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91.
Falini G Fermani S Ripamonti A Sabatino P Sparla F Pupillo P Trost P 《Biochemistry》2003,42(16):4631-4639
Photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Spinacia oleracea belongs to a wide group of GAPDHs found in most organisms displaying oxygenic photosynthesis, including cyanobacteria, green and red algae, and higher plants. As a major catalytic difference with respect to glycolytic GAPDH, photosynthetic GAPDH exhibits dual cofactor specificity toward pyridine nucleotides with a preference for NADP(H). Here we report the crystal structure of NAD-complexed recombinant A(4)-GAPDH (NAD-A(4)-GAPDH) from Spinacia oleracea, expressed in Escherichia coli. Its superimposition onto native A(4)-GAPDH complexed with NADP (NADP-A(4)-GAPDH) pinpoints specific conformational changes resulting from cofactor replacement. In photosynthetic NAD-A(4)-GAPDH, the side chain of Asp32 is oriented toward the coenzyme to interact with the adenine ribose diol, similar to glycolytic GAPDHs (NAD-specific). On the contrary, in NADP-A(4)-GAPDH Asp32 moves away to accommodate the additional 2'-phosphate group of the coenzyme and to minimize electrostatic repulsion. Asp32 rotation is allowed by the presence of the small residue Ala40, conserved in most photosynthetic GAPDHs, replacing bulky amino acid side chains in glycolytic GAPDHs. While in NADP-A(4)-GAPDH two amino acids, Thr33 and Ser188, are involved in hydrogen bonds with the 2'-phosphate group of NADP, in the NAD-complexed enzyme these interactions are lacking. The crystallographic structure of NAD-A(4)-GAPDH highlights that four residues, Thr33, Ala40, Ser188, and Ala187 (Leu, Leu, Pro, and Leu respectively, in glycolytic Bacillus stearothermophilus GAPDH sequence) are of primary importance for the dual cofactor specificity of photosynthetic GAPDH. These modifications seem to trace the minimum evolutionary route for a primitive NAD-specific GAPDH to be converted into the NADP-preferring enzyme of oxygenic photosynthetic organisms. 相似文献
92.
Matei E Miron S Blouquit Y Duchambon P Durussel I Cox JA Craescu CT 《Biochemistry》2003,42(6):1439-1450
Human centrin 2 (HsCen2) is an EF-hand protein that plays a critical role in the centrosome duplication and separation during cell division. We studied the structural and Ca(2+)-binding properties of two C-terminal fragments of this protein: SC-HsCen2 (T94-Y172), covering two EF-hands, and LC-HsCen2 (M84-Y172), having 10 additional residues. Both fragments are highly disordered in the apo state but become better structured (although not conformationally homogeneous) in the presence of Ca(2+) and depending on the nature of the cations (K(+) or Na(+)) in the buffer. Only the longer C-terminal domain, in the Ca(2+)-saturated state and in the presence of Na(+) ions, was amenable to structure determination by nuclear magnetic resonance. The solution structure of LC-HsCen2 reveals an open two EF-hand structure, similar to the conformation of related Ca(2+)-saturated regulatory domains. Unexpectedly, the N-terminal helix segment (F86-T94) lies over the exposed hydrophobic cavity. This unusual intramolecular interaction increases considerably the Ca(2+) affinity and constitutes a useful model for the target binding. 相似文献
93.
Constitutive versus thermoinducible expression of heterologous proteins in Escherichia coli based on strong PR,PL promoters from phage lambda 总被引:1,自引:0,他引:1
Constitutive and thermoinducible expression plasmids based on strong P(R),P(L) promoters from phage lambda were compared for production of TNF-alpha and its analogs under various conditions. Much higher accumulation of TNF was obtained in a constitutive system, so the wider applicability of such systems was studied. In constitutive systems, proteolytically susceptible proteins can be produced easily at low cultivation temperatures and the addition of expensive or toxic chemical inducers is not required. On the other hand, toxic proteins cannot be produced and selection pressure must be strictly maintained to ensure segregational stability of plasmids. Accumulation of TNF-alpha and various analogs at levels up to 25% of total soluble protein was repeatedly achieved, which was 2-3-fold higher than in a thermoinducible system. The stable behavior of the constitutive system in laboratory fermentors was also confirmed. We propose the constitutive system described here as a general model for many currently used expression systems containing strong but not completely repressed promoters. Such systems may be considered as constitutive ones with reduced promoter strengths, but still exhibiting all the intrinsic properties of constitutive expression systems. Although all modern expression systems are inducible, wider use of a constitutive system is evidently possible. 相似文献
94.
