磷酸烯醇式丙酮酸羧化酶cDNA在T7 RNA多聚酶/启动子系统中专一性表达的研究

用PT7和pGP1—2质粒偶联表达系统,通过温度诱导(30～42℃),使温度敏感基因CI875失活;加入利福霉素选择性抑制E.coliRNA多聚酶的表达,从而使外源PEP羧化酶cDNA得到专一性的表达。产物经SDS—PAGE和Wesern杂交分析,表明该系统表达出两条PEP羧化酶带,分子量分别是78kD和80kD。     

Two versatile shuttle vectors for Thermus thermophilus-Escherichia coli containing multiple cloning sites, lacZα gene and kanamycin or hygromycin resistance marker

Two versatile shuttle vectors for Thermus thermophilus and Escherichia coli were developed on the basis of the T. thermophilus cryptic plasmid pTT8 and E. coli vector pUC13. These shuttle vectors, pTRK1T and pTRH1T, carry a gene encoding a protein homologous to replication protein derived from pTT8, a replicon for E. coli, new multiple cloning sites and a lacZα gene from E. coli vector pUC13, and also have a gene encoding a thermostable protein that confers resistance to kanamycin or hygromycin, which can be used as a selection marker in T. thermophilus. These shuttle vectors are useful to develop enzymes and proteins of biotechnological interest. We also constructed a plasmid, pUC13T, which carries the same multiple cloning sites of pTRK1T and pTRH1T. These vectors should facilitate cloning procedures both in E. coli and T. thermophilus.     

Expression of Escherichia coli phosphoenolpyruvate carboxylase in a cyanobacterium. Functional complementation of Synechococcus PCC 7942 ppc.

The gene (ppc) coding for phosphoenolpyruvate carboxylase (PEPCase) in the cyanobacterium Synechococcus PCC 7942 has been inactivated via insertional mutagenesis while being functionally complemented by Escherichia coli ppc. Cyanobacterial cells functionally complemented by E. coli ppc showed decreased PEPCase activity in crude cell lysates and detergent-permeabilized whole cell assays. Decreased rates of growth, reduced levels of chlorophyll a, and decreased photosynthetic O2 evolution capacity per cell when compared to wild-type cyanobacterial cells were also observed. Phycobiliprotein levels were not affected. The results are discussed in terms of the impact of reduced PEPCase activity on cyanobacterial cell metabolism and the regulatory properties of the E. coli gene product.     

Regulatory seryl-phosphorylation of C4 phosphoenolpyruvate carboxylase by a soluble protein kinase from maize leaves

A reconstituted system composed of purified phosphoenolpyruvate carboxylase (PEP-Case) and a soluble protein kinase (PK) from green maize leaves was developed to critically assess the effects of in vitro protein phosphorylation on the catalytic and regulatory (malate sensitivity) properties of the target enzyme. The PK was partially purified from light-adapted leaf tissue by ammonium sulfate fractionation (0-60% saturation fraction) of a crude extract and blue dextran-agarose affinity chromatography. The resulting preparation was free of PEPCase. This partially purified protein kinase activated PEPCase from dark-adapted green maize leaves in an ATP-, Mg2+-, time-, and temperature-dependent fashion. Concomitant with these changes in PEPCase activity was a marked decrease in the target enzyme's sensitivity to feedback inhibition by L-malate. The PK-mediated incorporation of 32P from [gamma-32P]ATP into the protein substrate was directly correlated with these changes in PEPCase activity and malate sensitivity. The maximal molar 32P-incorporation value was about 0.25 per 100-kDa PEPCase subunit (i.e., 1 per holoenzyme). Phosphoamino acid analysis of the 32P-labeled target enzyme by two-dimensional thin-layer electrophoresis revealed the exclusive presence of phosphoserine. These in vitro results, together with our recent studies on the light-induced changes in phosphorylation status of green maize leaf PEPCase in vivo (J. A. Jiao and R. Chollet (1988) Arch. Biochem. Biophys. 261, 409-417), collectively provide the first unequivocal evidence that the seryl-phosphorylation of the dark-form enzyme by a soluble protein kinase is responsible for the changes in catalytic activity and malate sensitivity of C4 PEPCase observed in vivo during dark/light transitions of the parent leaf tissue.     

Cloning and sequence analysis of cDNA encoding active phosphoenolpyruvate carboxylase of the C4-pathway from maize.

