PubMed · 14945521
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G ANNONI, N TENTO. [Vitamin B complex metabolism].. https://pubmed.ncbi.nlm.nih.gov/14945521/
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The cyanobacterium Synechocystis sp. PCC 6803 contains four members of the FtsH protease family. One of these, FtsH (slr0228), has been implicated recently in the repair of photodamaged photosystem II (PSII) complexes. We have demonstrated here, using a combination of blue native PAGE, radiolabeling, and immunoblotting, that FtsH (slr0228) is required for selective replacement of the D1 reaction center subunit in both wild type PSII complexes and in PSII subcomplexes lacking the PSII chlorophyll a-binding subunit CP43. To test whether FtsH (slr0228) has a more general role in protein quality control in vivo, we have studied the synthesis and degradation of PSII subunits in wild type and in defined insertion and missense mutants incapable of proper assembly of the PSII holoenzyme. We discovered that, when the gene encoding FtsH (slr0228) was disrupted in these strains, the overall level of assembly intermediates and unassembled PSII proteins markedly increased. Pulse-chase experiments showed that this was due to reduced rates of degradation in vivo. Importantly, analysis of epitope-tagged and green fluorescent protein-tagged strains revealed that slr0228 was present in the thylakoid and not the cytoplasmic membrane. Overall, our results show that FtsH (slr0228) plays an important role in controlling the removal of PSII subunits from the thylakoid membrane and is not restricted to selective D1 turnover.
Measuring stable isotope fractionation of carbon, hydrogen, and other elements by Compound Specific Isotope Analysis (CSIA) is a new, innovative approach to assess organic pollutant degradation in the environment. Central to this concept is the Rayleigh equation which relates degradation-induced decreases in concentrations directly to concomitant changes in bulk (= average over the whole compound) isotope ratios. The extent of in situ transformation may therefore be inferred from measured isotope ratios in field samples, provided that an appropriate enrichment factor (epsilonbulk) is known. This epsilonbulk value, however, is usually only valid for a specific compound and for specific degradation conditions. Therefore, a direct comparison of epsilonbulk values for different compounds and for different types of reactions has in general not been feasible. In addition, it is often uncertain how robust and reproducible epsilonbulk values are and how confidently they can be used to quantify contaminant degradation in the field. To improve this situation and to achieve a more in-depth understanding, this critical review aims to relate fundamental insight about kinetic isotope effects (KIE) found in the physico(bio)chemical literature to apparent kinetic isotope effects (AKIE) derived from epsilonbulk values reported in environmentally oriented studies. Starting from basic rate laws, a quite general derivation of the Rayleigh equation is given, resulting in a novel set of simple equations that take into account the effects of (1) nonreacting positions and (2) intramolecular competition and that lead to position-specific AKIE values rather than bulk enrichment factors. Reevaluation of existing epsilonbulk literature values result in consistent ranges of AKIE values that generally are in good agreement with previously published data in the (bio)-chemical literature and are typical of certain degradation reactions (subscripts C and H indicate values for carbon and hydrogen): AKIEc = 1.01-1.03 and AKIEH = 2-23 for oxidation of C-H bonds; AKIEc = 1.03-1.07 for SN2-reactions; AKIEc = 1.02-1.03 for reductive cleavage of C-Cl bonds; AKIEc = 1.00-1.01 for C=C bond epoxidation; AKIEc = 1.02-1.03 for C=C bond oxidation by permanganate. Hence, the evaluation scheme presented bridges a gap between basic and environmental (bio)chemistry and provides insight into factors that control the magnitude of bulk isotope fractionation factors. It also serves as a basis to identify degradation pathways using isotope data. It is shown how such an analysis may be even possible in complex field situations and/or in cases where AKIE values are smaller than intrinsic KIE values, provided that isotope fractionation is measured for two elements simultaneously ("two-dimensional isotope analysis"). Finally, the procedure is used (1) to point outthe possibility of estimating approximate epsilonbulk values for new compounds and (2) to discuss the moderate, but non-negligible variability that may quite generally be associated with epsilonbulk values. Future research is suggested to better understand and take into account the various factors that may cause such variability.
Methodology was developed for specifically anchoring Escherichia coli 70S ribosomes onto a chemically modified, cysteine-reactive glass surface. Immobilized ribosomes maintain the capability of binding a polyuridylic acid (poly(U)) template, enabling investigation of mechanical properties of individual ribosome-poly(U) complexes using laser tweezers. Streptavidin-coated polystyrene microspheres bound specifically to the biotinylated 3' end of long (up to 10,000 bases) poly(U) strands. A novel optical method was built to control the position of the laser trap along the microscope optical axis at 2 nm resolution, facilitating measurement of the force-extension relationship for poly(U). Some immobilized ribosome-poly(U) complexes supported 100 pN of force applied at the 3' end of the mRNA. Binding of N-acetylated Phe-tRNA(Phe), an analog of the initiator fMet-tRNA(Met), enhanced the population of complexes that could withstand high forces. The persistence length of poly(U) RNA homopolymer, modeled as a worm-like chain, was found to be 0.79 +/- 0.05 nm and the backbone elasticity was 900 +/- 140 pN, similar to values for single-stranded DNA.