Glycerolipid labelling kinetics in isolated intact chloroplasts.
Ontology highlight
ABSTRACT:
SUBMITTER: Gardiner SE
PROVIDER: S-EPMC1144475 | biostudies-other | 1984 Dec
REPOSITORIES: biostudies-other
Similar Datasets
Project description:BackgroundPerforming chloroplast DNA (cpDNA) isolation is considered a major challenge among different plant groups, especially conifers. Isolating chloroplasts in conifers by such conventional methods as sucrose gradient and high salt has not been successful. So far, plastid genome sequencing protocols for conifer species have been based mainly on long-range PCR, which is known to be time-consuming and difficult to implement.Methodology/principal findingsWe developed a protocol for cpDNA isolation using three different conifer families: Araucaria angustifolia and Araucaria bidwilli (Araucariaceae), Podocarpus lambertii (Podocarpaceae) and Pinus patula (Pinaceae). The present protocol is based on high salt isolation buffer followed by saline Percoll gradient. Combining these two strategies allowed enhanced chloroplast isolation, along with decreased contamination caused by polysaccharides, polyphenols, proteins, and nuclear DNA in cpDNA. Microscopy images confirmed the presence of intact chloroplasts in high abundance. This method was applied to cpDNA isolation and subsequent sequencing by Illumina MiSeq (2×250 bp), using only 50 ng of cpDNA. Reference-guided chloroplast genome mapping showed that high average coverage was achieved for all evaluated species: 24.63 for A. angustifolia, 135.97 for A. bidwilli, 1196.10 for P. lambertii, and 64.68 for P. patula.ConclusionResults show that this improved protocol is suitable for enhanced quality and yield of chloroplasts and cpDNA isolation from conifers, providing a useful tool for studies that require isolated chloroplasts and/or whole cpDNA sequences.
| S-EPMC3879346 | biostudies-literature
Project description:1. Chloroplasts isolated from spinach leaves by using the low-ionic-strength buffers of Nakatani & Barber [(1977) Biochim. Biophys. Acta.461, 510-512] had higher rates of HCO(3) (-)-dependent oxygen evolution (up to 369mumol/h per mg of chlorophyll) and higher rates of [1-(14)C]acetate incorporation into long-chain fatty acids (up to 1500nmol/h per mg of chlorophyll) than chloroplasts isolated by using alternative procedures. 2. Acetate appeared to be the preferred substrate for fatty acid synthesis by isolated chloroplasts, although high rates of synthesis were also measured from H(14)CO(3) (-) in assays permitting high rats of photosynthesis. Incorporation of H(14)CO(3) (-) into fatty acids was decreased by relatively low concentrations of unlabelled acetate. Acetyl-CoA synthetase activity was present 3-4 times in excess of that required to account for rates of [1-(14)C]acetate incorporation into fatty acids, but pyruvate dehydrogenase was either absent or present in very low activity in spinach chloroplasts. 3. Rates of long-chain-fatty acid synthesis from [1-(14)C]acetate in the highly active chloroplast preparations, compared with those used previously, were less dependent on added cofactors, but showed a greater response to light. The effects of added CoA plus ATP, Triton X-100 and sn-glycerol 3-phosphate on the products of [1-(14)C]acetate incorporation were similar to those reported for less active chloroplast preparations. 4. Endogenous [(14)C]acetyl-CoA plus [(14)C]malonyl-CoA was maintained at a constant low level even when fatty acid synthesis was limited by low HCO(3) (-) concentrations. Endogenous [(14)C]acyl-(acyl-carrier protein) concentrations increased with increasing HCO(3) (-) concentration and higher rates of fatty acid synthesis, but were slightly lower in the presence of Triton X-100. It is proposed that rates of long-chain-fatty acid synthesis in isolated chloroplasts at saturating [1-(14)C]acetate concentrations and optimal HCO(3) (-) concentrations may be primarily controlled by rates of removal of the products of the fatty acid synthetase.
