Degradation of lindane by cell-free preparations of Clostridium sphenoides.

Cell-free preparation of Clostridium sphenoides degraded the insecticide lindane, the gamma-isomer of 1,2,3,4,5,6-hexachlorocyclohexane, to the gamma-isomer of 3,4,5,6-tetrachloro-1-cyclohexene. The activity appeared to be associated with the membrane fraction and required reduced glutathione. The tetrachlorocy-clohexene intermediate was further metabolized by the membrane fraction to unknown substances.     

Identification of intermediates formed during the degradation of hexachlorocyclohexanes by Clostridium sphenoides.

Washed cell suspensions of Clostridium sphenoides degraded the alpha-isomer of 1,2,3,4,5,6-hexachlorocyclohexane via delta-3,4,5,6-tetrachloro-1-cyclohexene and the gamma-isomer via gamma-3,4,5,6-tetrachloro-1-cyclohexene. Both intermediates were further metabolized to unknown substances. The tetrachlorocyclohexene intermediates were identified by gas chromatography and mass spectrometry.     

Identification of intermediates formed during the degradation of hexachlorocyclohexanes by Clostridium sphenoides.

Washed cell suspensions of Clostridium sphenoides degraded the alpha-isomer of 1,2,3,4,5,6-hexachlorocyclohexane via delta-3,4,5,6-tetrachloro-1-cyclohexene and the gamma-isomer via gamma-3,4,5,6-tetrachloro-1-cyclohexene. Both intermediates were further metabolized to unknown substances. The tetrachlorocyclohexene intermediates were identified by gas chromatography and mass spectrometry.     

Lindane effect upon the vasoactive intestinal peptide receptor/effector system in rat enterocytes

Isolated rat enterocytes exposed to the insecticide lindane (the gamma-isomer of hexachlorocyclohexane, HCCH) showed an important decrease in the efficiency of the neuropeptide vasoactive intestinal peptide (VIP) upon the stimulation of cyclic AMP accumulation. The effect of lindane was time- and dose-dependent, optimal conditions being reached after 5 min incubation of cells at 25 degrees C with 0.5 mM of this organochlorine compound. Lindane action exhibited an important degree of specificity since the isomer alpha-HCCH and endrin reproduced the same inhibitory pattern but beta-HCCH and dieldrin were inactive. The inhibition of VIP-induced cyclic AMP accumulation could not be explained by a lindane-dependent reduction in the binding of VIP to its specific receptors. Among various possibilities, the results suggest the modification of membrane fluidity by lindane and/or the activation of Ca2+-dependent protein kinase C by this compound leading to phosphorylation of Gs/adenylate cyclase.     

Affinity labeling of folate transport proteins with the N-hydroxysuccinimide ester of the gamma-isomer of fluorescein-methotrexate

Fluorescein-methotrexate, a derivative in which the fluorophore is linked via a diaminopentane spacer to either the alpha- or gamma-carboxyl group of the glutamate moiety in the drug [Gapski et al. (1975) J. Med. Chem. 18, 526-528], has been synthesized by an improved procedure and separated by DEAE-Trisacryl chromatography into the alpha- and gamma-isomers (alpha-F-MTX and gamma-F-MTX). Each isomer was characterized by mass spectrometry, elemental analysis, absorbance spectrum, TLC, and reversed-phase HPLC. Identity of the isomers was established by the following enzymatic criteria: (a) gamma-F-MTX (but not the alpha-isomer) was hydrolyzed at the pteroate-glutamate bond by carboxypeptidase G2 to yield 4-amino-4-deoxy-10-methylpteroate and gamma-glutamyldiaminopentane-fluorescein; and (b) gamma-F-MTX was a much better inhibitor of human dihydrofolate reductase than the alpha-isomer (Ki values of 0.079 and 4.6 nM). alpha- and gamma-F-MTX were comparable as inhibitors (Ki values of 1.6 and 0.6 microM) of the transport system for reduced folates and MTX in L1210 cells, but the transporter in Lactobacillus casei was inhibited only by the gamma-isomer (Ki = 4.3 microM). The gamma-isomer, therefore, was selected for covalent labeling of proteins. When L. casei folate transport protein (18 kDa) was treated with gamma-F-MTX that had been activated with N-hydroxysuccinimide (NHS), the protein was readily visualized as a fluorescent band on SDS-PAGE electrophoretograms. The probe was also able to detect the transporter in membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of actin polymerization by membrane fraction of platelets

