Evidence for water channels in renal proximal tubule cell membranes

Summary Water transport mechanisms in rabbit proximal convoluted cell membranes were examined by measurement of: (1) osmotic (P _f ) and diffusional (P _d ) water permeabilities, (2) inhibition ofP _f by mercurials, and (3) activation energies (E _a ) forP _f .P _f was measured in PCT brush border (BBMV) and basolateral membrane (BLMV) vesicles, and in viable PCT cells by stopped-flow light scattering;P _d was measured in PCT cells by proton NMR T_i relaxation times using Mn as a paramagnetic quencher. In BLMV,P _f (0.019 cm/sec, 23°C) was inhibited 65% by 5mm pCMBS and 75% by 300 m HgCl₂ (K _l =42 m);E _a increased from 3.6 to 7.6 kcal/mole (15–40°C) with 300 m HgCl₂. In BBMV,P _f (0.073 cm/sec, 23°C,E _a =2.8 kcal/mole, <33°C and 13.7 kcal/mole, >33°C) was inhibited 65% with HgCl₂ withE _a =9.4 kcal/mole (15–45°C). Mercurial inhibition in BLMV and BBMV was reversed with 10 m mercaptoethanol. Viable PCT cells were isolated from renal cortex by Dounce homogenization and differential seiving. Impedence sizing studies show that PCT cells are perfect osmometers (100–1000 mOsm). Assuming a cell surface-to-volume ratio of 25,000 cm^–1,P _f was 0.010±0.002 cm/sec (37°C) andP _d was 0.0032 cm/sec.P _f was independent of osmotic gradient size (25–1000 mOsm) withE _a 2.5 kcal/mole (<27°C) and 12.7 kcal/mole (>27°C). CellP _f was inhibited 53% by 300 m HgCl₂ (23°C) withE _a 6.2 kcal/mole. These findings indicate that cellP _f is not restricted by extracellular or cytoplasmic unstirred layers and that cellP _f is not flow-dependent. The high BLMV and BBMVP _f , inhibition by HgCl₂, lowE _a which increases with inhibition, and the measuredP _f /P _d >1 in cells in the absence of unstirred layers provide strong evidence for the existence of water channels in proximal tubule brush border and basolateral membranes. These channels are similar to those found in erythrocytes and are likely required for rapid PCT transcellular water flow.     

Diffusive water permeability in isolated kidney proximal tubular cells: Nature of the cellular water pathways

Summary The diffusive water permeability (P _d ) of the plasma membrane of proximal kidney tubule cells was measured using a¹H-NMR technique. The values obtained for the exchange time (T _ex) across the membrane were independent of the cytocrit and of the Mn²⁺ concentration (in the range 2.5 to 5mm). At 25°C the calculatedP _d value was (per cm² of outer surface area without taking into account membrane invaginations) 197±17 m/sec. This value equals 22.3±1.9 m/sec when the invaginations are taken into account. Cell exposure to 2.5mm parachloromercuribenzenesulfonic acid,pCMBS, (for 20 to 35 min) reducedP _d to 45% of its control value. Fivemm dithiothreitol, DTT, reverted this effect. The activation energy for the diffusive water flux was 5.2±1.0 kcal/mol under control conditions. It increased to 9.1±2.2 kcal/mol in the presence of 2.5mm pCMBS. Using our previous values for the osmotic water permeability (P _os) in proximal straight tubular cells theP _os/P _d ratio equals 18±1, under control conditions, and 3.2±0.3 in the presence ofpCMBS. These experimental results indicate the presence of pathways for water, formed by proteins, crossing these membranes, which are closed bypCMBS. Assuming laminar flow (within the pore), fromP _os/P _d of 13 to 18 an unreasonably large pore radius of 12 to 15 Å is calculated which would not hinder cell entry of known extracellular markers. Alternatively, for a single-file pore, 11 to 20 would be the number of water molecules which would be in tandem inside the pore. The water permeability remaining in the presence ofpCMBS indicates water permeation through the lipid bilayer. There are similarities between these results and those obtained in human red blood cells and in the apical cell membrane of the toad urinary bladder.     

