Phototransformation of oat phytochrome with millisecond and submillisecond flashes: The level of the photostationary Pfr/Ptot ratio is modified by photoreversible intermediates

Photoconversion of the red-absorbing form of phytochrome (P_r) to the far-red-absorbing form of phytochrome (P_fr) and vice versa has been measured spectrophotometrically at 10°C in immobilized and soluble phytochrome (118 kdalton), prepared from 5-day-old etiolated oat seedlings ( Avena saliva L. cv. Sol II). The photostationary equilibrium φ= P_frP_tot (with P_tot= total amount of phytochrome P_r+ P_fr) for red light depends on whether it is established by repetitive pulses (≥ 5 s) or by repetitive flashes (≥ 4 ms). In the wavelength region around 660 nm, a lower φ is reached with flashes as compared to that with pulses. This difference becomes negligible if the wavelength is shortened to the 600 nm region, and it also disappears if the fluence of each individual flash is reduced. In contrast, in long-wavelength red light and short-wavelength far-red light, a higher φ is reached with flashes than with pulses.
We relate the differences in φ for flash and pulse irradiation to photochromic systems between P_r and photoreversible intermediates in the phototransformation pathway P_r→ P_fr. Thus, light absorption by phytochrome intermediates can be limiting for the quantitative relationship between light signal and P_fr formed.     

Restoration of mitochondrial energy-linked reactions following dexamethasone treatment of rats infected with the liver fluke Fasciolahepatica

Mitochondria isolated from male Wistar rats experimentally infected with the common liver fluke, Fasciolahepatica, exhibit loss of respiratory control from 2 weeks post-infection (Rule, et al. (1989) Biochem. J. 260, 517–523). We now report that subcutaneous injections of the anti-inflammatory drug, dexamethasone, during the final week of infection prevented the mitochondrial uncoupling and restored respiratory control almost to the levels of uninfected controls. Further investigations have shown that mitochondria from infected rat livers are unable to synthesize ATP and that abnormal respiration is also evident in hepatocytes isolated from infected rats. These abnormalities were absent when infected rats were treated with dexamethasone. In addition, liver mitochondrial function in infected, congenitally athymic, nude rats (CBH/R nu/nu) was not significantly different from that in uninfected nude or Wistar controls. These results provide evidence that the mitochondrial dysfunction in fascioliasis is host-mediated and that T lymphocytes in particular may be involved.     

Reversible unfolding and refolding behavior of a monomeric aldolase from Staphylococcus aureus.

Thermal and GdmCl-induced unfolding transitions of aldolase from Staphylococcus aureus are reversible under a variety of solvent conditions. Analysis of the transitions reveals that no partially folded intermediates can be detected under equilibrium conditions. The stability of the enzyme is very low with a delta G0 value of -9 +/- 2 kJ/mol at 20 degrees C. The kinetics of unfolding and refolding of aldolase are complex and comprise at least one fast and two slow reactions. This complexity arises from prolyl isomerization reactions in the unfolded chain, which are kinetically coupled to the actual folding reaction. Comparison with model calculations shows that at least two prolyl peptide bonds give rise to the observed slow folding reactions of aldolase and that all of the involved bonds are presumably in the trans conformation in the native state. The rate constant of the actual folding reaction is fast with a relaxation time of about 15 s at the midpoint of the folding transition at 15 degrees C. The data presented on the folding and stability of aldolase are comparable to the properties of much smaller proteins. This might be connected with the simple and highly repetitive tertiary structure pattern of the enzyme, which belongs to the group of alpha/beta barrel proteins.     

Photointermediates of visual pigments

Much progress has been made in recent years toward understanding the interactions between various proteins responsible for visual transduction which are initiated by an activated state of visual pigments. However, the changes which take place in the visual pigments themselves to convert them to the activated state are more poorly understood. Many spectroscopic techniques have been applied to this problem in recent years and considerable progress has been made. A major goal of these efforts is to understand at which stages protein change occurs and to characterize its structural features. In the visual system evidence is accumulating, for example, that chromophore independent protein change begins immediately prior to lumirhodopsin formation. Considerable insight has been gained recently into the early intermediates of visual transduction and the stage is set to achieve similar understanding of the later intermediates leading to rhodopsin's activated state.     