We studied the interaction between the purified membrane fraction of human platelets and the polymerization of skeletal actin. The viscosity of actin was measured by the falling ball method. The fraction suppressed the polymerization of actin in the presence of 20 mM KCl and 0.4 mM EGTA. The addition of calcium ion or thrombin to the fraction did not cause suppression. A DNase I affinity column bound the membrane fraction in the presence of calcium ion. The frozen membrane fraction and the vesicles reconstituted with lipids from the platelet membrane enhanced the polymerization of actin. Trypsinized membrane fraction and the membrane fraction treated with phospolipase A2 enhanced the polymerization of actin, but membrane fraction treated with phospholipase C had no effect. The reconstituted membrane vesicles mentioned above lowered the critical concentration for actin polymerization. These findings suggested that the polymerization of intracellular actin is enhanced not only by the mobilization of calcium ion, but also by biochemical changes in the membrane lipids.     

Separation of inner and outer membranes of Rhodopseudomonas spheroides.

The separation of inner and outer membrane of Rhodopseudomonas spheroides has been achieved by means of sucrose density gradient (20%, 40%, 60%, w/w) centrifugation. The upper fraction of the gradient, with a specific density 1.181 (g/cm3), is high in cytochrome and succinate dehydrogenase activities, low in lipopolysaccharides and it is designated the inner membrane fraction. The bottom fraction of the gradient, with a specific density 1.240, is high in lipopolysaccharide and contains neither cytochrome nor succinate dehydrogenase activities. This fraction is the cell wall or outer membrane fraction. The intermediate band on the gradient is an unseparated fraction of inner and outer membrane fragments. This fraction has a specific denisty of 1.211 and represents less than 3% of total crude envelope. Thin sections of the vesicles of the inner membrane fraction and those of outer membrane provide morphological evidence for the identity of the individual membrane fractions. At least 22 protein bands are resolved by employing sodium dodecyl sulfate slab gel electrophoresis. Six bands are present only in the inner membrane and two bands are found exclusively in the outer membrane. Most of the remaining polypeptides are present in greater amounts in the inner membrane relative to the outer membrane fractions.     

Subcellular fractionation of porcine neutrophils by nitrogen cavitation and sucrose-density-gradient centrifugation

Neutrophils, isolated in large quantities from porcine blood were disrupted by nitrogen cavitation and separated by differential centrifugation into a nuclear fraction and a post-nuclear supernatant. The latter was subfractionated by sucrose density gradient centrifugation into cytosol, a fraction consisting of membrane vesicles and two granule-rich fractions. The membrane fraction accounted for 1.9% of the protein in the post-nuclear supernatant, the light granule fraction for 2.2% and the dense granule fraction for 4.2%. Catalase, lactate dehydrogenase and malate dehydrogenase were largely confined to the cytosol. The dense granule fraction contained the highest quantities of the hydrolytic enzymes, although the membrane fraction was also rich in alkaline and acid phosphatase and gamma-glutamyl transpeptidase activities. Electron microscopy of the membrane fraction showed intact membrane vesicles, whereas the granular fractions consisted of electron-dense, membrane-bound granules. Two granular fractions were isolated which contained granules of differing size and density. 3H-labeled wheat germ agglutinin bound to the surface of intact neutrophils and when these were disrupted and fractionated the membrane fraction showed a specific binding activity 16-times greater than that of the cavitated sample. The membrane fraction interacted with the detergent digitonin and as a result underwent density perturbation increasing from 1.13 g X cm-3 to 1.18 g X cm-3. Dodecylsulphate-polyacrylamide gel electrophoresis showed the membrane fraction to consist of at least 40 protein bands, with relative molecular masses ranging from 200 000-16 000. The granule fractions contained less protein bands, with a protein composition quite distinct from that of the membrane fraction.     

Cytoplasmic membrane vesicles of Escherichia coli. A simple method for preparing the cytoplasmic and outer membranes.

