色氨酸残基在磷酸烯醇式丙酮酸羧化酶催化功能中的作用

在pH7.5条件下,用NBS对PEP羧化酶中色氨酸残基进行共价修饰表明,PEP羧化酶中48个色氨酸残基均能被NBS修饰。用邹承鲁图解法求得,其中4个残基为酶表现催化活性所必需的。 PEP羧化酶的变构效应剂G6P、Gly及Mal分别与酶预保温后,再经NBS修饰,前两种处理中,同样浓度的NBS所用修饰的色氨酸残基数和处理后的残存酶活与对照相比有很大的差异,而用Mal处理的,两者与对照相差无几。     

马齿苋叶片PEP羧化酶的分子特性

马齿苋叶片PEPCase由四个相同的亚基组成,亚基分子量为83kD。远紫外CD光谱分析表明,此酶含有36.6％α—螺旋结构。马齿苋叶片PEPCase可被G6P激活,但不能被Gly、Ser激活。G6P可防止酶的尿素变性和枯草杆菌蛋白酶的作用。这种保护效应与G6P诱导的酶构象变化有关。 从酶对低温、高温及尿素的反应来看,马齿苋叶片PEPCase的稳定性高于高粱叶片PEP—Case,两者的免疫特性和电泳特性亦不同。     

A bioluminescent assay for the determination of phosphoenolpyruvate carboxykinase activity in nanogram-sized tissue samples

A highly specific and sensitive assay for the determination of phosphoenolpyruvate carboxykinase (PEPCK) in nanogram-sized tissue samples is described. This test system is based on the stoichiometric transformation of phosphoenolpyruvate into ATP. In a subsequent step ATP is quantified by bioluminescent techniques. The applicability of this assay system is shown by measurements in liver samples with normal and high PEPCK activity levels.     

Inheritance of two foreign genes co-introduced intoPetunia hybrida by direct gene transfer

In previous work, transformedPetunia hybrida plants were obtained by direct gene transfer, using two different genes on separate plasmids (NPT II gene and a cDNA of PEPC from green sorghum leaves). In this study, we have analysed the sexual transmission of the acquired genes by genetic crossing analysis of 2 of the transgenic petunias. The ploïdies of the two clones were determined by flow cytometric analysis showing that one was 2n and the other 4n. Self and back crosses show that the kanamycin character was inherited as a single dominant trait, and that the two clones were heterozygotes for this character. Therefore, the 4n clone probably arises from an endoploidization followed by a transformation event. Southern blot analyses show that all of the resistant progenies which were analysed harboured the kanamycin gene, and expressed the phosphorylation activity in vitro. The DNA of several progenies were also tested for the presence of co-transformed PEPC cDNA sequence. All of the kanamycin-resistant progenies tested contained the second coding sequence, indicating that the two foreign genes might be genetically co-inherited in the transgenic plants. The way in which the two genes are integrated into the genome is discussed.Abbreviations NPT neomycin phosphotransferase - PEG polyethyleneglycol - PEPC phosphoenolpyruvate carboxylase     

The role of acid phosphatases in plant phosphorus metabolism

Hydrolysis of phosphate esters is a critical process in the energy metabolism and metabolic regulation of plant cells. This review summarizes the characteristics and putative roles of plant acid phosphatase (APase). Although immunologically closely related, plant APases display remarkable heterogeneity with regards to their kinetic and molecular properties, and subcellular location. The secreted APases of roots and cell cultures are relatively non-specific enzymes that appear to be important in the hydrolysis and mobilization of P_i from extracellular phosphomonoesters for plant nutrition. Intracellular APases are undoubtedly involved in the routine utilization of P_i reserves or other P_i-containing compounds. A special class of intracellular APase exists that demonstrate a clear-cut (but generally nonabsolute) substrate selectivity. These APases are hypothesized to have distinct metabolic functions and include: phytase, phosphoglycolate phosphatase, 3-phosphoglycerate phosphatase, phosphoenolpyruvate phosphatase, and phosphotyrosyl-protein phosphatase. APase expression is regulated by a variety of developmental and environmental factors. P_i starvation induces de novo synthesis of extra- and intracellular APases in cell cultures as well as in whole plants. Recommendations are made to achieve uniformity in the analyses of the different APase isoforms normally encountered within and between different plant tissues.     

Cloning,sequence analysis and expression of a cDNA encoding active phosphoenolpyruvate carboxylase of the C3 plant Solanum tuberosum

A cDNA coding for phosphoenolpyruvate carboxylase (PEPC) was isolated from a cDNA library from Solanum tuberosum and the sequence of the cDNA was determined. It was inserted into a bacterial expression vector and a PEPC^- Escherichia coli mutant could be complemented by the cDNA construct. A functional fusion protein could be synthesized in E. coli. The properties of this PEPC protein clearly resembled those of typical C3 plant enzymes.     

