Interactions between anion exchange and other membrane proteins in rabbit kidney medullary collecting duct cells

Summary In separated outer medullary collecting duct (MCD) cells, the time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to the MCD cell analog of band 3, the red blood cell (rbc) anion exchange protein, can be measured by the stopped-flow method and the reaction time constant, _DBDS, can be used to report on the conformational state of the band 3 analog. In order to validate the method we have now shown that the ID_50,DBDS,MCD (0.5±0.1 m) for the H₂-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of _DBDS is in agreement with the ID_50,Cl ^–,_MCD (0.94±0.07 m) for H₂-DIDS inhibition of MCD cell Cl^– flux, thus relating _DBDS directly to anion exchange. The specific cardiac glycoside cation transport inhibitor, ouabain, not only modulates DBDS binding kinetics, but also increases the time constant for Cl^– exchange by a factor of two, from _Cl=0.30±0.02 sec to 0.56±0.06 sec (30mm NaHCO₃). The ID_50,DBDS,MCD for the ouabain effect on DBDS binding kinetics is 0.003±0.001 m, so that binding is about an order of magnitude tighter than that for inhibition of rbc K⁺ flux (K _I,K ⁺,_rbc=0.017 m). These experiments indicate that the Na⁺,K^–-ATPase, required to maintain cation gradients across the MCD cell membrane, is close enough to the band 3 analog that conformational information can be exchanged. Cytochalasin E (CE), which binds to the spectrin/actin complex in rbc and other cells, modulates DBDS binding kinetics with a physiological ID_50,DBDS,MCD (0.076±0.005 m); 2 m CE also more than doubles the Cl^– exchange time constant from 0.20±0.04 sec to 0.50±0.08 sec (30mm NaHCO₃). These experiments indicate that conformational information can also be exchanged between the MCD cell band 3 analog and the MCD cell cytoskeleton.     

Influence of ammonium and extracellular pH on the amino and organic acid contents of suspension culture cells of Acer pseudoplatanus

The effects of supplied ammonium and nitrate on the amino and organic acid contents and enzyme activities of cell suspension cultures of Acer pseudoplatanus L. were examined. Regardless of nitrogen source the pH of the culture medium strongly affected the malate and citrate contents of the cells; these organic acid pools declined at pH 5, but increased at pH 7 and 8. Over a period of two days, ammonium had little effect on the responses of the organic acid pool sizes to the pH of the medium. In contrast, ammonium had a strong influence on amino acid pool sizes, and this effect was dependent on the pH of the medium. At pH 5 there was no increase in cell ammonium or amino acid contents, but at higher pH values cellular ammonium content rose, accompanied by accumulation of glutamine, glutamate and asparagine. Over several days, supplied ammonium led to an increase in activity of glutamate dehydrogenase irrespective of any changes in internal ammonium and amino acid contents. If the pH of the medium was allowed to fall below pH 4 in the presence of ammonium, phosphoenolpyruvate (PEP) carboxylase activity declined to a very low value over several days; at higher pH, the activity of this enzyme, and that of NAD malic enzyme and NAD malate dehydrogenase, remained substantial irrespective of whether the nitrogen source was NH⁺₄ or NO-₃.     

Decomposition of cellulose,soil organic matter and plant litter in a temperate grassland soil

The formation of mor humus in an experimental grassland plot, which has been acidified by long-term fertiliser treatment, has been studied by comparing the rates of cellulose, soil organic matter and plant litter decay with those in an adjacent plot with near-neutral pH and mull humus. The decomposition of cellulose filter paper in litter bags of 5 mm, 1-mm and 45-μm mesh size buried at 3 to 4 cm depth the plots was followed by measuring the weight loss and changes in glucose content over a 6 month period. Soil pH was either 5.3 or 4.3. Decomposition of native soil organic matter and plant litter in soil from the same plots were followed using CO₂ evolution in laboratory microcosms. Cellulose weight loss at pH 5.3 was greatest from the 5-mm mesh bags and least from the 45-um mesh bags. At pH 4.3 there was little weight loss from bags and no significant differences in weight loss between bags with different sized mesh. There was, however, a reduction in the glucose content of the hydrolysed and derivatised filter paper with time. The decomposition rate of native soil organic matter in the low pH soil was increased to that observed in the less acid soil when the pH of the former was increased from 4.3 to 5.3. The increase in decomposition rate of added plant litter in the more acid soil as a result of CA(OH)₂ addition was only 60% of that observed in the soil with pH 5.3. These data support the hypothesis that the absence of soil animals and the restricted microbial decomposition in the acidic soil was responsible for mor humus formation.     

