Cercospora beticola Toxins (X. Inhibition of Plasma Membrane H+-ATPase by Beticolin-1)

Influxes of 13NH4+ across the root plasmalemma were measured in intact seedlings of Picea glauca (Moench) Voss. Two kinetically distinct uptake systems for NH4+ were identified. In N-deprived plants, a Michaelis-Menten-type high-affinity transport system (HATS) operated in a 2.5 to 350 [mu]M range of external NH4+ concentration ([NH4 +]o). The Vmax of this HATS was 1.9 to 2.4 [mu]mol g-1 h-1, and the Km was 20 to40 [mu]M. At [NH4+]o from 500 [mu]M to 50 mM, a linear low-affinity system (LATS) was apparent. Both HATS and LATS were constitutive. A time-dependence study of NH4+ influx in previously N-deprived seedlings revealed a small transient increase of NH4+ influx after 24 h of exposure to 100 [mu]M [NH4+]o. This was followed by a decline of influx to a steady-state value after 4 d. In seedlings exposed to 100 [mu]M external NO3- concentration for 3 d, the Vmax for NH4+ uptake by HATS was increased approximately 30% compared to that found in N-deprived seedlings, whereas LATS was down-regulated. The present study defines the much higher uptake capacity for NH4+ than for N03- in seedlings of this species.     

Ammonium Uptake by Rice Roots (III. Electrophysiology)

The transmembrane electrical potential differences ([delta][psi]) were measured in epidermal and cortical cells of intact roots of 3-week-old rice (Oryza sativa L. cv M202) seedlings grown in 2 or 100 [mu]M NH4+ (G2 or G100 plants, respectively). In modified Johnson's nutrient solution containing no nitrogen, [delta][psi] was in the range of -120 to -140 mV. Introducing NH4+ to the bathing medium caused a rapid depolarization. At the steady state, average [delta][psi] of G2 and G100 plants were -116 and -89 mV, respectively. This depolarization exhibited a biphasic response to external NH4+ concentration similar to that reported for 13NH4+ influx isotherms (M.Y. Wang, M.Y. Siddiqi, T.J. Ruth, A.D.M. Glass [1993] Plant Physiol 103: 1259-1267). Plots of membrane depolarization versus 13NH4+ influx were also biphasic, indicating distinct coupling processes for the two transport systems, with a breakpoint between two concentration ranges around 1 mM NH4+. The extent of depolarization was also influenced by nitrogen status, which was larger for G2 plants than for G100 plants. Depolarization of [delta][psi] due to NH4+ uptake was eliminated by a protonophore (carboxylcyanide-m-chlorophenylhydrazone), inhibitors of ATP synthesis (sodium cyanide plus salicylhydroxamic acid), or an ATPase inhibitor (diethylstilbestrol). The results of these observations are discussed in the context of the mechanisms of NH4+ uptake by high- and low-affinity transport systems operating across the plasma membranes of root cells.     

Ammonium Uptake by Rice Roots (II. Kinetics of 13NH4+ Influx across the Plasmalemma)

Short-term influxes of 13NH4+ were measured in intact roots of 3-week-old rice (Oryza sativa L. cv M202) seedlings that were hydroponically grown at 2, 100, or 1000 [mu]M NH4+. Below 1 mM external concentration ([NH4+]0), influx was saturable and due to a high-affinity transport system (HATS). For the HATS, Vmax values were negatively correlated and Km values were positively correlated with NH4+ provision during growth and root [NH4+]. Between 1 and 40 mM [NH4+]0, 13NH4+ influx showed a linear response due to a low-affinity transport system (LATS). The 13NH4+ influxes by the HATS, and to a lesser extent the LATS, are energy-dependent processes. Selected metabolic inhibitors reduced influx of the HATS by 50 to 80%, but of the LATS by only 31 to 51%. Estimated values for Q10 (the ratio of rates at temperatures differing by 10[deg]C) for HATS were greater than 2.4 at root temperatures from 5 to 10[deg]C and were constant at approximately 1.5 between 5 and 30[deg]C for the LATS. Influx of 13NH4+ by the HATS was insensitive to external pH in the range from 4.5 to 9.0, but influx by the LATS declined significantly beyond pH 6.0. The data presented are discussed in the context of the kinetics, energy dependence, and the regulation of ammonium influx.     

