Subcellular distribution of glycoside hydrolase and polysaccharide depolymerase enzymes in the rumen entodiniomorphid ciliatePolyplastron multivesiculatum

The distribution of 12 acid hydrolase and two polysaccharide depolymerase enzymes in the rumen entodiniomorphid ciliatePolyplastron multivesiculatum, isolated from the ovine rumen 2 h after feeding, was examined by differential and density-gradient centrifugation. Approximately 60%–70% of the recovered activity was sedimentable in fractions prepared by centrifugation at 10³ g for 10 min (F1) and 10⁴ g for 10 min (F2) with 25%–35% of the acid hydrolases and 15%–20% of acid phosphatase and the polysaccharidases remaining nonsedimentable (in fraction F5) after centrifugation at 10⁵ g for 60 min. Approximately 60% of the sedimentable activity was located in fraction F1. Latency of the hydrolase activity was demonstrated. After isopycnic centrifugation in sucrose density gradients, the hydrolytic enzymes cosedimented in acid phosphatase-containing, membrane-bound, pleomorphic lysosomelike vesicles 0.1–1.0 m in size, with a mean equilibrium density of 1.17 (1.15–1.19) g/ml.     

Studies on lysosomes. XI. Characterization of a hydrolase-rich fraction from human lymphocytes

Pure suspensions of human lymphocytes were separated from peripheral blood by means of nylon wool, homogenized in 0.34 M sucrose-0.01 M EDTA solution, and fractionated by differential centrifugation. The bulk of acid hydrolase activity was found to be concentrated in a 20,000 g x 20 min granular fraction, whereas nuclear, debris, and supernatant fractions contained lesser concentrations of hydrolases. Acid hydrolase activity present in the granular fraction showed appropriate "latency" as judged by its dose-dependent release into the 20,000 g x 20 min supernatant after exposure to membrane-disruptive agents such as streptolysin S, filipin, and lysolecithin. Heparin proved to be necessary in the suspending medium so that reproducible homogenization and cell fractionation could be obtained. Even excessive contamination of lymphocyte suspensions with platelets did not appreciably alter the acid hydrolase activity of lymphocyte homogenates or the distribution of enzymes in subcellular fractions. Discontinuous density-gradient centrifugation of a 500 g x 10 min supernatant, containing both acid hydrolase-rich organelles and mitochondria, resulted in partial resolution of hydrolase-rich organelles from mitochondria. Fine structural studies of the intact lymphocytes showed the presence of acid phosphatase-positive, membrane-bounded organelles. Electron microscopy of the "large granule" (20,000 g x 20 min) fraction of such lymphocytes demonstrated 80–90% mitochondria, 5–10% platelets, and 5–10% membrane-bounded acid phosphatase-positive structures. The data indicate the presence in human peripheral blood lymphocytes of acid hydrolase-rich granules which possess many of the biochemical and structural characteristics of lysosomes in other tissues.     

Glycoside hydrolases of rumen bacteria and protozoa

Sixteen strains of rumen bacteria and 21 protozoal preparations were screened for glycoside hydrolase and phosphatase activity, using 22 nitrophenyl glycoside substrates. The range and level of bacterial enzyme activities were species dependent, although, the glycosidases associated with plant cell wall breakdown were most active in the cellulolytic and hemicellulolytic species. Alkaline phosphatase occurred widely in the organisms examined, but was most active in the twoBacteroides ruminicola strains.A wide range of enzyme activities was also detected in the holotrich and Entodiniomorphid ciliates isolated from the rumen or cultured in vitro. The glycosidases involved in cellulose and hemicellulose breakdown were detected in all of the protozoa examined, and, with the exception ofEntodinium spp., were most active in the Entodiniomorphid protozoa; -l-arabinofuranosidase, an essential hemicellulolytic glycoside hydrolase, was particularly active in this latter group of ciliates.     

