Synthesis and conformational analysis of aib-containing peptide modelling for N-glycosylation site in N-glycoprotein

A tetrapetide containing an Aib residue, Boc-Asn-Aib-Thr-Aib-OMe, was synthesized as a peptide model for the N-glycosylation site in N-glycoproteins. Backbone conformation of the peptide and possible intramolecular interaction between the Asn and Thr side chains were elucidated by means of n.m.r. spectroscopy. Temperature dependence of NH proton chemical shift and NOE experiments showed that Boc-Asn-Aib-Thr-Aib-OMe has a tendency to form a β-turn structure with a hydrogen bond involving Thr and Aib⁴ NH groups. Incorporation of Aib residues in the peptide model promotes folding of the peptide backbone. With folded backbone conformation, carboxyamide protons of the Asn residue are not involved in hydrogen bond network, while the OH group of the Thr residue is a candidate for a hydrogen bond in DMSO-d₆ solution.     

Alterations in cardiac membrane Ca2+ transport during oxidative stress

Although cardiac dysfunction due to ischemia-reperfusion injury is considered to involve oxygen free radicals, the exact manner by which this oxidative stress affects the myocardium is not clear. As the occurrence of intracellular Ca²⁺ overload has been shown to play a critical role in the genesis of cellular damage due to ischemia-reperfusion, this study was undertaken to examine whether oxygen free radicals are involved in altering the sarcolemmal Ca²⁺-transport activities due to reperfusion injury. When isolated rat hearts were made globally ischemic for 30 min and then reperfused for 5 min, the Ca²⁺ -pump and Na⁺-Ca²⁺ exchange activities were depressed in the purified sarcolemmal fraction; these alterations were prevented when a free radical scavenger enzymes (superoxide dismutase plus catalase) were added to the reperfusion medium. Both the Ca²⁺- pump and Na⁺- Ca²⁺ exchange activities in control heart sarcolemmal preparations were depressed by activated oxygen-generating systems containing xanthine plus xanthine oxidase and H₂O₂; these changes were prevented by the inclusion of superoxide dismutase and catalase in the incubation medium. These results support the view that oxidative stress during ischemia-reperfusion may contribute towards the occurrence of intracellular Ca²⁺ overload and subsequent cell damage by depressing the sarcolemmal mechanisms governing the efflux of Ca²⁺ from the cardiac cell.     

Several new substrates for Desulfovibrio vulgaris strain Marburg and a spontaneous mutant from it

The ability of Desulfovibrio vulgaris strain Marburg (DSM 2119) to oxidize alcohols was surveyed in the presence and absence of hydrogen-scavenging anaerobes, Acetobacterium woodii and Methanospirillum hungatei. In the presence of sulfate, D. vulgaris grew not only on ethanol, 1-propanol, and 1-butanol, but also on isobutanol, 1-pentanol, ethyleneglycol, and 1,3-propanediol. Metabolism of these alcohols was simple oxidation to the corresponding acids, except with the last two substrates: ethyleneglycol was oxidized to glycolate plus acetate, 1,3-propanediol to 3-hydroxypropionate plus acetate. Experimental evidence was obtained, suggesting that 2-methoxyethanol was not utilized by all the cells of strain marburg, but by a spontaneous mutant. 2-Methoxyethanol was oxidized to methoxyacetate by the mutant. Co-culture of strain Marburg plus A. woodii grew on ethanol, 1-propanol, 1-butanol, and 1,3-propanediol in the absence of sulfate. Co-culture of strain Marburg plus M. hungatei grew on ethanol, 1-propanol, and 1-butanol, but not on ethyleneglycol and 1,3-propanediol, Co-culture of the mutant plus A. woodii or M. hungatei did not grow on 2-methoxyethanol.     

Influence of red light on the activity of phosphofructokinase in Chlorella kessleri

In crude extracts of the unicellular green alga Chlorella kessleri Fott et Novákóva grown in red light the activity of the glycolytic enzyme phosphofructokinase (PFK, EC 2.7.1.11) is about 40% higher compared to white light conditions giving the same dry matter production. Application of cycloheximide and density labelling with D₂O indicate that this increase depends on the de novo synthesis of the enzyme: Twelve h of illumination at a fluence rate of 7 × 10¹⁸ quanta m⁻² s⁻¹ (11.6 μmol m⁻² s⁻¹) suffice to saturate the effect. In autotrophically grown algae maximal increase in enzyme saturate the effect. In autotrophically grown algae maximal increase in enzyme activity is reached in light of 680 nm, while in 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU)-poisoned, glucose-fed cells, light of wavelengths around 727 nm is most effective. Involvement of a phytochrome-like photoreceptor is discussed.     

