Structure and function of human hemoglobin covalently labeled with periodate-oxidized adenosine triphosphate

Periodate-oxidized adenosine triphosphate (o-ATP), a ribose ring-opened dialdehyde derivative of ATP, reacts specifically with human deoxyhemoglobin to give a single major covalently modified product after reduction with sodium borohydride. This product, designated di-ATP Hb, was isolated using ion-exchange chromatography and shown to have incorporated two molecules of o-ATP/tetramer. Peptide mapping and x-ray crystallography at 2.8-A resolution indicate that a covalent adduct is formed between the ligand and residues Lys-82 EF6 of each beta chain in the organic phosphate-binding site of the molecule. di-ATP Hb exhibits a significantly decreased oxygen affinity (P50 = 20.8 mm Hg versus 5.8 mm Hg control; 50 mM 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol, pH 7.4, 0.1 M C, 20 degrees C). The subunit cooper-activity of di-ATP Hb is also reduced (nmax = 1.9 versus 2.7 control).     

A higher plant enzyme exhibiting broad acceptance of stereoisomers

An arginase, purified from the leaf of the jack bean, Canavalia ensiformis, can effectively hydrolyze both l- and d-arginine. Arginases, examined from a number of other plant and animal sources, exhibit marked substrate stereospecificity and fail to catabolize d-arginine. In order to provide essential nitrogen, jack bean leaf arginase also catabolizes l-canavanine, an arginine analog that is a predominant nitrogen-storing metabolite of this legume. The ability of arginase to metabolize both stereoisomers of arginine may result from the requirement for this enzyme to exhibit limited substrate specificity in order to hydrolyze both arginine and canavanine.     

High-resolution X-ray study of deoxyhemoglobin Rothschild 37 beta Trp----Arg: a mutation that creates an intersubunit chloride-binding site.

The mutation site in hemoglobin Rothschild (37 beta Trp----Arg) is located in the "hinge region" of the alpha 1 beta 2 interface, a region that is critical for normal hemoglobin function. The mutation results in greatly reduced cooperativity and an oxygen affinity similar to that of hemoglobin A [Gacon, G., Belkhodja, O., Wajcman, H., & Labie, D. (1977) FEBS Lett. 82, 243-246]. Crystal were grown under "low-salt" conditions [100 mM Cl- in 10 mM phosphate buffer at pH 7.0 with poly(ethylene glycol) as a precipitating agent]. The crystal structure of deoxyhemoglobin Rothschild and the isomorphous crystal structure of deoxyhemoglobin A were refined at resolutions of 2.0 and 1.9 A, respectively. The mutation-induced structural changes were partitioned into components of (1) tetramer rotation, (2) quaternary structure rearrangement, and (3) deformations of tertiary structure. The quaternary change involves a 1 degree rotation of the alpha subunit about the "switch region" of the alpha 1 beta 2 interface. The tertiary changes are confined to residues at the alpha 1 beta 2 interface, with the largest shifts (approximately 0.4 A) located across the interface from the mutation site at the alpha subunit FG corner-G helix boundary. Most surprising was the identification of a mutation-generated anion-binding site in the alpha 1 beta 2 interface. Chloride binds at this site as a counterion for Arg 37 beta. The requirement of a counterion implies that the solution properties of hemoglobin Rothschild, in particular the dimer-tetramer equilibrium, should be very dependent upon the concentration and type of anions present.     

理解运动行为抑制调控的新视角：乙酰胆碱门控氯离子通道受体

Acetylcholine, the first identified neurotransmitter, plays crucial roles in various brain functions. One well-known case is its involvement as an activating neurotransmitter in the regulation of locomotion. However, its inhibitory regulatory role, particularly in locomotion, remains poorly understood. In a study conducted by Polat et al., the authors investigated the inhibitory role of acetylcholine in locomotion in C. elegans. In this organism, the acetylcholine-gated chloride channel receptor consists of four subunits. The authors thoroughly examined the loss-of-function of each subunit in movement regulation. Interestingly, the mutant worms were still capable of performing various movements such as forward, backward crawling, and turning, suggesting that the overall movement was not significantly affected. However, quantitative behavior analysis revealed subtle yet significant differences in the timing and postures of the movement in these mutants. Furthermore, the authors employed optogenetics to stimulate a specific neuron involved in backward crawling and demonstrated that the loss-of-function of the receptors in individual neurons affects the transitioning between locomotion modes. This work provides evidence for the inhibitory regulatory role of acetylcholine in locomotion. The loss-of-function of acetylcholine-gated chloride channel receptors likely disrupts the balance of neuronal and circuit physiology, thereby affecting the regulation of locomotion. Moreover, this study highlights the powerful role of quantitative behavior analysis in discovering and understanding more sophisticated functions of neural circuits.     

The envelope glycoprotein of an amphotropic murine retrovirus binds specifically to the cellular receptor/phosphate transporter of susceptible species.