A simple preparative method is described for isolation of the cytoplasmic and outer membranes from E. coli. The characteristics of both membrane fractions were studied chemically, biologically, and morphologically. Spheroplasts of E. coli K-12 strain W3092, prepared by treating cells with EDTA-lysozyme [EC 3.2.1.17], were disrupted in a French press. The crude membrane fraction was washed with 3 mM EDTA-10% (w/v) sucrose, pH 7.2, and the cytoplasmic membranes and outer membranes were separated by sucrose isopycnic density gradient centrifugation. The crude membrane fraction contained approximately 10% of the protein of the whole cells, 0.3% of the DNA, 0.7% of the RNA, 0.3% of the peptidoglycan, and about 30% of the lipopolysaccharide. The cytoplasmic membrane fraction was rich in phospholipid, while the outer membrane fraction contained much lipopolysaccharide and carbohydrate; the relative contents of lipopolysaccharide and carbohydrate per mg protein in the cytoplasmic membrane fraction were 12 and 40%, respectively, of the contents in the outer membrane fraction. Cytochrome b1, NADH oxidase, D-lactate dehydrogenase [EC 1.1.1.28], succinate dehydrogenase [EC 1.3.99.1], ATPase [EC 3.5.1.3], and activity for concentrative uptake of proline were found to be localized mainly in the cytoplasmic membranes; their specific activities in the outer membrane fraction were 1.5 to 3% of those in the cytoplasmic membrane fraction. In contrast, a phospholipase A appeared to be localized mainly in the outer membranes and its specific activity in the cytoplasmic membrane fraction was only 5% of that in the outer membrane fraction. The cytoplasmic and outer membrane fractions both appeared homogeneous in size and shape and show vesicular structures by electron microscopy. The advantages of this method for large scale preparation of the cytoplasmic and outer membrane fractions are discussed.     

Characterization of membrane-bound spermidine dehydrogenase of Citrobacter freundii.

Spermidine dehydrogenase found in the membrane fraction of Citrobacter freundii IFO 12681 was solubilized with Triton X-100 and further purified to homogeneity. The properties of the membrane enzyme were almost identical to those obtained from the soluble fraction of the organism with respect to molecular and catalytic properties. Thus, binding properties of the enzyme to the bacterial membrane were checked. The ratio of enzyme activity found in the soluble fraction to the membrane fraction was dependent on salt concentration during cell disruption. A hydrophobic interaction was largely involved in anchoring the enzyme to the membrane fraction. Purified spermidine dehydrogenase from the soluble fraction was readily adsorbed into the membrane fraction in the presence of salt. Spermidine dehydrogenase appeared to be a membrane-bound enzyme localized in the cytoplasmic membranes in a manner that makes a partial release of the enzyme possible during mechanical cell disruption. When spermidine oxidation was done with the resting cells of C. freundii, a stoichiometric formation of two reaction products, 1,3-diaminopropane and gamma-aminobutyraldeyde, was observed without any lag time. These facts indicate that the enzyme is localized on the outer surface of the cytoplasmic membranes or in the periplasmic space of the organism.     

The isolation and characterisation of the plasma membrane fromHalobacterium salinarium

The plasma membrane ofHalobacterium salinarium, strain 1, has been isolated and characterised. A fraction containing cell envelope vesicles was isolated from a cell homogenate by centrifuging. A crude membrane fraction was obtained from the envelope fraction by dialysing it against distilled water, incubating with nucleases and centrifuging. A nucleotide-free purified membrane fraction, identified with the plasma membrane, was obtained by gel-filtration chromatography of the crude membrane fraction on Agarose. The nucleotide-free membrane-rich fraction contained all the cell lipid, including menaquinone and carotenoid, and cytochrome. No amino sugars could be detected. The action of the detergent, sodium dodecyl sulphate, on the nucleotide-free membrane-rich fraction broke up the membrane into smaller particles. The disaggregation occurred in at least two distinct steps. The disaggregated particles could be reaggregated to a fraction which resembled the original membrane by removing the detergent by dialysis or gel-filtration. A fraction which may be analogous to mitochondrial structural protein was isolated by ammonium sulphate fractionation of a preparation of the nucleotide-free membrane-rich fraction dissolved in a mixture of sodium deoxycholate, sodium cholate and sodium dodecyl sulphate. Protein fractions were separated from the nucleotide-free membrane-rich fraction during gel-filtration chromatography on Agarose in the presence of 6m urea. The authors would like to acknowledge the technical assistance of Miss C. Goode and Mrs. J. Wicks. We are indebted to Mrs. A. Flo of the Department of Biochemistry, The Technical University of Norway, Trondheim, for technical assistance in the preparation of samples for electron microscopy.     

