Purification and phosphorylation of initiation factor eIF-2 from rabbit skeletal muscle

Core particle DNA unfolding and refolding are followed by stopped-flow circular dichroism technique. When core particles are dissociated in the stopped-flow cuvette, the high CD deviation corresponding to the dissociated state is reached in the first millisecond, which means that the dissociation process is completed within the dead time of the apparatus which is ～1 ms. The same conclusion can be drawn when core particles are reassociated, since the low CD value, typical of the associated state, is immediately reached. Similarly histone release from chromatin is a very fast process. We also include some points of discussion about core particle assembly process.     

Tris对高LET碳重离子辐照下质粒DNA链断裂的保护作用

利用100 keV/μm碳离子束(初始能量为290 MeV/u)照射溶解于纯水、10 mmol/L Tris、1 mmol/L EDTA及TE 缓冲液中的pUC19质粒DNA.通过琼脂糖凝胶电泳技术分析了不同溶液中各种形态DNA分子所占份额,并计算得到不同剂量下平均每个质粒分子中单链断裂(SSB)及双链断裂(DSB)的数目.发现Tris通过抑制SSB和DSB的产生对碳重离子辐照下的质粒DNA有明显的保护作用,而EDTA能够加剧SSB的产生而抑制DSB的形成.     

Interactions of Hydroxyl Radicals with Tris (Hydroxymethyl) Aminomethane and Good's Buffers Containing Hydroxymethyl or Hydroxyethyl Residues Produce Formaldehyde

The production of formaldehyde from tris(hydroxymethyl) aminomethane(Tris) by interaction with hydroxyl radicals(.OH) was studied, since the reaction mixture from the Fenton reaction performed in Tris/HCl buffer was found to be color-developed by colorimetric determination of formaldehyde. The absorption spectrum of chromogens was identical to that of authentic formaldehyde. Color development, which required the presence of Tris, hydrogen peroxide and cupric ions in the Fenton reaction mixture, was inhibited by the addition of hydroxyl radical scavengers such as glucose or hyaluronic acid. These results indicated that formaldehyde was produced when Tris interacted with ·OH. With structures similar to Tris, Good's buffers were also found to produce formaldehyde by interaction with ·OH. Analysis of formaldehyde derived from these buffers may provide a simple and convenient assay for detecting ·OH generation. In evaluating effects of ·OH on the biological system in Tris/HCl buffer or certain Good's Buffers, ·OH loss may be due to interactions of ·OH with these buffers. The formaldehyde produced as a result of such interactions may affect biological systems.     

The effect of phospholipid vesicles on the kinetics of reduction of cytochrome c.

The rate of reduction of cytochrome c by ascorbate and by 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine was examined as a function of ionic strength and of binding to phospholipid vesicles (liposomes). Binding of cytochrome c to liposomes, which occursat low ionic strength, decreases the rate of reduction by ascorbate by a factor of up to 100, which can be primarily explained on electrostatic grounds. In the absence of liposomes, kinetics of reduction by the neutral pteridine derivative showed no ionic strength dependence. Binding of cytochrome c to liposomes increased the rate of reduction by pteridine. An estimation of the binding constant of cytochrome c to liposomes at 0.06 M ionic strength, pH 7, is given.     

Calmodulin antagonists inhibit formation of platelet-activating factor in stimulated human neutrophils

Conversion of native, 97-100 kDa rat liver microsomal HMG CoA reductase to membrane-bound 62 kDa and soluble 52-56 kDa catalytically active forms was catalyzed in vitro by the calcium-dependent, leupeptin- and calpastatin-sensitive protease calpain-II purified from rat liver cytosol. Cleavage of the native 97-100 kDa reductase was enhanced by pretreatment (inactivation) of microsomes with ATP(Mg2+) and liver reductase kinase (compared to protein phosphatase-pretreated controls). This was reflected in a loss of the 97-100 kDa species and an increase in the soluble 52-56 kDa species (total enzyme activity and specific immunoblot recovery).     

How does lysozyme penetrate through the bacterial outer membrane?