Influence of cytosolic pH changes on the organisation of the endoplasmic reticulum in epidermal cells of onion bulb scales: Acidification by loading with weak organic acids

Summary The anastomosing ER system of epidermal cells of onion bulb scales is composed of three modifications: lamellar and tubular elements, located in the cell periphery, and long tubular stands located deeper in the cytoplasm. Cytoplasmic acidification of epidermal cells by loading with weak organic acids like acetic or propionic acid causes the decay of the lamellar elements and the disappearance of long tubular strands. Organelle movement is also inhibited. The effects depend on the pH of the incubation medium and on the administered acid concentration, and are characterized by a distinct lag phase of about 7 min. The induced ER changes are transient with adaptation starting after about 50min. Buffer components alone have little influence on the cellular ER organization within a pH-range of 4.0–8.0. However, the pH of the medium strongly affects the time course of the effects as well as recovery after omitting the administered acid. Both modulation and recovery occur more rapidly at neutral or slightly alkaline pH. Actin filaments, which play a major role in ER organization and organelle movement, are not affected by cytosolic acidification.Dedicated to the memory of Professor Oswald Kiermayer     

The use of amphipathic maleimides to study membrane-associated proteins

A series of amphiphilic polymethylenecarboxymaleimides has been synthesized for use as sulfhydryl reagents applicable to membrane proteins. Physical properties of the compounds which are relevant to their proposed mode of action have been determined. By comparing rates of reaction in aqueous and aprotic solvents, the compounds have been shown to react exclusively with the thiolate ion. The effects of the reagents on three membrane-associated proteins are reported, and in two cases a comparative study has been made of the effects on the proteins in the absence of membranes. A mechanism is proposed whereby the reagents are anchored at the lipid/water interface by the negatively charged carboxyl group, thus siting the reactive maleimide in a plane whose depth is defined by the length of the reagent. Supporting evidence for this model is provided by the inability of the reagents to traverse membranes, and variation of their inhibitory potency with chain length when the proteins are embedded in the membrane, but not when extracted into solution. As examples of general use of the reagents to probe sulfhydryl groups in membrane proteins, the reagents have been used to (a) determine the depths in the membrane at which two populations of sulfhydryl groups occur in the mitochondrial phosphate transporter; (b) locate a single sulfhydryl associated with the active site ofD--hydroxybutyrate dehydrogenase in the inner mitochondrial membrane; (c) examine sulfhydryl groups in theD-3-glyceraldehyde phosphate dehydrogenase associated with the human red blood cell membrane.     

Sulfur dynamics in mineral horizons of two northern hardwood soils. A column study with 35S