Phospholipid surface density determines the partitioning and permeability of acetic acid in DMPC:cholesterol bilayers

Relationships between the permeability coefficient (P_HA) and partition coefficient (K _m/w) of acetic acid and the surface density of DMPC:cholesterol bilayers have been investigated. Permeability coefficients were measured in large unilamellar vesicles by NMR line broadening. Bilayer surface density, , was varied over a range of 0.5–0.9 by changing cholesterol concentration and temperature. The temperature dependence of P_HA for acetic acid exhibits Arrhenius behavior with an average apparent activation energy (E _a ) of 22±3 kcal/mole over a cholesterol mole fraction range of 0.00–0.40. This value is much greater than the enthalpy change for acetic acid partitioning between bulk decane and water (H° = 4.8±0.8 kcal/mole) and the calculated E _a (= 8.0 kcal/mole) assuming a bulk phase permeability model which includes the enthalpy of transfer from water to decane and the temperature dependence of acetic acid's diffusion coefficient in decane. These results suggest that dehydration, previously considered to be a dominant component, is a minor factor in determining E _a . Values of 1n P_HA decrease linearly with the normalized phospholipid surface density with a slope of = -12.4±1.1 (r = 0.90). Correction of P_HA for those temperature effects considered to be independent of lipid chain order (i.e., enthalpy of transfer from water to decane and activation energy for diffusion in bulk hydrocarbon) yielded an improved correlation ( = -11.7±0.5 (r = 0.96)). The temperature dependence of K_m/w is substantially smaller than that for P_HA and dependent on cholesterol composition. Values of 1n K_m/w decrease linearly with the surface density with a slope of = -4.6±0.3 (r = 0.95), which is 2.7-fold smaller than the slope of the plot of 1n P_HA vs. . Thus, chain ordering is a major determinant for molecular partitioning into and transport across lipid bilayers, regardless of whether it is varied by lipid composition or temperature.This work was supported by grants from Glaxo, INTERx/Merck, and University of Utah Research Committee.     

Very low osmotic water permeability and membrane fluidity in isolated toad bladder granules

Summary Osmotic water permeability of the apical membrane of toad urinary epithelium is increased greatly by vasopressin (VP) and is associated with exocytic addition of granules and aggrephores at the apical surface. To determine the physiological role of granule exocytosis, we measured the osmotic water permeability and membrane fluidity of isolated granules, surface membranes and microsomes prepared from toad bladder in the presence and absence of VP.P _f was measured by stopped-flow light scattering and membrane fluidity was examined by diphenylhexatriene (DPH) fluorescence anisotropy. In response to a 75mm inward sucrose gradient, granule size decreased with a single exponential time constant of 2.3±0.1 sec (sem, seven preparations, 23°C), corresponding to aP _f of 5×10^–4 cm/sec; the activation energy (E _a ) forP _f was 17.6±0.8 kcal/mole. Under the same conditions, the volume of surface membrane vesicles decreased biexponentially with time constants of 0.13 and 1.9 sec; the fast component comprised 70% of the signal. Granule, surface membrane and microsome time constants were unaffected by VP. However, in surface membranes, there was a small decrease (6±2%) in the fraction of surface membranes with fast time constant. DPH anisotropies were 0.253 (granules), 0.224 (surface membrane fluidity is remarkably lower than that of surface and microsomal membranes, and (4) rapid water transport occurs in surface membrane vesicles. The unique physical properties of the granule suggests that apical exocytic addition of granule membrane may be responsible for the low water permeability of the unstimulated apical membrane.     

Effect of Geometrical and Chemical Constraints on Water Flux across Artificial Membranes

Studies have been made on the temperature dependence of both the hydraulic conductivity, L_p, and the THO diffusion coefficient, ω, for a series of cellulose acetate membranes (CA) of varying porosity. A similar study was also made of a much less polar cellulose triacetate membrane (CTA). The apparent activation energies, E_a, for diffusion across CA membranes vary with porosity, being 7.8 kcal/mole for the nonporous membrane and 5.5 kcal/mole for the most porous one. E_a for diffusion across the less polar CTA membrane is smaller than E_a for the CA membrane of equivalent porosity. Classical viscous flow, in which the hydraulic conductivity is inversely related to bulk water viscosity, has been demonstrated across membranes with very small equivalent pores. Water-membrane interactions, which depend upon both chemical and geometrical factors are of particular importance in diffusion. The implication of these findings for the interpretation of water permeability experiments across biological membranes is discussed.     