Intracellular distribution of molecular forms of acetylcholinesterase in rat brain and changes after diisopropylfluorophosphate treatment

The subcellular distribution of acetylcholinesterase activities was studied in the striatum and cerebellum of rat brain. The highest percentage of the enzyme activity was found in the crude synaptosomal (P₂) fraction, with striatum much higher than cerebellum. On sucrose density gradient centrifugation analyses all the particulate fractions (P₁, P₂, and P₃) showed a major peak of the 10 S form of acetylcholinesterase activity with very little activity of the 4 S form of the enzyme. The 10 S/4 S ratio was much higher in striatum than in cerebellum. In the soluble fraction (100,000g supernatant) the 10 S form was less than the 4 S form in the adult rat brain, but this was reversed in the 6-day-old rat brain. After diisopropylfluorophosphate administration the recovery of acetylcholinesterase molecular forms in various subcellular fractions differed at different recovery periods. These results indicate that the distribution of molecular forms of acetylcholinesterase in rat brain differs in various subcellular fractions, and also the pattern of distribution differs in different regions of the brain as well as in adult and developing brains.     

γ-Glutamyltranspeptidase in echinoderm eggs and larvae: fertilization-dependent and developmentally-induced changes in specific activity

• 1.1. γ-Glutamyltranspeptidase is present in echinoderm eggs and larvae: in homogenates the level of activity is comparable to that of rat cerebral cortex.
• 2.2. In eggs of Lytechinus pictus, fertilization induces an early rapid and sustained (5 min–6 hr) 37% increase in the activity of γ-glutamyltranspeptidase in homogenate fractions.
• 3.3. Relative to these homogenate levels, the specific activity of γ-glutamyltranspeptidase are ≈60% lower in 40,000 g supernatant fractions and 2.7-fold higher in 40,000 g particulate fractions in both unfertilized and 15 min post-fertilized Lytechinus pictus eggs.
• 4.4. The subcellular distribution of γ-glutamyltranspeptidase is the same in both unfertilized and 15-min post-fertilized Lytechinus pictus eggs: 78% in 40,000 g particulate fractions, 22% in 40,000 g soluble fractions.
• 5.5. In both unfertilized and 15 min post-fertilized eggs of Lytechinus pictus the enzyme responds to heat (50 vs 37°C) by activation in a similar manner: 1.72- and 1.68-fold homogenates; 2.6- and 3.0-fold in supernatants; 1.97- and 1.90-fold in particulate fractions.
• 6.6. In homogenates of Pisaster ochraceous larvae, γ-glutamyltranspeptidase activity increases steadily during the course of larval development: relative to the low activity at day 5, activities exhibit an increase of 1.2-, 2.0-, 3.1- and 5.4-fold at days 10, 16, 22 and 28, respectively.

     

Subcellular and regional localization ofγ-glutamyl transpeptidase in sheep brain

Studies of the subcellular distribution of-glutamyl transpeptidase from sheep brain by discontinuous sucrose density gradient centrifugation showed that 40% of the transpeptidase activity associated with the mitochondrial-synaptosomal fraction was localized with the synaptosomal-enriched fraction. The microsomal fraction was found to have the highest specific activity when-glutamylp-nitroanalide was used as substrate. This activity, however, represented only 5% of the total-glutamyl transpeptidase activity. Approximately 90% of the total enzyme activity was apparently associated with the fraction containing cell debris and membrane fragments.The 160,000g supernatant fluid (soluble supernatant fraction) represented the least total activity, with only 1.2% recovery; however, this fraction contained two apparent forms of the enzyme. One form had a highK _mand the other a lowK _m for the substrate,-glutamylp-nitroanilide.It was observed that the enzyme-glutamyl transpeptidase was not evenly distributed in all areas of brain when the homogenate was used as the enzyme source. The brain region with the highest enzyme activity was the thalamus, which was able to form 1.10 molp-nitroanaline/min/g wet brain tissue. The cortex was found to have the lowest activity. The 40,000g supernatant fluid from each region, however, exhibited only slight distribution differences.     

