Biosorption of cadmium,cobalt, nickel,and strontium by aBacillus simplex strain isolated from the vadose zone

A subsurface Gram-positive, endospore-forming, filamentous bacterium, designated ZAN-044, was isolated from a depth of 96.2 m in the vadose zone of the Hanford Site in Washington State. A phylogenetic analysis of the 16S rRNA gene sequence of strain ZAN-044 revealed it to be 99.5% similar toBacillus simplex strain DSM 1321, indicating that they may be members of the same species.B. simplex ZAN-044 was studied along withBacillus subtilis 168, andEscherichia coli K-12 (AB264), two well-characterized metal-sorbing bacteria, for the binding of Cd²⁺, Co²⁺, Ni²⁺, and Sr²⁺. There was rapid (less than 1 h) uptake of 1 M metal by the three bacteria in the order Cd>NiCo>Sr. Binding followed a saturation isotherm at cation concentrations from 0.1 M to 1 mM. Cation binding was pH-dependent, with less binding at low pH.B. simplex ZAN-044 bound more metal thanB. subtilis orE. coli, demonstrating that subsurface microorganisms can remove significant quantities of metals from solution and may be able to influence radionuclide and metal transport in the subsurface.     

The Na+-independentd-glucose transporter in the enterocyte basolateral membrane: Orientation and cytochalasin B binding characteristics

Summary Phloridzin-insensitive, Na⁺-independentd-glucose uptake into isolated small intestinal epithelial cells was shown to be only partially inhibited by trypsin treatment (maximum 20%). In contrast, chymotrypsin almost completely abolished hexose transport. Basolateral membrane vesicles prepared from rat small intestine by a Percoll^® gradient procedure showed almost identical susceptibility to treatment by these proteolytic enzymes, indicating that the vesicles are predominantly oriented outside-out. These vesicles with a known orientation were employed to investigate the kinetics of transport in both directions across the membrane. Uptake data (i.e. movement into the cell) showed aK _t of 48mm and aV _max of 1.14 nmol glucose/mg membrane protein/sec. Efflux data (exit from the cell) showed a lowerK _t of 23mm and aV _max of 0.20 nmol glucose/mg protein/sec.d-glucose uptake into these vesicles was found to be sodium independent and could be inhibited by cytochalasin B. TheK _t for cytochalasin B as an inhibitor of glucose transport was 0.11 m and theK _D for binding to the carrier was 0.08 m.d-glucose-sensitive binding of cytochalasin B to the membrane preparation was maximized withl- andd-glucose concentrations of 1.25m. Scatchard plots of the binding data indicated that these membranes have a binding site density of 8.3 pmol/mg membrane protein. These results indicate that the Na⁺-independent glucose transporter in the intestinal basolateral membrane is functionally and chemically asymmetric. There is an outward-facing chymotrypsin-sensitive site, and theK _t for efflux from the cell is smaller than that for entry. These characteristics would tend to favor movement of glucose from the cell towards the bloodstream.     

Detection of different adenosine triphosphatases in human placental brush border membranes

The microvillous membrane of human placental syncytiotrophoblast cells contains a high ATPase activity. The purpose of this study was to characterize this activity and to investigate the presence of vacuolar type H⁺ ATPase in this membrane. Intact brush border membrane vesicles strongly hydrolyzed ATP, reflecting the presence of ATPase on the external side of the membrane. The ATPase activity was entirely Mg²⁺ dependent and increased with pH. At pH 7.5, Vmax was 31.0 ± 1.7 mol/mg/20 min and Km 0.18 ± 0.03 mm ATP. Hydrolysis of ATP was not influenced by the presence of bicarbonate or alkaline phosphatase inhibitors, but at pH 8 it decreased by half following addition of 100 m dicyclohexylcarbodiimide (DCCD). At pH 7.5, 1 mm N-ethylmaleimide (NEM) depressed this activity by less than 5%. Opening the membrane vesicles with 0.1% desoxycholate (DOC) or Triton-X neither revealed any additional ATPase activity nor altered the low sensitivity to NEM. Treatment of these membranes with 1% cholate decreased the ATPase activity by more than 70% and did not enhance the sensitivity of ATP hydrolysis to NEM. 10^–7 m Bafilomycin, which reduced by 56 ± 9% the ATPase activity in dog kidney brash border membranes treated with 0.1% DOC, had no effect on placental brush border membranes subjected to the same procedure. Finally, neither immunocytochemical staining using monoclonal antibody to the Mr 31000 subunit of V-type H⁺ ATPase, nor electron microscopic examination detected the presence of H⁺ ATPase in placental membranes.In conclusion, the placental brush border membrane is the site of a strong ecto ATPase activity which is partially DCCD sensitive. However, our results did not detect the presence of any vacuolar type H⁺ ATPase activity in these membranes.This study was supported by the MRC grant N MA 9565 and by an extramural grant from Baxter Health Care Corporation. The authors are indepted to Dr. Patrick Vinay for this help in providing Bafilomycin and dog kidney membranes, as well as for his insightful discussion.     