Intracellular localization of photosynthetic membrane growth initiation sites in Rhodopseudomonas sphaeroides

Putative membrane invagination sites at which intracytoplasmic photosynthetic membrane growth is initiated in Rhodopseudomonas sphaeroides can be isolated in an upper pigmented fraction by rate-zone sedimentation. The intracellular localization of membranes present in the isolated fraction was investigated with the impermeant surface-labeling reagent pyridoxal 5'-phosphate, which has been shown to diffuse into the periplasmic space and to label proteins of both the peripheral cytoplasmic membrane and the mature intracytoplasmic membrane. A comparison of the extent of labeling at 25 and 0 degrees C was consistent with the possibility that membranes present in the upper pigmented fraction arise from sites near the cell periphery. Pronase digestion of the surface-labeled membranes suggested further that the purified upper fraction consisted largely of open membrane fragments and that the majority of the intracytoplasmic membrane is labeled by this procedure. The pigmented membrane growth initiation sites were separated partially from undifferentiated respiratory cytoplasmic membrane also present in the upper fraction.     

A partial resolution and reconstitution of the ammonia-oxidizing system of Nitrosomonas europaea: role of cytochrome c554

The cell-free ammonia-oxidizing system of Nitrosomonas europaea was resolved into three major fractions: a membrane fraction containing cytochrome a1 and c-type cytochromes, a fraction with hydroxylamine-cytochrome c reductase and a cytochrome c fraction. The ammonia-oxidizing activity was reconstituted by the combination of these three fractions. The activity was more consistently reconstituted by adding Nitrosomonas cytochrome c554 to the membrane fraction. The hydroxylamine-cytochrome c reductase activity of the membrane fraction increased with the addition of cytochrome c554, but the oxidation of hydroxylamine to nitrite required a further addition of cytochrome c552. The ammonia oxidation by the membrane plus cytochrome c554 was affected by the concentration of phosphate and the addition of bovine serum albumin, spermine, or MgCl2.     

Comparison of Proteins Involved with Cyclic AMP Metabolism Between Synaptic Membrane and Postsynaptic Density Preparations Isolated from Canine Cerebral Cortex and Cerebellum

Synaptic membrane and postsynaptic density (PSD) fractions isolated from canine cerebral cortex and cerebellum were assayed for the following proteins: adenylate cyclase and phosphodiesterase (PDE) activities against cyclic AMP and cyclic GMP, the regulatory subunit of the cyclic AMP-dependent protein kinase, and the substrate proteins for this kinase. The results were expressed on the basis of both the protein content of the fractions and the number of synapses in the synaptic membrane fractions. The number of synapses on a constant protein content basis was about three times higher in the cerebral cortex synaptic membrane fraction than in the comparable cerebellar fraction. Adenylate cyclase activity was from 3.4 to 5.6 times higher in the cerebral cortex membrane fraction than in the cerebellar membrane fraction based on protein content but only slightly higher based on synapse counts. PSD fractions had no adenylate cyclase activity. The cyclic AMP-PDE activity was from 17 to 27 times higher in the cerebral cortex membrane fraction than in the cerebellar membrane fraction based on protein content, and about five times higher based on synapse counts. By doing PDE histochemistry at the electron microscopy level it was found that all the cerebral cortex PSDs in the isolated fraction contained PDE activity, none being found associated with the broken-up material in the fraction. The amount of the regulatory subunit of the cyclic AMP-dependent protein kinase was about equal in the two fractions based on protein, but about one-third lower in cerebral cortex fraction than in cerebellar fractions. In the cerebral cortex membrane fraction the primary substrate for the cyclic AMP-dependent protein kinase is synapsin I, with much lower amounts in the cerebellar membrane fraction. The PSD fraction from the two sources also showed these differences in synapsin I content. In the cerebellar membrane fraction, the primary substrate for the enzyme is a approximately 245,000 Mr protein not found in the cerebral cortex membrane fraction. The findings that the turnover of cyclic AMP is much higher in cerebral cortex synapses than in cerebellar synapses, and that differences are found between the cerebral cortex and cerebellum with regard to the substrate proteins for the cyclic AMP-dependent protein kinase indicate a divergence in the effect of cyclic AMP between cerebral cortex and cerebellar synapses.     