Some properties of adenylate kinase from chemolithotrophically grown Thiobacillus novellus

Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) from Thiobacillus novellus was purified 54-fold. The preparation was, on the basis of densitometer scans of polyacrylamide gels, at least 85% pure. The optimum pH in the forward direction (2 ADP ATP+AMP) was about 8.7, and in the reverse 8.2 The enzyme was specific for AMP, ADP and ATP with apparent K _m values of 0.04, 0.34 and 0.09 mM respectively. A double reciprocal plot of specific activity vs. (ADP)² was linear. Both AMP and ATP inhibited the forward reaction with AMP the more inhibitory of the two. AMP inhibition was competitive with respect to ADP with a K _i of 0.125 mM. Although Mg²⁺ was necessary for maximal activity, about 20% of this was obtained in its absence. Co²⁺ and Mn²⁺ at similar concentrations gave 46% and 26% respectively of the activity found with Mg²⁺. Apparent K _m for Mg²⁺ was about 0.054 mM in the forward and 0.15 mM in the reverse direction. pHMB and HgCl₂ were potent inhibitors. Inhibition by pHMB but not HgCl₂ was reversible by GSH or cysteine. Arrhenius plots gave an E _a of 3.25 kcal/mole/degree C in the forward direction without discontinuity. In the reverse, there was a break at 26.7°C with an E _a of 3.62 kcal/mole/degree C for lower temperatures and 3.92 kcal/mole/degree C for higher temperatures.Molecular weights of the enzyme were 46,300±300 by SDS PAGE and 47,800±200 by SDS PAGE after treatment with 5 M urea and about 40,000 by sucrose density gradient centrifugation.Abbreviations APS adenosine-5-phosphosulfate - DEAE diethylaminoethyl - DTT dithiothreitol - E_a energy of activation - ECTEOLA epichlorohydrin triethanolamine - G6P glucose-6-phosphate - GSH glutathione (reduced) - PAGE Polyacrylamide gel electrophoresis - pHMB parahydroxymercuribenzoate - PEP phosphoenolpyruvate - PPi inorganic pyrophosphate - SDS sodium dodecyl sulfate - TEAE triethylaminoethyl Supported by an operating grant to A.M.C. from NSERCSummer student research trainee     

Effect of temperature on nonelectrolyte permeation across the toad urinary bladder

Summary The permeability of the toad urinary bladder to 22 nonelectrolytes was obtained from measurements of radioactive tracer fluxes. The permeability coefficients (P's), after suitable corrections for unstirred layers, were proportional to the olive oil/water partition coefficients for the majority of the molecules (P K_oil ^1.3). In the absence of chain branching, inductive effects, and intramolecular hydrogen bonding effects, a hydroxyl group reducedP an average 500-fold and a methylene group increasedP an average four fold. Branched chain solutes were less permeable than their straight chain isomers, and small solutes, polarand nonpolar, exhibited higher rates of permeation than expected from the relationship betweenP and K_oil. (Over the molecular size range 18–175 cc/moleP (Molecular Volume)^–2.7.) The high rates of permeation of small molecules are consistent with diffusion through a highly organized lipid structure. Large polar solutes, e.g., sucrose, appear to pass across the epithelium via an extracellular shunt pathway. The apparent activation energies (E _a ) for the permeation of 16 select molecules were obtained from permeability measurements over the temperature range 2–32°C. Linear Arrhenius plots (i. e., logP/T ^–1) were obtained for all molecules after unstirred layer corrections. In the absence of these corrections phase transitions were seen for molecules with very highP's (P>300×10^–7 cm/sec), but these are simply due to diffusion limited permeation.E _a increased by 2.5–3.6 kcals/mole with the introduction of each additional methylene group into a molecule, and decreased by up to 9 kcals/mole for the addition of a hydroxyl group. Qualitatively similar results were obtained in preliminary studies of olive oil/water partition coefficients. Arrhenius plots of the toad bladder conductance over the temperature range 2–32°C yield apparent activation energies of 4–5 kcals/mole which is identical to that found previously for leaky epithelia.     