| S-EPMC1161752 | biostudies-other
Project description:RNA levels detected at steady state are the consequence of multiple dynamic processes within the cell. In addition to synthesis and decay, many transcripts undergo processing. For example, in the case of intron-containing transcripts, there is splicing to take into the equation. Metabolic tagging with a nucleotide analogue is one way of determining the relative contributions of synthesis, decay and conversion processes globally. By using a much refined method of 4-thiouracil labelling in Saccharomyces cerevisiae we can isolate RNA produced during as little as 1 min, allowing the detection of RNA species with high turn-over rates, including intron-containing pre-mRNAs and short-lived non-coding RNAs. Nascent RNA labelled for 1.5, 2.5 or 5 minutes was isolated and analysed by reverse transcriptase-quantitative PCR and RNA sequencing. From these data we measured the relative stability of pre-mRNA species with different high turn-over rates and investigated potential correlations with intron features. This extremely brief metabolic labeling method enables the isolation of short-lived RNA species and the production of transcriptome-wide high-resolution kinetic data with some unexpected results . Previous studies using reporter genes suggested that secondary structure in introns is favourable for efficient splicing and may affect splice site usage. In contrast, our data reveal that ribosomal protein transcripts with intron secondary structures that are predicted to be less stable, splice faster. These data, in combination with previous results, indicate that there is an optimal range of stability of intron secondary structures that allows for rapid splicing.
2015-11-19 | GSE70378 | GEO
Project description:Isolated pea chloroplasts undergo both cyclic and non-cyclic electron flow. Both processes are coupled to photophosphorylation. During non-cyclic flow the rate of oxygen production showed ADP-governed ;photosynthetic control' analogous to respiratory control of isolated mitochondria. Measurements of ADP/O and photosynthetic control ratios yielded values of 1-1.3 and 2-5.7 respectively. ;Photosynthetic control' was shown to be dependent on the intactness of the chloroplasts.
| S-EPMC1186938 | biostudies-other
Project description:The mechanism and kinetics of electron transfer in isolated D1/D2-cyt(b559) photosystem (PS) II reaction centers (RCs) and in intact PSII cores have been studied by femtosecond transient absorption and kinetic compartment modeling. For intact PSII, a component of approximately 1.5 ps reflects the dominant energy-trapping kinetics from the antenna by the RC. A 5.5-ps component reflects the apparent lifetime of primary charge separation, which is faster by a factor of 8-12 than assumed so far. The 35-ps component represents the apparent lifetime of formation of a secondary radical pair, and the approximately 200-ps component represents the electron transfer to the Q(A) acceptor. In isolated RCs, the apparent lifetimes of primary and secondary charge separation are approximately 3 and 11 ps, respectively. It is shown (i) that pheophytin is reduced in the first step, and (ii) that the rate constants of electron transfer in the RC are identical for PSII cores and for isolated RCs. We interpret the first electron transfer step as electron donation from the primary electron donor Chl(acc D1). Thus, this mechanism, suggested earlier for isolated RCs at cryogenic temperatures, is also operative in intact PSII cores and in isolated RCs at ambient temperature. The effective rate constant of primary electron transfer from the equilibrated RC* excited state is 170-180 ns(-1), and the rate constant of secondary electron transfer is 120-130 ns(-1).
| S-EPMC1458990 | biostudies-literature
Project description:Amyloid formation is involved in a wide range of neurodegenerative diseases including Alzheimer's and prion diseases. Structural understanding of the amyloid is critical to delineate the mechanism of aggregation and its pathological spreading. Site-directed spin labelling has emerged as a powerful structural tool in the studies of amyloid structures and provided structural evidence for the parallel in-register β-sheet structure for a wide range of amyloid proteins. It is generally accepted that spin labelling does not disrupt the structure of the amyloid fibrils, the end product of protein aggregation. The effect on the rate of protein aggregation, however, has not been well characterized. Here, we employed a scanning mutagenesis approach to study the effect of spin labelling on the aggregation rate of 79 spin-labelled variants of the Ure2 prion domain. The aggregation of Ure2 protein is the basis of yeast prion [URE3]. We found that all spin-labelled Ure2 mutants aggregated within the experimental timeframe of 15 to 40 h. Among the 79 spin-labelled positions, only five residue sites (N23, N27, S33, I35 and G42) showed a dramatic delay in the aggregation rate as a result of spin labelling. These positions may be important for fibril nucleation, a rate-limiting step in aggregation. Importantly, spin labelling at most of the sites had a muted effect on Ure2 aggregation kinetics, showing a general tolerance of spin labelling in protein aggregation studies.