An inclusion complex between imazalil (IMZ), a selected fungicide, and cyclomaltoheptaose (beta-cyclodextrin, betaCD) was obtained using supercritical fluid carbon dioxide. The best preparation conditions were determined, and the inclusion complex was investigated by means of 1H NMR spectroscopy in aqueous solution and 13C CPMAS NMR spectroscopy in the solid state. Information on the geometry of the betaCD/IMZ complex was obtained from ROESY spectroscopy, while the dynamics of the inclusion complex in the kilohertz range was obtained from the proton spin-lattice relaxation times in the rotating frame, T(1rho) (1H). 相似文献
95.
Scheggi S Leggio B Masi F Grappi S Gambarana C Nanni G Rauggi R De Montis MG 《Journal of neurochemistry》2002,83(4):895-903
Stressful events are accompanied by modifications in dopaminergic transmission in distinct brain regions. As the activity of the neuronal dopamine (DA) transporter (DAT) is considered to be a critical mechanism for determining the extent of DA receptor activation, we investigated whether a 3-week exposure to unavoidable stress, which produces a reduction in DA output in the nucleus accumbens shell (NAcS) and medial prefrontal cortex (mPFC), would affect DAT density and DA D1 receptor complex activity in the NAcS, mPFC and caudate-putamen (CPu). Rats exposed to unavoidable stress showed a decreased DA output in the NAcS accompanied by a decrease in the number of DAT binding sites, and an increase in the number of DA D1 binding sites and Vmax of SKF 38393-stimulated adenylyl cyclase. In the mPFC, stress exposure produced a decrease in DA output with no modification in DAT binding or in DA D1 receptor complex activity. Moreover, in the CPu stress exposure induced no changes in DA output or in the other neurochemical variables examined. This study shows that exposure to a chronic unavoidable stress that produces a decrease in DA output in frontomesolimbic areas induced several adaptive neurochemical modifications selectively in the nucleus accumbens. 相似文献
96.
Poggi A Zocchi MR Carosio R Ferrero E Angelini DF Galgani S Caramia MD Bernardi G Borsellino G Battistini L 《Journal of immunology (Baltimore, Md. : 1950)》2002,168(12):6071-6077
We have previously reported that the Vdelta2(+)TCRgammadelta(+) T lymphocyte subset, expressing the NK receptor protein 1a (NKRP1a; CD161), is expanded in patients with relapsing-remitting multiple sclerosis and uses this molecule to migrate through endothelium. In this work, we show that Vdelta1(+) and Vdelta2(+) gammadelta T lymphocytes use distinct signal transduction pathways to accomplish this function. Indeed, we have found that Vdelta1(+) cells lack NKRP1a and selectively express the platelet endothelial cell adhesion molecule 1 (PECAM1; CD31), which drives transendothelial migration of this cell subset, at variance with Vdelta2(+) T cells, which are PECAM1 negative and use NKRP1a for transmigration. Interestingly, when Vdelta2(+) T cells were pretreated with two specific inhibitors of the calcium calmodulin-dependent kinase II KN62 and KN93, but not with the inactive compound KN92, the number of migrating cells and the rate of transmigration were significantly decreased. In turn, the phosphatidylinositol 3 kinase blockers wortmannin and LY294002 exerted a dose-dependent inhibition of Vdelta1(+) cell migration. Finally, NKRP1a and PECAM1 engagement led to activation of different signal transduction pathways: indeed, oligomerization of NKRP1a on Vdelta2(+) T cells activates calcium calmodulin-dependent kinase II, while occupancy of PECAM1 on Vdelta1(+) cells triggers the phosphatidylinositol 3 kinase-dependent Akt/protein kinase Balpha activation. These findings suggest that subsets of gammadelta T lymphocytes may migrate to the site of lesion in multiple sclerosis using two different signaling pathways to extravasate. 相似文献
97.