A recombinant clone, pM52, containing cDNA for maize phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) was isolated from a maize leaf cDNA library constructed using an expression vector in Escherichia coli. The screening of the clone was conveniently performed through its ability to complement the phenotype (glutamate requirement) of PEPCase-negative mutant of E. coli. The enzyme encoded by this clone was identical with the major PEPCase in maize, a key enzyme in the C4-pathway, as judged from its allosteric properties and immunological reactivity. The cloned cDNA (3093 nucleotides in length) contained an open reading frame of 2805 nucleotides, the 3'-untranslated region of 222 nucleotides and the poly(dA) tract of 64 nucleotides. The deduced amino acid sequence (935 residues) of the enzyme showed higher homology with that of an enterobacterium, E. coli (43%) than that of a cyanobacterium (blue-green alga), Anacystis nidulans (33%).     

Multiple forms of phosphoenolpyruvate carboxylase in mesophyll, epidermal and guard cells of Vicia faba

The distribution of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) in different leaf‐cell‐types and tissues of Vicia faba L. cv. 3‐fach Weiße was studied. The highest specific PEPCase activity was found in guard cell protoplasts (16.3 µmol mg⁻¹ protein h⁻¹) whereas for epidermal and mesophyll protoplasts remarkably lower specific activities were found (1.6 and 1.0 µmol mg⁻¹ protein h⁻¹, respectively). On chlorophyll and protoplast basis, a similar distribution of enzyme activity was observed. Compared with epidermal extracts, the specific PEPCase activity of mesophyll tissue was 17‐fold lower. Immunological studies with polyclonal antibodies to PEPCase indicated 3 immunoreactive proteins in epidermal tissue and guard cell protoplasts with molecular masses of 107 000, 110 000, and 112 000. Only the M_r 107 000 protein was found in extracts of mesophyll and epidermis protoplasts. Western immunoblots after native electrophoresis of epidermal and mesophyll proteins showed a significant difference in PEPCase mobility. It is assumed, that the immunostained proteins of M_r 110 000 and 112 000 represent isoforms or subunits of the PEPCase and that they are involved in stomatal movements.     

Light regulation of phosphoenolpyruvate carboxylase in barley mesophyll protoplasts is modulated by protein synthesis and calcium, and not necessarily correlated with phosphoenolpyruvate carboxylase kinase activity

The regulation of phosphoenolpyruvate carboxylase (PEPCase, EC. 4.1.1.31) and PEPCase kinase was investigated using barley (Hordeum vulgare L.) mesophyll protoplasts. Incubation of protoplasts in the light resulted in a reduction in the sensitivity of PEPCase to the inhibitor L-malate; PEPCase from protoplasts incubated in the light for 1 h was inhibited 48±2% by 2mM malate, whereas the enzyme from protoplasts incubated for 1 h in the dark was inhibited by 67±2%. Light-induced reduction of sensitivity of PEPCase to malate was decreased by cycloheximide (CHM), indicating the involvement of protein synthesis. The PEPCase kinase in protoplasts increased with time after isolation in darkness, and increased still further following light treatment. The increase in kinase activity in the light was sensitive to CHM. When protoplasts were illuminated in the presence of EGTA and the calcium ionophore A23187 to reduce intracellular Ca²⁺, the reduction in the senstivity of PEPCase to malate was enhanced, though no more PEPCase kinase activity was detected than in protoplasts illuminated in the absence of EGTA and A23187. Incubation with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) had no effect on the light-induced reduction of sensitivity of PEPCase to malate inhibition or on light-activation of PEPCase kinase. These results indicate that there is a constitutive PEPCase kinase activity in C₃ leaf tissue, that there is another kinase which is light-activated in a CHMsensitive way, that the sensitivity of PEPCase to its inhibitor may not always be correlated with apparent PEPCase kinase actvity, and that PEPCase and PEPCase kinase are regulated in a different manner in C₃ protoplasts than in C₄ protoplasts or leaf tissue.Abbreviations CAM Crassulacean acid metabolism - Chl chlorophyll - CHM cycloheximide - DCMU 3-(3,4-dichloro-phenyl)-1,1-dimethylurea - PEP phosphoenolpyruvate - PEPCase PEP carboxylase     

Phosphoenolpyruvate carboxylase in mistletoe leaves: Regulation of gene expression, protein content, and covalent modification

Seasonal changes in the activity of phospho enol pyruvate carboxylase (PEPCase, EC 4.1.1.31), a key enzyme in the interaction of carbohydrate and nitrogen metabolism, were studied in leaves of the C₃ semiparasitic mistletoe, Viscum album, growing on different host trees. Maximum extractable PEPCase activities were higher in leaves of mistletoes growing on Betula pendula and Alnus glutinosa hosts compared with those on the conifers, Abies alba and Larix decidua . Independent of host, maximum extractable PEPCase activities were high in spring and autumn while low in summer. Samples with higher PEPCase activities showed higher amounts of PEPCase protein and higher PEPCase mRNA levels. A curvilinear correlation between leaf total nitrogen content and the maximum extractable PEPCase activity as well as PEPCase mRNA level suggested that nitrogen might affect the activity of PEPCase of mistletoe by up-regulating gene expression. In addition to extractable activity, seasonal changes of the PEPCase activation state, the ratio of activities resulting from limited:non-limited assays, were found, which was correlated to the variation of malate content in leaves of mistletoe. ATP-dependent activation of PEPCase was characterized by an increase in I_0.5( l -malate), indicating that PEPCase of leaves of mistletoes is probably regulated via phosphorylation.     