A rat cDNA (rRam-1), which was cloned on the basis that it enables Chinese hamster ovary (CHO) cells to be infected by amphotropic host range murine retroviruses, was recently found to encode a widely expressed Na(+)-phosphate symporter (M. P. Kavanaugh, D. G. Miller, W. Zhang, W. Law, S. L. Kozak, D. Kabat, and A. D. Miller, Proc. Natl. Acad. Sci. USA 91:7071-7075, 1994). CHO cells express the hamster homolog of Ram-1 but are resistant to amphotropic retroviruses. Although the amphotropic envelope glycoprotein gp70 bound weakly onto control CHO cells, CHO/rRam-1 cells had novel high-affinity binding sites, and the resulting strongly adsorbed gp70 was only slowly removed from cell surfaces, with a half-life of greater than 6 h. CHO/rRam-1 cells were also specifically and efficiently killed by exposure to amphotropic gp70 followed by antiserum to gp70 in the presence of complement. Infection with an appropriately pseudotyped form of amphotropic retrovirus 4070A did not perturb control CHO cells or inhibit their phosphate transport. In contrast, 4070A infection of CHO/rRam-1 cells caused major alterations including cell-cell fusions, a specific 40% down-modulation of the rRam-1 component of phosphate transport, and complete interference to super-infection by amphotropic viruses. The 4070A virus-infected CHO/rRam-1 cells retained a substantial cell surface pool of rRam-1 that functioned as a phosphate transporter but not as a viral receptor. We conclude that amphotropic gp70 binds more strongly to rRam-1 than to the homologous hamster protein and that this stable attachment is necessary for infection, interference, membrane fusion, and pathogenesis.     

Functional expression of a myo-inositol/H+ symporter from Leishmania donovani.

The vast majority of surface molecules in such kinetoplastid protozoa as members of the genus Leishmania contain inositol and are either glycosyl inositol phospholipids or glycoproteins that are tethered to the external surface of the plasma membrane by glycosylphosphatidylinositol anchors. We have shown that the biosynthetic precursor for these abundant glycolipids, myo-inositol, is translocated across the parasite plasma membrane by a specific transporter that is structurally related to mammalian facilitative glucose transporters. This myo-inositol transporter has been expressed and characterized in Xenopus laevis oocytes. Two-electrode voltage clamp experiments demonstrate that this protein is a sodium-independent electrogenic symporter that appears to utilize a proton gradient to concentrate myo-inositol within the cell. Immunolocalization experiments with a transporter-specific polyclonal antibody reveal the presence of this protein in the parasite plasma membrane.     

Activity and flexibility of alcohol dehydrogenase in organic solvents

The oxidation of cinnamyl alcohol to cinnamaldehyde by horse liver alcohol dehydrogenase (LADH) was carried out in nearly anhydrous organic solvents and in solvents containing from 0.1 to 10% added water. In nearly anhydrous solvents containing less than 0.02% water, the oxidation rate increased as the water solubility in the solvent decreased, but the reaction did not require active LADH. Moreover, the highest activity in nearly anhydrous heptane was obtained by lyophilizing the enzyme from a solution of pH 2.0, even though LADH exhibits virtually no enzymatic activity in water at this pH. The catalytic activity of LADH was restored and increased dramatically as small amounts of water were added to each solvent. In conjunction with the activity measurements, electron paramagnetic resonance (EPR) spectroscopy and two active-site directed spin labels were used to examine solvent-dependent structural features of LADH. The EPR spectra indicated that LADH became more rigid as the dielectric constant of the solvent decreased. The degree of rigidity also depended on the pH from which the enzyme was lyophilized, indicating that the ionization state of the enzyme can have an important influence on its dynamics in organic solvents. Finally, adding 1% water to organic solvents had no apparent effect on the enzyme's conformation or flexibility near the spin label, even though enzyme activity was an order of magnitude higher when 1% water was present.     

栓孔菌属漆酶高产菌株的初步筛选及其产酶条件的优化

利用显色反应对栓孔菌属(Trametes)进行了漆酶高产菌株的筛选,并对目标菌株的产酶条件进行了优化,在添加愈创木酚的固体培养基中,通过显色反应初步筛选出漆酶高产菌株东方栓孔菌Trametes orientalis Cui 6300;进一步通过单因子分析、正交试验和ABTS法确定了菌株Cui 6300的最适产酶条件:麦芽糖15 g/L,蛋白胨3 g/L,pH 4.8,Cu2+2.0 mmol/L,培养温度28°C,接种饼直径1.5 cm,此时酶活最高可达19.923 U/mL;同时探索了Cu2+浓度及添加时间对其菌丝生物量和漆酶活力的影响。研究表明,Cu2+最适添加浓度为2.0 mmol/L,添加时间为接种后第3天。     

瘦蛋白与皮肤创伤愈合

自1994年瘦蛋白（leptin）被发现以来,其生理作用得到广泛的研究。近年的研究表明,在高等动物的皮肤创伤愈合过程中,胶原与血管的生成、肉芽组织与上皮的再生以及组织的重建等因子,对皮肤的创伤愈合发挥着至关重要的作用,了解瘦蛋白与上述因子之间的相互关系与作用机制,有助于指导治疗,改善预后。