Lateral diffusion of wheat germ agglutinin-labeled glycoconjugates in the membrane of differentiating HL-60 and U-937 cells assessed with fluorescence recovery after photobleaching (FRAP)

The promyelocytic leukemia cell line HL-60 and the histiocytic cell line U-937 were grown in suspension culture. They were induced to differentiate during 5-d cultivation in the presence of dimethylsulfoxide (DMSO; 1.3% w/v) or phorbol-12-myristate-acetate (PMA; 10(-7) M), which yields granulocyte- and macrophage-like cells, respectively. Differentiation was evidenced by increased capacity to recognize and phagocytize IgG- or complement-coated yeast particles. Aliquots taken from the cultures with and without DMSO (or PMA) were spun down directly on glass microscope slides, washed, labeled with fluoresceinated wheat germ agglutinin (WGA), and directly examined at room temperature for the rate of fluorescence recovery after photobleaching (FRAP). It was found that cultivation of the HL-60 and the U-937 cells in the presence of DMSO, which yields granulocyte-like cells, reduced the average value of lateral diffusion coefficient D (X 10(10] from 1.72 +/- 0.13 cm2s-1 to 0.97 +/- 0.13 cm2s-1 and from 1.77 +/- 0.11 cm2s-1 to 0.82 +/- 0.13 cm2s-1, respectively. U-937 cells grown with PMA also showed a reduction of D(X 10(10] to 0.88 +/- 0.10 cm2s-1. There was a larger immobile fraction of fluorescence in the HL-60 cells than in the U-937 cells, viz., 70-80% compared to 10-50%. The total number of binding sites for WGA was not altered, but the surface density changed, since the HL-60 and the U-937 cells became smaller and larger, respectively, when grown in the presence of DMSO. It is concluded that differentiation reduces the average lateral mobility of the WGA-binding membrane component by a factor around 2.     

Reconstituting the barrier properties of a water-tight epithelial membrane by design of leaflet-specific liposomes

To define aspects of lipid composition and bilayer asymmetry critical to barrier function, we examined the permeabilities of liposomes that model individual leaflets of the apical membrane of a barrier epithelium, Madin-Darby canine kidney type 1 cells. Using published lipid compositions we prepared exofacial liposomes containing phosphatidylcholine, sphingomyelin, glycosphingolipids, and cholesterol; and cytoplasmic liposomes containing phosphatidylethanolamine, phosphatidylserine, and cholesterol. The osmotic permeability of cytoplasmic liposomes to water (P(f)), solutes, and NH(3) was 18-90-fold higher than for the exofacial liposomes (P(f(ex)) = 2.4 +/- 0.4 x 10(-4) cm/s, P(f(cy)) = 4.4 +/- 0.3 x 10(-3) cm/s; P(glycerol(ex)) = 2.5 +/- 0.3 x 10(-8) cm/s, P(glycerol(cy)) = 2.2 +/- 0.02 x 10(-6) cm/s; P(NH3(ex)) = 0. 13 +/- 0.4 x 10(-4) cm/s, P(NH3(cy)) = 7.9 +/- 1.0 x 10(-3) cm/s). By contrast, the apparent proton permeability of exofacial liposomes was 4-fold higher than cytoplasmic liposomes (P(H+(ex)) = 1.1 +/- 0. 1 x 10(-2) cm/s, P(H+(cy)) = 2.7 +/- 0.6 x 10(-3) cm/s). By adding single leaflet permeabilities, we calculated a theoretical P(f) for a Madin-Darby canine kidney apical membrane of 4.6 x 10(-4) cm/s, which compares favorably with experimentally determined values. In exofacial liposomes lacking glycosphingolipids or sphingomyelin, permeabilities were 2-7-fold higher, indicating that both species play a role in barrier function. Removal of cholesterol resulted in 40-280-fold increases in permeability. We conclude: 1) that we have reconstituted the biophysical properties of a barrier membrane, 2) that the barrier resides in the exofacial leaflet, 3) that both sphingomyelin and glycosphingolipids play a role in reducing membrane permeability but that there is an absolute requirement for cholesterol to mediate this effect, 4) that these results further validate the hypothesis that each leaflet offers an independent resistance to permeation, and 5) that proton permeation was enhanced by sphingolipid/cholesterol interactions.     