Water permeability of plant cuticles

Using the system vapor/membrane/liquid, permeability coefficients of cuticular transpiration (P _ct) were determined as functions of water activity in the vapor (a _wv). Enzymatically isolated cuticular membranes (CM) of Citrus aurantium L. and nonisolated CM of onion bulb scales and eggplant fruits were investigated. P _ct of Citrus and eggplant CM decreased with decreasing a _wv, while permeability coefficients of CM of onion were independent of a _wv. Extraction of soluble cuticular lipids (SCL) from the CM of Citrus increased permeability coefficients by a factor of approximately 500. This extraction had no effect on the dependence of P _ct on a _wv.Treating cuticular membranes as a resistance network consisting of SCL and the polymer matrix, it is shown that the permeability of onion CM is determined by the resistance of the SCL arranged in series with the polymer matrix. In this type of CM liquid and vapor are separated by a continuous, nonporous layer of SCL, and the driving force of transpiration is the gradient of partial pressure of water vapor across the SCL layer. In the CM of Citrus and eggplant, the SCL layer is traversed by polar pores that swell or shrink depending on a _wv. However, liquid continuity is maintained across these membranes down to a _wv=0.22, the lowest value used. In this type of membrane the driving force of transpiration is the water potential gradient across the membrane.Abbreviations CM cuticular membrane - MX polymer matrix - SCL soluble cuticular lipids - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MES (N-morpholino)ethane sulfonic acid - SADH succinic acid 2,2-dimethyl hydrazide     

Oxygen equilibria and structural characteristics of the tetrameric and polymeric intracellular hemoglobins from the bivalve molluscBarbatia reeveana

Summary The arcid bivalveBarbatia reeveana contains within its erythrocytes two hemoglobins with remarkably different structures and oxygen equilibrium properties. A tetrameric hemoglobin (M _r about 60,000) with non-identical subunits (₂₂) constitutes about 60% of the erythrocytic heme protein. This hemoglobin has a relatively low oxygen affinity (P ₅₀=19 Torr at 20°C, pH 7.2), shows cooperativityn _H=2.2, shows no Bohr effect between pH 6.8 and 7.6 and a heat of oxygenation (H) of –5.4 kcal/mole between 15 and 35°C. Its oxygen affinity appears to be insensitive to ATP.B. reeveana erythrocytes also contain another hemoglobin withM _r=430,000, the largest intracellular hemoglobin known in any organism. The subunit of this hemoglobin is unusual, having aM _r of 32–34,000 and two heme oxygen binding sites per polypeptide chain. The large hemoglobin has a very low oxygen affinity (P ₅₀=33 Torr at 20°C, pH 7.2), shows slight cooperativity,n _H=1.8, and no Bohr effect (Grinich and Terwilliger 1980). The H at pH 7.2 equals –2.9 kcal/mole, a low value for most hemoglobins, and its O₂ affinity appears to be insensitive to ATP. The two hemoglobins ofB. reeveana, so different in their structure, are also different in their functional properties.     

Hydraulic Conductivity (Lp) and Its Activation Energy (Ea), Cryoprotectant Agent Permeability (Ps) and ItsEa, and Reflection Coefficients (?) for Golden Hamster Individual Pancreatic Islet Cell Membranes

Long-term cryopreservation of islets of Langerhans would be advantageous to a clinical islet transplantation program. Fundamental cryobiology utilizes knowledge of basic biophysical characteristics to increase the understanding of the preservation process and possibly increase survival rate. In this study several of these previously unreported characteristics have been determined for individual islet cells isolated from Golden hamster islets. Using an electronic particle counting device and a temperature control apparatus, dynamic volumetric response of individual islet cells to anisosmotic challenges of 1.5 M dimethyl sulfoxide (DMSO) and 1.5 M ethylene glycol (EG) were recorded at four temperatures (8, 22, 28, and 37°C). The resulting curves were fitted using Kedem and Katchalsky equations which describe water flux and cryoprotectant agent (CPA) flux based on hydraulic conductivity (L_p), CPA permeability (P_s), and reflection coefficient (?) for the membrane. For Golden hamster islet cells,L_p,P_s, and ? for DMSO at 22°C were found to be 0.23 ± 0.06 μm/min/atm, 0.79 ± 0.32 × 10⁻³cm/min, and 0.55 ± 0.37 (n= 11) (mean ± SD), respectively. For EG at 22°C,L_pequaled 0.23 ± 0.06 μm/min/atm,P_sequaled 0.63 ± 0.20 × 10⁻³cm/min, and ? was 0.75 ± 0.17 (n= 9). Arrhenius plots (lnL_por lnP_sversus 1/temperature (K)) were created by adding the data from the other three temperatures and the resulting linear regression yielded correlation coefficients (r) of 0.99 for all four plots (L_pandP_sfor both CPAs). Activation energies (E_a) ofL_pandP_swere calculated from the slopes of the regressions. The values for DMSO were found to be 12.43 and 18.34 kcal/mol forL_pandP_s(four temperatures, totaln= 52), respectively. For EG,E_aofL_pwas 11.69 kcal/mol andE_aofP_swas 20.35 kcal/mol (four temperatures, totaln= 58).     