Ion permeability of maize root membrane vesicles: Studies with light scattering

A possible modulation of permeabilities of membrane vesicles to anions and cations was explored by light scattering techniques, evaluated by measuring the capacity of the vesicles to shrink and swell in response to changes of the osmolarity of the incubation medium. Membrane fractions were obtained by phase partition. Purity was evaluated by detection and quantification of membrane enzyme markers: vanadate-sensitive ATPase for the plasma membrane, nitrate-sensitive ATPase for the tonoplast and azide-sensitive ATPase for mitochondria. Membrane vesicles (250 g protein) were exposed to hypertonic solutions of salts (0.6 osmolar). Kinetics of the changes in apparent absorbance at 546 nm were observed by the addition of potassium, nitrate and chloride salts. The diffusion of ions into vesicles was induced by an osmotic gradient across the membrane and brought about volume changes of vesicles. Upon addition of vesicles to the different solutions the following ion permselectivity sequences were observed: PNO ₃ ^– >PCl ^–>PSO ₄ ^2– and PK ⁺PNa ⁺>PNH ₄ ⁺.Abbreviations ATP adenosine 5-triphosphate - EDTA ethylene diaminetetraacetic acid - Tris-Mes (Tris[hydroxymethyl]aminomethane, Mes-(2-[N-Morpholino]ethanesulfonic acid) - PEG polyethylene glycol     

Cloning of the cyclomaltodextrinase gene fromBacillus subtilis high-temperature growth transformant H-17

The cyclomaltodextrinase gene fromBacillus subtilis high-temperature growth transformant H-17 was cloned on separatePstI,BamHI, andEcoRI fragments into the plasmid vector pUC18, but was expressed in an inactive form in the host,Escherichia coli DH5. High level constitutive expression of the gene product was also detrimental to theE. coli host, which led to structural instability of the recombinant plasmid. The cyclomaltodextrinase gene was cloned on a 3-kbEcoRI fragment into the plasmid vector pPL708, and the fragment was structurally maintained in the hostB. subtilis YB886. The cloned gene product was synthesized in an enzymatically active form in theB. subtilis host; however, expression was at a low level. Subcloning of the 3-kbEcoRI fragment into pUC18 and transformation intoE. coli XL1-Blue (FlacI^q) indicated that the cyclomaltodextrinase gene was cloned with its own promoter, since expression of the gene occurred in the absence of IPTG. Subcloning of the cyclomaltodextrinase gene downstream from theBacillus temperate phage SPO2 promoter of pPL708 may increase expression of this gene.Florida Agricultural Experiment Station Journal Series No. R-02177     

Solubilization and reconstitution of a vanadate-sensitive H+-ATPase from the plasma membrane ofBeta vulgaris

Summary A vanadate-sensitive H⁺-translocating ATPase isolated from red beet plasma membrane has been solubilized in active form and successfully reconstituted into artificial proteoliposomes. The H⁺-ATPase was solubilized in active form with deoxycholate, CHAPSO or octylglucoside in the presence of glycerol. Following detergent removal by gel filtration and reconstitution into proteoliposomes, ATP:Mg-dependent H⁺ transport could be measured as ionophore-reversible quenching of acridine orange fluorescence. Solubilization resulted in a three-to fourfold purification of the plasma membrane ATPase, with some additional enrichment of specific activity following reconstitution. H⁺ transport activity was inhibited half-maximally between 1 and 5 M vanadate (Na₃VO₄) and nearly abolished by 100 M vanadate. ATPase activity of native plasma membrane showed aK _i for vanadate inhibition of 9.5 M, and was inhibited up to 80% by 15 to 20 M vanadate (Na₃VO₄). ATPase activity of the reconstituted vesicles showed aK _i of 2.6 M for vanadate inhibition. The strong inhibition by low concentrations of vanadate indicates a plasma membrane rather than a mitochondrial or tonoplast origin for the reconstituted enzyme.     