Do amiloride and ouabain affect ammonia fluxes in perfused Carcinus gill epithelia?

The effects of inhibitors on the efflux of ammonia (from the basolateral to the apical side, Jb----a) were studied in preparation of isolated Carcinus gills immersed in dilute seawater (DSW) that was identical to the perfusion solution. Adding 10(-4) M amiloride to the solution bathing the gill preparations reduces the efflux of ammonia by 29% relative to the control value. Under experimental conditions, it appears that only about 1% of the amiloride-sensitive influx of Na+ (Ja----b) can be exchanged with NH4+ on an equimolar basis. The ammonium ion is apparently transported at the basolateral side by a carrier-mediated process. Kinetic analyses of the influx of ammonium ions revealed a Km of 36.99 microM and a maximum velocity (Vmax) equal to 19.6 mumol g-1.h-1. Basolaterally applied ouabain (5 x 10(-3) M) and NaCN (10(-3) M) reduced the efflux of ammonia by 46.7 and 42.2%, respectively, suggesting an interaction of NH4+ with the basolaterally located Na+/K+ exchanger in which NH4+ appears to be able to substitute for K+.     

Alleviation of rapid, futile ammonium cycling at the plasma membrane by potassium reveals K+-sensitive and -insensitive components of NH4+ transport

Futile plasma membrane cycling of ammonium (NH4+) is characteristic of low-affinity NH4+ transport, and has been proposed to be a critical factor in NH4+ toxicity. Using unidirectional flux analysis with the positron-emitting tracer 13N in intact seedlings of barley (Hordeum vulgare L.), it is shown that rapid, futile NH4+ cycling is alleviated by elevated K+ supply, and that low-affinity NH4+ transport is mediated by a K+-sensitive component, and by a second component that is independent of K+. At low external [K+] (0.1 mM), NH4+ influx (at an external [NH4+] of 10 mM) of 92 micromol g(-1) h(-1) was observed, with an efflux:influx ratio of 0.75, indicative of rapid, futile NH4+ cycling. Elevating K+ supply into the low-affinity K+ transport range (1.5-40 mM) reduced both influx and efflux of NH4+ by as much as 75%, and substantially reduced the efflux:influx ratio. The reduction of NH4+ fluxes was achieved rapidly upon exposure to elevated K+, within 1 min for influx and within 5 min for efflux. The channel inhibitor La3+ decreased high-capacity NH4+ influx only at low K+ concentrations, suggesting that the K+-sensitive component of NH4+ influx may be mediated by non-selective cation channels. Using respiratory measurements and current models of ion flux energetics, the energy cost of concomitant NH4+ and K+ transport at the root plasma membrane, and its consequences for plant growth are discussed. The study presents the first demonstration of the parallel operation of K+-sensitive and -insensitive NH4+ flux mechanisms in plants.     

Responses to reversed NH3 and NH4+ gradients in a teleost (Ictalurus punctatus), an elasmobranch (Raja erinacea), and a crustacean (Callinectes sapidus):evidence for NH4+/H+ exchange in the teleost and the elasmobranch

Ammonia excretion rates of channel catfish, Ictalurus punctatus, little skate (Raja erinacea), and blue crab (Callinectes sapidus) were measured in experimental regimes which permitted simultaneous assessment of the partial pressure gradients for nonionized NH3 and the chemical concentration gradients of NH4+. Under conditions of low external ammonia, the average ammonia excretion was +295 microM kg-1 h-1 for catfish, +149 microM kg-1 h-1 for blue crabs, and +59 microM kg-1 h1 for skates with partial pressure gradients of +72.5 mu Torr, +413 mu Torr, and +24.4 mu Torr, respectively; and [NH4+] gradients of +189 microM l-1, +643 microM l-1, and +107 microM l-1 (positive indicating greater from inside to medium). When the external ammonia was increased to 1.15 mM l-1, both gradients were reversed, and the net ammonia movement was initially from the external water into all three species. In the catfish the inward movement ceased, however, and ammonia excretion eventually resumed in the face of reversed gradients of both NH3 partial pressure and [NH4+]. Unidirectional Na+ influx, indicative of a Na+/NH4+ exchange, did not increase. The ammonia data, changes in titratable acidity, and net apparent H+ efflux were all consistent with a linked extrusion of internal NH4+ for external H+. Incorporation of such an exchange into a computer simulation model of the ammonia equilibrium and exchange system duplicated the experimental data. Other hypotheses failed to match experimental data, or failed to predict internal ammonia levels lower than outside. In the crab, internal ammonia levels rose rapidly to concentrations and partial pressures above the external medium until excretion was reestablished, with no evidence of maintenance of a reversed gradient. In the skate, internal concentrations rose appreciably in the first hour and continued to rise for 6-8 h, with no resumption of ammonia excretion. The interspecies differences appear to be due at least partly to differences in ammonia permeability of the gills.     