Diurnal and Seasonal Patterns of Water Potential,Photosynthesis, Evapotranspiration and Water Use Efficiency of Clusterbean

Diurnal patterns of leaf water potential (_W), canopy net photosynthetic rate (P _N), evapotranspiration rate (E), canopy temperature (T_c), and water use efficiency (WUE) of clusterbean [Cyamopsis tetragonoloba (L.) Taub., cv. Desi] were studied at six phenological stages of plant development under field conditions at CCS Haryana Agricultural University, Hisar. The highest P _N, E, and WUE were observed at pod initiation stage (61 DAS). Daily maxima of P _N were usually between 11:00 to 14:00 h while those of E and WUE between 12:30 and 16:00 h. P _N was mainly dependent on photosynthetically active radiation and E on air temperature (T_a) but the relationships varied at different growth stages. WUE declined with the increase in T _a. At mid-day, _W was highest during pod initiation.     

Purification and properties of extracellularβ-fructofuranosidases fromAureobasidium sp. ATCC 20524

Summary Two extracellular -fructofuranosidases (E-1 andE-2) fromAureobasidium sp. ATCC 20524, producing 1-kestose (1^F--fructofuranosyl-sucrose) from sucrose, were purified to homogeneity. Molecular weights of the enzymes were estimated to be about 304000 (E-1) and 315000 (E-2) Da by gel filtration. The enzymes contained 33% (w/w) (E-1) and 27% (w/w) (E-2) carbohydrate. TheK _m values for sucrose ofE-1 andE-2 andE-2 were 0.34 and 0.28 M, respectively. were 0.34 and 0.28 M, respectively. The enzymatic profiles of these enzymes were almost identical to intracellular enzymesP-1 andP-2 except for the differences in carbohydrate content andK _m values ofE-2 andP-2.     

Pathways for alanine transport in intestinal basal lateral membrane vesicles

Summary Membrane vesicles obtained from the basal lateral membranes of the rat intestinal epithelium were used to study the pathways for neutral amino acid transport.In the absence of sodium there was a stereospecific uptake ofl-alanine which exhibited saturation kinetics (K _m 0.73mm andV _max 5.3 nmol/mg min at 22°C). The activation energy for this process was 8.1 kcal/mole between 5 and 25°C. Preloading the vesicles with alanine increased the unidirectional influx of alanine into the vesicle. Competition experiments indicated that the affinity of the sodium-independent transport system was glutamine > threonine > alanine > phenylalanine > valine > methionine > glycine > histidine > proline, N-MeAIB. These are the characteristics of the classical L transport system.External sodium increased the rate of the stereospecificl-alanine uptake. The Na-dependent flux had aK _m of 0.04mm and aV _max of 0.26 nmol/mg min at 22°, and an activation energy of 9.1 kcal/mole between 5 and 25°C. Competition experiments suggest the existence of three separate pathways for alanine transport in the presence of sodium. A major pathway is shared by all other amino acids tested (i.e., threonine, glutamine, methionine, phenylalanine, valine, proline and N-MeAIB). This resembles the classical A system. A second pathway is unavailable to either phenylalanine or N-MeAIB; this is reminiscent of the classical ASC system; and the third is a novel pathway which is shared by N-MeAIB but not phenylalanine.The sodium-independent and the sodium-dependent transport ofl-alanine was blocked by PCMBS and significantly inhibited by DTP and NEM. It is concluded that the sodium-independent system (the L-like system) accounts for the efflux of neutral amino acids from the epithelium to the blood during the absorption of amino acids from the gut, and that the sodium-dependent transport processes may play an important role in the supply of amino acids to the epithelium in the absence of amino acids from the gut lumen.     

Proton conductance through phospholipid bilayers: Water wires or weak acids?