Formation of Hydroxyl Radicals in Biological Systems. Does Myoglobin Stimulate Hydroxyl Radical Formation from Hydrogen Peroxide?

Incubation of horse-heart oxymyoglobin or metmyoglobin with excess H₂O₂ causes formation of myoglobin(IV), followed by haem degradation. At the time when haem degradation is observed, hydroxyl radicals (.OH) can be detected in the reaction mixture by their ability to degrade the sugar deoxyribose. Detection of hydroxyl radicals can be decreased by transferrin or by OH scavengers (mannitol, arginine, phenylalanine) but not by urea. Neither transferrin nor any of these scavengers inhibit the haem degradation. It is concluded that intact oxymyoglobin or metmyoglobin molecules do not react with H₂O₂ to form OH detectable by deoxyribose, but that H₂O₂ eventually leads to release of iron ions from the proteins. These released iron ions can react to form OH outside the protein or close to its surface. Salicylate and the iron chelator desferrioxamine stabilize myoglobin and prevent haem degradation. The biological importance of OH generated using iron ions released from myoglobin by H₂O₂ is discussed in relation to myocardial reoxygenation injury.     

Helix packing of leucine-rich peptides: a parallel leucine ladder in the structure of Boc-Aib-Leu-Aib-Aib-Leu-Leu-Leu-Aib-Leu-Aib-OMe.

The packing of peptide helices in crystals of the leucine-rich decapeptide Boc-Aib-Leu-Aib-Aib-Leu-Leu-Leu-Aib-Leu-Aib-OMe provides an example of ladder-like leucylleucyl interactions between neighboring molecules. The peptide molecule forms a helix with five 5----1 hydrogen bonds and two 4----1 hydrogen bonds near the C terminus. Three head-to-tail NH ... O = C hydrogen bonds between helices form continuous columns of helices in the crystal. The helicial columns associate in an antiparallel fashion, except for the association of Leu ... Leu side chains, which occurs along the diagonal of the cell where the peptide helices are parallel. The peptide, with formula C56H102N10O13, crystallizes in space group P2(1)2(1)2(1) with Z = 4 and cell parameters a = 16.774(3) A, b = 20.032(3) A and c = 20.117(3) A; overall agreement factor R = 10.7% for 2014 data with magnitude of F(obs) greater than 3 sigma (F); resolution 1.0 A.     

Diel water movement between parenchyma and chlorenchyma of two desert CAM plants under dry and wet conditions

Abstract. Electric-circuit analogue models of the water relations of crassulacean acid metabolism (CAM) succulents such as Agave deserti and Ferocactus acanthodes have predicted diel movement of water between the water-storage parenchyma and the photo-synthetic chlorenchyma. Injection of tritiated water into either tissue in the laboratory confirmed substantial and bidirectional water movements, especially under conditions of wet soil. For A. deserti , water movement from the water-storage parenchyma to the chlorenchyma increased at night as the chlorenchyma osmotic pressure increased. Although nocturnal osmotic pressure increases and transpiration for both species were minimal in the field under dry conditions, diel changes in the deuterium: hydrogen ratio (expressed as ΔD) were similar for the water-storage parenchyma and the chlorenchyma. Such indication of [substantial mixing of water between the tissues over a 24-h cycle was more evident under wet conditions in the field. For A. deserti , ΔD then increased by 32%o from the afternoon to midnight and was essentially identical in the water-storage parenchyma and the chlorenchyma. For F. acanthodes , the diel changes in ΔD were one-third those of A. deserti , and ΔD was always slightly higher for the chlorenchyma than for the water-storage parenchyma, apparently reflecting the lower surface-to-volume ratio of A. deserti. In summary, data obtained using radioactive and stable isotopes strongly supported model predictions concerning diel cycles of internal water distribution for these CAM species.     

Crystal structure of the Y52F/Y73F double mutant of phospholipase A2: increased hydrophobic interactions of the phenyl groups compensate for the disrupted hydrogen bonds of the tyrosines.