Water permeabilities and salt reflection coefficients of luminal, basolateral and intracellular membrane vesicles isolated from rabbit kidney proximal tubule

The mechanisms of water transport across the rabbit renal proximal convoluted tubule were approached by measuring osmotic permeabilities and solute reflection coefficients of the brush-border and the basolateral membranes. Plasma and intracellular membrane vesicles were isolated from rabbit renal cortex by centrifugation on a Percoll gradient. Three major turbidity bands were obtained: a fraction of purified basolateral membranes (BLMV), the two others being brush-border (BBMV) and endoplasmic reticulum (ERMV) membrane vesicles. The osmotic permeability (Pf) of the three types of vesicle was measured using stop-flow techniques and their geometry was determined by quasi-elastic light scattering. Pf was equal to 123 +/- 8 microns/s (n = 10) for BBMV, 166 +/- 10 microns/s (n = 10) for BLMV and 156 +/- 9 microns/s (n = 4) for ERMV (T = 26 degrees C). A transcellular water permeability, per unit of apical surface area, of 71 microns/s was calculated considering that the luminal and the basolateral membranes act as two conductances in series. This value is in close agreement, after appropriate normalizations, with previously reported transepithelial water permeabilities obtained using in vitro microperfusion techniques thus supporting the hypothesis of a predominantly transcellular route for water flow across rabbit proximal convoluted tubule. The addition of 0.4 mM HgCl2, a sulfhydryl reagent, decreased Pf about 60% in three types of membrane providing evidence for the existence of proteic pathways. NaCl and KCl reflection coefficients were measured and found to be close to one for plasma and intracellular membranes suggesting that the water channels are not shared by salts.     

Vasopressin-enhanced urea transport by rat inner medullary collecting duct cells in culture

The distal inner medullary collecting duct (IMCD) is critical in the urinary concentrating process, in part because it is the site of vasopressin (AVP)-regulated permeability to urea. The purpose of these experiments was to develop a cell culture model of the IMCD on permeable structure and to characterize the responsiveness to AVP. Rat IMCD cells were grown to confluence on collagen-coated Millipore filters glued onto plastic rings. To assess the time required to achieve confluence, the transepithelial resistance was measured periodically and was found to be stable after 2 weeks, at a maximal value of 595 +/- 22 omega cm2. In separate monolayers the effect of AVP on inulin and urea permeability was determined. While inulin permeability was unchanged after AVP, urea permeability increased from 6.0 +/- 0.4 to peak values of 16.0 +/- 3.8 (10 nM), 23.1 +/- 3.9 (1 microM) and 28.1 +/- 4.9 (10 microM) x 10(-6) cm s-1 (n = 24). In 10 other monolayers, after the addition of 1 mM 8-Br-cAMP, urea permeability increased from 5.1 +/- 0.3 to 8.1 +/- 1.6 x 10(-6) cm s-1 and, after 8-Br-cAMP + 3-isobutyl-1-methylxanthine, to 12.2 +/- 0.7 x 10(-6) cm s-1. We conclude that rat IMCD cells grown in culture exhibit the characteristics of a 'tight' epithelium. Inulin and urea permeability are not different in the absence of AVP, consistent with high resistance junctional complexes. Furthermore, IMCD cells retain the capacity for AVP-regulated urea permeability, a characteristic feature of this nephron segment in vivo.     