The proton/hydroxide (H⁺/OH^–) permeability of phospholipid bilayer membranes at neutral pH is at least five orders of magnitude higher than the alkali or halide ion permeability, but the mechanism(s) of H⁺/OH^– transport are unknown. This review describes the characteristics of H⁺/OH^– permeability and conductance through several types of planar phospholipid bilayer membranes. At pH7, the H⁺/OH^– conductances (G _H/OH) range from 2–6 nS cm^–2, corresponding to net H⁺/OH^– permeabilities of (0.4–1.7)×10^–5 cm sec^–1. Inhibitors ofG _H/OH include serum albumin, phloretin, glycerol, and low pH. Enhancers ofG _H/OH include chlorodecane, fatty acids, gramicidin, and voltages >80 mV. Water permeability andG _H/OH are not correlated. The characteristics ofG _H/OH in fatty acid (weak acid) containing membranes are qualitatively similar to the controls in at least eight different respects. The characteristics ofG _H/OH in gramicidin (water wire) containing membranes are qualitatively different from the controls in at least four different respects. Thus, the simplest explanation for the data is thatG _H/OH in unmodified bilayers is due primarily to weakly acidic contaminants which act as proton carriers at physiological pH. However, at low pH or in the presence of inhibitors, a residualG _H/OH remains which may be due to water wires, hydrated defects, or other mechanisms.     

Osmotic water permeabilities of brush border and basolateral membrane vesicles from rat renal cortex and small intestine

Summary The osmotic water permeabilityP _f of brush border (BBM) and basolateral (BLM) membrane vesicles from rat small intestine and renal cortex was studied by means of stopped-flow spectrophotometry. Scattered light intensity was used to follow vesicular volume changes upon osmotic perturbation with hypertonic mannitol solutions. A theoretical analysis of the relationship of scattered light intensity and vesicular volume justified a simple exponential approximation of the change in scattered light intensity. The rate constants extracted from fits to an exponential function were proportional to the final medium osmolarity as predicted by theory. For intestinal membranes, computer analysis of optical responses fitted well with a single-exponential treatment. For renal membranes a double-exponential treatment was needed, implying two distinct vesicle populations.P _f values for BBM and BLM preparations of small intestine were equal and amount to 60 m/sec. For renal preparations,P _f values amount to 600 m/sec for the fast component, BBM as well as BLM, and to 50 (BBM) and 99 (BLM) m/sec for the slow component. The apparent activation energy for water permeation in intestinal membranes was 13.3±0.6 and in renal membranes, 1.0±0.3 kCal/mole, between 25 and 35°C. The mercurial sulfhydryl reagentpCMBS inhibited completely and reversibly the highP _f value in renal brush border preparations. These observations suggest that in intestinal membranes water moves through the lipid matrix but that in renal plasma membranes water channels may be involved. From the highP _f values of renal membrane vesicles a transcellular water permeability for proximal tubules can be calculated which amounts to 1 cm/sec. This value allows for an entirely transcellular route for water flow during volume reabsorption.     

Ordered substrate binding and evidence for a thermally induced change in mechanism for E. coli aspartate transcarbamylase

Isotopic exchange kinetics at equilibrium for E. coli native aspartate transcarbamylase at pH 7.8, 30 °C, are consistent with an ordered BiBi substrate binding mechanism. Carbamyl phosphate binds before l-Asp, and carbamyl-aspartate is released before inorganic phosphate. The rate of [¹⁴C]Asp C-Asp exchange is much faster than [³²P]carbamyl phosphate P_i exchange. Phosphate, and perhaps carbamyl phosphate, appears to bind at a separate modifier site and prevent dissociation of active-site bound P_i or carbamyl phosphate. Initial velocity studies in the range of 0–40 °C reveal a biphasic Arrhenius plot for native enzyme: E_a (>15 °C) = 6.3 kcal/ mole and E_a (<15 °C) = 22.1 kcal/mole. Catalytic subunits show a monophasic plot with E_a ? 20.2 kcal/mole. This, with other data, suggests that with native enzyme a conformational change accompanying aspartate association contributes significantly to rate limitation at t > 15 °C, but that catalytic steps become definitively slower below 15 °C. Model kinetics are derived to show that this change in mechanism at low temperature can force an ordered substrate binding system to produce exchange-rate patterns consistent with a random binding system with all exchange rates equal. The nonlinear Arrhenius plot also has important consequences for current theories of catalytic and regulatory mechanisms for this enzyme.     