Lack of Control in Inorganic Phosphate Uptake by Catharanthus roseus (L.) G. Don Cells (Cytoplasmic Inorganic Phosphate Homeostasis Depends on the Tonoplast Inorganic Phosphate Transport System?)

Inorganic phosphate (Pi) uptake by Catharanthus roseus (L.) G. Don cells was studied in relation to its apparent uncontrolled uptake using 31P-nuclear magnetic resonance spectroscopy. Kinetics of Pi uptake by the cells indicated that apparent Km and Vm were about 7 [mu]M and 20 [mu]mol g-1 fresh weight h-1, respectively. Pi uptake in Murashige-Skoog medium under different Pi concentrations and different initial cell densities followed basically the same kinetics. When supplied with abundant Pi, cells absorbed Pi at a constant rate (Vm) for the first hours and accumulated it in the vacuole. As the endogenous pool expanded, the rate of Pi uptake gradually decreased to nil. Maximum Pi accumulation was 100 to 120 [mu]mol g-1 fresh weight if cell swelling during Pi uptake (about 2-fold in cell volume) was not considered. Results indicated that (a) the rate of Pi uptake by Catharanthus cells was independent of initial cell density and was constant over a wide range of Pi concentrations (2 mM to about 10 [mu]M) unless the cells were preloaded with excess Pi, and (b) there was no apparent feedback control over the Pi uptake process in the plasma membrane to avoid Pi toxicity. The importance of the tonoplast Pi transport system in cytoplasmic Pi homeostasis is discussed.     

Ammonium and nitrate uptake by soybean during recovery from nitrogen deprivation

Soybean [Glycine max (L.) Merrill] plants that had been subjected to 15 d of nitrogen deprivation were resupplied for 10 d with 1.0 mol m-3 nitrogen provided as NO3-, NH4+, or NH4(+) + NO3- in flowing hydroponic culture. Plants in a fourth hydroponic system received 1.0 mol m-3 NO3- during both stress and resupply periods. Concentrations of soluble carbohydrates and organic acids in roots increased 210 and 370%, respectively, during stress. For the first day of resupply, however, specific uptake rates of nitrogen, determined by ion chromatography as depletion from solution, were lower for stressed than for non-stressed plants by 43% for NO3- resupply, by 32% for NH4(+) + NO3- resupply, and 86% for NH4+ resupply. When specific uptake of nitrogen for stressed plants recovered to rates for non-stressed plants at 6 to 8 d after nitrogen resupply, carbohydrates and organic acids in their roots had declined to concentrations lower than those of non-stressed plants. Recovery of nitrogen uptake capacity of roots thus does not appear to be regulated simply by the content of soluble carbon compounds within roots. Solution concentrations of NH4+ and NO3- were monitored at 62.5 min intervals during the first 3 d of resupply. Intermittent 'hourly' intervals of net influx and net efflux occurred. Rates of uptake during influx intervals were greater for the NH4(+)-resupplied than for the NO3(-)-resupplied plants. For NH4(+)-resupplied plants, however, the hourly intervals of efflux were more numerous than for NO3(-)-resupplied plants. It thus is possible that, instead of repressing NH4+ influx, increased accumulation of amino acids and NH4+ in NH4(+)-resupplied plants inhibited net uptake by stimulation of efflux on NH4+ absorbed in excess of availability of carbon skeletons for assimilation. Entry of NH4+ into root cytoplasm appeared to be less restricted than translocation of amino acids from the cytoplasm into the xylem.     