The localisation of succinic dehydrogenase in the muscles of Nereis virens and Homarus gammarus

Summary The localisation of succinic dehydrogenase and cytochrome oxidase in body muscles of Nereis virens and in tail muscles of Homarus gammarus was studied. Pig heart muscle was used for some comparisons.Electron microscopic studies on tissue sections generally showed well developed and independent mitochondria in Homarus gammarus. A lower degree of independence characterised the less developed mitochondria of Nereis virens.Sections were stained with nitro-BT. Light microscopic studies showed a distinct and selective staining of the mitochondria in sections of Homarus gammarus. In addition to the few mitochondria of Nereis virens strings within the cytoplasm became distinctly blue. Electron microscopic studies on Nereis virens showed a higher electron density along the membranes of the vesicular sarcotubular system in incubated than in non-incubated sections.The fractions obtained on centrifugation of the homogenised tissues were used for combined enzyme studies and electron microscopic investigation. Similarly prepared fractions from the two invertebrates showed a similar electron microscopic appearance. The supernatants obtained by centrifugation at 12,000 g for 10 minutes contained vesicles different from the majority of those in the mitochondrial fractions. These supernatants had rather considerable activities of succinate-cytochrome c reductase and of cytochrome c oxidase. The activity of succinate-cytochrome c reductase was most pronounced in the supernatants of Nereis virens and much greater than the cytochrome c oxidase activity in these fractions. The ratio between succinate-cytochrome c reductase activity and cytochrome c oxidase activity in the supernatants of Nereis virens was about three times that in the corresponding fractions of Homarus gammarus.Manometric studies were performed to get the effect of added succinate on the O₂ uptake of the supernatants obtained by centrifugation at 12,000 g for 10 minutes. A distinctly larger increase in oxygen consumption characterised the supernatants of Nereis virens.The results presented indicate the occurrence of an extra-mitochondrial succinic dehydrogenase in Nereis virens. This conclusion was related to the occurrence of alternative oxidative systems in the muscles of this invertebrate.The literature dealing with an extra-mitochondrial localisation of succinic dehydrogenase is briefly reviewed as well as the electron microscopic studies concerning transformations between the membrane structures of cells.     

Subcellular localization of H+-ATPase from pumpkin hypocotyls (Cucurbita maxima L.) by membrane fractionation

A new method of preparing sealed vesicles from membrane fractions of pumpkin hypocotyls in ethanolamine-containing buffers was used to investigate the subcellular localization of H⁺-ATPase measured as nigericin-stimulated ATPase. In a fluorescence-quench assay, the H⁺ pump was directly demonstrated. The H⁺ pump was substrate-specific for Mg·ATP and 0.1 mM diethylstilbestrol completely prevented the development of a pH. The presence of unsupecific phosphatase hampered the detection of nigericin-stimulated ATPase. Unspecific phosphatases could be demonstrated by comparing the broad substrate specificity of the hydrolytic activities of the fractions with the clear preference for Mg·ATP as the substrate for the proton pump. Inhibitor studies showed that neither orthovanadate nor molybdate are absolutely specific for ATPase or acid phosphatase, respectively. Diethylstilbestrol seemed to be a specific inhibitor of ATPase activity in fractions containing nigericin-stimulated ATPase, but it stimulated acid phosphatase which tended to obscure its effect on ATPase activity. Nigericin-stimulated ATPase had its optimum at pH 6.0 and the nigericin effect was K⁺-dependent. The combination of valinomycin and carbonylcyanide m-chlorophenylhydrazone had a similar effect to nigericin, but singly these ionophores were much less stimulatory. After prolonged centrifugation on linear sucrose gradients, nigericin-stimulated ATPase correlated in dense fractions with plasma membrane markers but a part of it remained at the interphase. This lessdense part of the nigericin-stimulated ATPase could be derived from tonoplast vesicles because -mannosidase, an enzyme of the vacuolar sap, remained in the upper part of the gradient. Nigericinstimulated ATPase did not correlate with the mitochondrial marker, cytochrome c oxidase, whereas azide inhibition of ATPase activity did.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DES dethyltilbestrol     

Intracellular localization of N-formyl chemotactic receptor and Mg dependent ATPase in human granulocytes