Lysozyme fails to penetrate through the outer membrane of stationary phase cells of Escherichia coli when it is simply added to suspensions of plasmolyzed cells. Lysozyme penetrates the outer membrane only when these cells are exposed to a mild osmotic shock in the presence of EDTA and lysozyme.In the presence of Mg²⁺, the outer membrane is stabilized sufficiently so that there is no lysozyme penetration during osmotic shock. If Mg²⁺ is added after an osmotic shock has been used to cause lysozyme to penetrate a destabilized outer membrane, the outer membrane is stabilized once again. In this case however, cells are converted to spheroplasts by the lysozyme which has gained access to the murein layer prior to the addition of Mg²⁺. Mg²⁺ stabilizes the outer membranes of these spheroplasts sufficiently so that they remain immune to lysis even in the absence of osmotic stabilizers such as sucrose.These results are discussed in terms of current information on the structure of the murein layer and the outer membrane.     

Light-induced Mg2+ ATPase activity of coupling factor in intact chloroplasts

Intense illumination of isolated, intact, spinach chloroplasts triggers the well known proton-pumping Mg²⁺ ATPase activity of coupling factor, which can be assayed in subsequently lysed chloroplasts by monitoring ATP-driven quenching of 9-aminoacridine fluorescence. The light-triggered ATPase activity decays slowly in the dark and is inhibited by N,N′-dicyclohexylcarbodiimide. After osmotic lysis and washing of the chloroplasts, preillumination no longer triggers maximal proton-pumping ATPase until methylviologen and dithiothreitol are added to the medium. It is suggested that intact organelles contain soluble or loosely bound cofactors necessary for light-triggering of coupling factor ATPase. On osmotic lysis, these endogenous cofactors are diluted or inactivated and must be replaced by addition of a dithiol reagent and an electron acceptor.     

Neocarzinostatin: controlled release of chromophore and its interaction with DNA

A nonagglutinating derivative of wheat germ agglutinin has been prepared that binds to platelets and precipitates an antibody to the lectin. Platelets treated with this inactive derivative released serotonin when exposed to bivalent F(ab′)₂, but not monovalent Fab, fragments of the lectin antibody. Bridging of platelet-bound Fab by an antibody again induced secretion. The F(ab′)₂ or Fab fragments plus IgG, without the derivative, did not induce secretion. This secretion was not affected by indomethacin showing a direct activation of platelets. Platelets treated with con A followed by F(ab′)₂ to con A did not secrete. In addition, lentil lectin failed to release platelet serotonin. The receptors of the lectin derivative are mobile on the platelet surface and their redistribution may lead to secretion.     

Silver(I) complexes of fluorinated scorpionates: Ligand effects in silver catalyzed carbene insertion into C-H bonds in alkanes

Activation of C-H bonds of hydrocarbons via intermolecular carbene insertion has been investigated using tris(pyrazolyl)boratosilver(I) catalysts [MeB(3-(CF₃)Pz)₃]Ag(C₂H₄), [MeB(3-(C₂F₅)Pz)₃]Ag(C₂H₄) and [HB(3,5-(CF₃)₂Pz)₃]Ag(C₂H₄). Cyclopentane, 2-methylbutane, and 2,3-dimethylbutane were used as substrates. Carbenes derived from ethyl and tert-butyl diazoacetates have effectively been inserted into tertiary, secondary, as well as primary C-H bonds of hydrocarbons at room temperature using these catalysts. Tertiary C-H bonds in these substrates get preferentially activated over secondary C-H followed by primary C-H bonds. However, it is possible to increase the amount of primary C-H bond activated product by utilizing catalysts with increasingly acidic silver sites and sterically bulky tris(pyrazolyl)borate ligands. The carbene insertion into primary C-H bonds increases in the order: [MeB(3-(CF₃)Pz)₃]Ag(C₂H₄) < [MeB(3-(C₂F₅)Pz)₃]Ag(C₂H₄) < [HB(3,5-(CF₃)₂Pz)₃]Ag(C₂H₄). The carbene derived from tert-butyl diazoacetate with these catalysts shows slightly lower selectivity for primary C-H bonds compared to the ethyl diazoacetate-based carbene.     

The possible role of diodoquin as a zinc ionophore in the treatment of acrodermatitis enteropathica.

The mechanism by which 5,7-di-iodo-8-hydroxy quinoline (Diodoquin) enhances zinc absorption in the zinc deficiency disorder acrodermatitis enteropathica was investigated using liposomes. This compound increased the permeability of the pure lipid membranes to ⁶⁵Zn and it is proposed that the therapeutic effect of Diodoquin and related compounds may derive from their ability to act as ionophores.     