Palmitate binding to and efflux kinetics from human erythrocyte ghost

At 0 degrees C, pH 7.3, palmitate (PA) binds to human erythrocyte ghosts suspended in 0.2% bovine serum albumin (BSA) solution with molar ratios of PA to BSA, v, between 0.2 and 1.3. The binding depends on the water phase PA concentration, measured in equilibrium experiments, using BSA-filled ghosts as semipermeable bags. The saturable binding has a capacity of 19.4 +/- 7.5 nmol g-1 packed ghosts (7.2 x 10(9) cells) and Kd = 13.5 +/- 5 nM. PA exchange efflux kinetics to 0.2% BSA is recorded from ghosts without and with 0.2% BSA with a resolution time of about 1 s. Data are analyzed in terms of compartmental models. Using BSA-free ghosts the kinetics is essentially monoexponential. The rate constant is 0.0287 +/- 0.0022 s-1. Using ghosts with BSA, the kinetics is biexponential with widely different rate constants. Extrapolated zero-time values reflect, according to additional investigations, 'instantaneous' release of PA from the outer surface of the ghosts. Analyses of the biexponential curve up to about 55% tracer efflux assign unequivocally values to three model parameters. (1) k1, the dissociation rate constant of the PA-BSA complex is (1.47 +/- 0.03) x 10(-3) s-1 and (2.56 +/- 0.08) x 10(-3) s-1 and (4.08 +/- 0.13) x 10(-3) s-1 at v = 0.2, 0.6 and 1.4, respectively. (2) k3*, the overall rate constant of PA transport from the inside of the ghost membrane to the medium is 0.0269 +/- 0.0020 s-1 independent of v. (3) Qkin, the ratio of PA on the inside of the membrane to PA on BSA within the ghosts is v dependent and smaller than a corresponding ratio Qeq measured in equilibrium by a value corresponding to PA on the outer surface. This fraction is released with a rate constant, k5, which is of the order of 1 s-1. The data suggest a maximum PA transport capacity, Jmax, of 2 pmol min-1 cm-2, 0 degrees C, pH 7.3.     

Permeability of ammonia,methylamine and ethylamine in the cyanobacterium,Synechococcus R-2 (Anacystis nidulans) PCC 7942

Summary Permeabilities of ammonia (NH₃), methylamine (CH₃NH₂) and ethylamine (CH₃CH₂NH₂) in the cyanobacterium (cyanophyte)Synechococcus R-2 (Anacystis nidulans) have been measured. Based on net uptake rates of DCMU (dichlorophenyldimethylurea) treated cells, the permeability of ammonia was 6.44±1.22 m sec^–1 (n=13). The permeabilities of methylamine and ethylamine, based on steady-state¹⁴C labeling were more than ten times that of ammonia (P _methylamine=84.6±9.47 m sec^–1 (76),P _ethylamine=109±11 m sec^–1 (55)). The apparent permeabilities based on net uptake rates of methylamine and ethylamine uptake were significantly lower, but this effect was partially reversible by ammonia, suggesting that net amine fluxes are rate limited by proton fluxes to an upper limit of about 700 nmol m^–2 sec^–1. Increasing concentrations of amines in alkaline conditions partially dissipated the pH gradient across the cell membrane, and this property could be used to calculate the relative permeabilities of different amines. The ratio of ethylamine to methylamine permeabilities was not significantly different from that calculated from the direct measurements of permeabilities; ammonia was much less effective in dissipating the pH gradient across the cell membrane than methylamine or ethylamine. An apparent permeability of ammonia of 5.7±0.9 m sec^–1 could be calculated from the permeability ratio of ammonia to methylamine and the experimentally measured permeability of methylamine. The permeability properties of ammonia and methylamine are very different; this poses problems in the interpretation of experiments where¹⁴C-methylamine is used as an ammonia analogue.     

Cadmium and thallous ion permeabilities through lipid bilayer membranes

Cadmium (Cd2+) and thallous ion (Tl+) permeabilities were measured in planar (Mueller-Rudin) lipid bilayer membranes made from diphytanoylphosphatidylcholine in decane. Permeabilities of the electroneutral Cl- complexes, measured with tracers (109Cd and 204Tl), were about 10(-8) cm X s-1 for CdCl2 and 10(-6) cm X s-1 for TlCl. Electrical conductance measurements showed that permeabilities to Cd2+ and Tl+ were approx. 10(-11) cm X s-1, similar to the Na+ permeability. The low permeabilities to both Cd2+ and CdCl2 are consistent with biological studies which suggest that Cd transport and toxicity are protein mediated and correlated with Cd2+, not CdCl2, concentration. However, the low bilayer permeability to Tl+ raises questions about recent reports that Tl+ is a lipid permeable cation in biological membranes and liposomes. An alternative explanation for the lipid permeable behavior of Tl+ is presented, based on the diffusion of TlCl and other complexes of Tl+ with inorganic and organic anions.     