Characterization of the superoxide dismutase of the denitrifying bacterium,Bacillus halodenitrificans

Summary Bacillus halodenitrificans produced a dimeric, manganese-containing superoxide dismutase constitutively when grown either aerobically or as a denitrifier. The molecular mass of the enzyme was determined by sedimentation equilibrium to be 41.4±3 kDa with each subunit estimated at 26 kDa. Plasma emission spectroscopy indicated the presence of 1.22 mol manganese atoms/mol holoenzyme. The electronic absorption spectrum displayed a broad band centered at approximately 474 nm (=560 mM^–1 · cm^–1) and a shoulder at 595 nm. In the ultraviolet range, the spectrum exhibited split maxima at 278 nm and 283 nm and a shoulder at 291 nm, thus resembling the spectra of superoxide dismutase fromBacillus subtilis andEscherichia coli. The amino acid composition of theB. halodenitrificans enzyme differed slightly quantitatively but little qualitatively from counterpart enzymes from other sources. Like the superoxide dismutases ofMycobacterium lepraemurium and human mitochondria, theB. halodenitrificans enzyme exhibited several cysteine residues. As expected from the capacity superoxide dismutase exhibits for protecting NO as neutrophil cytotoxicity factor, theB. halodenitrificans superoxide dismutase did not interfere with accumulation of NO produced by the organism's nitrite reductase.     

The effect of endotoxin on thin lipid bilayer membranes

Summary The effect of endotoxin fromSalmonella typhimurium orEscherichia coli was studied on bilayer lipid membranes (1% lecithin–1% cholesterol in n-decane) formed in buffered 0.1m NaCl solution (pH 6.8). Endotoxin was added to the buffered solution either prior to membrane formation or after stable membranes were formed. In both cases, concentrations of 110 to 720 g/ml endotoxin initiated a decrease in the electrical resistance of the membranes followed by their rupture. A 50 g/ml concentration of the agent was unable to elicit any response. Also, the addition of an equal volume of buffer solution, serving as a control, caused no decrease in membrane resistance or survival time. Treatment of the endotoxin with alkaline hydroxylamine to remove esterand amide-bound fatty acids likewise abolished the membrane effect. This is the first report of an endotoxin effect on lipid bilayer membranes. The potential correlation of this interaction of bilayer and endotoxin with the diverse biologic effects of endotoxin is discussed.     

<Superscript>65</Superscript>Zn<Superscript>2+</Superscript> transport by lobster hepato-pancreatic baso-lateral membrane vesicles

The lobster (Homarus americanus) hepato-pancreatic epithelial baso-lateral cell membrane possesses three transport proteins that transfer calcium between the cytoplasm and hemolymph: an ATP-dependent calcium ATPase, a sodium-calcium exchanger, and a verapamil-sensitive cation channel. We used standard centrifugation methods to prepare purified hepato-pancreatic baso-lateral membrane vesicles and a rapid filtration procedure to investigate whether ⁶⁵Zn²⁺ transfer across this epithelial cell border occurs by any of these previously described transporters for calcium. Baso-lateral membrane vesicles were osmotically reactive and exhibited a time course of uptake that was linear for 10–15 s and approached equilibrium by 120 s. In the absence of sodium, ⁶⁵Zn²⁺ influx was a hyperbolic function of external zinc concentration and followed the Michaelis-Menten equation for carrier transport. This carrier transport was stimulated by the addition of 150 M ATP (increase in K_m and J_max) and inhibited by the simultaneous presence of 150 mol l^–1 ATP+250 mol l^–1 vanadate (decrease in both K_m and J_max). In the absence of ATP, ⁶⁵Zn²⁺ influx was a sigmoidal function of preloaded vesicular sodium concentration (0, 5, 10, 20, 30, 45, and 75 mmol l^–1) and exhibited a Hill Coefficient of 4.03±1.14, consistent with the exchange of 3 Na⁺/1Zn²⁺. Using Dixon analysis, calcium was shown to be a competitive inhibitor of baso-lateral membrane vesicle ⁶⁵Zn²⁺ influx by both the ATP-dependent (K_i=205 nmol l^–1 Ca²⁺) and sodium-dependent (K_i=2.47 mol l^–1 Ca²⁺) transport processes. These results suggest that zinc transport across the lobster hepato-pancreatic baso-lateral membrane largely occurred by the ATP-dependent calcium ATPase and sodium-calcium exchanger carrier proteins.Communicated by: I.D. Hume     