Sulfur dynamics of two Spodosols were ascertained using soil columns constructed from homogenized mineral soil from nothern hardwood ecosystems at the Huntington Forest (HF) in the Adirondack Mountains of New York and Bear Brook Watershed in Maine (BBWM). Columns were leached for 20 weeks with a simulated throughfall solution with³⁵SO₄ ^2-. Sulfur constituents were similar to those of other Spodosols, with the organic S fractions (C-bonded S and ester sulfate) constituting over 90% of total S. HF soil columns had higher total S (14.9 mol S g^-1) than that for the BBWM soil columns (7.4 mol g^-1) primarily due to higher C-bonded S in the former.Initially, adsorbed SO₄ ^- accounted for 5 and 4% of total S for the BBWM and HF soil columns, respectively. After 20 weeks, adsorbed SO₄ ^2- decreased (81%) in BBWM and increased (33%) in HF soil columns. For both HF and BBWM soil columns, C-bonded S increased and ester sulfate decreased, but only for HF columns was there a net mineralization of organic S (5.6% of total S). The greatest decrease in ester sulfate occurred at the top of the columns.Leaching of³⁵S was less than 0.5% of the³⁵S added due to its retention in various S constituents. There was an exponential decrease in³⁵S with column depth and most of the radioisotope was found in C-bonded S (70–88 and 70–91% for BBWM and HF, respectively). The rapid turnover of adsorbed SO^2- ₄ was reflected in its high specific activity (834 and 26 kBq mol^-1 S for BBWM and HF, respectively). The lower specific activity of adsorbed SO₄ ^2- in HF was attributable to greater isotopic dilution by non-radioactive SO^2- ₄ derived from greater organic S mineralization in the HF versus the BBWM columns.Both soil columns initially had high levels of NO^- ₃ which resulted in the generation of H⁺ and net retention of SO₄ ^2- in the early phase of the experiment due to pH dependent sulfate adsorption; later NO₃ ^- decreased and SO₄ ^2- was desorbed. Leaching of NIO₃ ^- and SO₄ ^2- was correlated with losses of Mg²⁺ and Ca²⁺ of which the latter was the dominant cation.Analyses using both S mass balances and radioisotopes corroborate that for BBWM soil columns, SO^2- ₄ adsorption-desorption dominated the S biogeochemistry while in HF soil columns, organic S mineralization-immobilization processes were more important. It is suggested that similar techniques can be applied to soils in the field to ascertain the relative importances of SO₄ ^2- adsorption processes and organic S dynamics.     

Aluminum geochemistry in peatland waters

The chemical speciation of aluminum was examined in surface water samples from Sphagnum peatlands in north-central Minnesota, from peatlands along the Canadian east coast, and from bogs in the Pennine Mountain area of England. In highly organic ([DOC] 50 mg L^–1 ), low pH waters, 80–90% of total dissolved Al was complexed with organic matter (OM), while in waters with low DOC ([DOC] 5 mg L^–1) 54–86% of total dissolved Al existed as Al⁺³ or other inorganic Al species. Batch titrations of OM with Al revealed a high Al binding capacity, 1.4–2.8 mol (mg DOC)^–1, that generally was unsaturated with Al. Titrations of OM with Al in conjunction with a continuous distribution model were used to determine Al-OM conditional stability constants. Binding capacity (mol Al (mg DOC)^–1) and strength (formation constant) increased from pH 3 to 5 but decreased above pH 5 due to formation of AI-hydroxy species including A1(OH)₃ (s). The high binding capacity of OM in bog waters facilitates metal mobility, especially in low pH (< 5) wetlands where metal solubility is high and OM concentrations are highest. Results showed that the relative degree of organic matter saturation with metal ions was important in modeling AI speciation in bog waters.     

Development of cytochrome b558 and oxidative metabolism in human granulocytes, monocytes and during differentiation of HL-60 and U 937 cells

The development of cytochrome b558 (Cyt b) as determined spectrophotometrically, was investigated in human polymorphonuclear neutrophils (PMN), monocytes (MN) and during differentiation of HL-60 and U 937 cells induced by retinoic acid (RA) alone or in combination with IFN gamma. O2- release in response to a panel of stimulating agents, ie latex particles, opsonised zymosan, PMA, Con A and fMLP, was monitored by lucigenin-amplified chemiluminescence (CL). In parallel the expression of myeloperoxidase (MPO) was investigated and its catalytic activity on H2O2 related to luminol-amplified CL responses. In mature PMN and MN phagocytes, regardless of the stimulating agent, the O2- production is closely related to Cyt b but not to MPO specific contents. In differentiated HL-60 and U 937 cells, the oxidative metabolism increases in parallel with Cyt b specific contents, both being enhanced by the addition of IFN gamma to the RA treatment. However, marked differences in the O2- production intensities are observed depending on the stimulating agent tested and the state of differentiation considered. The PMA-stimulated O2- production is rather low ie 100 and 20 times less in granulocytic HL-60 and monocyto-macrophagic U 937 cells than in PMN and MN respectively. Latex, zymosan and Con A stimulated responses are close to those of MN, in monocyte-macrophagic U 937 cells. In conclusion, these data show that during differentiation; 1), Cyt b plays a critical role in O2- production; 2), the pathways leading to NADPH oxidase activation are diversely modulated following phagocyte differentiation with IFN gamma and/or with RA.     