Labelling of cardiolipin in vitro.

A method for labelling the polar head groups of cardiolipin is described. Labelling was carried out on sonicated cardiolipin/water suspensions. The free hydroxyl group of cardiolipin was oxidised with an excess of p-(diazonium) benzenesulfonic acid (DABS) and then reduced with NaB3H4. Isopropanol was oxidised in the presence of DABS to test the reactivity of the diazonium salts, and the reaction product was analysed by means of gas-chromatography. Labelled cardiolipin, identified by thin-layer chromatography (TLC), was chromatographically pure and identical to untreated cardiolipin. The hydrolysis of cardiolipin confirmed that the labelling was at the level of polar head groups.     

Effectiveness of a tris-based extender (SHOTOR diluent) for the preservation of Bactrian camel (Camelus bactrianus) semen

The development of a suitable semen extender is required to extend artificial breeding programs and to preserve the genetic potential of Bactrian camel. Experiments were conducted to provide the optimal osmolality and pH of tris-based extender and to compare that with available extenders for short-term preservation of Bactrian camel semen at 4 degrees C during 24 h. In experiments I and II, the effects of varying osmolalities (270, 300, 330, 360, and 390 mOsm/kg) and pHs (5.5, 6, 6.9, 7.5, 7.9, and 8.9) of tris-based extender on sperm viability were investigated. In experiment III, the efficiency of tris-based extender (SHOTOR diluent) in preserving Bactrian camel semen was compared with lactose (10%), sucrose (10%) and Green buffer. Viability parameters including progressive forward motility (PFM), plasma membrane integrity and the percentage of live spermatozoa were assessed. The data were analyzed using general linear model procedure. In the majority of assessments using tris-based extender, the viability of spermatozoa was superior at the osmolality of 330 mOsm/kg and pH of 6.9. PFM was significantly greater at the time of semen dilution in tris-based (65.5%) and Green buffer (60.5%) compared to that of lactose (31%) and sucrose (28%) extenders (P<0.05), and remained elevated throughout the experiment. There was no significant difference in other viability parameters among 4 extenders (P>0.05). In conclusion, the utilization of a tris-based extender, having the osmolality of 330 mOsm/kg and pH of 6.9, favors the short-term preservation of the Bactrian camel spermatozoa under chilled condition.     

Calelectrins are a ubiquitous family of Ca2+-binding proteins purified by Ca2+-dependent hydrophobic affinity chromatography by a mechanism distinct from that of calmodulin

The calelectrins, a heterogeneous group of three new Ca2+-binding proteins of M 67 000, 35 000 and 32 500, copurify with calmodulin during Ca2+-dependent hydrophobic affinity chromatography (Südhof et al., Biochemistry, in press, 1984). This property is exploited for the rapid purification of all three calelectrins including for the first time the Mr 35 000, from commercially available acetone powders from several bovine tissues (heart, liver, brain, pancreas and testis). The nature of the Ca2+-dependent interaction of the calelectrins with hydrophobic affinity matrices has been investigated. As with calmodulin, the Ca2+-binding sites of all three purified calelectrins can be probed with Tb3+ which binds to them in a stoichiometric, saturable and Ca2+-displaceable manner. However, using several hydrophobic fluorescence probes which bind to the proteins, contrary to calmodulin no Ca2+-dependent exposure of hydrophobic sites could be detected in any of the three purified proteins. Therefore the Ca2+-dependent purification of the calelectrins on hydrophobic affinity columns seems not to involve the surface exposure of hydrophobic sites and the calelectrins have in this respect little similarity to calmodulin.     

Contents to volume 164

Cytochrome b₆ from spinach chloroplasts (either within the purified cytochrome b₆f complex, or in its isolated form) exhibits two spectral species, which correspond to two midpoint potentials. This can be demonstrated by low temperature difference spectroscopy at fixed redox potentials. The high potential form of cytochrome b₆ has a split α-peak at 557.5 and 561.5 nm, the low potential form has a symmetrical α-peak at 560.5 nm. Similar results were obtained with cytochrome b₆ in the isolated cytochrome b₆f complex from the cyanobacterium Anabaena variabilis.