Human granulocytes were disrupted by nitrogen cavitation and the lysates fractionated by sucrose density gradient centrifugation at 83 000 × g for 20 min (rate zonal) or 3.5 h (isopycnic). The distribution of marker enzymes allowed the identification of the following subcellular components: plasma membrane, Golgi, endoplasmic reticulum, azurophil granules, specific granules, mitochondria and cytosol. Examination of the gradient fractions by electron microscopy confirmed the biochemical marker analysis. The protocol permitted isolation of vesicles highly enriched in either plasma membrane or Golgi (galactosyl transferase) activities. Absolute plasma membrane yields of 40–60% were achieved with a 20–70-fold increase in specific activity of surface marker over the cells. Plasma membrane sedimented to an average density of 1.14 g·cm⁻³. Galactosyl transferase activity was bimodal in distribution. The denser peak cosedimanted with specific granules (g9 = 1.19). The lighter peak sedimented to unique position at an average density of 1.11, was enriched 18-fold over the low speed supernatant, and contained structures resembling Golgi. N-Formyl-Met-Leu-Phe binding and Mg²⁺ -ATPase activities cosedimented with the plasma membrane as well as specific granule and/or high density galactosyl transferase fractions. These findings suggest that Mg²⁺ -ATPase and N-formyl chemotactic peptide receptor activities may be localized in an internal pool of membranes as well as in the plasma membrane and that Golgi may have been a contaminant of previous granulocyte plasma membrane or specific granule preparations.     

Hemicellulose-degrading enzymes in rumen ciliate protozoa

Hemicellulose-degrading enzymes were detected in cell-free extracts of protozoa representing ten genera of rumen entodiniomorphid and holotrich ciliates. The enzyme preparations released monosaccharides, disaccharides, and oligomers fromLolium perenne hemicellulose B and oat spelt xylan; the activity was present both in cells isolated directly from rumen contents and in those cultured in vitro. The specific activities were higher in the cellulolytic entodiniomorphid genera (Polyplastron, Diploplastron, Eremoplastron, Epidinium, Ophryoscolex, Eudiplodinium) than in the holotrich ciliates (Dasytrichia ruminantium, Isotricha intestinalis/I. prostoma) and the entodinia examined (Entodinium bursa, E. simplex, E. caudatum). The rate of hemicellulose-B degradation to alcohol-soluble products was approximately 5–10 times higher than the rate of reducing sugar accumulation; this indicates an initial depolymerization to intermediate oligosaccharide fragments. Examination of the hemicellulose degradation products by thin-layer and gas-liquid chromatography confirmed oligosaccharide formation, revealed markedly different rates of arabinose and xylose release, and indicated that the mode of polysaccharide degradation was similar in the protozoal preparations examined.     

Studies on the biosynthesis of tobacco mosaic virus VI. On the subcellular localization of double-stranded viral RNA

Summary Tobacco leaves were homogenized and separated by differential centrifugation into four subcellular fractions. Each fraction was characterized by the presence of certain marker substances and enzymes. It was found that nuclei and chloroplasts were highly enriched in the 2500 ×g sediment. Mitochondria were especially enriched in the 6500 × g g sediment, but were also found in the 20000 ×g sediment. The activity of a marker enzyme for cytomembranes (inosinediphosphatase) was high in the 6500 ×g and the 20000 ×g sedi-ments. In TMV infected material complementary viral R~qA (minus strands) was enriched in the 6500 ×g and the 20000 ×g sediments. Also, after labeling infected leaves with tritiated uridine, RNase resistant radioactivity was enriched in these two fractions. The results suggest that the TMV RNA replication complex is bound to cytoplasmic membranes.     

