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1.
Conformation of the glucotriose unit in the lipid-linked oligosaccharide precursor for protein glycosylation. 总被引:1,自引:0,他引:1
The conformation of the glucotriose unit of the protein glycosylation precursor Glc3Man9GlcNAc2 was assessed by deuterium exchange studies on the model tetrasaccharide alpha Glc----2 alpha Glc----3 alpha Glc----3 alpha Man----OCH2CH2CH3 dissolved in deuterated dimethyl sulfoxide. The hydroxyl proton on C-2 of the nonreducing end glucose and on C-4 of the glucose attached to mannose both show dramatic isotope shifts indicative of a strong hydrogen bond between these two hydroxyl groups. Such a hydrogen bond requires a fixed conformation of the glucotriose unit that brings these hydroxyl groups within 3 A of each other, a conformation that is supported by molecular modeling based on hard-sphere exo-anomeric (HSEA) calculations. The temperature dependence of the hydroxyl proton chemical shifts supports the postulated hydrogen bond, and the torsional angles between the three glucose units derived from the HSEA calculations are consistent with results from related studies on other saccharides. The results support a model for biochemical function in which the glucotriose unit could modulate the activity of the oligosaccharyltransferase by binding in a fixed conformation to a specific effector site in the enzyme. 相似文献
2.
Electroneutral, HCO3(-)-independent, pH gradient-dependent uphill transport of Cl- by ileal brush-border membrane vesicles. Possible role in the pathogenesis of chloridorrhea.
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By applying a rapid filtration technique to isolated brush border membrane vesicles from guinea pig ileum, 36Cl uptake was quantified in the presence and absence of electrical, pH and alkali-metal ion gradients. A mixture of 20 mM-Hepes and 40 mM-citric acid, adjusted to the desired pH with Tris base, was found to be the most suitable buffer. Malate and Mes could be used to replace the citrate, but succinate, acetate and maleate proved to be unsuitable. In the absence of a pH gradient (pHout:pHin = 7.5:7.5), Cl- uptake increased slightly when an inside-positive membrane potential was applied, but uphill transport was never observed. A pH gradient (pHout:pHin = 5.0:7.5) induced both a 400% increase in the initial Cl- influx rate and a long-lasting (20 to 300 s) overshoot, indicating that a proton gradient can furnish the driving force for uphill Cl- transport. Under pH gradient conditions, initial Cl- entry rates had the following characteristics. (1) They were unaffected by cis-Na+ and/or -K+, indicating the absence of Cl-/K+, Cl-/Na+ or Cl-/K+/Na+ symport activity. (2) Inhibition by 20-100 mM-trans-Na+ and/or -K+ occurred, independent of the existence of an ion gradient. (3) Cl- entry was practically unaffected by short-circuiting the membrane potential with equilibrated potassium and valinomycin. (4) Carbonyl cyanide m-chlorophenylhydrazone was strongly inhibitory and so, to a lesser extent, was 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid [(SITS)], independent of the sign and size of the membrane potential. (5) Cl- entry was negligibly increased (less than 30%) by either trans-Cl- or -HCO3-, indicating the absence of an obligatory Cl-/anion antiport activity. In contrast, the height of the overshoot at 60 s was increased by trans-Cl-, indicating time-dependent inhibition of 36Cl efflux. That competitive inhibition of 36Cl fluxes by anions is involved here is supported by initial influx rate experiments demonstrating: (1) the saturability of Cl- influx, which was found to exhibit Michaelis-Menten kinetics; and (2) competitive inhibition of influx by cis-Cl- and -Br-. Quantitatively, the conclusion is warranted that over 85% of the total initial Cl- uptake energized by a pH gradient involves an electroneutral Cl-/H+ symporter or its physicochemical equivalent, a Cl-/OH- antiporter, exhibiting little Cl- uniport and either Cl-/Cl- or Cl-/HCO3- antiport activities.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
3.
