3-磷酸甘油醛脱氢酶胍变性时的活力及构象变化

酵母3-磷酸甘油醛脱氢酶在盐酸胍溶液中的内源荧光及剩余活力的变化结果提示:apo酶及holo酶的活力在胍浓度为0.5M左右可完全丧失.同时伴有内源荧光强度的下降,光谱宽度的增加和335nm最大发射峰的红移(提示了色氨酸残基的暴露).与已经报导的肌肉酶(内源荧光强度在胍浓度为0.4—1.2M范围相对稳定)不同,酵母酶内源荧光在此浓度范围内表现为逐渐降低.在0.7M胍溶液中,内源荧光变化动力学过程只能测出一相,而酶失活动力学过程为快慢两相,快相动力学速度常数至少大于内源荧光降低速度常数三个数量级以上.以上结果提示:低浓度胍可引起该酶的完全失活,活性部位的空间构象比酶分子的构象更易受到变性剂的扰乱;有一个色氨酸残基位于或靠近酶的活性部位.     

Factors affecting chloride conductance in apical membrane vesicles from human placenta

Summary Apical membrane vesicles from human term placenta were isolated using a magnesium precipitation technique, and the purity of the vesicles was assessed morphologically using scanning and transmission electron microscopy, and biochemically, using marker enzymes. The vesicles were found to be morphologically intact and significantly enriched in enzymes associated with apical membranes. ³⁶Cl^– uptake into these vesicles was studied in the presence of an outwardly directed Cl^– gradient. This uptake was found to be time dependent, with an initial rapid uptake tending to peak between 10 and 20 min and thereafter decline. Uptake was found to be voltage dependent since 5 m valinomycin caused a decrease in uptake. The effects of N-phenylanthranilic acid (NPA) and 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS) and bumetanide on the initial rate of Cl^– were examined in the presence and absence of 5 m valinomycin. NPA and DIDS inhibited isotope uptake strongly with IC₅₀ values of 0.83±0.35 m and 3.43±0.37 m, respectively, in the absence of valinomycin. Although valinomycin reduced ³⁶Cl^– uptake by about 80% when added before the isotope, DIDS reduced the uptake which remained in a concentration-dependent fashion with an IC₅₀ of 5.6±2.1 m. Under these conditions, NPA was without effect at concentrations below 100 m. Bumetanide was without effect at the concentrations used in the absence of valinomycin. However, following valinomycin pretreatment, bumetanide reduced ³⁶Cl^– uptake significantly at 100 m concentration. Vesicle diameter, as assessed by flow cytometry, did not change under the conditions employed.The effects of some fatty acids were also investigated. Arachidonic acid and linoleic acid inhibited Cl^– uptake with IC₅₀ values of 37.6±14.9 m and 4.59±0.51 m, respectively. Arachidonyl alcohol and elaidic acid were found to be without effect. These studies show that human placental brush border membrane vesicles possess a chloride conductance channel, the activity of which can be measured in the presence of an outwardly directed Cl^– gradient and this channel is sensitive to Cl^– channel inhibitors, especially N-phenylanthranilic acid, and can be inhibited by unsaturated fatty acids such as arachidonic acid and linoleic acid.This work was supported in part by the Cystic Fibrosis Association of Ireland and Eolas, The Irish Science and Technology Agency. The technical assistance of Mr. Cormac O' Connell in the preparation of the electron micrographs and of Mr. Roddy Monks in the flow cytometric analysis is gratefully acknowledged.     

Inhibition of ileal brush-border chloride conductance by specific antibody

Summary Antibody raised in mice was used in attempting to identify proteins responsible for the conductive chloride transport that can be measured in porcine ileal brush border membrane vesicles. Ileal brush-border membrane vesicle protein from pig was separated into five different molecular mass fractions by preparative SDS polyacrylamide disc gel electrophoresis. Separated protein fractions were used to immunize mice. Antibody was screened for reactivity with antigen by Western blotting, and for effects on conductive chloride transport in ileal brush border membrane vesicles. Immunization with brush-border protein from fraction I proteins (>110 kDa) produced polyclonal antisera which specifically inhibited the conductive component of chloride uptake by ileal brush border vesicle preparations. Western blotting of the antigen showed the presence of several protein species of molecular mass >100 kDa that were recognized by immune serum. Spleen cells from a mouse producing antiserum that inhibited conductive chloride transport were fused with a myeloma cell line. The resulting hybridoma colonies produced antibody that reacted with at least seven distinct protein bands by Western blot assay and inhibited chloride conductance in brush-border membrane vesicles.     