| S-EPMC8074925 | biostudies-literature
Project description:The incorporation of [2-(14)C,(5R)-5-(3)H(1)]MVA* and [2-(14)C,5-(3)H(2)]MVA into geranylgeraniol and phytoene by a preparation of ;non-aqueous' bean leaf chloroplasts has been studied. In the formation of phytoene from two molecules of geranylgeranyl pyrophosphate, the loss of hydrogen is stereospecific, the hydrogen atom lost from C-1 of each molecule of geranylgeranyl pyrophosphate being that which was originally the pro-S hydrogen atom from C-5 of mevalonate. All the pro-R hydrogen atoms from C-5 of mevalonate are retained. These results with a cell-free system confirm and extend the observations made in previous work with tomato slices.
| S-EPMC1184938 | biostudies-other
Project description:Antibody-conjugated nanomaterials have attracted much attention because of their applications in nanomedicine and nanotheranostics, and amplification of detection signals. For many of these applications, the nanoconjugates must bind with a cell membrane receptor (antigen) specifically before entering the cells and reaching the final target, which is thus important but not well understood. Here, a plasmonic imaging study of the binding kinetics of antibody-conjugated gold nanoparticles with antigen-expressing cells is presented, and the results are compared with that of the nanoparticle-free antibody. It is found that the nanoconjugates can significantly affect the binding kinetics compared with free antibody molecules, depending on the density of the antibody conjugated on the nanoparticles, and expressing level of the antigen on the cell membrane. The results are analyzed in terms of a transition from monovalent binding model to a bivalent binding model when the conjugation density and expressing level increase. These findings help optimize the design of functional nanomaterials for drug delivery and correct interpretation of data obtained with nanoparticle signal amplification.
| S-EPMC4552349 | biostudies-literature
Project description:Stromules are stroma-containing tubules that have been observed to emanate from the main plastidic body in vivo. These structures have been shown to require cytoskeletal components for movement. Though numerous studies have shown a close association with the endoplasmic reticulum, nucleus, mitochondria, and other plastids, the mechanism of formation and their overall function remain unknown. A limiting factor in studying these structures has been the lack of a reconstituted system for in vitro stromule formation. In this study, stromule formation was induced in vitro by adding a plant extract fraction that is greater than 100 kDa to a population of isolated chloroplasts. Kinetic measurements show that stromule formation occurs within ~10 seconds after the addition of the plant extract fraction. Heat inactivation and apyrase treatment reveal that the stromule stimulating compound found in the extract fraction is a protein or protein complex 100 kDa or greater. The formation of the stromules in vitro with isolated chloroplasts and a concentrated fraction of cell extract opens an avenue for the biochemical dissection of this process that has heretofore been studied only in vivo.
| S-EPMC4739594 | biostudies-literature
Project description:The moss Physcomitrella patens is increasingly being used as a model for plant systems biology studies. While genomic and transcriptomic resources are in place, tools and experimental conditions for proteomic studies need to be developed. In the present study we describe a rapid and efficient protocol for the simultaneous isolation of chloroplasts and mitochondria from moss protonema. Routinely, 60-100 ?g mitochondrial and 3-5 mg chloroplast proteins, respectively, were obtained from 20 g fresh weight of green moss tissue. Using 14 plant compartment marker antibodies derived from seed plant and algal protein sequences, respectively, the evolutionary conservation of the compartment marker proteins in the moss was demonstrated and purity and intactness of the extracted organelles confirmed. This isolation protocol and these validated compartment markers may serve as basis for sub-cellular proteomics in P. patens and other mosses.
| S-EPMC3020298 | biostudies-literature