Regulation of pentose phosphate pathway dehydrogenases by NADP+/NADPH ratios.

Equilibrium dialysis indicates that rat liver glucose-6-P dehydrogenase binds two molecules of NADP⁺ per subunit with a dissociation constant of 0.6 × 10^?6 M. The NADP⁺ free enzyme will not bind glucose-6-P indicating a compulsory order of substrate binding. Development of an isotopic assay allowed a direct measurement of the effect of physiological alterations in the NADP⁺/NADPH ratio on the activity of glucose-6-P and 6-phosphogluconate dehydrogenases. A combination of enzyme induction and altered NADP⁺/NADPH ratios could produce 30–50 fold changes in the capacity of these enzymes to produce NADPH during alterations in the nutritional state of the animal.     

Quantitation of messenger RNA levels for rat liver 6-phosphogluconate dehydrogenase

Liver poly(A)-containing RNA isolated from rats in different dietary states was translated in a cell free protein synthesizing system employing reticulocyte lysates. Immunoprecipitation of the cell-free reaction products with goat anti-6-phosphogluconate dehydrogenase followed by sodium dodecyl sulfate-urea-gel electrophoresis showed that the induction of this lipogenic enzyme was accompanied by a corresponding increase in the concentration of its specific translatable mRNA.     

Versatile design of biohybrid light-harvesting architectures to tune location,density, and spectral coverage of attached synthetic chromophores for enhanced energy capture

Biohybrid antennas built upon chromophore–polypeptide conjugates show promise for the design of efficient light-capturing modules for specific purposes. Three new designs, each of which employs analogs of the β-polypeptide from Rhodobacter sphaeroides, have been investigated. In the first design, amino acids at seven different positions on the polypeptide were individually substituted with cysteine, to which a synthetic chromophore (bacteriochlorin or Oregon Green) was covalently attached. The polypeptide positions are at –2, –6, –10, –14, –17, –21, and –34 relative to the 0-position of the histidine that coordinates bacteriochlorophyll a (BChl a). All chromophore–polypeptides readily formed LH1-type complexes upon combination with the α-polypeptide and BChl a. Efficient energy transfer occurs from the attached chromophore to the circular array of 875 nm absorbing BChl a molecules (denoted B875). In the second design, use of two attachment sites (positions –10 and –21) on the polypeptide affords (1) double the density of chromophores per polypeptide and (2) a highly efficient energy-transfer relay from the chromophore at –21 to that at –10 and on to B875. In the third design, three spectrally distinct bacteriochlorin–polypeptides were prepared (each attached to cysteine at the –14 position) and combined in an ~1:1:1 mixture to form a heterogeneous mixture of LH1-type complexes with increased solar coverage and nearly quantitative energy transfer from each bacteriochlorin to B875. Collectively, the results illustrate the great latitude of the biohybrid approach for the design of diverse light-harvesting systems.     

Determination of the rate and yield of B-side quinone reduction in Rhodobacter capsulatus reaction centers

In the native purple bacterial reaction center (RC), light-driven charge separation utilizes only the A-side cofactors, with the symmetry related B-side inactive. The process is initiated by electron transfer from the excited primary donor (P*) to the A-side bacteriopheophytin (P* --> P+ H(A)-) in approximately 3 ps. This is followed by electron transfer to the A-side quinone (P+ H(A)- --> P+ Q(A)-) in approximately 200 ps, with an overall quantum yield of approximately 100%. Using nanosecond flash photolysis and RCs from the Rhodobacter capsulatus F(L181)Y/Y(M208)F/L(M212)H mutant (designated YFH), we have probed the decay pathways of the analogous B-side state P+ H(B)-. The rate of the P+ H(B)- --> ground-state charge-recombination process is found to be (3.0 +/- 0.8 ns)(-1), which is much faster than the analogous (10-20 ns)(-1) rate of P+ H(A)- --> ground state. The rate of P+ H(B)- --> P+ Q(B)- electron transfer is determined to be (3.9 +/- 0.9 ns)(-1), which is about a factor of 20 slower than the analogous A-side process P+ H(A)- --> P+ Q(A)-. The yield of P+ H(B)- --> P+ Q(B)- electron-transfer calculated from these rate constants is 44%. This value, when combined with the known 30% yield of P+ H(B)- from P in YFH RCs, gives an overall yield of 13% for B-side charge separation P* --> P+ H(B)- --> P+ Q(B)- in this mutant. We determine essentially the same value (15%) by comparing the P-bleaching amplitude at approximately 1 ms in YFH and wild-type RCs.     

Increased Platelet Reactivity Is Associated with Circulating Platelet-Monocyte Complexes and Macrophages in Human Atherosclerotic Plaques

Objective

Platelet reactivity, platelet binding to monocytes and monocyte infiltration play a detrimental role in atherosclerotic plaque progression. We investigated whether platelet reactivity was associated with levels of circulating platelet-monocyte complexes (PMCs) and macrophages in human atherosclerotic carotid plaques.