Gravitropic bending of cress roots without contact between amyloplasts and complexes of endoplasmic reticulum

The polar arrangement of cell organelles in Lepidium root statocytes is persistently converted to a physical stratification during lateral centrifugation (the centrifugal force acts perpendicular to the root long axis) or by apically directed centrifugation combined with cytochalasin-treatment. Lateral centrifugation (10 min, 60 min at 10\g or 50\g) causes displacement of amylplasts to the centrifugal anticlinal cell wall and shifting of the endoplasmic reticulum (ER) complex to the centripetal distal cell edge. After 60 min of lateral centrifugation at 10\g or 50\g all roots show a clear gravitropic curvature. The average angle of curvature is about 40° and corresponds to that of roots stimulated gravitropically in the horizontal position at 1\g in spite of the fact that the gravistimulus is 10-or 50-fold higher. Apically directed centrifugation combined with cytochalasin B (25 g\ml^-1) or cytochalasin D (2.5 g\ml^-1) incubation yields statocytes with the amyloplasts sedimented close to the centrifugal periclinal cell wall and ER cisternae accumulated at the proximal cell pole. Gravitropic stimulation for 30 min in the horizontal position at 1\g and additional 3 h rotation on a clinostat result in gravicurvature of cytochalasin B-treated centrifuged (1 h at 50\g) roots, but because of retarded root growth the angle of curvature is lower than in control roots. Cytochalasin D-treatment during centrifugation (20 min at 50\g) does not affect either root growth or gravicurvature during 3 h horizontal exposure to 1\g relative to untreated roots. As lateral centrifugation enables only short-term contact between the amyloplasts and the distal ER complex at the onset of centrifugation and apically directed centrifugation combined with cytochalasin-treatment even exclude any contact the integrity of the distal cell pole need not necessarily be a prerequisite for graviperception in Lepidium root statocytes.Abbreviations CB cytochalasin B - CD cytochalasin D - ER endoplasmic reticulum - g gravitational acceleration     

Insulin degradation V. Unmasking of glutathione-insulin transhydrogenase in rat liver microsomal membrane

Glutathione-insulin transhydrogenase (glutathione:protein disulfide oxidoreductase, EC 1.8.4.2) inactivates insulin by cleaving its disulfide bonds. The distribution of GSH-insulin transhydrogenase in subcellular fractions of rat liver homogenates has been studied. From the distribution of insulin-degrading activity and marker enzymes (glucose-6-phosphatase and succinate-INT reductase) (INT, 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride) after cell fractionation by differential centrifugation, the immunological analysis of the isolated subcellular fractions with antibody to purified rat liver GSH-insulin transhydrogenase, and chromatographic analysis (on a column of Sephadex G-75 in 50% acetic acid) of the products formed from ¹²⁵I-labelled insulin after incubation with the isolated subcellular fractions, it is concluded that GSH-insulin transhydrogenase is located primarily in the microsomal fraction of rat liver homogenate. An enzyme(s) that further degrades insulin by proteolysis is located mainly in the soluble fraction; a significant amount of the protease(s) activity is also present in the mitochondrial fraction. The possibility has been discussed that the protease(s) acts upon the intermediate product of insulin degradation, A and B chains of insulin, rather than upon the intact insulin molecule itself.The GSH-insulin transhydrogenase in intact microsomes occurs in a latent state; it is readily released from the microsomal membrane and its activity is greatly increased by treatments which affect the lipoprotein membrane structure of microsomal vesicles. There include homogenization with a Polytron homogenizer, sonication, freezing and thawing, alkaline pH, the nonionic detergent Triton X-100, and phospholipases A and C.     

Subcellular distribution of [H]-prostaglandin E2 binding sites in porcine gastric mucosa

The distribution of PGE₂ binding sites in four subcellular fractions (F1–F4) from porcine fundic mucosa obtained by gradient centrifugation was examined. Binding of HPGE₂ to fractions F2–F4 was specific, dissociable, saturable and pH dependent. A significant degree of specific binding was not evident in F1. The Scatchard analysis of binding to F2 and F3 revealed heterogenous populations of binding sites with similar dissociation constants but greater concentrations of binding sites than was evident in the initial 30,000 xg homogenate protein. A single class of low affinity binding sites was evident in F4. The ratio of total: nonspecific binding was approximately equal in F2 and F3. The ratio was considerably smaller in F4. The activity of 5' nucleotidase the marker enzyme for plasma membranes followed this ratio. There was no correlation between the binding ratio and marker enzyme activities for mitochondrial membranes and endoplasmic reticulum. These data suggest that high affinity PGE₂ binding sites occur predominantly on the plasma membrane from gastric mucosal tissue.     