Serial permeability barriers to water transport in human placental vesicles

Summary Microvillous vesicles were prepared from term human placenta by shearing, differential centrifugation and Mg²⁺ precipitation. Vesicles were purified further on a sucrose density gradient producing two bands with densities of 1.16 to 1.18 g/ml (C1) and 1.13 to 1.15 g/ml (C2). The C2 fraction, which had a 24-fold enrichment of alkaline phosphatase and a three-fold reduction in Na⁺, K⁺-ATPase activity compared to homogenates, was used to measure osmotic water (P _f ) permeability.P _f was measured from the time course of scattered light intensity following exposure of vesicles to specified gradients of impermeant solutes.P _f decreased from 3.0×10^–3 to 0.6×10^–3 cm/sec with increasing gradient size (65 to 730mm; 23°C). Four possible causes of this behavior were examined theoretically and experimentally: an unstirred layer, saturation of water transport, large changes in the vesicle surface area with changes in volume and a structural restriction to vesicle volume change. The measured dependence ofP _f on gradient size and the effect of the channel-forming ionophore gramicidin onP _f fit best to the theoretical dependences predicted by a structural restriction mechanism. This finding was supported by experiments involving the effects onP _f of increased solution viscosity, initial vesicle volume, the magnitude of transmembrane volume flow, and the effects of gradient size on activation energy (E _a) forP _f . The decreasedP _f resulting from a structural restriction limiting vesicle volume change was modeled mathematically as a second barrier in series with the vesicle membrane.E _a measured using a 250-mm inwardly directed sucrose gradient was 5.4±0.6 kcal/mol (T>27°C) and 10.0±0.6 kcal/mol (T<27°C).E _a above 27°C is in the range normally associated with transmembrane passage of water via aqueous channels. Water transport was not inhibited byp-chloromercuribenzenesulfonate.     

Analog circuit of theAcetabularia membrane

Summary The high membrane potential ofAcetabularia (E _m=–170 mV) is due to an electrogenic pump in parallel with the passive diffusion system (E _d=–80 mV) which could be studied separately in the cold, when the pump is blocked. Electrical measurements under normal conditions show that the pump pathway consists of its electromotive forceE _p with two elementsP ₁ andP ₂ in series;P ₂ is shunted by a large capacitance (C _p=3 mF cm^–2). The nonlinear current-voltage relationship ofP ₁ (light- and temperature-sensitive) could be determined separately; it reflects the properties of a carrier-mediated electrogenic pump. The value ofE _p (–190 mV) indicates a stoichiometry of 21 between electrogenically transported charges and ATP. The electrical energy, normally stored inC _p, compares well with the metabolic energy, stored in the ATP pool. The nonlinear current-voltage relationship ofP ₂ (attributed to phosphorylating reactions) is also sensitive to light and temperature and is responsible for the region of negative conductance of the overall current-voltage relationship. The power of the pump (1 W cm^–2) amounts to some percent of the total energy turnover. The high Cl^– fluxes (1 nmol cm^–2 sec^–1) and the electrical properties of the plasmalemma are not as closely related as assumed previously. For kinetic reasons, a direct and specific Cl^– pathway between the vacuole and outside is postulated to exist.     

The Synthesis of Phosphates of Long-Chain ω-Hydroxyalkyl Esters of 11-Deoxyprostaglandin E1

Di(p-methylbenzyl) phosphates of -hydroxyalkyl esters of 11-deoxyprostaglandin E₁ were synthesized from disubstituted 1,10-decane and 1,22-docosane derivatives for studying the permeability of bilayer membranes.     

Brassinolide may control aquaporin activities in Arabidopsis thaliana

 It is usually assumed that aquaporins present in the cellular membranes could be an important route in the control of water flux in plants, but evidence for this hypothesis is scarce. In this paper, we report measurements of the osmotic permeability (P _os ) of protoplasts isolated from hypocotyls of wild-type and mutant Arabidopsis thaliana (L.) Heynh. Mutants were affected in their growth and exhibited different sensitivities to the phytohormone, brassinolide. For the two mutants studied (cpd: constitutive photomorphogenesis and dwarfism; bri1: brassinosteroid insensitive), hypocotyl length was correlated to P _os for the protoplasts. Under experimental conditions where hypocotyl growth had ceased, restoration of root, hypocotyl and petiole growth by brassinolide was correlated with an increase in P _os of the hypocotyl protoplasts. We consider that the increase in P _os of the hypocotyl cells was needed because these cells were part of the transcellular water pathway of the plant. This is the first time, to our knowledge, that brassinolide has been shown to be involved in the modification of the water-transport properties of cell membranes. Our results also emphasize the importance of aquaporins and the transcellular pathway in water transport under normal growth conditions. Received: 15 January 2000 / Accepted: 18 May 2000