Kinetics of NO3- Influx in Spruce

Influxes of 13NO3- across the root plasmalemma were measured in intact seedlings of Picea glauca (Moench) Voss. Three kinetically distinct uptake systems for NO3- were identified. In seedlings not previously exposed to external NO3-, a single Michaelis-Menten-type constitutive high-affinity transport system (CHATS) operated in a 2.5 to 500 [mu]M range of external NO3- [NO3-]o. The Vmax of this system was 0.1 [mu]mol g-1 h-1, and the Km was approximately 15 [mu]M. Following exposure to NO3- for 3 d, this CHATS activity was increased approximately 3-fold, with no change of Km. In addition, a NO3--inducible high-affinity system became apparent with a Km of approximately 100[mu]M. The combined Vmax for these discrete saturable components was 0.7 [mu]mol g-1 h-1. In both uninduced and induced plants a linear low-affinity system, additive to CHATS and an NO3--inducible high-affinity system, operated at [NO3-]o [greater than or equal to] 1 mM. The time taken to achieve maximal rates of uptake (full induction) was 2 d from 1.5 mM [NO3-]o and 3 d from 200 [mu]M [NO3-]o.     

盐度与体重对台湾罗非鱼耗氧率的影响

在盐度为淡水、7、14、2 1、2 8和 35的条件下 ,测定了 3个体重组 (1.5 7～ 4.87g ,7.0 7～ 18.2 3g和31.5 0～ 5 2 .41g)的台湾红罗非鱼的耗氧率 ,方差分析表明 ,盐度对台湾红罗非鱼的耗氧率有极显著的影响(P <0 .0 1) .体重范围为 1.5 7～ 18.2 4g时 ,盐度 7实验组的耗氧率最高 ,分别为 0 .41mgO2 ·g-1·h-1(1.5 7～ 4.78g)和 0 .34mgO2 ·g-1·h-1(7.0 7～ 18.2 3g) ,体重范围为 31.5 0～ 5 2 .41g时 ,耗氧率最高值出现在盐度 35组 ,为 0 .30mgO2 ·g-1·h-1.耗氧率最低值也因体重范围的不同而出现在不同的盐度 ,体重范围为1.5 7～ 4.78g时 ,盐度 14组的耗氧率最低 ,为 0 .2 8mgO2 ·g-1·h-1,体重范围在 7.0 7～ 5 2 .41g时 ,耗氧率的最低值均出现在盐度 2 1组 ,其中体重范围 7.0 7～ 18.2 3g的最低值为 0 .2 2mgO2 ·g-1·h ,而体重范围31 5 0～ 5 2 .41g的最低耗氧率为 0 .13mgO2 ·g-1·h-1.协方差分析表明 ,盐度和体重对台湾红罗非鱼的耗氧率存在极显著的交互作用 (P <0 .0 1) .     

The Mechanism of Amino Acid Efflux from Seed Coats of Developing Pea Seeds as Revealed by Uptake Experiments

The uptake of amino acids by excised seed coat halves of developing seeds of pea (Pisum sativum L.) was characterized. The influx of L-valine and L-glutamic acid was proportional to their external concentration, with coefficients of proportionality (k) of 11.0 and 7.1 [mu]mol g-1 fresh weight min-1 M-1, respectively. The influx of L-lysine could be analyzed into a component with linear kinetics (k = 8.1 [mu]mol g-1 fresh weight min-1 M-1) and one with saturation kinetics (Michaelis constant = 6.5 mM), but the latter may have resulted from the mutual interaction between the influx of the cationic lysine and the membrane potential. The influx of the amino acids was not affected by 10 [mu]M carbonylcyanide m-chlorophenylhydrazone, but was inhibited by about 50% in the presence of 2.5 mM p-chloromercuribenzene sulfonic acid. Conservative estimates of the permeability coefficients of the plasma membrane of seed coat parenchyma cells for lysine, glutamic acid, and several neutral amino acids were all in the range of 4 x 10-7 cm s-1 to 9 x 10-7 cm s-1, which is 4 to 5 orders of magnitude greater than those reported for artificial lipid bilayers. It is concluded that nonselective pores constitute a pathway in the plasma membrane for passive transport of amino acids. It is argued that this pathway is also used for the efflux of endogenous amino acids, the process by which nitrogen becomes available for the embryo.     