Role of facilitative glucose transporters in diffusional water permeability through J774 cells

We have reported previously that in the presence of an osmotic gradient, facilitative glucose transporters (GLUTs) act as a transmembrane pathway for water flow. Here, we find evidence that they also allow water passage in the absence of an osmotic gradient. We applied the linear diffusion technique to measure the diffusional permeability (Pd) of tritiated water (3H-H2O) through plasma membranes of J774 murine macrophage-like cells. Untreated cells had a Pd of 30.9 +/- 1.8 microns/s; the inhibitors of facilitative glucose transport cytochalasin B (10 microM) and phloretin (20 microM) reduced that value to 15.3 +/- 1.8 (50%) and 11.0 +/- 0.7 (62%) microns/s, respectively. In contrast, no significant effect on Pd was observed in cells treated with dihydrocytochalasin B (Pd = 28.4 +/- 1.5 microns/s). PCMBS (3 mM) inhibited glucose uptake by greater than 95%, and 3H-H2O diffusion by approximately 30% (Pd = 22.9 +/- 1.5 microns/s). The combination of cytochalasin B plus pCMBS reduced Pd by about 87% (Pd = 3.9 +/- 0.3 microns/s). Moreover, 1 mM pCMBS did not affect the osmotic water permeability in Xenopus laevis oocytes expressing the brain/erythroid form of facilitative glucose transporters (GLUT1). These results indicate for the first time that about half of the total Pd of J774 cells may be accounted for by water passage across GLUTs. Hence, they highlight the multifunctional properties of these transporters serving as conduits for both water and glucose. Our results also suggest for the first time that pCMBS blocks glucose transport without affecting water permeation through GLUTs. Lastly, because pCMBS decreases the Pd of J774 cells, this suggests the presence in their plasma membranes of another protein(s) exhibiting water channel properties.     

The Permeability of Ammonia, Methylamine and Ethylamine in the Charophyte Chara corallina (C. australis)

Ritchie, R. J. 1987. The permeability of ammonia, methylamineand ethylamine in the charophyte Chara corallina (C. australis).—J.exp. Bot. 38: 67–76 The permeabilities of the amines, ammonia (NH₃), methylamine(CH₃NH₂) and ethylamine (CH₃CH₂NH₂) in the giant-celled charophyteChara corallina (C. australis) R.Br. have been measured andcompared. The permeabilities were corrected for uptake fluxesof the amine cations. Based on net uptake rates, the permeabilityof ammonia was 6?4?0?93 µm s^–1 (n = 38). The permeabilitiesof methylamine and ethylamine were measured in net and exchangeflux experiments. The permeabilities of methylamine were notsignificantly different in net and exchange experiments, norto that of ammonia (P_methylamine = 6?0?0?49 µm s^–1(n = 44)). In net flux experiments the apparent permeabilityof ethylamine was slightly greater than that of ammonia andmethylamine (P_{ethylamine, net} = 8?4?1?2 µm s^–1 (n= 40)) but the permeability of ethylamine based on exchangeflux data was significantly higher (P_{ethylamine, exchange} =14?1?2 µm s^–1 (n = 20)). Methylamine can be validlyused as an ammonium analogue in permeability studies in Chara. The plasmalemma of Chara has acid and alkaline bands; littlediffusion of uncharged amines would occur across the acid bands.The actual permeability of amines across the alkaline bandsis probably about twice the values quoted above on a whole cellbasis i.e. the permeability of ammonia across the permeablepart of the plasmalemma is probably about 12 µm s^–1. Key words: Chara, permeability, ammonia, methylamine     

