The acceptor quinone complex of Rhodopseudomonas viridis reaction centers

The acceptor complex of isolated reaction centers from Rhodopseudomonas viridis contains both menaquinone and ubiquinone. In a series of flashes the ubiquinone was observed to undergo binary oscillations in the formation and disappearance of a semiquinone, indicative of secondary acceptor (QB) activity. The oscillating signal, Q-B, was typical of a ubisemiquinone anion with a peak at 450 nm (delta epsilon = 6 mM-1 X cm-1) and a shoulder at 430 nm. Weak electrochromic bandshifts in the infrared were also evident. The spectrum of the reduced primary acceptor (Q-A) exhibited a major peak at 412 nm (delta epsilon = 10 mM-1 X cm-1) consistent with the assignment of menaquinone as QA. The Q-A spectrum also had minor peaks at 385 and 455 nm in the blue region. The same spectrum was recorded after quantitative removal of the secondary acceptor, when only menaquinone was present in the reaction centers. Spectral features in the near-infrared due to Q-A were attributed to electrochromic effects on bacteriochlorophyll (BChl) b and bacteriopheophytin (BPh) b pigments resulting in a distinctive split peak at 810 and 830 nm (delta epsilon = 8 mM-1 X cm-1). The menaquinone was identified as 2-methyl-3-nonylisoprenyl-1,4-naphthoquinone (menaquinone-9). The native QA activity was uniquely provided by this menaquinone and ubiquinone was not involved. QB activity, on the other hand, displayed at least a 40-fold preference for ubiquinone (Q-10) as compared to menaquinone. Thus, both quinone-binding sites display remarkable specificity for their respective quinones. In the absence of donors to P+, charge recombination of the P+Q-A and P+Q-B pairs had half-times of 1.1 +/- 0.2 and 110 +/- 20 ms, respectively, at pH 9.0, indicating an electron-transfer equilibrium constant (Kapp2) of at least 100 for Q-AQB in equilibrium QAQ-B. Also observed was a slow recombination of the cytochrome c-558+ Q-A pair, with t 1/2 = 2 +/- 0.5 s at pH 6.     

MHC class II invariant chains in antigen processing and presentation

Most protein antigens cannot elicit a T-cell response unless they are processed to peptides, which are then presented to T lymphocytes by surface MHC class II molecules. Recent evidence supports an essential role of the invariant chain associated with class II MHC polypeptides in antigen processing.     

Inhibition and labelling of isolated reaction centers from Rhodobacter sphaeroides by dicyclohexylcarbodiimide

The effect of dicyclohexylcarbodiimide (DCCD) on electron transfer in the acceptor quinone complex of reaction centers (RC) from Rhodobacter sphaeroides is reported. DCCD covalently labelled the RC over a wide concentration range. At low concentrations (<10 M) the binding was specific for the L subunit. At relatively high concentrations (>100 M) DCCD accelerated the rate of charge recombination of the P⁺Q_B ^- state, consistent with a decrease in the equilibrium constant between Q_A ^-Q_B and Q_AQ_B ^-. At similar concentrations, in the presence of cytochrome c as exogenous donor, turnover of the RC was inhibited such that only three cytochromes were oxidized in a train of flashes. Both these inhibitory effects were fully reversed by dialysis, indicating that stable covalent binding was not involved. Possible mechanisms of action are discussed in terms of the putative role of specific residues in proton transfer and protonation and release of quinol from the RC.     

Investigations on the influence of headgroup substitution and isoprene side-chain length in the function of primary and secondary quinones of bacterial reaction centers

The contributions of headgroup and side-chain in the binding and function of the primary (QA) and secondary (QB) quinones of isolated reaction centers (RCs) from Rhodobacter sphaeroides were investigated. Various ubiquinones and structurally similar quinones were reconstituted into RCs depleted of one (1Q-RCs) or both (0Q-RCs) quinones. The influence of partition coefficients on the apparent binding affinities was minimized by expressing dissociation constants in terms of the mole fraction of quinone partitioned into the detergent. It was then apparent that the size of the isoprenyl side-chain was of little consequence in determining the binding affinity or the functional competence of either QA or QB, although an alkyl chain of equivalent size was a poor substitute. The degree of substitution of the headgroup, however, was a sensitive determinant of binding. For both quinone sites, the trisubstituted plastoquinones bond more weakly than the fully substituted ubiquinones. Similarly, for binding to the QA site, duroquinone (tetramethylbenzoquinone) bound much more strongly than trimethylbenzoquinone. The affinity of the QA site for ubiquinones was about 20-times stronger than the QB site, but the QB site is probably not more specific than the QA site. However, QB function depends on a suitable redox free-energy drop from QA as well as binding, and of all the quinones tested only the ubiquinones simultaneously supported full QA and QB activity. Even plastoquinone-A, which fills both roles in Photosystem II, was unable to do so in bacterial RCs, although it did bind. The unique ability of ubiquinones to both bind and provide the appropriate redox span is discussed. The temperature dependence of binding of the isoprenyl ubiquinones at the QA site changed markedly with chain length. For Q-10-Q-7, the binding enthalpy was positive and net binding was entirely driven by entropic factors. For the shorter-chain ubiquinones, Q-6-Q-1, both entropy and enthalpy of binding were favorable. This strong entropy-enthalpy compensation is suggested to arise from antagonistic interactions (anticooperativity) between headgroup and tail binding. For QB function by hydrophobic quinones, the temperature dependence of the micelle properties prevented easy access to thermodynamic parameters. However, for water-soluble Q-0, binding to the QB site was determined to be enthalpically driven.(ABSTRACT TRUNCATED AT 400 WORDS)     

Electron acceptors of bacterial photosynthetic reaction centers. II. H+ binding coupled to secondary electron transfer in the quinone acceptor complex.

