative as phosphoenolpyruvate carboxylase levels increase, unless there are offsetting changes in stomatal aperture. The principal effect of ribulose bisphosphate carboxylase/oxygenase on δ¹³C should occur through its effect on the extent of leakage of CO₂ from the bundle sheath cells. In this case, δ¹³C values should become more positive as ribulose bisphosphate carboxylase levels increase. Accordingly, the variation in δ¹³C values seen in maize leaves appears to be the result of variations in the level of phosphoenolpyruvate carboxylase.     

Effects of Nitrate and Ammonium on Gene Expression of Phosphoenolpyruvate Carboxylase and Nitrogen Metabolism in Maize Leaf Tissue during Recovery from Nitrogen Stress

We previously showed that the selective accumulation of phosphoenolpyruvate carboxylase (PEPC) in photosynthetically maturing maize (Zea mays L.) leaf cells induced by nitrate supply to nitrogen-starved plants was primarily a consequence of the level of its mRNA (B Sugiharto, K Miyata, H Nakamoto, H Sasakawa, T Sugiyama [1990] Plant Physiol 92: 963-969). To determine the specificity of inorganic nitrogen sources for the regulation of PEPC gene expression, nitrate (16 millimolar) or ammonium (6 millimolar) was supplied to plants grown previously in low nitrate (0.8 millimolar), and changes in the level of PEPC and its mRNA were measured in the basal region of the youngest, fully developed leaves of plants during recovery from nitrogen stress. The exogenous supply of nitrogen selectively increased the levels of protein and mRNA for PEPC. This increase was more pronounced in plants supplemented with ammonium than with nitrate. The accumulation of PEPC during nitrogen recovery increased in parallel with the increase in the activity of glutamine synthetase and/or ferredoxin-dependent glutamate synthase. Among the major amino acids, glutamine was the most influenced during recovery, and its level increased in parallel with the steady-state level of PEPC mRNA for 7 hours after nitrogen supply. The administration of glutamine (12 millimolar) to nitrogen-starved plants increased the steady-state level of PEPC mRNA 7 hours after administration, whereas 12 millimolar glutamate decreased the level of PEPC mRNA. The results indicate that glutamine and/or its metabolite(s) can be a positive control on the nitrogen-dependent regulation of PEPC gene expression in maize leaf cells.     

Evidence for Light-stimulated Synthesis of Phosphoenolpyruvate Carboxylase in Leaves of Maize

Illumination (22,000 lumens per meter²) of etiolated maize plants for 80 hours brings about a 5-fold increase in phosphoenolpyruvate carboxylase activity per unit of protein. An increase in carboxylase protein and incorporation of [³⁵S]methionine into the protein occurs simultaneously with the activity increase. In green plants, the level of phosphoenolpyruvate carboxylase protein and enzyme activity is dependent on the intensity of light during growth. These results are consistent with the conclusion that the activity increase results from light-stimulated de novo synthesis of phosphoenolypyruvate carboxylase protein.     

HIV-1基因表达的转录调控

高效抗逆转录病毒治疗（HAART）可以有效地抑制人类免疫缺陷病毒Ⅰ型（HIV-1）的复制及血浆病毒载量,延缓发病进程,改善、提高患者的生活质量和存活时间。但是,一旦停止治疗就会导致血浆病毒血症迅速反弹,HIV-1以原病毒的形式在静息记忆CD4⁺T等细胞中的持续存在是清除HIV-1的一个障碍。HIV-1基因转录的激活与阻抑决定了受感染细胞进入产毒性感染或潜伏感染。本文从原病毒整合位置与转录干扰、细胞转录因子与HIV-1启动子相互作用招募RNA聚合酶起始转录、转录的表观遗传调控和反式激活因子Tat及其相关蛋白促进转录延伸等方面探讨了HIV-1原病毒转录调控机制。     

Oligomerization and the Affinity of Maize Phosphoenolpyruvate Carboxylase for Its Substrate

When two different forms of phosphoenolpyruvate carboxylase (PEPC) from maize (Zea mays L.) leaves are present in an assay it is possible to estimate the ratio of Vmax to Km (V/K) for the two forms separately. This measure of the binding of the substrate by the enzyme permits evaluation of the effects of various treatments on the relative substrate-binding velocity of the enzyme. PEPC diluted 1/20 is present in a mixture of a tetrameric form with a high affinity for phosphoenolpyruvate and a dimeric form with a low affinity (M.-X. Wu, C.R. Meyer, K.O. Willeford, R.T. Wedding [1990] Arch Biochem Biophys 281: 324-329). Malate at 5 mM reduced (V/K)1,[mdash]the V/K of the probable tetrameric form[mdash]almost to zero, but reduced (V/K)2[mdash]the V/K of the probable dimer[mdash]by only about 80%. Glucose-6-phosphate (Glc-6-P) at 5 mM increased (V/K)1 to 155% of the control but had no effect on (V/K)2. Glycerol (20%) alone increased both V/Ks, and its effects are additive to the Glc-6-P effects, implying different mechanisms for activation by Glc-6-P and glycerol.     

In Vivo Regulation of Wheat-Leaf Phosphoenolpyruvate Carboxylase by Reversible Phosphorylation

Regulation of C3 phosphoenolpyruvate carboxylase (PEPC) and its protein-serine/threonine kinase (PEPC-PK) was studied in wheat (Triticum aestivum) leaves that were excised from low-N-grown seedlings and subsequently illuminated and/or supplied with 40 mM KNO3. The apparent phosphorylation status of PEPC was assessed by its sensitivity to L-malate inhibition at suboptimal assay conditions, and the activity state of PEPC-PK was determined by the in vitro 32P labeling of purified maize dephospho-PEPC by [[gamma]-32P]ATP/Mg. Illumination ([plus or minus]NO3-) for 1 h led to about a 4.5-fold increase in the 50% inhibition constant for L-malate, which was reversed by placing the illuminated detached leaves in darkness (minus NO3-). A 1 -h exposure of excised leaves to light, KNO3, or both resulted in relative PEPC-PK activities of 205, 119, and 659%, respectively, of the dark/0 mM KNO3 control tissue. In contrast, almost no activity was observed when a recombinant sorghum phosphorylation-site mutant (S8D) form of PEPC was used as protein substrate in PEPC-PK assays of the light plus KNO3 leaf extracts. In vivo labeling of wheat-leaf PEPC by feeding 32P-labeled orthophosphate showed that PEPC from light plus KNO3 tissue was substantially more phosphorylated than the enzyme in the dark minus-nitrate immunoprecipitates. Immunoblot analysis indicated that no changes in relative PEPC-protein amount occurred within 1 h for any of the treatments. Thus, C3 PEPC activity in these detached wheat leaves appears to be regulated by phosphorylation of a serine residue near the protein's N terminus by a Ca2+ -independent protein kinase in response to a complex interaction in vivo between light and N.