Characterization of ammonium (methylammonium)/potassium antiport in Escherichia coli

The energetics of ammonium ion transport by Escherichia coli have been studied using [14C]methylammonium as a substrate. Rapid assays for uptake allowed kinetic parameters (CH3NH3+ Km = 36 microM; Vmax = 4 nmol X s-1 X mg-1 to be determined in the absence of CH3NH3+ metabolism. Cells cultured in media containing 1 mM NH4+ failed to express CH3NH3+ transport activity. Methylammonium accumulated at levels which were 100-fold higher than those of the medium. This accumulation was dependent upon the addition of glucose or pyruvate. The entry of CH3NH3+ supported by glucose oxidation in an F1F0-ATPase-deficient mutant was blocked by uncoupler. Transport by wild-type cells under similar conditions was significantly inhibited by arsenate. Thus, CH3NH3+ uptake requires both ATP and an electrochemical H+ gradient. This transport activity was lost upon exposure of E. coli to osmotic shock, but could be recovered by incubation of shocked cells with boiled shock fluid or with glucose plus K+ in the presence of chloramphenicol. Similar reconstitution was observed in K+-depleted parental strains, but not in a mutant defective in K+ transport, demonstrating a requirement for internal K+. However, external K+ proved to be a noncompetitive inhibitor (Ki = 1 mM) of CH3NH3+ uptake by K+ -replete bacteria. External Na+ had no effect on transport. The addition of NH4+ or CH3NH3+ induced a rapid exodus of intracellular 86Rb+, an analog which was able to substitute for K+. The molar ratio of CH3NH3+ uptake to Rb+ exit was 1.12 +/- 0.11. These findings support a mechanism for CH3NH3+ (NH4+) accumulation which requires K+ antiport (exchange) and is driven by the electrochemical K+ gradient.     

Ammonium and methylammonium transport in Rhodobacter sphaeroides.

Rhodobacter sphaeroides maintained intracellular ammonium pools of 1.1 to 2.6 mM during growth in several fixed nitrogen sources as well as during diazotrophic growth. Addition of 0.15 mM NH4+ to washed, nitrogen-free cell suspensions was followed by linear uptake of NH4+ from the medium and transient formation of intracellular pools of 0.9 to 1.5 mM NH4+. Transport of NH4+ was shown to be independent of assimilation by glutamine synthetase because intracellular pools of over 1 mM represented NH4+ concentration gradients of at least 100-fold across the cytoplasmic membrane. Ammonium pools of over 1 mM were also found in non-growing cell suspensions in nitrogen-free medium after glutamine synthetase was inhibited with methionine sulfoximine. In NH4+-free cell suspensions, methylammonium (14CH3NH3+) was taken up rapidly, and intracellular concentrations of 0.4 to 0.5 mM were maintained. The 14CH3NH3+ pool was not affected by methionine sulfoximine. Unlike NH4+ uptake, 14CH3NH3+ uptake in nitrogen-free cell suspensions was repressed by growth in NH4+. These results suggest that R. sphaeroides may produce an NH4+-specific transport system in addition to the NH4+/14CH3NH3+ transporter. This second transporter is able to produce normal-size NH4+ pools but has very little affinity for 14CH3NH3+ and is not repressed by growth in high concentrations of NH4+.     

Purification and some properties of carboxynorspermidine synthase participating in a novel biosynthetic pathway for norspermidine in Vibrio alginolyticus