Stimulation of Nitrate and Nitrite Efflux by Ammonium in Barley (Hordeum vulgare L.) Seedlings

The inhibitory effect of NH4+ on net NO3- uptake has been attributed to an enhancement of efflux and, recently, to an inhibition of influx. To study this controversy, we devised treatments to distinguish the effects of NH4+ on these two processes. Roots of intact barley (Hordeum vulgare L.) seedlings, uninduced or induced with NO3- or NO2-, were used. Net uptake and efflux, respectively, were determined by following the depletion and accumulation in the external solutions. In roots of both uninduced and NO2- -induced seedlings, NO3- efflux was negligible; hence, the initial uptake rates were equivalent to influx. Under these conditions, NH4+ had little effect on NO3- uptake (influx) rates by either the low- or high-Km uptake systems. In contrast, in plants preloaded with NO3-, NH4+ and its analog CH3NH3+ decreased net uptake, presumably by enhancing NO3- efflux. The stimulatory effect of NH4+ on NO3- efflux was a function of external NH4+ and internal NO3- concentration. These results were corroborated by the absence of any effect of NH4+ on NO2- uptake unless the roots were preloaded with NO2-. In this case NH4+ increased efflux and decreased net uptake. Hence, the main effect of NH4+ on net NO3- and NO2- uptake appears to be due to enhancement of efflux and not to inhibition of influx.     

Compartmentation Analysis of Paraquat Fluxes in Maize Roots as a Means of Estimating the Rate of Vacuolar Accumulation and Translocation to Shoots

Efflux analysis conducted after five loading periods of various lengths (2, 6, 12, 18, or 24 h) was used to investigate uptake, compartmentation, and translocation of [14C]paraquat in maize (Zea mays L.) seedlings. The time course for net paraquat uptake (paraquat concentration in uptake solution = 25[mu]M) into maize roots was linear (56.7 nmol g-1 root fresh weight h-1) for 24 h. Estimates of changes in paraquat content in the vacuole, cytoplasm, and cell wall after 2-, 6-, 12-, 18-, and 24-h loading periods indicated that the cell wall saturated rapidly, whereas accumulation of paraquat into the vacuole increased linearly (12.4 nmol g-1 root fresh weight h-1) over 24 h. In contrast to vacuolar accumulation, cytoplasmic paraquat content appeared to approach saturation. The half-time for paraquat efflux from the cell wall (16.6 min [plus or minus] 1.2 SD) and cytoplasm (58.8 min [plus or minus] 8.9 SD remained relatively constant regardless of the length of the loading period, whereas the half-time for efflux from the vacuole was considerably longer and increased linearly with increased loading time (6.1-18.7 h). The time course for paraquat translocation to the shoot was linear within a 24-h exposure to radiolabeled herbicide, but translocation did not begin until 5 h after initiation of treatment. The experimental approach used in these experiments provides a valuable method for examining the movement of paraquat in maize seedlings. Results indicate that the herbicide slowly accumulates in the vacuole of root cells but is also translocated to the shoot.     

Growth characteristics of Escherichia coli HB101[pGEc47] on defined medium

This paper shows that differences in growth behavior of Escherichia coli strain HB101 and strain HB101[pGEc47] can be related to yeast extract-enriched medium rather than plasmid properties. An optimal medium for growth of E. coli HB101[pGEc47] was designed based on the individual yield coefficients for specific medium components (NH4+ 6 g g-1, PO43- 14 g g-1, SO42- 50 g g-1). The yield coefficient for L-leucine depends on the glucose content of the medium (20 g g-1 for 3% glucose, 40 g g-1 for 1% glucose) and the yield coefficient for L-proline depends on the cultivation mode (20 g g-1 for batch cultivation, 44 g g-1 for continuous cultivation). Growth on defined medium after medium optimization is as rapid as on complex medium (0. 42-0.45 h-1). The critical dilution rate (DR) in the defined medium above which undesired production of acetic acid occurs is in the range of 0.23-0.26 h-1.     