Subcellular distribution of [3H]-prostaglandin E2 binding sites in porcine gastric mucosa

The distribution of PGE₂ binding sites in four subcellular fractions (F1–F4) from porcine fundic mucosa obtained by gradient centrifugation was examined. Binding of HPGE₂ to fractions F2–F4 was specific, dissociable, saturable and pH dependent. A significant degree of specific binding was not evident in F1. The Scatchard analysis of binding to F2 and F3 revealed heterogenous populations of binding sites with similar dissociation constants but greater concentrations of binding sites than was evident in the initial 30,000 xg homogenate protein. A single class of low affinity binding sites was evident in F4. The ratio of total: nonspecific binding was approximately equal in F2 and F3. The ratio was considerably smaller in F4. The activity of 5' nucleotidase the marker enzyme for plasma membranes followed this ratio. There was no correlation between the binding ratio and marker enzyme activities for mitochondrial membranes and endoplasmic reticulum. These data suggest that high affinity PGE₂ binding sites occur predominantly on the plasma membrane from gastric mucosal tissue.     

Estrogen synthetase in choriocarcinoma cell culture. stimulation by dibutyryl cyclic adenosine monophosphate and theophylline

The stimulation of estrogen biosynthesis by N⁶, O² -dibutyryl adenosine 3':5'-cyclic monophosphate and theophylline (dbT) in cultures of the JAr line of choriocarcinoma cells was investigated by measuring the specific activity and kinetic constants of estrogen synthetase (aromatase) in the various subcellular fractions after differential centrifugation of homogenized cells in isotonic sucrose. The low speed (900x$\text{g}$) pellet,from cells grown with or without dbT and homogenized in isotonic sucrose,contains the majority of the aromatase activity and the highest aromatase specific activity. The aromatase specific activity in the homogenate of cells grown with dbT and in the various subcellular fractions is 4- to 10-fold higher than in cells grown without dbT. The Vmax of androstenedione (4-androstene-3,17-dione) aromatization in homogenates from dbT-stimulated cells (6.9 pmol estrogen/min per mg protein) is significantly increased over that measured in the absence of dbT (1.5 pmol estrogen/min per mg protein); the Km values, however, are not significantly different (average of 43.8nM in dbT-stimulated fractions; 53.2nM in control fractions). These results suggest that the increased aromatase specific activity in dbT-stimulated cells results from an increase in amount of active enzyme, rather than from an increase in affinity of the enzyme for its substrate.     

Isolation of Relaxing Particles from Rat Skeletal Muscles in Zonal Centrifuges

Supernatants of rat skeletal muscle homogenates were fractionated by differential centrifugation and by zonal centrifugation in sucrose density gradients. Cytochrome oxidase was employed as an enzymatic marker for locating mitochondria. The subcellular fractions were also assayed for their ability to prevent the ATP-induced contraction of myofibrils. Both the mitochondrial and microsomal fractions obtained by differential fractionation were found to be rich in such relaxing activity, and the microsomal fraction was appreciably contaminated by mitochondria. In contrast to this, when fractionation was carried out by means of zonal centrifugation (4200 RPM x 205 min. to 40,000 RPM x 60 min.), relaxing activity was found to be associated only with particles having the sedimentation characteristics of microsomes (s _20,w estimated to be between 370 and 1880S). Relaxing activity was not detected in the regions of the gradient containing either the starting sample zone (soluble phase) or the mitochondrial peak. The microsomal relaxing particles showed negligible cytochrome oxidase activity.     

Tyrosine aminotransferase activity of frog (Rana esculenta) liver—III. A circannual study

• 1.1. A circannual study of tyrosine aminotransferase and other metabolic enzymes in frog liver is reported. The subcellular distribution of all enzymatic activities under investigation was also studied.
• 2.2. Results show significant oscillations of all enzymatic activities throughout the year; in particular tyrosine aminotransferase has a marked summer maximum.
• 3.3. The subcellular distribution of tyrosine aminotransferase shows significant variations: the soluble activity of the enzyme presents a bimodal circannual distribution, which has its counterpart in an increased activity of heavier fractions.

     

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.