Carboxynorspermidine synthase, mediates the nicotinamide-nucleotide-linked reduction of the Schiff base H2N(CH2)3N = CHCH2CH(NH2)COOH. This is formed from L-aspartic beta-semialdehyde (ASA) and 1,3-diaminopropane (DAP) and is reduced to carboxynorspermidine [H2N(CH2)3NH(CH2)2CH(NH2)COOH], an intermediate in the novel pathway for norspermidine (NSPD) biosynthesis. The enzyme was purified to apparent homogeneity from Vibrio alginolyticus and characterized. The overall purification was about 1800-fold over the crude extract, with a yield of 33%. The enzyme displayed an apparent Mr of 93500 +/- 1000 by gel filtration and 45100 +/- 500 by SDS-PAGE, indicating that the native form is probably composed of two subunits of similar size. The specific activity of the purified enzyme was 31.0 mumol carboxynorspermidine produced min-1 (mg protein)-1. The enzyme was activated by dithiothreitol, and inhibited by SH-reactive compounds. The pH and temperature optima were 7.25-7.5 and 37 degrees C, respectively. The Km value for the Schiff base was 4.68 mM, measured by varying the ASA concentration while keeping the DAP concentration constant. Putrescine was slightly active as a substrate, forming carboxyspermidine (at about 7% of the rate of DAP), but ethylenediamine, cadaverine and D-ASA were inert. The Km value for NADPH was 1.51 mM. NADH was a much poorer cofactor than NADPH. When V. alginolyticus was grown in the presence of 5 mM-NSPD, the specific activity of this enzyme was reduced by approximately 70%. NSPD also repressed two other enzymes responsible for its biosynthesis, 2,4-diaminobutyrate decarboxylase and carboxynorspermidine decarboxylase.     

Water permeability differs between growing and non-growing barley leaf tissues

A pressure probe technique and an osmotic swelling assay were used to compare water transport properties between growing and non-growing tissues of leaf three of barley. The epidermis was analysed in planta by pressure probe, whereas (predominantly) mesophyll protoplasts were analysed by osmotic swelling. Hydraulic conductivity (Lp) and, by implication, water permeability (Pf) of epidermal cells was 31% higher in the leaf elongation zone (Lp=0.5+/-0.2 microm s-1 MPa-1; Pf=65+/-25 microm s-1; means+/-SD of n=17 cells) than in the, non-growing, emerged leaf zone (Lp=0.4+/-0.1 microm s-1 MPa-1; Pf=50+/-15 microm s-1; n=24; P<0.05). Similarly, water permeability of mesophyll protoplasts was by 55% higher in the elongation compared with emerged leaf zone (Pf=13+/-1 microm s-1 compared with 8+/-1 microm s-1; n=57 and 36 protoplasts, respectively; P<0.01). Within the leaf elongation zone, a small population of larger-sized protoplasts could be distinguished. These protoplasts, which originated most likely from parenchymateous bundle sheath or midrib parenchyma cells, had a three-fold higher water permeability (P<0.001) as mesophyll protoplasts. The effect on Lp and Pf of known aquaporin inhibitors was tested with the pressure probe (Au+, Ag+, Hg2+, phloretin) and the osmotic swelling assay (phloretin). Only phloretin, when applied to protoplasts in the swelling assay caused an average decrease in Pf, but the effect varied between isolations. Technical approaches and cell-type and growth-specific differences in water transport properties are discussed.     

Motility response of Rhodobacter sphaeroides to chemotactic stimulation.

Tethered rotating cells of Rhodobacter sphaeroides varied widely in their stopping frequency; 45% of cells showed no stops of longer than 1 s, whereas others showed stops of up to several seconds. Individual cells alternated between stops and rotation at a fairly constant rate, without continuous variation. Addition of the chemoattractant propionate to free-swimming cells of R. sphaeroides increased the mean population swimming speed from 15 to 23 microns s-1. After correction for nonmotile cells, the percentage swimming at less than 5 microns s-1 dropped from approximately 22 to 8, whereas the percentage swimming at greater than 50 microns s-1 increased from 6 to 15. However, cells already swimming did not swim faster after propionate addition; the increase in the mean population speed after propionate addition was caused by an increase in the mean run length between stops from 25 to 101 microns. The increased run length was the result of a drop in both the stopping frequency and the length of a stop. Addition of propionate over the range of 10 microM to 1 mM decreased the stopping frequency; this decrease was almost entirely blocked by benzoate, a competitive inhibitor of propionate transport. The chemoattractants acetate and potassium had the same effect as propionate on the distribution of stopping frequency, which demonstrated that this is a general behavioral response to chemotactic stimulation. Adaptation to propionate stimulation was slow and very variable, cultures frequently showing little adaptation over 30 min. This characteristic may be the result of the lack of a highly specific chemosensory system in R. sphaeroides.     

