Multiple molecular recognition properties of the lipocalin protein family

The lipocalins, a diverse family of small extracellular ligand proteins, display a remarkable range of different molecular properties. While their binding of small hydrophobic molecules, and to a lesser extent their binding to cell surface receptors, is well known, it is shown here that formation of macromolecular complexes is also a common feature of this family. Analysis of known crystallographic structures reveals that the lipocalins process a conserved common structure: an antiparallel β-barrel with a repeated +1 topology. Comparisons show that within this overall similarity the structure of individual proteins is specifically adapted to bind their particular ligands, forming a binding site from an internal cavity (within the barrel) and/or an external loop scaffold, which gives rise to different binding modes that reflects the need to accommodate ligands of different shape, size, and chemical structure. The architecture of the lipocalin fold suggests that the both the ends and sides of this barrel are topologically distinct, differences also apparent in analyses of structural and sequence variation within the family. These different can be linked to experimental evidence suggesting a possible functional dichotomy between the two ends of the lipocalin fold. The structurally invariant end of the molecule may be implicated in general binding small ligands and forming macromolecular complexes via an exposed binding surface.     

Mitochondrial DNA sequence variation and genetic stock structure of Atlantic cod (Gadus morhua) from bay and offshore locations on the Newfoundland continental shelf

Bay cod, Atlantic cod (Gadus morhua) that over-winter in the deep-water bays of northeastern Newfoundland, have historically been regarded as distinct in migration and spawning behaviour from offshore (Grand Bank) cod stocks. To investigate their genetic relationships, we determined the DNA sequence of a 307-base-pair portion of the mitochondrial cytochrome b gene for 236 adult cod taken from the waters off northeastern Newfoundland, including fish found over-wintering and spawning in Trinity Bay. Although 17 genotypes were found, a single common genotype occurs at a frequency of greater than 80% in all samples, and no alternative genotype occurs at a frequency of greater than 3%. Genotype proportions did not differ significantly among samples. Measures of genetic subdivision among sampling locations are nil. Cod over-wintering in Trinity Bay are not genetically distinct from offshore cod. In combination with tagging and physiological studies, these data suggest that there is sufficient movement of cod between bay and offshore locations to prevent the development or maintenance of independent inshore stocks. Adult cod that over-winter in Trinity Bay appear to represent an assemblage of temporarily nonmigratory fish that have become physiologically acclimated to cold-water inshore environments. The pattern of genetic variation in northern cod suggests a recent population structure characterized by extensive movement of contemporary individuals superimposed on an older structure characterized by a bottleneck in the population size of cod in the north-western Atlantic.     

Superoxide dismutase mimetic activity of a cyclic tetrameric Schiff base N-coordinated Cu(II) complex

A pulse radiolytic study using the cyclic tetrameric Schiff base N-coordinated copper complex Cu(TAAB)²⁺ has been performed. The reaction of the Cu(TAAB)²⁺ complex with superoxide revealed pseudo first-order characteristics with the rate constant of k ₂ = (2.9 ± 0.5) × 10⁸ mol^–1 s^–1 dm³. The complex survive presence of competing serum albumin in physiological concentrations. The complex stability constant K = 1.15 × 10¹⁸ (log K = 18.06) is two orders of magnitude higher than that of Cu(II)-serum albumin (log K = 16.2). Transient changes of the stability during the oxidation/reduction process and in the presence of 600 /mol l^–1 albumin did not affect significantly either the electronic absorption of the complex or its catalytic activity.     

细胞色素bc_1复合物的喇曼光谱研究

对提纯的细胞色素ｂｃ１复合物的氧化态和底物琥珀酸还原态两个样品进行了共振喇曼和富立叶喇曼光谱测定和比较。琥珀酸还原态与氧化态的共振谱比较明显有变化,而富立叶红外谱没有什么差别。说明呼吸链的电子传递体在氧化态与还原态交替变化进行电子传递时,蛋白总体构象不发生大的改变,而活性中心血红素辅基局部构象变化很大。     

Biogenesis of mitochondria: Defective yeast H+-ATPase assembled in the absence of mitochondrial protein synthesis is membrane associated

We have investigated the extent to which the assembly of the cytoplasmically synthesized subunits of the H⁺-ATPase can proceed in a mtDNA-less (rho°) strain of yeast, which is not capable of mitochondrial protein synthesis. Three of the membrane sector proteins of the yeast H⁺-ATPase are synthesized in the mitochondria, and it is important to determine whether the presence of these subunits is essential for the assembly of the imported subunits to the inner mitochondrial membrane. A monoclonal antibody against the cytoplasmically synthesized -subunit of the H⁺-ATPase was used to immunoprecipitate the assembled subunits of the enzyme complex. Our results indicate that the imported subunits of the H⁺-ATPase can be assembled in this mutant, into a defective complex which could be shown to be associated with the mitochondrial membrane by the analysis of the Arrhenius kinetics of the mutant mitochondrial ATPase activity.This paper is No. 61 in the seriesBiogenesis of Mitochondria. For paper No. 60, see Novitskiet al. (1984).     