The enzyme phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 ester bond of membrane phospholipids. The highly conserved Tyr residues 52 and 73 in the enzyme form hydrogen bonds to the carboxylate group of the catalytic Asp-99. These hydrogen bonds were initially regarded as essential for the interfacial recognition and the stability of the overall catalytic network. The elimination of the hydrogen bonds involving the phenolic hydroxyl groups of the Tyr-52 and -73 by changing them to Phe lowered the stability but did not significantly affect the catalytic activity of the enzyme. The X-ray crystal structure of the double mutant Y52F/Y73F has been determined at 1.93 A resolution to study the effect of the mutation on the structure. The crystals are trigonal, space group P3(1)21, with cell parameters a = b = 46.3 A and c = 102.95 A. Intensity data were collected on a Siemens area detector, 8,024 reflections were unique with an R(sym) of 4.5% out of a total of 27,203. The structure was refined using all the unique reflections by XPLOR to a final R-factor of 18.6% for 955 protein atoms, 91 water molecules, and 1 calcium ion. The root mean square deviation for the alpha-carbon atoms between the double mutant and wild type was 0.56 A. The crystal structure revealed that four hydrogen bonds were lost in the catalytic network; three involving the tyrosines and one involving Pro-68. However, the hydrogen bonds of the catalytic triad, His-48, Asp-99, and the catalytic water, are retained. There is no additional solvent molecule at the active site to replace the missing hydroxyl groups; instead, the replacement of the phenolic OH groups by H atoms draws the Phe residues closer to the neighboring residues compared to wild type; Phe-52 moves toward His-48 and Asp-99 of the catalytic diad, and Phe-73 moves toward Met-8, both by about 0.5 A. The closing of the voids left by the OH groups increases the hydrophobic interactions compensating for the lost hydrogen bonds. The conservation of the triad hydrogen bonds and the stabilization of the active site by the increased hydrophobic interactions could explain why the double mutant has activity similar to wild type. The results indicate that the aspartyl carboxylate group of the catalytic triad can function alone without additional support from the hydrogen bonds of the two Tyr residues.     

Ferritin enhances production of DNA strand breaks by 6-hydroxydopamine,ascorbic acid and H2O2 in CCC PM2 bacteriophage DNA

Damage of CCC PM2 DNA by 6-hydroxydopamine (6-OHDA) and ascorbic acid (AA), compounds that are both able to release iron from ferritin, was significantly enhanced in the presence of ferritin. H₂O₂, a product of 6-OHDA autoxidation, did not induce DNA strand breaks in the absence of ferritin and only to a minor extent in the presence of ferritin. DNA damage by 6-OHDA and AA could be reduced by the hydroxyl radical scavenger mannitol, the iron chelator desferrioxamine, and, partly, by a combination of superoxide dismutase and catalase. These inhibitory effects were clearly less pronounced in the presence of ferritin. Ferritin obviously played an important role as a source of iron in the pro-oxidative processes of 6-OHDA and AA. These features might be of importance in cancer therapy since many tumor cells contain elevated ferritin levels.     

固氮蓝藻高光放氢突变种的筛选和放氢特点

作者以前报道过几种快速生长的固氮蓝藻在某种条件下能好气光放氢,其速度可以达到光合放氧速度的10—15%,但这种活性只有在不积累氢气的流动气相下或在短时间内发生。本文报道用亚硝基胍诱变所得到的Anabaena spp。Strain CA的高光放氢突变种——N9A和18A——的筛选和氢代谢特点。在达生长饱和光照以后,野生型的光放氢活性与光照强度的增加成正相关,而其吸氢活性则与之成负相关,显示高光照强度可能抑制吸氢酶的活性。无论在什么光强下,均测不到两个突变种的吸氢活性,暗示在突变种中,吸氢酶或有关系统受损伤。把细胞固相化在琼脂上,在密闭系统中,高光强下培养50个小时,两个突变种光释放和积累的氢分别为野生型的2倍(N9A)和6倍(18A),后者等于氢占气相(1%CO_2的空气)的1.8%。两个突变种在生长速度、叶绿素含量、乙炔还原活性以及光合放氧方面与野生型无明显不同。当以含50—100nM的镍离子的培养基培养时,野生型的好气净产氢活性完全丢失,其吸氢活性却增加约10倍。培养基中镍离子的存在,对两个突变种的高光放氢活性则毫无影响,而且在此情况下,仍测不出其吸氢活性。实验结果表明,这两个突变种系吸氢酶缺陷型突变种。