Aldosterone regulation of active sodium chloride transport in the lizard colon (Gallotia galloti)

Summary Bioelectrical parameters and unidirectional sodium and chloride fluxes were measured under voltageclamp conditions in groups of lizards submitted to single or chronic aldosterone treatment. Both acute (AT) and chronic (CT) treatment induced significant increases in the short-circuit current (I _sc), as well as in the mucosa-to-serosa (J _m-s ^Na ) and net sodium flux (J _net ^Na ). In AT tissues, aldosterone did not change net chloride flux (J _net ^Cl ) but did so in CT tissues. Amiloride reduced the aldosterone-increased I _sc in AT and CT tissues, inhibited J _net ^Na in AT tissues and abolished it in CT colons. J _net ^Cl was also reduced by the diuretic in the group of AT colons, whereas no changes were observed in the CT tissues. Addition of luminal DIDS reduced Na⁺ absorption and totally inhibited Cl^- absorption in the AT tissues, but did not change I _sc. However, in CT tissues neither Na⁺ nor Cl^- transport were affected by DIDS. A good relationship between I _sc and J _m-s ^Na was apparent after DIDS treatment in AT tissues. In this group, simultaneous addition of DIDS and amiloride totally abolished J _net ^Na and reduced I _sc to untreated control values. Addition of serosal ouabain abolished I _sc and Na⁺ absorption in AT and CT colons, but Cl^- absorption was only altered in AT tissues. These results support the hypothesis that aldosterone induces an electrogenic, amiloride-sensitive sodium absorption, and in a dose-dependent fashion suppresses electroneutral NaCl absorption in the lizard colon.Abbreviations AT acutely treated - CT chronically treated animals - DIDS 4-4-diisothiocyanatostibene-2-2-disulfonic acid - DMSO dimethylsulphoxide - G _t tissue conductance - I _sc short circuit current - PD transepithelial potential difference - SITS 4-acetamido-4-isothiocyanatostilbene-2-2-disulfonic acid - UC untreated controls Preliminary results of this paper were presented at the X ^th meeting of the European Intestinal Transport Group (EITG), Askov Hojskole, Denmark, 16–19 September 1990     

The effects of lodging and a paclobutrazol — chlormequat chloride mixture on the yield and quality of oilseed rape

The effects of lodging and a plant growth regulator mixture on oilseed rape cv. Ariana were studied in three field experiments. Natural and artificially induced lodging treatments varying in time of imposition and severity were compared to a supported control. A mixture of paclobutrazol and chlormequat chloride was applied either as a spray or as paclobutrazol granules followed immediately by a chlormequat chloride spray.
In 1987, severe lodging treatments reduced yield by up to 52%. Yield penalties varied with the time at which lodging was imposed. Yield was inversely correlated with the ground cover of volunteers growing from shed seed under lodged crops.
In 1988, two experiments showed increased incidence of disease and decreases in seed yield and quality in lodged crops. Yield reductions were related to the severity of lodging. Lodging decreased oil contents and increased glucosinolate levels. PGR treatments reduced lodging and maintained yield at a level not significantly different to a supported control treatment. Oil contents were also similar in seed from PGR treated and control plots. Glucosinolate levels in PGR treated seed were similar to control levels in one experiment and intermediate to those from control and artificially lodged plots in another experiment.
The results are discussed in relation to the use of PGRs to prevent lodging in 'double zero' varieties of oilseed rape, and the potential losses from using ground vehicles to apply pesticides after flowering.     