Isolation of the plasma membrane of a trypanosomatid flagellate: General characterization and lipid composition

A technique is described for the isolation of a fraction that contains the plasma membrane of the trypanosomatid flagellate Leptomonas collosoma. This fraction has been investigated by electron microscopy and has been shown to be mostly membranes associated with microtubules, a known plasma membrane marker in this organism. The fraction is enriched in Mg²⁺-dependent ATPase but has a decreased specific activity of succinate dehydrogenase. Lipid has been extracted from whole cells and the isolated plasma membrane fraction. A fraction of the total lipid that is eluted from a silicic acid column by acetone is found to be concentrated in the plasma membrane. Also enriched in the plasma membrane fraction is a 5,7-diene sterol identified as ergosterol. The major phospholipids of the whole cell and the plasma membrane are phosphatidylethanolamine and phosphatidylcholine. Approximately 60% of the fatty acids of the cell and plasma membrane have a carbon chain length of eighteen, and half of this is in the form of the mono-unsaturated fatty acid.     

Isolation of plasma membrane and tonoplast fractions from spinach leaves by preparative free-flow electrophoresis and effect of photoinduction

A membrane fraction enriched in plasma membrane and tonoplast vesicles was isolated from green leaves of Spinacia oleracea L. and subjected to subfractionation by free-flow electrophoresis. The most electronegative membrane vesicle fraction collected after the free-flow electrophoretic separation was identified as derived from tonoplast, while the least electronegative fraction was identified as derived from plasma membrane. The identification of the fractions was based on membrane morphology, and on the presence or absence of biochemical markers. The plasma membrane fraction was enriched in thick (9–11 nm) membranes which bound N-1-naphthylphthalamic acid (NPA), and reacted with phosphotungstic acid at low pH on thin sections for electron microscopy. The tonoplast fraction was enriched in vesicles with 7–9 nm thick membranes that neither bound NPA nor reacted with phosphotungstic acid at low pH. Both the plasma membrane and the tonoplast fraction were about 90% pure, with a cross-contamination of not more than 2%. Membrane vesicles originating from dictyosomes, endoplasmic reticulum, mitochondria, plastids, or peroxisomes contaminated the plasma membrane and the tonoplast fractions by a few % only. In leaves of photoinduced plants (24 h light period), the plasma membranes were thicker than in control leaves (8 h light, 16 h dark). The plasma membrane fraction obtained from photo-induced leaves by free-flow electrophoresis retained this increase in thickness, showing not only that photoinduction alters plasma membrane structure, but also that this change is stable to isolation.     

Preparation and characterization of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts. I. Electrophoretic and immunochemical analyses

We have developed a fast and reliable method for the separation of two membrane fractions respectively enriched in outer and inner envelope membranes from isolated, intact, purified spinach chloroplasts kept in a hypertonic medium (0.6 M mannitol). This separation was achieved by osmotically shrinking the inner envelope membrane, thus widening the intermembrane space, and then subsequently removing the "loosened" outer envelope membrane by applying low pressure to the shrunken chloroplasts and slowly extruding them through the small aperture of a Yeda press under controlled conditions. By centrifugation of the mixture obtained through a discontinuous sucrose gradient, we were able to separate two membrane fractions having different densities (fraction 2 or light fraction, d = 1.08 g/cm3, and fraction 3 or heavy fraction, d = 1.13 g/cm3). The recent characterization of polypeptides localized on the outer envelope membrane from spinach chloroplasts, E10 and E24 (Joyard, J., Billecocq, A., Bartlett, S. G., Block, M. A., Chua, N.-H., and Douce, R. J. Biol. Chem., 258, 10000-10006) enabled us to characterize our two membrane fractions. Analyses of the polypeptides by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis and immunoblotting have shown that fraction 2 (light fraction) was completely devoid of polypeptide E30, which is involved in the transport of phosphate across the inner envelope membrane, but was enriched in polypeptides E10 and E24. The reverse was true for fraction 3 (heavy fraction). Under these conditions, it is clear that fraction 2 is strongly enriched in outer envelope membrane whereas fraction 3 consisted mostly of inner envelope membrane. Indeed, by immunoelectrophoresis, we were able to demonstrate that, on a protein basis, fraction 2 contained about 90% of outer membrane, whereas fraction 3 contained about 80% of inner membrane. Further characterization of the outer envelope membrane was achieved by using thermolysin, a nonpenetrant protease.     