Constancy of nitrogen turnover kinetics in the plant cell: insights into the integration of subcellular N fluxes

Compartmental analysis with 13N was used to determine cytosolic nitrate (NO3-) pools, and their turnover rates, in roots of intact barley (Hordeum vulgare L. cv Klondike) seedlings. Influx, efflux, flux to the vacuole and assimilation, and flux to the xylem, varied as much as 300-fold over a wide range of external NO3- conditions. By contrast, the kinetic constant kc describing cytosolic NO3- turnover varied by less than 4% from a mean value of 0.0407 min(-1). Accordingly, cytosolic NO3- pools varied linearly with influx. A literature survey showed that kc constancy is observed with both NO3- and ammonium (NH4+) fluxes in many plant species, including H. vulgare, Arabidopsis thaliana, Picea glauca, and Oryza sativa. The regulatory system implied by this phenomenon is fundamentally different from that of potassium (K+) fluxes, in which cytosolic pool size is held constant while kc varies with external K+ concentrations. We further present data showing that barley plants, grown on one steady-state concentration of NH4+, restore kc within minutes of exposure to new, non-steady-state, NH4+ concentrations. We propose the existence of a high-fidelity mechanism governing the timing of cytosolic N turnover, and discuss its implications for attempts to improve plants biotechnologically.     

Urea synthesis from ammonia in periportal and pericentral regions of the liver lobule. Effect of oxygen

Rates of urea synthesis were determined in periportal and pericentral regions of the liver lobule in perfused liver from fed, phenobarbital-treated rats by measuring the extra O2 consumed upon infusion of NH4Cl with miniature O2 electrodes and from decreases in NADPH fluorescence detected with micro-light-guides. Urea synthesis by the perfused rat liver supplemented with lactate (5 mM), ornithine (2 mM) and methionine sulfoximine (0.15 mM), an inhibitor of glutamine synthetase, was stimulated by stepwise infusion of NH4Cl at doses ranging from 0.24 mM to 3.0 mM. A good correlation (r = 0.92) between decreases in NADPH fluorescence and urea production was observed when the NH4Cl concentration was increased. Sublobular rates of O2 uptake were determined by placing miniature oxygen electrodes on periportal or pericentral regions of the lobule on the liver surface, stopping the flow and measuring decreases in oxygen tension. From such measurements local rates of O2 uptake were calculated in the presence and absence of NH4Cl and local rates of urea synthesis were calculated from the extra O2 consumed in the presence of NH4Cl and the stoichiometry between O2 uptake and urea formation. Rates of urea synthesis were also estimated from the fractional decrease in NADPH fluorescence, caused by NH4Cl infusion in each region, measured with micro-light-guides and the rate of urea synthesis by the whole organ. When perfusion was in the anterograde direction, maximal rates of urea synthesis, calculated from changes in fluorescence, were 177 +/- 31 mumol g-1 h-1 and 61 +/- 24 mumol g-1 h-1 in periportal and pericentral regions, respectively. When perfusion was in the retrograde direction, however, rates were 76 +/- 23 mumol g-1 h-1 in periportal areas and 152 +/- 19 mumol g-1 h-1 in pericentral regions. During perfusion in the anterograde direction, urea synthesis, calculated by changes in O2 uptake, was 307 +/- 76 mumol g-1 h-1 and 72 +/- 34 mumol g-1 h-1 in periportal and pericentral regions, respectively. When perfusion was in the retrograde direction, urea was synthesized at rates of 54 +/- 17 mumol g-1 h-1 and 387 +/- 99 mumol g-1 h-1 in periportal and pericentral regions, respectively. Thus, maximal rates of urea synthesis were dependent upon the direction of perfusion. In addition, rates of urea synthesis were elevated dramatically in periportal regions when the flow rate per gram liver was increased (e.g. 307 versus 177 mumol g-1 h-1).(ABSTRACT TRUNCATED AT 400 WORDS)     

The influence of cellular and lipoprotein cholesterol contents on the flux of cholesterol between fibroblasts and high density lipoprotein