Regeneration of sensory axons within the injured spinal cord induced by intraganglionic cAMP elevation 总被引:47,自引:0,他引:47
The peripheral branch of primary sensory neurons regenerates after injury, but there is no regeneration when their central branch is severed by spinal cord injury. Here we show that microinjection of a membrane-permeable analog of cAMP in lumbar dorsal root ganglia markedly increases the regeneration of injured central sensory branches. The injured axons regrow into the spinal cord lesion, often traversing the injury site. This result mimics the effect of a conditioning peripheral nerve lesion. We also demonstrate that sensory neurons exposed to cAMP in vivo, when subsequently cultured in vitro, show enhanced growth of neurites and an ability to overcome inhibition by CNS myelin. Thus, stimulating cAMP signaling increases the intrinsic growth capacity of injured sensory axons. This approach may be useful in promoting regeneration after spinal cord injury. 相似文献
98.
Sarnataro D Paladino S Campana V Grassi J Nitsch L Zurzolo C 《Traffic (Copenhagen, Denmark)》2002,3(11):810-821
PrP(C) is a glycosylphosphatidylinositol-anchored protein expressed in neurons as well as in the cells of several peripheral tissues. Although the normal function of PrP(C) remains unknown, a conformational isoform called PrP(Sc) (scrapie) has been proposed to be the infectious agent of transmissible spongiform encephalopathies in animals and humans. Where and how the PrP(C) to PrP(Sc) conversion occurs in the cells is not yet known. Therefore, dissecting the intracellular trafficking of the wild-type prion protein, as well as of the scrapie isoform, can be of major relevance to the pathogenesis of the diseases. In this report we have analyzed the exocytic pathway of transfected mouse PrP(C) in thyroid and kidney polarized epithelial cells. In contrast to the majority of glycosylphosphatidylinositol-anchored proteins, we found that PrP(C) is localized mainly on the basolateral domain of the plasma membrane of both cell lines. This is reminiscent of the predominant somatodendritic localization found in neurons. However, similarly to apical glycosylphosphatidylinositol-proteins, PrP(C) associates with detergent-resistant microdomains, which have been suggested to have a role in apical sorting of glycosylphosphatidylinositol-proteins, as well as in the conversion process of PrP(C) to PrP(Sc). In order to discriminate whether detergent-resistant microdomains have a direct role in PrP(Sc) conversion, or whether they are involved in the transport of the protein to the site of its conversion, we have examined the effect of disruption of detergent-resistant microdomain association on PrP(C) intracellular traffic. Consistent with the unusual basolateral localization of this glycosylphosphatidylinositol-linked protein, our data exclude a classical role for detergent-resistant microdomains in the post-trans-Golgi network sorting and transport of PrP(C) to the plasma membrane. 相似文献
99.
100.
DNA-based methods for the detection and the identification of phytoplasmas in insect vector extracts
DNA extraction and storage methods have been evaluated with laboratory-reared leafhoppers and/or field-collected leafhoppers
and psyllids. Detection of four different phytopathogenic phytoplasmas, belonging to three taxonomic groups, has been achieved
by several direct or nested polymerase chain reaction (PCR) methods with such DNA extracts. Reactions differed in both the
16/23S ribosomal primer pairs used and the specific assay and cycling conditions. Merits and possible hindrances of the various
primer pairs, in relation to insect DNA extracts, are discussed. However, identification of the phytoplasma(s) necessarily
relied on comparison of the polymorphism in length of the amplified DNA fragments obtained by restriction with appropriate
endonucleases. Endonuclease digestion is crucial for determining the identity (subgroup affiliation) of phytoplasmas of the
same groups that can be carried by an individual vector. 相似文献