Intracellular distribution of lipophilic fluorescent probes in mammalian cells

The intracellular distribution of several hydrophobic fluorescent probes (1,6-diphenyl-1,3,5-hexatriene (DPH), perylene, and $\text{2-p-}\text{toluidinyl-6-naphthalene}$ sulfonate (TNS)) in mouse lymphocytes and a fibroblast cell line was examined using radiolabeled fluorescent probes and the technique of high resolution EM autoradiography. Following a short term incubation, DPH and perylene were found largely internalized in cells, while TNS was localized predominantly at the cell surface. These findings suggest that fluorescence polarization studies using such probes with intact cells do not necessarily monitor only the cell surface membrane and must be interpreted with caution.     

The effects of cell proliferation on the lipid composition and fluidity of hepatocyte plasma membranes.

The fluorescence probe, 1,6-diphenyl-1,3,5-hexatriene, has been used to investigate the effects of controlled and uncontrolled growth on the dynamic properties of the lipid regions of hepatocyte plasma membranes. DPH was incubated with plasma membranes derived from quiescent and regenerating liver and Morris hepatoma 7777, and the resulting systems were studied by fluorescence polarization spectroscopy. Membranes from the rapidly growing hepatoma exhibited a significantly lower fluorescence polarization than observed in quiescent liver, suggesting the presence of a more fluid membrane lipid domain. Membranes from regenerating liver exhibited a time-dependent increase in membrane fluidity, reaching a maximum 12 h after growth stimulation. A close correspondence between membrane fluidity and the cholesterol-phospholipid ratio was also observed where a decrease in this ratio resulted in a more fluid lipid matrix. These results suggest that cell cycling, as observed in regenerating liver and Morris hepatoma 7777, results in significant increases in membrane fluidity, a property which may play an important regulatory role in various cell functions.     

Fluorescence polarization study on the increase of membrane fluidity of human erythrocyte ghosts induced by synthetic water-soluble polymers

The effect of water-soluble polymers on the membrane fluidity of human erythrocyte ghosts was investigated and was compared with that of concanavalin A by means of the fluorescence polarization technique. 8-Anilino-1-naphthalene sulfonic acid sodium salt and 1,6-diphenyl-1,3,5-hexatriene were used as probe molecules. The membrane fluidity was increased by the addition of polycations with concentrations of less than $\text{2 · 10}{}^{\mathrm{?3}}$ wt% 60 min after mixing. The fluidity changes were affected by the chemical structure (hydrophobicity, charge density, etc.) of polycations. Thus, the membrane fluidity increased markedly with increasing charge density on the chain backbone of polycations. On the other hand, nonionic polymers such as poly(ethylene glycol) and $\text{poly}\text{(N-}\text{vinyl}\text{-2-}\text{pyrrolidone}$) changed the membrane fluidity in a biphasic manner. That is, the fluidity of human erythrocyte ghost was temporarily increased and then decrease. For example, 20 wt% of poly(ethylene glycol) gave a maximum fluidity 15 min after mixing with erythrocyte ghosts. A similar fluidity change was observed by adding concanavalin A. Such fluidity changes were not observed when lipid bilayer vesicles were used instead of cell membranes. These results suggested that the increase of membrane fluidity resulted from the intramembraneous aggregation of membrane-bound proteins which was induced by the added polymers. Cell agglutination was also induced by the addition of a large amount of polymers. This agglutination was considered to be due to the intermembraneous aggregation of membrane-bound proteins.     

Regulation of ribonucleotide reductase activity in intact mammalian cells

An intact cell assay system based upon Tween-80 permeabilization was used to investigate the regulation of ribonucleotide reductase activity in Chinese hamster ovary cells. Models used to explain the regulation of the enzyme have been based upon work carried out with cell-free extracts, although there is concern that the properties of such a complex enzyme would be modified by extraction procedures. We have used the intact cell assay system to evaluate, within whole cells, the current model of ribonucleotide reductase regulation. While some of the results agree with the proposals of the model, others do not. Most significantly, it was found that ribonucleotide reductase within the intact cell could simultaneously bind the nucleoside triphosphate activators for both CDP and ADP reductions. According to the model based upon studies with cell-free preparations, the binding of one of these nucleotides should exclude the binding of others. Also, studies on intracellular enzyme activity in the presence of combinations of nucleotide effectors indicate that GTP and perhaps dCTP should be included in a model for ribonucleotide reductase regulation. For example, GTP has the unique ability to modify through activation both ADP and CDP reductions, and synergistic effects were obtained for the reduction of CDP by various combinations of ATP and dCTP. In general, studies with intact cells suggest that the in vivo regulation of ribonucleotide reductase is more complex than predicted from enzyme work with cell-free preparations. A possible mechanism for the in vivo regulation of ribonucleotide reductase, which combines observations of enzyme activity in intact cells and recent reports of independent substrate-binding subunits in mammalian cells is discussed.     

Influence of membrane fluidity on transport mediated by ubiquinones through phospholipid vesicles

Coenzymes Q₁₀ and Q₃ are incorporated into dipalmitoylphosphatidylcholine and egg yolk lecithin liposomes. Dithionite reduction of ferricyanide trapped inside these phospholipid vesicles is taken as a measure of ubiquinone-mediated transport of reducing equivalents. The reaction shows complex pattern with a high order for CoQ. The initial transport rates are very sensitive to the membrane physical state, being considerably reduced at temperatures below the phase transition of the pure dipalmitoylphosphatidylcholine, both for CoQ₁₀ and CoQ₃ reconstituted with this phospholipid. It is suggested that a different reaction mechanism operates in fluid and rigid membranes. This suggestion is related to the possible organization of CoQs in phospholipid membranes.     