Methods

Platelet reactivity was determined by measuring platelet P-selectin expression after platelet stimulation with increasing concentrations of adenosine diphosphate (ADP), in two independent cohorts: the Circulating Cells cohort (n = 244) and the Athero-Express cohort (n = 91). Levels of PMCs were assessed by flow cytometry in blood samples of patients who were scheduled for percutaneous coronary intervention (Circulating Cells cohort). Monocyte infiltration was semi-quantitatively determined by histological examination of atherosclerotic carotid plaques collected during carotid endarterectomy (Athero-Express cohort).

Results

We found increased platelet reactivity in patients with high PMCs as compared to patients with low PMCs (median (interquartile range): 4153 (1585–11267) area under the curve (AUC) vs. 9633 (3580–21565) AUC, P<0.001). Also, we observed increased platelet reactivity in patients with high macrophage levels in atherosclerotic plaques as compared to patients with low macrophage levels in atherosclerotic plaques (mean±SD; 8969±3485 AUC vs. 7020±3442 AUC, P = 0.02). All associations remained significant after adjustment for age, sex and use of drugs against platelet activation.

Conclusion

Platelet reactivity towards ADP is associated with levels of PMCs and macrophages in human atherosclerotic carotid plaques.     

Collagen stimulation of platelets induces a rapid spatial response of cAMP and cGMP signaling scaffolds

Intracellular communication is tightly regulated in both space and time. Spatiotemporal control is important to achieve a high level of specificity in both dimensions. For instance, cAMP-dependent kinase (PKA) attains spatial resolution by interacting with distinct members of the family of A-kinase anchoring proteins (AKAPs) that position PKA at specific loci within the cell. To control the cAMP induced signal in time, distinct signal terminators such as phosphodiesterases and phosphatases are often co-localized at the AKAP scaffold. In platelets, high levels of cAMP/cGMP maintain the resting state to allow free circulation. Exposure to collagen, for instance when the vessel is damaged, triggers platelet activation through initiation of the GPVI (glycoprotein VI)/FcRγ-chain forming the onset of a plethora of signaling pathways. Consequently overall intra-platelet cAMP and cGMP levels drop, however detail on how PKA, but also cGMP-dependent protein kinase (PKG) respond in relation to their localized signaling scaffolds is currently missing. To investigate this, we employed a quantitative chemical proteomics approach in activated human platelets enabling the specific enrichment of cAMP/cGMP signaling nodes. Our data reveal that within a few minutes several specific PKA and PKG signaling nodes respond significantly to the activating signal, whereas others do not, suggesting a rapid adaption of specific localized cAMP and cGMP pools to the stimulus. Using protein phosphorylation data gathered we touch upon the potential cross-talk between protein phosphorylation and signaling scaffold function as a general theme in platelet spatiotemporal control.     

New insights into the photochemistry of carotenoid spheroidenone in light-harvesting complex 2 from the purple bacterium <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Light-harvesting complex 2 (LH2) from the semi-aerobically grown purple phototrophic bacterium Rhodobacter sphaeroides was studied using optical (static and time-resolved) and resonance Raman spectroscopies. This antenna complex comprises bacteriochlorophyll (BChl) a and the carotenoid spheroidenone, a ketolated derivative of spheroidene. The results indicate that the spheroidenone-LH2 complex contains two spectral forms of the carotenoid: (1) a minor, “blue” form with an S₂ (1¹B _u ⁺ ) spectral origin band at 522 nm, shifted from the position in organic media simply by the high polarizability of the binding site, and (2) the major, “red” form with the origin band at 562 nm that is associated with a pool of pigments that more strongly interact with protein residues, most likely via hydrogen bonding. Application of targeted modeling of excited-state decay pathways after carotenoid excitation suggests that the high (92%) carotenoid-to-BChl energy transfer efficiency in this LH2 system, relative to LH2 complexes binding carotenoids with comparable double-bond conjugation lengths, derives mainly from resonance energy transfer from spheroidenone S₂ (1¹B _u ⁺ ) state to BChl a via the Q_x state of the latter, accounting for 60% of the total transfer. The elevated S₂ (1¹B _u ⁺ ) → Q_x transfer efficiency is apparently associated with substantially decreased energy gap (increased spectral overlap) between the virtual S₂ (1¹B _u ⁺ ) → S₀ (1¹A _g ^? ) carotenoid emission and Q_x absorption of BChl a. This reduced energetic gap is the ultimate consequence of strong carotenoid–protein interactions, including the inferred hydrogen bonding.     