The photoreduction of ubiquinone in the electron acceptor complex (QIQII) of photosynthetic reaction centers from Rhodopseudomonas sphaeroides, R26, was studied in a series of short, saturating flashes. The specific involvement of H+ in the reduction was revealed by the pH dependence of the electron transfer events and by net H+ binding during the formation of ubiquinol, which requires two turnovers of the photochemical act. On the first flash QII receives an electron via QI to form a stable ubisemiquinone anion (QII-); the second flash generates QI-. At low pH the two semiquinones rapidly disproportionate with the uptake of 2 H+, to produce QIIH2. This yields out-of-phase binary oscillations for the formation of anionic semiquinone and for H+ uptake. Above pH 6 there is a progressive increase in H+ binding on the first flash and an equivalent decrease in binding on the second flash until, at about pH 9.5, the extent of H+ binding is the same on all flashes. The semiquinone oscillations, however, are undiminished up to pH 9. It is suggested that a non-chromophoric, acid-base group undergoes a pK shift in response to the appearance of the anionic semiquinone and that this group is the site of protonation on the first flash. The acid-base group, which may be in the reaction center protein, appears to be subsequently involved in the protonation events leading to fully reduced ubiquinol. The other proton in the two electron reduction of ubiquinone is always taken up on the second flash and is bound directly to QII-. At pH values above 8.0, it is rate limiting for the disproportionation and the kinetics, which are diffusion controlled, are properly responsive to the prevailing pH. Below pH 8, however, a further step in the reaction mechanism was shown to be rate limiting for both H+ binding electron transfer following the second flash.     

Application of an optimized marker amplification protocol in immunohistochemistry — a valuable tool for visualizing antigenic sites of low signal strength or sparse distribution

Amplification of immunohistochemical markers received considerable attention during the 1980s and 1990s. The amplification approach was largely abandoned following the development of antigen retrieval and reporter amplification techniques, because the latter were incorporated more easily into high throughput automated procedures in industrial and diagnostic laboratories. There remain, however, a number of instances where marker amplification still has much to offer. Consequently, we examined experimentally the utility of an optimized marker amplification technique in diagnostically relevant tissue where either the original signal strength was low or positive sites were visible, but sparsely distributed. Marker amplification in the former case not only improved the visibility of existing positive sites, but also revealed additional sites that previously were undetectable. In the latter case, positive sites were rendered more intense and therefore more easily seen during low magnification examination of large areas of tissue.     

EFFECT OF VITAMIN E SUPPLEMENTATION AND PARTIAL SUBSTITUTION OF POLY- WITH MONO-UNSATURATED FATTY ACIDS IN PIG DIETS ON MUSCLE,AND MICROSOME EXTRACT a-TOCOPHEROL CONCENTRATION AND LIPID OXIDATION

The experiment was organized in a 3×2 factorial arrangement with three dietary fat blends and a basal (20 mg kg^?1 diet) or supplemented (220 mg kg^?1) level of α-tocopheryl acetate. Dietary vitamin E and monounsaturated to polyunsaturated fatty acid ratio (dietary MUFA/PUFA) affected muscle α-tocopherol concentration (α-tocopherol [log μg g^?1]=0.18 (±0.105)+0.0034 (±0.0003)·dietary α-tocopherol [mg kg^?1 diet] (P<0.0001)+0.39 (±0.122)·dietary MUFA/PUFA (P<0.0036)). An interaction between dietary α-tocopherol and dietary MUFA/PUFA exists for microsome α-tocopherol concentration (α-tocopherol [log μg g^?1]=1.14 (±0.169) (P<0.0001)+0.0056 (±0.00099)·dietary α-tocopherol [mg kg^?1 diet] (P<0.0001)+0.54 (±0.206)·dietary MUFA/PUFA (P<0.0131)?0.0033 (±0.0011)·dietary α-tocopherol [mg kg^?1)]×dietary MUFA/PUFA (P<0.0067)), and hexanal concentration in meat (hexanal [ng·g^?1]=14807.9 (±1489.8)?28.8 (±10.6) dietary α-tocopherol [mg·kg^?1] (P<0.01)?8436.6 (±1701.6)·dietary MUFA/PUFA (P<0.001)+24.0 (±11.22)·dietary α-tocopherol·dietary MUFA/PUFA (P<0.0416)). It is concluded that partial substitution of dietary PUFA with MUFA lead to an increase in the concentration of α-tocopherol in muscle and microsome extracts. An interaction between dietary α-tocopherol and fatty acids exists, in which at low level of dietary vitamin E inclusion, a low MUFA/PUFA ratio leads to a reduction in the concentration of α-tocopherol in microsome extracts and a concentration of hexanal in meat above the expected values.     

Quirks of dye nomenclature. 5. Rhodamines

Rhodamines were first produced in the late 19^th century, when they constituted a new class of synthetic dyes. These compounds since have been used to color many things including cosmetics, inks, textiles, and in some countries, food products. Certain rhodamine dyes also have been used to stain biological specimens and currently are widely used as fluorescent probes for mitochondria in living cells. The early history and current biological applications are sketched briefly and an account of the ambiguities, complications and confusions concerning dye identification and nomenclature are discussed.