AUT凝胶电泳在牛胰核糖核酸酶折叠研究中的应用

目的:牛胰核糖核酸酶是一种用于蛋白折叠研究的经典模式蛋白,在折叠研究过程中主要使用高效液相色谱用于分离检测不同阶段的蛋白折叠中间体。高效液相色谱具有自动化、分离效果好、样品可回收等优点,同时也存在检测通量较低、仪器设备较为昂贵等不足。AUT凝胶电泳简便、快捷、检测通量较高,本文尝试将其应用于牛胰核糖核酸酶的折叠研究。方法:使用AUT凝胶电泳、酶活性检测、质谱对牛胰核糖核酸酶还原变性过程及产生的折叠中间体进行检测;通过高效液相色谱和质谱对折叠中间单体进行分离检测,并分别进行AUT凝胶电泳检测以解析各折叠中间单体在电泳中的条带位置;通过AUT凝胶电泳和酶切后质谱鉴定各折叠中间单体的二硫键配对方式。结果:AUT凝胶电泳可以有效区分不同条件下的牛胰核糖核酸酶还原变性过程,检测结果与酶活性、质谱结果相符,并可以很好地区分牛胰核糖核酸酶还原变性过程折叠中间体。高效液相色谱将牛胰核糖核酸酶还原变性过程折叠中间体分离为13个色谱峰,并与AUT凝胶电泳中的11个条带位置进行匹配。确认牛胰核糖核酸酶还原变性过程折叠中间单体的二硫键配对方式,并与AUT凝胶电泳条带进行匹配,Cys58-Cys110和Cys26-Cys84构象熵减作用强于Cys40-Cys95和Cys65-Cys72。结论:AUT凝胶电泳适用于检测牛胰核糖核酸酶折叠中间体,可以与高效液相色谱、质谱等检测技术相互补充,共同应用于牛胰核糖核酸酶的折叠研究。     

Co-translational Folding Intermediate Dictates Membrane Targeting of the Signal Recognition Particle Receptor

Much of our knowledge on the function of proteins is deduced from their mature, folded states. However, it is unknown whether partially synthesized nascent protein segments can execute biological functions during translation and whether their premature folding states matter. A recent observation that a nascent chain performs a distinct function, co-translational targeting in vivo, has been made with the Escherichia coli signal recognition particle receptor FtsY, a major player in the conserved pathway of membrane protein biogenesis. FtsY functions as a membrane-associated entity, but very little is known about the mode of its targeting to the membrane. Here we investigated the underlying structural mechanism of the co-translational FtsY targeting to the membrane. Our results show that helices N_2–4, which mediate membrane targeting, form a stable folding intermediate co-translationally that greatly differs from its fold in the mature FtsY. These results thus resolve a long-standing mystery of how the receptor targets the membrane even when deleted of its alleged membrane targeting sequence. The structurally distinct targeting determinant of FtsY exists only co-translationally. Our studies will facilitate further efforts to seek cellular factors required for proper targeting and association of FtsY with the membrane. Moreover, the results offer a hallmark example for how co-translational nascent intermediates may dictate biological functions.     

Enhanced lipid peroxidation by extracellular ATP in PC12 cells

Recently we have demonstrated that extracellular ATP acts as an excitatory neurotransmitter and enhances cell death in the presence of ferrous ions. By using a newly developed cis-parinaric acid fluorescence technique, we demonstrated that ATP, in a dose dependent manner, enhanced the increased membrane lipid peroxidation in PC12 cells when cells were incubated with micromolar FeCl₂/DTP. P₂ purinoceptor agonists, α,β-methylene ATP and 2-methylthio-ATP, induced PC12 cell lipid peroxidation, but to a lesser extent than ATP. ATP-induced Ca²⁺ influx via P₂ purinoceptor activation significantly increased the intracellular Ca²⁺ concentration, which may have triggered a free radical generating cascade(s), and led to membrane lipid peroxidation and cell death. Since oxidative stress has been implicated in certain neurodegenerative diseases such as aging, extracellular ATP may contribute to neuronal cell death by an oxidative mechanism involving lipid peroxidation.