Hydrogen fluoride effects on plasma membrane composition,ATPase activity and cell structure in needles of eastern white pine (Pinus strobus) seedlings

Eastern white pine (Pinus strobus L.) seedlings were grown in controlled environment growth cabinets and fumigated with 0.4 and 1.6 g m^–3 hydrogen fluoride for 2–28 days. Plasma membranes were isolated from needles of treated and control seedlings and their chemical composition and ATPase activity examined to determine early effects of hydrogen fluoride action. In plants treated for 2 days with both fluoride levels, ratios of plasma membrane free sterols:phospholipids and sterols:proteins were drastically higher than ratios in control plants. Seedlings treated with hydrogen fluoride for 8 days contained plasma membranes with elevated phospholipid:protein and sterol:protein ratios and their plasma membrane ATPase activity was higher than that of control plants. Prolonged, 28-day hydrogen fluoride treatment with 1.6 g m^–3 level was the only treatment which produced a drastic inhibition of plasma membrane ATPase activity. During the initial stages of hydrogen fluoride treatment, treated cells did not show alterations of ultrastructure which were previously shown in cells of plants treated with soil applied sodium fluoride. The results of the present study indicate that the plasma membranes may be among the initial sites of hydrogen fluoride injury to plants as well as initial sites of defense reaction.     

Secretion of β-lactamase by Escherichia coli in vivo and in vitro: effect of cerulenin

The effect of cerulenin on the production of -lactamase and other periplasmic proteins was studied in Escherichia coli IA199 carrying plasmid pBR322. Cerulenin (10 to 25 g/ml) had almost no effect on the growth rate of E. coli but it decreased the amount of -lactamase and other periplamic proteins in shock fluid. Higher amounts of the antibiotic (40 to 100 g/ml)decreased turbidity and almost completely prevented synthesis of -lactamase and other periplasmic proteins. Cerulenin decreased incorporation of l-[³⁵S]methionine into membranes during growth as well. Spheroplasts secreted -lactamase into the external medium, but during a 3-h incubation in the presence of cerulenin (25 g/ml) this secretion was prevented by more than 90%. -Lactamase was secreted into the isolated membrane vesicles from E. coli IA199. However, only 5% of the total amount of pre--lactamase was secreted and processed by the membranes in vitro. Cerulenin did not prevent processing in vitro but the membranes prepared from the cells grown in the presence of cerulenin (25 g/ml) did not catalyze processing of pre--lactamase at all. Membrane preparations from Bacillus subtilis did not process pre--lactamase either in the absence or in the presence of cerulenin.     

Effects of equisetin on rat liver mitochondria: Evidence for inhibition of substrate anion carriers of the inner membrane

The effect of equisetin, an antibiotic produced byFusarium equiseti, has been studied on mitochondrial functions (respiration, ATPase, ion transport). Equisetin inhibits the DNP-stimulated ATPase activity of rat liver mitochondria and mitoplasts in a concentration-dependent manner; 50% inhibition is caused by about 8 nmol equisetin/mg protein. The antibiotic is without effect either on the ATPase activity of submitochondrial particles or on the purified F₁-ATPase. It inhibits both the ADP- or DNP-activated oxygen uptake by mitochondria in the presence of glutamate + malate or succinate as substrates, but only the ADP-stimulated respiration is inhibited if the electron donors are TMPD + ascorbate. It does not affect the NADH or succinate oxidation of submitochondrial particles. Equisetin inhibits in a concentration-dependent manner the active Ca²⁺-uptake of mitochondria energized both by ATP or succinate without affecting the Ca²⁺-uniporter itself. The antibiotic inhibits the ATP-uptake by mitochondria (50% inhibition at about 8 nmol equisetin/mg protein) and the P_i and dicarboxylate carrier. It does not lower the membrane potential at least up to 200 nmol/mg protein concentration. The data presented in this paper indicate that equisetin specifically inhibits the substrate anion carriers of the mitochondrial inner membrane.Abbreviations EGTA ethyleneglycol bis/-aminoethylether/-N, N-tetraacetic acid - DNP 2, 4-dinitrophenol - TMPD N,N,N,N,tetramethyl-p-phenylenediamine - CCP carbonylcyanide-m-chlorophenyl hydrazone - TPP tetraphenyl-phosphonium - Hepes /4,(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid/     

l-Alanine uptake in brush border membrane vesicles from the gill of a marine bivalve