Altered kinetic properties of tyrosine-183 to cysteine mutation in glutamine synthetase of anabaena variabilis strain SA1 is responsible for excretion of ammonium ion produced by nitrogenase

A L-methionine- D, L-sulfoximine-resistant mutant of the cyanobacterium Anabaena variabilis, strain SA1, excreted the ammonium ion generated from N(2) reduction. In order to determine the biochemical basis for the NH(4)(+)-excretion phenotype, glutamine synthetase (GS) was purified from both the parent strain SA0 and from the mutant. GS from strain SA0 (SA0-GS) had a pH optimum of 7.5, while the pH optimum for GS from strain SA1 (SA1-GS) was 6.8. SA1-GS required Mn(+2) for optimum activity, while SA0-GS was Mg(+2) dependent. SA0-GS had the following apparent K(m) values at pH 7.5: glutamate, 1.7 m M; NH(4)(+), 0.015 m M; ATP, 0.13 m M. The apparent K(m) for substrates was significantly higher for SA1-GS at its optimum pH (glutamate, 9.2 m M; NH(4)(+), 12.4 m M; ATP, 0.17 m M). The amino acids alanine, aspartate, cystine, glycine, and serine inhibited SA1-GS less severely than the SA0-GS. The nucleotide sequences of glnA (encoding glutamine synthetase) from strains SA0 and SA1 were identical except for a single nucleotide substitution that resulted in a Y183C mutation in SA1-GS. The kinetic properties of SA1-GS isolated from E. coli or Klebsiella oxytoca glnA mutants carrying the A. variabilis SA1 glnA gene were also similar to SA1-GS isolated from A. variabilis strain SA1. These results show that the NH(4)(+)-excretion phenotype of A. variabilis strain SA1 is a direct consequence of structural changes in SA1-GS induced by the Y183C mutation, which elevated the K(m) values for NH(4)(+) and glutamate, and thus limited the assimilation of NH(4)(+) generated by N(2) reduction. These properties and the altered divalent cation-mediated stability of A. variabilis SA1-GS demonstrate the importance of Y183 for NH(4)(+) binding and metal ion coordination.     

Purification and characterization of acyl carrier protein from two cyanobacteria species

The acyl carrier protein (ACP), an essential protein cofactor for fatty acid synthesis, has been isolated from two cyanobacteria: the filamentous, heterocystous, Anabaena variabilis (ATCC 29211) and the unicellular Synechocystis 6803 (ATCC 27184). Both ACPs have been purified to homogeneity utilizing a three-column procedure. Synechocystis 6803 ACP was purified 1800-fold with 67% yield, while A. variabilis ACP was purified 1040-fold with 50% yield. Yields of 13.0 micrograms ACP/g Synechocystis 6803 and 9.0 micrograms ACP/g A. variabilis were achieved. Amino acid analysis indicated that these ACPs were highly charged acidic proteins similar to other known ACPs. Sequence analysis revealed that both cyanobacterial ACPs were highly conserved with both spinach and Escherichia coli ACP at the phosphopantetheine prosthetic group region. Examining the probability of alpha-helix and beta-turn regions in various ACPs, showed that cyanobacterial ACPs were more closely related to E. coli ACP than spinach ACP I. Immunoblot analysis and a competitive binding assay for ACP illustrated that both ACPs bound poorly to spinach ACP I antibody. SDS/PAGE and native PAGE of Synechocystis 6803 ACP and A. variabilis ACP showed that cyanobacteria ACPs co-migrated with E. coli ACP and had relative molecular masses of 18,100 and 17,900 respectively. Both native and urea gel analysis of acyl-ACP products from fatty acid synthase reactions demonstrated that bacterial ACPs and plant ACP gave essentially the same metabolic products when assayed using either bacterial or plant fatty acid synthase. A. variabilis and Synechocystis 6803 ACP could be acylated using E. coli acyl ACP synthetase.     