The effects of hyperthermia and hyperthermia plus microwaves on rat brain energy metabolism

The effects of hyperthermia, alone and in conjunction with microwave exposure, on brain energetics were studied in anesthetized male Sprague-Dawley rats. The effect of temperature on adenosine triphosphate concentration [ATP] and creatine phosphate concentration [CP] was determined in the brains of rats that were maintained at 35.6, 37.0, 39.0, and 41.0 degrees C. At 37, 39, and 41 degrees C brain [ATP] and [CP] were down 6.0, 10.8, and 29.2%, and 19.6, 28.7, and 44%, respectively, from the 35.6 degrees C control concentrations. Exposure of the brain to 591-MHz radiation at 13.8 mW/cm2 for 0.5, 1.0, 3.0, and 5.0 min caused further decreases (below those observed for 30 degrees C hyperthermia only) of 16.0, 29.8, 22.5, and 12.3% in brain [ATP], and of 15.6, 25.1, 21.4, and 25.9% in brain [CP] after 0.5, 1.0, 3.0, and 5.0 min, respectively. Recording of brain reduced nicotinamide adenine dinucleotide (NADH) fluorescence before, during, and after microwave exposure showed an increase in NADH fluorescence during microwave exposure that returned to preexposure levels within 1 min postexposure. Continuous recording of brain temperatures during microwave exposures showed that brain temperature varied between -0.1 and +0.05 degrees C. Since the microwave exposures did not induce tissue hyperthermia, it is concluded that direct microwave interaction at the subcellular level is responsible for the observed decrease in [ATP] and [CP].     

Heterogeneity in photosystem I. Evidence from cation-induced decrease in Photosystem I electron transport

The rate of electron transfer through Photosystem I (reduced 2,6-dichlorophenol indophenol (DCIPH₂ → methylviologen) in a low-salt thylakoid suspension is inhibited by Mg²⁺ both under light-limited and the light-saturated conditions, the magnitude of inhibition being the same. The 2,6-dichlorophenol indophenol (DCIP) concentration dependence of the light-saturated rate in the presence and in the absence of Mg²⁺ shows that the overall rate constant of the photoreaction is not altered by Mg²⁺. With N,N,N′,N′-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethylphenylenediamine as electron donor only the light-limited rate, not the light-saturated rate, is inhibited by Mg²⁺ and the magnitude of inhibition is the same as with DCIP as donor. The results are interpreted in terms of heterogeneous Photosystem I, consisting of two types, PS I-A and PS I-B, where PS I-A is involved in cation-regulation of excitation energy distribution and becomes unavailable for DCIPH₂ → methyl viologen photoelectron transfer in the presence of Mg²⁺.     

Energy metabolism in the cyanobacterium Plectonema boryanum. Participation of the thylakoid photosynthetic electron transfer chain in the dark respiration of NADPH and NADH

The oxidation of NADPH and NADH was studied in the light and in the dark using sonically derived membrane vesicles and osmotically shocked spheroplasts. These two types of cell-free membrane preparations mostly differ in that the cell and thylakoid membranes are scrambled in the former type and that they are more or less separated in the latter type of preparations. In the light, using both kinds of preparations, each of NADPH and NADH donates electrons via the plastoquinone-cytochrome $\text{b}\text{c}$ redox complex (Qbc redox complex) to the thylakoid membrane-bound cytochrome c-553 preoxidized by a light flash and to methylviologen via Photosystem I. NADPH donates electrons to the thylakoid membrane via a weakly rotenone-sensitive dehydrogenase to a site that is situated beyond the 3(3′,4′-dichlorophenyl)-1,1-dimethylurea sensitive site and before plastoquinone. Ferredoxin and easily soluble cytoplasmic proteins are presumably not involved in light-mediated NADPH oxidation. Inhibitors of electron transfer at the Qbc redox complex as the dinitrophenylether of 2-iodo-4-nitrothymol, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone and 2-n-heptyl-4-hydroxy-quinone-N-oxide are effective, but antimycin A and KCN are not. The oxidation of NADH showed comparable sensitivity to these inhibitors. However, the oxidation of NADH is antimycin-A-sensitive regardless of the kind of membrane preparation used, indicating that in this case electrons are donated to a different site on the thylakoid membrane. In the dark, NADPH and NADH donate electrons at sites that behave similar to those of light-mediated oxidation, indicating that the initial steps of electron transfer are situated at the thylakoid membranes. However, NADPH oxidation is in some cases not sensitive to inhibitors active at the Qbc redox complex. It is concluded that O₂ reduction takes place at two different sites, one partly developed in vitro, situated near the rotenone-sensitive NADPH dehydrogenase, and another, highly KCN-sensitive one, situated beyond the Qbc redox complex and used in vivo. The terminal oxygen-reducing step of NADPH and NADH oxidation in the dark showed a preparation-dependent sensitivity for KCN, more than 80% inhibition in sonically derived membrane vesicles and less than 30% inhibition in osmotically shocked spheroplasts. From this result we tentatively conclude that the highly KCN-sensitive oxidase is not necessarily located at the thylakoid membrane and could be located at the cytoplasmic membrane.     

Magnetic field dependence of radical-pair decay kinetics and molecular triplet quantum yield in quinone-depleted reaction centers

Radical-pair decay kinetics and molecular triplet quantum yields at various magnetic fields are reported for quinone-depleted reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides R26. The radical-pair decay is observed by picosecond absorption spectroscopy to be a single exponential to within the experimental uncertainty at all fields. The decay time increases from 13 ns at zero field to 17 ns at 1 kG, and decreases to 9 ns at 50 kG. The orientation averaged quantum yield of formation of the molecular triplet of the primary electron donor, ³P, drops to 47% of its zero-field value at 1 kG and rises to 126% at 50 kG. Combined analysis of these data gives a singlet radical-pair decay rate constant of 5 · 10⁷s^?1, a lower limit for the triplet radical-pair decay rate constant of 1 · 10⁸s^?1 and a lower limit for the quantum yield of radical-pair decay by the triplet channel of 38% at zero field. The upper limit of the quantum yield of ³P formation at zero field is measured to be 32%. In order to explain this apparent discrepancy, decay of the radical pair by the triplet channel must lead to some rapid ground state formation as well as some ³P formation. It is proposed that the triplet radical pair decays to a triplet charge-transfer state which is strongly coupled to the ground state by spin-orbit interactions. Several possibilities for this charge-transfer state are discussed.