Isolation of a cloned DNA segment containing a ribosomal protein gene of Drosophila melanogaster

A Drosophila genomic DNA library in the vector Charon 4 was screened using cDNA derived from the small (6S-12S) poly(A)+ mRNA of 2-6-h-old Drosophila embryos. This fraction of mRNA is enriched for ribosomal protein-coding sequences. The selected recombinants were hybridized to total mRNA under conditions which allowed for isolation of homologous mRNAs. The mRNA from these RNA/DNA hybrids was eluted and translated in vitro. The translation products were analyzed by one- and two-dimensional electrophoresis with authentic ribosomal proteins as standards. One cloned DNA segment was found to contain a ribosomal protein gene, and a sequence which hybridizes strongly to at least 5 other ribosomal protein mRNAs.     

Chloride distribution in the proximal convoluted tubule ofNecturus kidney

Summary To assess the mechanism(s) by which intraluminal chloride concentration is raised above equilibrium values, intracellular Cl^– activity ( _i ^Cl ) was studied in the proximal tubule ofNecturus kidney. Paired measurements of cell membrane PD (V _BL) and Cl-selective electrode PD (V _BL ^Cl ) were performed in single tubules, during reversible shifts of peritubular or luminal fluid composition. Steadystate _i ^Cl was estimated at 14.6±0.6 mmol/liter, a figure substantially higher than that predicted for passive distribution. To determine the site of the uphill Cl^– transport into the cell, an inhibitor of anion transport (SITS) was added to the perfusion fluid. Introduction of SITS in peritubular perfusate decreased _i ^Cl , whereas addition of the drug in luminal fluid slightly increased _i ^Cl ; both results are consistent with basolateral membrane uphill Cl^– transport from interstitium to the cell. TMA⁺ for Na⁺ substitutions in either luminal or peritubular perfusate had no effect on _i ^Cl . Removal of bicarbonate from peritubular fluid, at constant pH (a situation increasing HCO ₃ ^– outflux), resulted in an increase of _i ^Cl , presumably related to enhanced Cl^– cell influx: we infer that Cl^– is exchanged against HCO ₃ ^– at the basolateral membrane. The following mechanism is suggested to account for the rise in luminal Cl^– concentration above equilibrium values: intracellular CO₂ hydration gives rise to cell HCO ₃ ^– concentrations above equilibrium. The passive exit of HCO ₃ ^– at the basolateral membrane energizes an uphill entry of Cl^– into the cell. The resulting increase of _i ^Cl , above equilibrium, generates downhill Cl^– diffusion from cell to lumen. As a result, luminal Cl^– concentration also increases.C.N.R.S. Greco 24. Part of this work was presented at the 12^th annual meeting of the American Society of Nephrology, Boston, Mass. (Edelman et al., 1979).     

Active sulfate absorption in rabbit ileum: Dependence on sodium and chloride and effects of agents that alter chloride transport