Previous studies indicate that free cholesterol moves passively between high density lipoprotein (HDL) and cell plasma membranes by uncatalyzed diffusion of cholesterol molecules in the extracellular aqueous phase. By this mechanism, the rate constants for free cholesterol influx (Cli) and efflux (ke) should not be very sensitive to the free cholesterol content of cells or HDL. Thus, at a given HDL concentration, the unidirectional influx and efflux of cholesterol mass (Fi, Fe) should be proportional to the cholesterol content of HDL and cells, respectively, and net efflux of cholesterol mass (Fe-Fi greater than 0) should occur when either cells are enriched with cholesterol or HDL is depleted of cholesterol. We have examined the influence of cell and HDL free cholesterol contents on the bidirectional flux of free cholesterol between HDL and human fibroblasts and also attempted to detect some dependence of flux on the binding of HDL to the cells. In the range of HDL concentrations from 1 to 1000 micrograms of protein/ml, ke for cell free cholesterol approximately doubled for every 10-fold increase in HDL concentration, reaching 0.04 h-1 at 1000 micrograms of HDL/ml. ke and Cli were not influenced by the doubling of fibroblast free cholesterol content (from 31 +/- 5 to 62 +/- 13 micrograms of cholesterol/mg of protein). There was an approximate exchange of cholesterol between HDL and the unenriched fibroblasts (e.g. at [HDL] = 100 micrograms/ml, Fe and Fi = 3.2 and 3.0 micrograms of cholesterol/[4 h.mg of cell protein], respectively). In contrast, there was substantial net efflux from the enriched cells (at [HDL] = 100 micrograms/ml, Fe and Fi = 5.5 and 3.1 micrograms of cholesterol/[4 h.mg of cell protein], respectively). The rate constants for cholesterol flux were not influenced by changing the free cholesterol content of HDL, so that there was net efflux of cell cholesterol in the presence of cholesterol-depleted HDL and net influx from cholesterol-rich HDL. The Kd of HDL binding to fibroblasts was reduced from 1.7 to 0.9 micrograms/ml by the enrichment of the cells with free cholesterol; this increase in affinity for HDL was not reflected in enhanced rate constants for cholesterol flux. The inhibition of specific HDL binding by treatment of the lipoprotein with dimethyl suberimidate did not affect cholesterol flux using either control or cholesterol-rich cells at any HDL concentration in the range 1-1000 micrograms/ml. The above results are consistent with the concept that net movement of free cholesterol between cells and HDL occurs by passive, mass-action effects.(ABSTRACT TRUNCATED AT 400 WORDS)     

Detoxification of Formaldehyde by the Spider Plant (Chlorophytum comosum L.) and by Soybean (Glycine max L.) Cell-Suspension Cultures

The phytotoxicity of formaldehyde for spider plants (Chlorophytum comosum L.), tobacco plants (Nicotiana tabacum L. cv Bel B and Bel W3), and soybean (Glycine max L.) cell-suspension cultures was found to be low enough to allow metabolic studies. Spider plant shoots were exposed to 7.1 [mu]L L-1 (8.5 mg m-3) gaseous [14C]-formaldehyde over 24 h. Approximately 88% of the recovered radioactivity was plant associated and was found to be incorporated into organic acids, amino acids, free sugars, and lipids as well as cell-wall components. Similar results were obtained upon feeding [14C]formaldehyde from aqueous solution to aseptic soybean cell-suspension cultures. Serine and phosphatidylcholine were identified as major metabolic products. Spider plant enzyme extracts contained two NAS+-dependent formaldehyde dehydrogenase activities with molecular mass values of about 129 and 79 kD. Only the latter enzyme activity required glutathione as an obligatory second cofactor. It had an apparent Km value of 30 [mu]M for formaldehyde and an isoelectric point at pH 5.4. Total cell-free dehydrogenase activity corresponded to 13 [mu]g formaldehyde oxidized h-1 g-1 leaf fresh weight. Glutathione-dependent formaldehyde dehydrogenases were also isolated from shoots and leaves of Equisetum telmateia and from cell-suspension cultures of wheat (Triticum aestivum L.) and maize (Zea mays L.). The results obtained are consistent with the concept of indoor air decontamination with common room plants such as the spider plant. Formaldehyde appears to be efficiently detoxified by oxidation and subsequent C1 metabolism.