水分胁迫对露花磷酸烯醇式丙酮酸羧化酶表达水平及特性的影响

水分胁迫能引起露花叶片PEP羧化酶的活力、酶蛋白和mRNA水平的提高。复水后,叶片PEP羧化酶表达量降低;茎中的PEP羧化酶在水分胁迫和恢复水分供应过程中变化情况与叶片相似,兼性CAM植物的碳代谢类型转变发生在植物的绿色组织中。露花叶片中除了250kD的PEP羧化酶同功酶外,还有300kD同功酶;主茎的叶片叶位越低,PEP羧化酶活力越高。     

Identification and cloning of the gene coding for lysophospholipase L2 of E. coli K-12

E. coli bearing hybrid plasmid pKOl (Oeda et al. (1981) Mol. Gen. Genet. 184, 191-199) expressed a large amount of lysophospholipase L2 activity. When a mutant which was defective in lysophospholipase L2 activity was transformed with plasmid pKOl, it overproduced lysophospholipase L2 activity. The gene responsible for the lysophospholipase L2 activity was designated as pld B. On the same hybrid plasmid another gene (pld A) coding for detergent-resistant phospholipase A (DR-phospholipase A) was also identified. These facts together with the results of a Pl transduction experiment revealed that the pld B gene must be between the pld A and met E genes on the E. coli chromosome.     

Short-term nitrogen-induced modulation of phosphoenolpyruvate carboxylase in tobacco and maize leaves

Untransformed maize and tobacco plants and tobacco plants constitutively expressing nitrate reductase were grown with sufficient NO(3)- to support maximal growth. Four days prior to treatment the tobacco plants were deprived of nitrogen. Excised maize leaves and tobacco leaf discs were fed with either 40 mM KNO(3) or 40 mM KCl (control) in the light. Phosphoenolpyruvate (PEP) carboxylase (Case) activity was measured at 0.3 mM and 3 mM PEP. The light- induced increase in PEPCase V(max) was greater in maize than tobacco. Furthermore light decreased malate sensitivity in maize (which was N-replete) but not in N-deficient tobacco. NO(3)- treatment increased PEPCase V:(max) values in both species and decreased the sensitivity to inhibition by malate, but effects of NO(3)- were much more pronounced in tobacco than maize. PEPCase kinase activity was, however, greater in maize leaves NO(3)- than in the Cl(-)-treated controls, suggesting that it is responsive to leaf nitrogen supply. A correlation between foliar glutamine content and PEPCase activity was observed. It is concluded that PEPCase is sensitive to N metabolites which favour increased flow through the anapleurotic pathway in both C(3) and C(4) plants.     

Partition functions of unit-copy plasmids can stabilize the maintenance of plasmid pBR322 at low copy number.

The maintenance of plasmid pBR322 is highly unstable in a polA12 strain of Escherichia coli at 29 degrees C due to severely reduced copy number. Under these conditions, introduction of the par (partition) locus of plasmid P1 or the par (sop) region of F into pBR322 stabilizes it. A region with similar activity was detected in the P7 plasmid. The activity of the P1 par locus was dependent on the P1 parA gene product and was sensitive to par-specified incompatibility.     

Activity of phosphoenolpyruvate carboxylase of an anthracycline-producing streptomycete

During fermantation studies on the production of anthracycline antibiotics by Streptomyces C5, it was observed that among the intermediate metabolism enzymes tested, only phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) increased significantly in specific activity during stationary phase. The specific activity of the Streptomyces C5 PEPCase increased ca. 3-fold during antibiotic production phase from the logarithmic phase levels. To characterize the regulation of the enzyme further, the Streptomyces C5 PEPCase was purified 150-fold from crude extracts. Acetyl-CoA and Mg2+ were shown to be required for PEPCase activity. The activity of the partially purified PEPCase was stimulated slightly by fructose 1,6-bisphosphate and AMP, and was inhibited severely by oxaloacetate, aspartate, malate, succinate, ATP, citrate, and CoASH.     

多变鱼腥藻ATCC 29413基因的一步插入失活

多变鱼腥藻ATCC 29413有潜力发展为异养生长产异形胞蓝藻的研究模式种, 但由于细胞内的限制-修饰系统等原因, 其基因转移效率极低。研究克隆了该藻株的两个甲基化酶(M. AvaⅠ和M. AvrⅡ)基因ava_3181和ava_4359, 构建了辅助质粒pHB6088, 对运载质粒进行甲基化保护以避免被细胞中的限制酶切割。以ava_1237和ava_4412基因为例, 研究证明利用该辅助质粒和接合转移系统可通过双交换对多变鱼腥藻ATCC 29413的基因实现一步插入失活。