Relationship between altered structure and photochemistry in mutant reaction centers in which bacteriochlorophyll replaces the photoactive bacteriopheophytin

Qy-excitation resonance Raman (RR) spectra are reported for two mutant reaction centers (RCs) from Rhodobacter capsulatus in which the photoactive bacteriopheophytin (BPhL) is replaced by a bacteriochlorophyll (BChl) molecule, designated beta. The pigment change in both mutants is induced via introduction of a histidine residue near the photoactive cofactor. In one mutant, L(M212)H, the histidine is positioned over the core of the cofactor and serves as an axial ligand to the Mg+2 ion. In the other mutant, F(L121)H/F(L97)V, the histidine is positioned over ring V of the cofactor, which is nominally too distant to permit bonding to the Mg+2 ion. The salient observations are as follows: (1) The beta cofactor in F(L121)H/F(L97)V RCs is a five-coordinate BChl molecule. However, there is no evidence for the formation of a Mg-His bond. This bond is either much weaker than in the L(M212)H RCs or completely absent, the latter implying coordination by an alternative ligand. The different axial ligation for beta in the F(L121)H/F(L97)V versus L(M212)H RCs in turn leads to different conformations of the BChl macrocycles. (2) The C9-keto group of beta in F(L121)H/F(L97)V RCs is free of hydrogen bonding interactions, unlike the L(M212)H RCs in which the C9-keto of beta is hydrogen bonded to Glu L104. The interactions between other peripheral substituents of beta and the protein are also different in the F(L121)H/F(L97)V RCs versus L(M212)H RCs. Accordingly, the position and orientation of beta in the protein is different in the two beta-containing RCs. Nonetheless, previous studies have shown that the primary electron transfer reactions are very similar in the two mutants but differ in significant respects compared to wild-type RCs. Collectively, these observations indicate that changes in the conformation of a photoactive tetrapyrrole macrocycle or its interactions with the protein do not necessarily lead to significantly perturbed photochemistry and do not underlie the altered primary events in beta-type RCs.     

Engineering of B800 bacteriochlorophyll binding site specificity in the Rhodobacter sphaeroides LH2 antenna

The light-harvesting 2 complex (LH2) of the purple phototrophic bacterium Rhodobacter sphaeroides is a highly efficient, light-harvesting antenna that allows growth under a wide-range of light intensities. In order to expand the spectral range of this antenna complex, we first used a series of competition assays to measure the capacity of the non-native pigments 3-acetyl chlorophyll (Chl) a, Chl?d, Chl?f or bacteriochlorophyll (BChl) b to replace native BChl?a in the B800 binding site of LH2. We then adjusted the B800 site and systematically assessed the binding of non-native pigments. We find that Arg_?10 of the LH2 β polypeptide plays a crucial role in binding specificity, by providing a hydrogen-bond to the 3-acetyl group of native and non-native pigments. Reconstituted LH2 complexes harbouring the series of (B)Chls were examined by transient absorption and steady-state fluorescence spectroscopies. Although slowed 10-fold to ~6?ps, energy transfer from Chl?a to B850 BChl?a remained highly efficient. We measured faster energy-transfer time constants for Chl?d (3.5?ps) and Chl?f (2.7?ps), which have red-shifted absorption maxima compared to Chl?a. BChl?b, red-shifted from the native BChl?a, gave extremely rapid (≤0.1?ps) transfer. These results show that modified LH2 complexes, combined with engineered (B)Chl biosynthesis pathways in vivo, have potential for retaining high efficiency whilst acquiring increased spectral range.     

Consequences of saturation mutagenesis of the protein ligand to the B-side monomeric bacteriochlorophyll in reaction centers from Rhodobacter capsulatus

In bacterial reaction centers (RCs), photon-induced initial charge separation uses an A-side bacteriochlorophyll (BChl, B_A) and bacteriopheophytin (BPh, H_A), while the near-mirror image B-side B_B and H_B cofactors are inactive. Two new sets of Rhodobacter capsulatus RC mutants were designed, both bearing substitution of all amino acids for the native histidine M180 (M-polypeptide residue 180) ligand to the core Mg ion of B_B. Residues are identified that largely result in retention of a BChl in the B_B site (Asp, Ser, Pro, Gln, Asn, Gly, Cys, Lys, and Thr), ones that largely harbor the Mg-free BPh in the B_B site (Leu and Ile), and ones for which isolated RCs are comprised of a substantial mixture of these two RC types (Ala, Glu, Val, Met and, in one set, Arg). No protein was isolated when M180 is Trp, Tyr, Phe, or (in one set) Arg. These findings are corroborated by ground state spectra, pigment extractions, ultrafast transient absorption studies, and the yields of B-side transmembrane charge separation. The changes in coordination chemistries did not reveal an RC with sufficiently precise poising of the redox properties of the B_B-site cofactor to result in a high yield of B-side electron transfer to H_B. Insights are gleaned into the amino acid properties that support BChl in the B_B site and into the widely observed multi-exponential decay of the excited state of the primary electron donor. The results also have direct implications for tuning free energies of the charge-separated intermediates in RCs and mimetic systems.