Summary Brush border membrane vesicles were prepared from mussel gills using differential and sucrose density gradient centrifugation. These vesicles contained both the maximal Na⁺-dependent alanine transport activity found in the gradient and the maximal activities of -glutamyl transpeptidase and alkaline phosphatase. Electron micrographs showed closed vesicles of approximately 0.1–0.5 m diameter. Transport experiments using these vesicles demonstrated a transient 18-fold overshoot in intravesicular alanine concentration in the presence of an inwardly directed Na⁺ gradient, but not under Na⁺ equilibrium conditions. A reduced overshoot (10-fold) was seen with an inwardly directed K⁺ gradient. Further studies revealed a broad cation selectivity, with preference for Na⁺, which was characteristic of alanine transport but not glucose transport in these membranes. The apparent amino acid specificity of the uptake pathway(s) was similar to that of intact gills and supported the idea of at least four separate pathways for amino acid transport in mussel gill brush border membranes. The apparent Michaelis constant for alanine uptake was approximately 7m, consistent with values forK _t determined with intact tissue.     

Characterization of Na+−Ca2+ exchange activity in plasma membrane vesicles from postmortem human brain

Procedures were developed for measurement of Na⁺/Ca²⁺ exchange in resealed plasma membrane vesicles from postmortem human brain. The vesicle preparation method permits use of stored frozen tissue with minimal processing required prior to freezing. Vesicles prepared in this manner transport Ca²⁺ in the presence of a Na⁺ gradient. The kinetic characteristics of the Na⁺/Ca²⁺ exchange process were determined in membrane vesicles isolated from hippocampus and cortex. The K_act for Ca²⁺ was estimated to be 32 M for hippocampal and 17 M for cortical tissue. The maximal rate of Ca²⁺ uptake (V_max) was 3.5 nmol/mg protein/15 sec and 3.3 nmol/mg protein/15 sec for hippocampal and cortical tissue, respectively. Exchange activity was dependent on the Na⁺ gradient, and was optimal in the high pH range. Therefore, membranes in which Na⁺-dependent o Ca²⁺ transport activity is preserved can be isolated from postmortem human brain and could be used to determine the influence of pathological conditions on this transport system.     

A 31P NMR study of the interaction of amphibian antimicrobialpeptides with the membranes of live bacteria

Summary Amphibian skin is a rich source of peptides that are specific to pathogens and act by disrupting bacterial membranes. Three antimicrobial peptides were isolated from the skin glands of Australian tree frogs,Litoria caerulea andLitoria genimaculata. NMR spectroscopy was used to observe changes induced by these peptides in the³¹P resonances of bacterial membranes in vivo. Caerin 1.1 and maculatin 1.1, both wide-spectrum antibiotics disrupted the membranes ofBacillus cereus andStaphylococcus epidermidis (Gram-positive), leading to an increase in the isotropic³¹P NMR signal. Caerin 4.1, a narrow-spectrum antibiotic, however, did not affect the³¹P spectra of these organisms. The results demonstrate the use of³¹P NMR to study the effects of membrane-disrupting agents on the membranes of live bacteria.     

Antimicrobial mechanism of β-glycyrrhetinic acid isolated from licorice, Glycyrrhiza glabra

-Glycyrrhetinic acid isolated from Glycyrrhiza glabra had an antibacterial activity of 7.6 and 12.5 g ml^–1 against Bacillus subtilis and Staphylococcus epidermidis without causing hemolysis of human erythrocytes, whereas it was not inhibitory against Escherichia coli, Proteus vulgaris and various fungi. Confocal microscopy showed that -glycyrrhetinic acid was located within the bacteria but had not caused membrane disruption. It then inhibited synthesis of DNA, RNA and protein.     

Nucleotide sequence of a cDNA coding for mitochondrial fumarase from human liver

The nucleotide sequence of a 1.46 kb cDNA, selected from a human liver library by the expression of fumarase antigenic determinants, was determined using the dideoxy chain termination method. The cDNA contained an open reading frame extending from the extreme 5-base and coding for a protein with 468 amino acids. This protein, with the exception of an N-terminal methionine, was identified as mitochondrial fumarase. The protein showed a high degree of identity of structure with the fumarase fromBacillus subtilis (56.6 %) and a fumarase fromEscherichia coli (product of thefumC gene, 59.3 %), and a lower degree of identity with the aspartase ofE. coli (37.2 %).     