Summary Unidirectional fluxes of³⁵SO₄ across and into rabbit ileal epithelium were measured under short-circuit conditions, mostly at a medium SO₄ concentration of 2.4mm. Unidirectional mucosa (m)-to-serosa (s) ands-to-m fluxes (J _ms,J _sm) were 0.456 and 0.067 moles hr^–1 cm^–2, respectively.J _ms was 2.7 times higher in distal ileum than in mid-jejunum. Ouabain abolished net SO₄ transport (J _net) by reducingJ _ms. Epinephrine, a stimulus of Cl absorption, had no effect on SO₄ fluxes. Theophylline, a stimulus of Cl secretion, reducedJ _ms without affectingJ _sm, causing a 33% reduction inJ _net. Other secretory stimuli (8-Br-cAMP, heat-stable enterotoxin, Ca-ionophore A23187) had similar effects. Replacement of all Cl with gluconate markedly reducedJ _net through both a decrease inJ _ms and an increase inJ _sm. The anion-exchange inhibitor, 4-acetoamido-4-isothiocyano-2,2-sulfonic acid stilbene (SITS), when added to the serosal side, reducedJ _ms by 94%, nearly abolishingJ _net. SITS also decreasedJ _sm by 75%. Mucosal SITS (50 m) was ineffective. 4,4-diisothiocyano-2,2-sulfonic acid stilbene (DIDS) had effects similar to SITS but was less potent. Measurements of initial rates of epithelial uptake from the luminal side (J _me) revealed the following: (1)J _me is a saturable function of medium concentration with aV _max of 0.94 moles hr^–1 cm^–2 and aK _1/2 of 1.3mm; (2) replacing all Na with choline abolishedJ _me; (3) replacing all Cl with gluconate increasedJ _me by 40%; (4) serosal SITS had no effect onJ _me; and (5) stimuli of Cl secretion had no effect onJ _me or increased it slightly. Determination of cell SO₄ with³⁵SO₄ indicated that, at steady-state, the average mucosal concentration is 1.1 mmoles per liter cell water, less than half the medium concentration. Cell SO₄ was increased to 3.0mm by adding SITS to the serosal side. Despite net transport rates greater than 1.4 Eq hr^–1 cm^–2, neither addition of SO₄ to the SO₄-free medium nor addition of SITS to SO₄-containing medium altered short-circuit current. The results suggest that (1) ileal SO₄ absorption consists of Na-coupled influx (symport) across the brush border and Cl-coupled efflux (antiport) across the basolateral membrane; (2) the overall process is electrically neutral; (3) the medium-to-cell Cl concentration difference may provide part of the driving force for net SO₄ absorption; and (4) since agents affecting Cl fluxes (both absorptive and secretory) have little effect on SO₄ fluxes, the mechanisms for their transcellular transports are under separate regulation.     

Cyanamide mediated syntheses of peptides containing histidine and hydrophobic amino acids

Summary Using the model of a primitive earth evaporation pond, the synthesis of three histidyl peptides in yields of up to 11% was demonstrated when aqueous solutions of histidine, leucine, ATP, cyanamide, and MgCl₂ were evaporated and heated for 24 h at 80°C. In addition, peptides were formed in yields of up to 56%, 35%, and 21%, respectively for phenylalanine, leucine, and alanine when aqueous solutions of the appropriate amino acid were evaporated and heated with cyanamide and one or more of the following components: ATP, AMP, 4-amino-5-imidazole carboxamide, or MgCl₂. The greatest peptide yield occurred at pH 3. But peptide formation was demonstrated for a system of Leu, cyanamide, and MgCl₂ adjusted to pH 7 with NH₄OH.Peptide synthesis was also studied in the presence of CaCl₂, ZnCl₂, different adenosine nucleotides, and UTP to compare their effects on peptide synthesis. The optimum conditions for cyanamide mediated peptide synthesis were also studied in terms of pH, reaction time, reaction temperature, and cyanamide concentration. The major side product in nearly all reactions studied appears to be an amino acid-cyanamide adduct. Peptides were analyzed and identified by thin layer chromatography, acid hydrolysis, and enzymatic degradation.     

The manipulation of polar head group composition of phospholipids in the wheat Miranovskaja 808 affects frost tolerance

Caryopses of the frost-resistant cultivar of the wheat Triticum aestivum L., Miranovskaja 808, were germinated and grown in the presence of various concentrations of choline chloride. Changes in the composition of leaf total phospholipids and leaf total fatty acids at two extreme temperatures (25°C and 2°C) as well as changes in frost resistance were followed. A choline chloride concentration-dependent accumulation of phosphatidyl choline was observed in the leaves. Seedlings grown at 2°C accumulated more phosphatidyl choline at each choline chloride concentration than those grown at 25°C. There was an inverse relationship between the contents of phosphatidyl choline and phosphatidic acid in the leaves. Neither the temperature nor choline chloride seemed to affect fatty-acid composition. Modification of polar-head group composition of phospholipids affected frost tolerance: Seedlings grown in the presence of 15 mM choline chloride at 25°C exhibited a freezing resistance equal to that of hardened controls. The data indicate that the polar-head group composition of membrane phospholipids in plants can be easily manipulated and point to the importance of phosphatidyl choline in cold adaptation processes.