An Ustilago maydis ergosterol biosynthesis mutant (A14) which is partially blocked in sterol 14alpha-demethylase (P45014DM) activity is described. This mutant accumulated the abnormal 14alpha-methyl sterols, eburicol, 14alpha-methylfecosterol, and obtusifoliol, along with significant amounts of ergosterol. Although the A14 mutant grew nearly as well as the wild type, it was impaired in cell extension growth, which indicated a dysfunction in apical cell wall synthesis. The mutant was also found to be hypersensitive to the azole fungicides penconazole and tebuconazole. 相似文献
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F De Venanzi F Pe?a O Jiménez H De Alvarado 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1976,152(1):47-51
Isolated perfused fed rat livers spontaneously liberated glucose and orthophosphate to the medium; 24-hr fasted rat livers did not exhibit these phenomena. In perfused fed rat livers, glucagon (2 mug) increased glucose output and promoted orthophosphate incorporation. In perfused fed rat livers, insulin (250 or 500 mU) inhibited the spontaneous liberation of glucose and orthophosphate. Comparable doses of insulin significantly reduced the glucagon (2 mug)-induced increase in glucose output from perfused fed rat liver, but did not affect orthophosphate uptake by the organ. 相似文献
6.
JA Kiernan 《Biotechnic & histochemistry》2013,88(5-6):203-210
Hexazonium pararosaniline is a valuable reagent that has been used in enzyme activity histochemistry for 50 years. It is an aqueous solution containing the tris-diazonium ion derived from pararosaniline, an aminotriarylmethane dye, and it contains an excess of nitrous acid that was not consumed in the diazotization reaction. Other investigators have found that immersion for 2 min in an acidic (pH 3.5) 0.0015 M hexazonium pararosaniline solution can protect cryostat sections of unfixed animal tissues from the deleterious effects of aqueous reagents such as buffered solutions used in immunohistochemistry, while preserving specific affinities for antibodies. In the present investigation hexazonium pararosaniline protected lymphoid tissue and striated muscle against the damaging effects of water or saline. The same protection was conferred on unfixed sections treated with dilute nitrous or hydrochloric acid in concentrations similar to those in hexazonium pararosaniline solutions. Model tissues (solutions, gels or films containing gelatin and/or bovine albumin) responded predictably to well known cross-linking (formaldehyde) or coagulant (mercuric chloride) fixatives. Hexazonium pararosaniline solutions prevented the dissolution of protein gels in water only after 9 or more days of contact, during which time considerable swelling occurred. It is concluded that there is no evidence for a “fixative” action of hexazonium pararosaniline. The protective effect on frozen sections of unfixed tissue is attributable probably to the low pH of the solution. 相似文献
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8.
In this study, autometallography and immunohistochemistry were used to localize and quantify cadmium and metallothionein (MT) levels, respectively, in cellular compartments of turbot liver on exposure to cadmium for 7 days and further depuration treatment for 14 days. Metals weakly bound to proteins (i.e. MTs) in hepatocyte lysosomes were visualized as black silver deposits (BSDs) using a light microscope. With the aid of a newly developed immunohistochemical procedure, MTs were localized and semi-quantified in both the cytosolic and the lysosomal compartments of hepatocytes. The BSD extent in the lysosomes of hepatocytes increased significantly as a result of cadmium exposure. This response was evidenced after 1h. Further, a progressive increase in the volume density of BSDs occurred up to the seventh day. Total MT immunohistochemical levels increased at a lower rate, starting after 1 day of cadmium exposure. BSD extent values recovered after depuration, whilst MT levels remain unchanged. It is possible that the detoxification rate of metals via lysosomes was diminished, whilst MT levels remained unchanged, at least after 14 days of depuration. It can be concluded that autometallography and MT immunohistochemistry are good tools for clarifying metal and metal-MT trafficking routes in hepatocytes, and also that BSD extent and MT immunohistochemical levels in the lysosomes and cytosol of fish hepatocytes can be considered to be useful biomarkers of metal exposure. 相似文献
9.
Alvarado Claudia Ramírez Jose Martín Herrera-López Enrique J. Cortez-Valladolid Diego Ramírez Gerardo 《Biological trace element research》2020,193(1):226-233
Biological Trace Element Research - Fluoride accumulates and is toxic to bones. Clinical bone lesions occur in a phased manner, being less severe early in the natural course of skeletal fluorosis.... 相似文献
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