Catalysis by Laccase (From Coriolus uersicolor) in Microheterogeneous Media of the Water/Organic Solvent/Surfactant Type

Catalysis by laccase from Coriolus uersicolor solubilized in the ternary systems of surfactant/water/organic solvent type, namely, Aerosol OT/water/octane, Brij 56/water/cyclohexane and egg lecithin/water/octane + pentanol + methanol mixture, has been studied. The laccase activity is found to depend, in principle, not only on the water/surfactant molar ratio, but on the surfactant concentration (with its hydration degree being constant) as well. The following inferences should be emphasized. Firstly, in all the systems under study, the catalytic activity (k_cat) of laccase entrapped into surfactant reversed micelles increases more than 50 times (when the surfactant concentration is extrapolated to zero) compared with the k_cat value in aqueous solution. Secondly, the catalytic activity (k_cat) of laccase entrapped in hydrated Aerosol OT aggregates, having lamellar, reversed cylindrical (hexagonal) and reversed micellar structure, depends greatly on the aggregate type. In other words, the phase transitions, i.e. an alteration in the packing of hydrated Aerosol OT molecules, evokes a sharp reversible change in the enzymatic activity. Thirdly, in the same phase, the catalytic activity of the solubilized enzyme depends on the linear dimensions of water cavities inside the surfactant aggregates (i.e. on the water content in the system under study). All these effects, regulating enzymatic activity, are probably caused by an alteration of the conformational mobility of laccase molecules incorporated into the inner polar cavities inside the surfactant aggregates.     

Chloride-induced Ca2+ Release from the Sarcoplasmic Reticulum of Chemically Skinned Rabbit Psoas Fibers and Isolated Vesicles of Terminal Cisternae

There is increasing evidence that Ca²⁺ release from sarcoplasmic reticulum (SR) of mammalian skeletal muscle is regulated or modified by several factors including ionic composition of the myoplasm. We have studied the effect of Cl⁻ on the release of Ca²⁺ from the SR of rabbit skeletal muscle in both skinned psoas fibers and in isolated terminal cisternae vesicles. Ca²⁺ release from the SR in skinned fibers was inferred from increases in isometric tension and the amount of release was assessed by integrating the area under each tension transient. Ca²⁺ release from isolated SR was measured by rapid filtration of vesicles passively loaded with ⁴⁵Ca²⁺. Ca²⁺ release from SR was stimulated in both preparations by exposure to a solution containing 191 mm choline-Cl, following pre-equilibration in Ca²⁺-loading solution that had propionate as the major anion. Controls using saponin (50 μg/ml), indicated that the release of Ca²⁺ was due to direct action of Cl⁻ on the SR rather than via depolarization of T-tubules. Procaine (10 mm) totally blocked Cl⁻- and caffeine-elicited tension transients recorded using loading and release solutions having ([Na⁺] + [K⁺]) × [Cl⁻] product of 6487.69 mm ² and 12361.52 mm ², respectively, and blocked 60% of Ca²⁺ release in isolated SR vesicles. Surprisingly, procaine had only a minor effect on tension transients elicited by Cl⁻ and caffeine together. The data from both preparations suggests that Cl⁻ induces a relatively small amount of Ca²⁺ release from the SR by activating receptors other than RYR-1. In addition, Cl⁻ may increase the Ca²⁺ sensitivity of RYR-1, which would then allow the small initial release of Ca²⁺ to facilitate further release of Ca²⁺ from the SR by Ca²⁺-induced Ca²⁺ release. Received: 6 February 1996/Revised: 17 July 1996