Nitrogenase reactivity: methyl isocyanide as substrate and inhibitor

We have examined the interaction of methyl isocyanide with the purified component proteins of Azotobacter vinelandii nitrogenase (Av1 and Av2). CH3NC was shown to be a potent reversible inhibitor (Ki = 158 microM) of total electron flow, apparently uncoupling magnesium adenosine 5'-triphosphate hydrolysis from electron transfer to substrate. CH3NC is a substrate (Km = 0.688 mM at Av2/Av1 = 8), and extrapolation of the data indicates that at high enough CH3NC concentration, H2 evolution can be eliminated. The products are methane plus methylamine (six electrons) and dimethylamine (four electrons). There is an excess (relative to methane) of methylamine formed, which may arise by hydrolysis of a two-electron intermediate. A rapid high-performance liquid chromatography/fluorescence method was developed for methylamine determination. The products C2H4 and C2H6 appear to be formed via a reduction followed by an insertion mechanism. CH3NC appears to be reduced at an enzyme state more oxidized than the one responsible for H2 evolution or N2 reduction. Other substrates (C2H2 greater than N2 congruent to azide greater than N2O) all both relieve CH3NC inhibition and inhibit CH3NC reduction. Both effects occur in the same relative order, implying productive (substrate) and nonproductive (inhibitor) modes of binding of CH3NC to the same site.     

Reactions of lumiflavin and lumiflavin radicals with .CO2- and alcohol radicals

The kinetics of reaction of oxidized lumiflavin (F0) with the radicals .CO2(-), CH3CHOH, and (CH3)2COH have been investigated at pH 7 and 24 +/- 1 degree C by the pulse radiolysis technique. The radicals have been shown to react with lumiflavin with second-order rate constants of 36 +/- 4, 26 +/- 3, and 20 +/- 3 in units of 10(8) M-1 s-1, respectively. These rate constants are close to the diffusion limit. The main product in each case was the lumiflavin semiquinone radical FH.. By utilization of long pulses (approximately 100 mus), it was shown that the reaction FH. + .AH(alpha) leads to FH- + A(alpha) + H+ [.AH(alpha) = .CO2(-), CH3CHOH, or (CH3)2COH] proceeded for all three types of .AH(alpha) radical with second-order rate constants of 17 (+4,-3), 9 (+5,-3), and 9 (+4,-3), respectively, in the above units. The beta-carbon radical .CH2CH(OH)CH3 added to .FH, forming an alkylated flavin, while the .CH2CH2OH radical appeared to be capable of addition or hydrogen atom donation to .FH.     

空气 NH3增高和不同氮源供应下大叶相思叶片光合参数的变化

生长在空气 NH3增高下 45 d的 NOˉ3- N大叶相思植株 ,其光饱和光合速率较对照的植株高 ;而生长在空气 NH3增高下的 NH 4- N和 NH4 NO3- N的大叶相思 ,当光强在 70 0 μmol·m- 2 ·s- 1左右时 Pn 达到最大值 ,较对照植株的要高。而当光强 >70 0 μmol·m- 2·s- 1时 ,Pn 降低 ,且较生长在对照条件下的低。表明在空气 NH3增高下生长的 NH 4- N和 NH4 NO3- N植株 ,其净光合速率 Pn会受到强光抑制。空气 NH3增高并不明显改变光呼吸 ( Rd)和无光呼吸下的 CO2 补充点 (Γ* )。无论生长在何种氮源下的大叶相思 ,其最大Ru BP饱和羧化速率 ( Vcmax)和最大电子传递速率 ( Jmax)均较生长在对照植株的高 ( P<0 .0 5 ) ,其叶氮含量亦较高 ( P<0 .0 5 ) ,其碳氮比较对照的低。在空气 NH3增高下 ,无论何种氮源生长的大叶相思 ,其 PR和 PB明显高于对照的植株 ,表明大叶相思能从空气 NH3中摄取和同化氮 ,增加氮积累和有利于 Rubisco和电子传递组分的合成 ,增高光合速率。空气 NH3增高可能有利于 Rubisco和电子传递组分的合成 ,在较低光强下能增高光合速率。空气 NH3增高可能有利于退化生态系统的生态恢复过程中的氮积累和先锋植物的早期生长。     

Cytochrome aa3 of Rhodobacter sphaeroides as a model for mitochondrial cytochrome c oxidase. Purification, kinetics, proton pumping, and spectral analysis.