Enhancement of Antigenic Site Detection with Gold Labeled Secondary and Tertiary Antibodies Using the Immunogold-Silver Staining Method

We report a modification of the immunogold-silver staining method (IGSS) for localizing hepatic phosphoenolpyruvate carboxykinase (PEPCK) in tissue sections, and we compare the efficacy of localizing the primary antibody with either a 5 nm gold labeled secondary antibody or 5 nm gold labeled secondary and tertiary antibodies. Light microscope examination of 10 μm frozen sections demonstrated that the use of combined secondary and tertiary gold labeled antibodies was superior to using a secondary gold labeled antibody alone. The increased labeling density (number of colloidal gold particles/antigenic site/cell) achieved by combined gold labeled antibodies was confirmed by electron microscopy. The increased labeling density resulted in a two-thirds reduction in the time needed for the IGSS physical development of the silver shells and less background. We achieved intense specific staining of hepatocytes expressing PEPCK while minimizing background staining. The use of combined secondary and tertiary gold labeled antibodies enhances the signal-to-noise ratio, achieves high resolution and is a suitable method for use in both light and electron microscopy.     

Diurnal changes in phosphoenolpyruvate carboxylase and pyruvate, orthophosphate dikinase properties in the natural environment: interplay of light and temperature in a C4 thermophile

Activities of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) were measured in leaf extracts of field grown Amaranthus paniculatus L. (C₄) during a natural diurnal irradiance and temperature pattern. Enzyme assays were run at both fixed (30°C) and the corresponding leaf temperature at the time of harvest. Light activation of PEP carboxylase (PEPCase) at fixed assay temperatures was expressed as a decrease in S_0–5 (PEP) after a threshold (> 330 μmol m^–2 s^–1) photon fluence rate was surpassed at noon. Earlier in the morning, increase in apparent enzyme affinity for PEP was observed when the assay was run at leaf temperature, indicating a physiologically meaningfull effect of temperature on S^0.5 (PEP). The 3.3-fold increase in PEPCase activity at low PEP and fixed assay temperature between the minimal and maximal irradiance and temperature hours of the day, became 12.8-, 11.5- and 7.4-fold when assays were run at the corresponding leaf temperature during three diurnal cycles with respective temperature differences (max minus min) of 9.0, 8.3 and 7.4°C. The extent of malate inhibition was the same for both day and night forms of PEPCase assayed at 35°C, but increased considerably with night enzyme at 25°C. The results indicate that light increases the apparent affinity of PEPCase for PEP and that at lower temperatures malate becomes more inhibitory. Pyruvate orthophosphate dikinase activity started to increase immediately after sunrise and the 10-fold increase at fixed temperature became 14.8-, 14.2- and 13.1-fold when assays were run at the above leaf temperatures. This indicates that the light effect predominates with pyruvate, orthophosphate dikinase, while with phosphoenolpyravate carboxylase, light and temperature co-operate to increase the day enzyme activities.     

Synthesis of citrate from phosphoenolpyruvate and acetylcarnitine by mitochondria from rabbit, pigeon and rat liver: Implications for lipogenesis

Rabbit, pigeon and rat liver mitochondria convert exogenous phosphoenolpyruvate and acetylcarnitine to citrate at rates of 14, 74 and 8 nmol/15 min/mg protein. Citrate formation is dependent on exogenous HCO3, is increased consistently by exogenous nucleotides (GDP, IDP, GTP, ADP, ATP) and inhibited strongly by 3-mercaptopicolinate and 1,2,3-benzenetricar☐ylate. Citrate is not made from pyruvate alone or combined with acetylcarnitine. Pigeon and rat liver mitochondria make large amounts of citrate from exogenous succinate, suggesting the presence of an endogenous source of acetyl units or a means of converting oxalacetate to acetyl units. Citrate synthesis from succinate by pigeon and rabbit mitochondria is increased significantly by exogenous acetylcarnitine. Pigeon and rat liver contain 80 and 15 times, respectively, more ATP:citrate lyase activity than does rabbit liver. Data suggest that mitochondrial phosphoenolpyruvate car☐ykinasein vivo could convert glycolysis-derived phosphoenolpyruvate to oxalacetate that, with acetyl CoA, could form citrate for export to support cytosolic lipogenesis as an activator of acetyl CoA car☐ylase, a carbon source via ATP:citrate lyase and NADPH via NADP: malate dehydrogenase or NADP: isocitrate dehydrogenase.