Causes of nondiffusing lipid in the plasma membrane of mammalian spermatozoa

In the plasma membranes of most mammalian somatic cells, lipid is nearly completely free to diffuse laterally in the plane of the membrane. In mammalian spermatozoa and certain other highly polarized mammalian cells, a significant fraction of the plasma membrane lipid is not free to diffuse laterally. Using the technique of fluorescence recovery after photobleaching, we have demonstrated that a variety of fluorescent lipid analogues exhibit a nondiffusing fraction in the plasma membrane of the anterior region of the ram sperm head. The possible causes of this nondiffusing fraction were investigated. The nondiffusing lipid fraction is not the result of lipid oxidation during handling, and it is not released by extensive enzymatic digestion of the membrane surface proteins or the "bleeding" of the membrane by hypoosmotic shock. When lipid bilayers were prepared from protein-free lipid extracts of the plasma membranes of spermatozoa, most of the nondiffusing fraction was retained. These results suggest that the nondiffusing lipid fraction results from lipid factors such as lateral phase separations, which can cause such a nondiffusing fraction in model systems.     

Characterization of Membrane-bound Spermidine Dehydrogenase of Citrobacter freundii

Spermidine dehydrogenase found in the membrane fraction of Citrohacter freundii IFO 12681 was solubilized with Triton X-100 and further purified to homogeneity. The properties of the membrane enzyme were almost identical to those obtained from the soluble fraction of the organism with respect to molecular and catalytic properties. Thus, binding properties of the enzyme to the bacterial membrane were checked. The ratio of enzyme activity found in the soluble fraction to the membrane fraction was dependent on salt concentration during cell disruption. A hydrophobic interaction was largely involved in anchoring the enzyme to the membrane fraction. Purified spermidine dehydrogenase from the soluble fraction was readily adsorbed into the membrane fraction in the presence of salt. Spermidine dehydrogenase appeared to be a membrane-bound enzyme localized in the cytoplasmic membranes in a manner that makes a partial release of the enzyme possible during mechanical cell disruption. When spermidine oxidation was done with the resting cells of C. freundii, a stoichiometric formation of two reaction products, 1,3-diaminopropane and γ-aminobutyraldeyde, was observed without any lag time. These facts indicate that the enzyme is localized on the outer surface of the cytoplasmic membranes or in the periplasmic space of the organism.     

Lipid Characterization of an Enriched Plasma Membrane Fraction of Dunaliella salina Grown in Media of Varying Salinity

We have developed a rapid procedure for isolating a fraction enriched in plasma membrane from Dunaliella salina using an aqueous two-phase system (dextran/polyethylene glycol, 6.7%/6.7%). An enriched plasma membrane fraction, free of chloroplast and mitochondrial contamination, could be obtained in 2.5 hours. Plasma membrane proteins, which accounted for approximately 1% of the total membrane protein, contained a number of unique proteins compared with the other cell fractions, as shown by gel electrophoresis. The lipids of the plasma membrane fraction from 1.7 molar NaCl-grown cells were extracted and characterized. Phosphatidylethanolamine and phosphatidylcholine were the two most prevalent phospholipids, at 20.6% and 6.0% of the total lipid, respectively. In addition, inositol phospholipids were a significant component of the D. salina plasma membrane fraction. Phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate accounted for 5.2% and 1.5% of the plasma membrane phospholipid, respectively. Diacylglyceryltrimethylhomoserine accounted for 7.9% of the plasma membrane total lipid. Free sterols were the major component of the plasma membrane fraction, at 55% of the total lipid, and consisted of ergosterol and 7-dehydroporiferasterol. Sterol peroxides were not present in the plasma membrane fraction. The lipid composition of enriched plasma membrane fractions from cells grown at 0.85 molar NaCl and 3.4 molar NaCl were compared with those grown at 1.7 molar NaCl. The concentration of diacylglyceryltrimethylhomoserine and the degree of plasma membrane fatty acid saturation increased in 3.4 molar plasma membranes. The relative concentration of sterols in the plasma membrane fraction was similar in all three NaCl concentrations tested.