Aerobically grown Rhodobacter sphaeroides synthesizes a respiratory chain similar to that of eukaryotes. We describe the purification of the aa3-type cytochrome c oxidase of Rb. sphaeroides as a highly active (Vmax > or = 1800 s-1), three-subunit enzyme from isolated, washed cytoplasmic membranes by hydroxylapatite chromatography and anion exchange fast protein liquid chromatography. The purified oxidase exhibits biphasic kinetics of oxidation of mammalian cytochrome c, similar to mitochondrial oxidases, and pumps protons efficiently (H+/e- = 0.7) following reconstitution into phospholipid vesicles. A membrane-bound cytochrome c is associated with the aa3-type oxidase in situ, but is removed during purification. The EPR spectra of the Rb. sphaeroides enzyme suggest the presence of a strong hydrogen bond to one or both of the histidine ligands of heme a. In other respects, optical, EPR, and resonance Raman analyses of the metal centers and their protein environments demonstrate a close correspondence between the bacterial enzyme and the structurally more complex bovine cytochrome c oxidase. The results establish this bacterial oxidase as an excellent model system for the mammalian enzyme and provide the basis for site-directed mutational analysis of its energy transducing function.     

Inverted behavioural responses in wild-type Rhodobacter sphaeroides to temporal stimuli

Both aerobically and photosynthetically grown wild-type Rhodobacter sphaeroides swarmed through soft nutrient agar. However, individual aerobically and photosynthetically grown tethered cells showed different responses to steps in concentrations of some attractants. Photosynthetically grown cells showed little response to a step-up in attractant, but large response to a step-down. Aerobically grown cells showed a large but opposite response to a step-up of chemoeffectors such as succinate and aspartate. The responses in che operon deletion mutants were also investigated and indicated that the aerobic response may depend on the protein products of che operon 1.     

Cell volume measured by total internal reflection microfluorimetry: application to water and solute transport in cells transfected with water channel homologs.

Total internal reflection (TIR) microfluorimetry was established as a method to measure continuously the volume of adherent cells and applied to measure membrane permeabilities in cells transfected with water channel homologs. Cytosol was labeled with the membrane-impermeant fluorophore calcein. Fluorescence was excited by the TIR evanescent field in a thin section of cytosol (approximately 150 nm) adjacent to the cell-substrate interface. Because cytosolic fluorophore number per cell remains constant, the TIR fluorescence signal should be inversely related to cell volume. For small volume changes in Sf-9 and LLC-PK1 cells, relative TIR fluorescence was nearly equal to inverse relative cell volume; deviations from the ideal were modeled theoretically. To measure plasma membrane osmotic water permeability, Pf, the time course of osmotically induced cell volume change was inferred from the TIR fluorescence signal. LLC-PK1 cells expressing the CHIP28 water channel had an HgCl2-sensitive, threefold increase in Pf compared to nontransfected cells (Pf = 0.0043 cm/s at 10 degrees C). Solute permeability was measured from the TIR fluorescence time course in response to solute gradients. Glycerol permeability in Sf-9 cells expressing the water channel homolog GLIP was (1.3 +/- 0.2) x 10(-5) cm/s (22 degrees C), greater than that of (0.36 +/- 0.04) x 10(-5) cm/s (n = 4, p < 0.05) for control cells, indicating functional expression of GLIP. Water and urea permeabilities were similar in GLIP-expressing and control cells. The TIR method should be applicable to the study of water and solute permeabilities and cell volume regulation in cells of arbitrary shape and size.