Characterization of a plasma membrane H+-ATPase from the extremely acidophilic algaDunaliella acidophila

Summary Dunaliella acidophila is an unicellular green alga which grows optimally at pH 0–1 while maintaining neutral internal pH. A plasma membrane preparation of this algae has been purified on sucrose density gradients. The preparation exhibits vanadatesensitive ATPase activity of 2 mol P_i/mg protein/min, an activity 15 to 30-fold higher than that in the related neutrophilic speciesD. salina. The following properties suggest that the ATPase is an electrogenic plasma membrane H⁺ pump. (i) ATP induces proton uptake and generates a positive-inside membrane potential as demonstrated with optical probes. (ii) ATP hydrolysis and proton uptake are inhibited by vanadate, diethylstilbestrol, dicyclohexylcarbodiimide and erythrosine but not by molybdate, azide or nitrate. (iii) ATP hydrolysis and proton uptake are stimulated by fussicoccin in a pH-dependent manner as found for plants plasma membrane H⁺-ATPase. Unusual properties of this enzyme are: (i) theK _m for ATP is around 60 M, considerably lower than in other plasma membrane H⁺-ATPases, and (ii) the ATPase activity and proton uptake are stimulated three to fourfold by K⁺ and to a smaller extent by other monovalent cations. These results suggest thatD. acidophila possesses a vanadate-sensitive H⁺-ATPase with unusual features enabling it to maintain the large transmembrane pH gradient.     

A T7 promoter-specific, inducible protein expression system forBacillus subtilis

The adaptation and application of theEscherichia coli T7 RNA polymerase system for regulated and promoter-specific gene expression inBacillus subtilis is reported. The expression cassette used inBacillus subtilis was tightly regulated and T7 RNA polymerase (T7 RNAP) appeared 30 min after induction. The efficiency of T7 promoter-specific gene expression inB. subtilis was studied using one secretory and two cytosolic proteins of heterologous origin. The accumulation ofE. coli -galactosidase, as well as a 1,4--glucosidase fromThermoanaerobacter brockii inB. subtilis after T7 RNAP induction was strongly enhanced by rifampicin inhibition of host RNAP activity. The-amylase ofThermoactinomyces vulgaris, a secretory protein, was found to accumulate in the culture supernatant up to levels of about 70 mg/l 10–20 h after T7 RNAP induction, but was also deposited in cellular fractions. The addition of rifampicin inhibited-amylase secretion, but unexpectedly, after a short period, also prevented its further (intra)cellular accumulation     

Rabbit distal colon epithelium: I. Isolation and characterization of basolateral plasma membrane vesicles from surface and crypt cells

Summary A method has been developed for the simultaneous isolation of basolateral plasma membrane vesicles from surface and crypt cells of rabbit distal colon epithelium by sequential use of differential sedimentation, isopycnic centrifugation and Ficoll 400 barrier centrifugation. The protein yield was high (total 0.81 mg/g mucosa) and surface and crypt cell-derived basolateral membrane fractions have been purified 34- and 9-fold with respect to the homogenate. The pattern of marker enzyme enrichments revealed only minor contamination by subcellular organelles. Latency of ouabain-sensitive (Na⁺, K⁺)-ATPase activity prior and after trypsin treatment of membranes indicated a vesicle configuration of sealed right side-out: sealed inside-out: leaky of approximately 211. The presence of sealed vesicles was also evident from the osmotic sensitivity of thed-[1-¹⁴C] mannitol equilibrium space determined with either fraction. Although considerably different in protein profile, surface and crypt basolateral membranes were similar in cholesterol to phospholipid molar ratio and membrane fluidity as determined by steady-state fluorescence polarization.Stopped-flow light scattering experiments revealed a rather low water permeability of the membranes with a permeability coefficient of 6 m/sec at 35°C, which is one order of magnitude lower than reported for small intestinal plasma membranes. Both membrane fractions have been shown to effectively generate outward uphill potassium ion gradients, a process that is energized by ATP and inhibited by the membrane-permeant cardiacglycoside digitoxin. These characteristics are consistent with the activity of a (Na⁺, K⁺) pump operating in inside-out vesicles.