Release of the surface coat from the plasma membrane of intact bloodstream forms of Trypanosoma brucei requires Ca2+

A-antigenicity; Glycocalyx; Hydrolases; Membrane glycoproteins     

Lateral mobility of plasma membrane lipids in dividing Xenopus eggs

The lateral mobility of plasma membrane lipids was analyzed during first cleavage of Xenopus laevis eggs by fluorescence photobleaching recovery (FPR) measurements, using the lipid analogs 5-(N-hexadecanoyl)aminofluorescein ("HEDAF") and 5-(N-tetradecanoyl)aminofluorescein ("TEDAF") as probes. The preexisting plasma membrane of the animal side showed an inhomogeneous, dotted fluorescence pattern after labeling and the lateral mobility of both probes used was below the detection limits of the FPR method (D much less than 10(-10) cm2/sec). In contrast, the preexisting plasma membrane of the vegetal side exhibited homogeneous fluorescence and the lateral diffusion coefficient of both probes used was relatively high (HEDAF, D = 2.8 X 10(-8) cm2/sec; TEDAF, D = 2.4 X 10(-8) cm2/sec). In the cleaving egg visible transfer of HEDAF or TEDAF from prelabeled plasma membrane to the new membrane in the furrow did not occur, even on the vegetal side. Upon labeling during cleavage, however, the new membrane was uniformly labeled and both probes were mobile, as in the vegetal preexisting plasma membrane. These data show that the membrane of the dividing Xenopus egg comprises three macrodomains: (i) the animal preexisting plasma membrane; (ii) the vegetal preexisting plasma membrane; (iii) the new furrow membrane.     

Disruption of solid tissue for plasma membrane preparation

A simple press is described for disrupting solid tissue for preparing plasma membrane. It can handle large amounts of tissue (about 20 g wet weight) per stroke, is readily applicable to the disruption of lymph node, spleen, and placenta, and can be adapted to the breakage of soft nonfibrous tissues such as brain. Plasma membrane was isolated from the disrupted tissue by differential and discontinuous sucrose density gradient centrifugation in yields of between 15 and 25% (relative to theoretical yield). The purified plasma membrane was not contaminated with significant amounts of other subcellular fractions.     

Rapid isolation and lipid characterization of plasma membranes from normal and malignant lymphoid cells of mouse

A rapid isolation method was developed for plasma membranes from mouse lymphoid cells such as lymph node lymphocytes, thymocytes, radiation-induced thymoma cells and L1210 cells. Lysates of these lymphoid cells were prepared by Dounce homogenization under hypotonic conditions and directly layered on sucrose step density gradients containing 2 mM CaCl₂ and 5 mM MgCl₂, and centrifuged at $\text{52 000 ×}\text{g}$ for 1 h. Plasma membrane fractions appeared at the interface between 20 and 42% sucrose in the gradients. The procedure permitted purified membranes from cells to be obtained within 3 h, and the preparations appeared to be uniform by electron microscopy. Specific activities of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATpase}$, Mg²⁺-ATPase and 5′-nucleotidase of the isolated plasma membranes were enriched 23- to 61-fold, 12- to 15-fold and 18- to 34-fold, respectively, in comparison with those of the corresponding cell homogenates. Cholesterol content of the malignant cell membranes was lower than that of the normal membranes and the molar ratio of cholesterol to phospholipid of the malignant cell membranes was also lower than that of the normal membranes. A decreased plasmalogen content was observed in the malignant plasma membranes, together with a higher percentage of phosphatidylethanolamine and a lower percentage of phosphatidylserine. In the normal cell membranes, thymocytes contained a higher percentage of phosphatidylcholine and a lower percentage of sphingomyelin than those of the lymph node lymphocytes. At all temperature ranges (5 to 40°C) the plasma membranes of the malignant cells had lower microviscosity than those of the normal cells.     

A simple method for routine determination of adenylate cyclase activity in intact fat cells

The role of metal ions in the biological activity of natural and synthetic metalloproteins is an area of active scientific interest. It would be advantageous in such work to be able to analyze for many elements simultaneously. Methods for this purpose should be rapid, require little or no elaborate pretreatment, and consume only small amounts of material. Of particular interest is the resulting metal-ion distribution in synthetically prepared preparations where the metal-free form of the enzyme can act as a scavenger for unwanted elements during the synthesis.Proton-induced X-ray emission analysis (PIXEA) has been applied, in our laboratory and in others, to the analysis of trace amounts of metallic and nonmetallic elements in a variety of matrices (1–3).In general, the sensitivity in terms of μg/g of this method is comparable to conventional tube-excited X-ray fluorescence, but PIXEA offers the advantage of a higher absolute sensitivity. The technique is nondestructive in the sense that the sample is not consumed in the process and, in addition, a broad range of elements can be simultaneously detected at the submicrogram level. Of particular significance is the small total-sample requirement in that, for the studies reported here, only milligram amounts of protein were required for a total analysis.     

The effect of growth temperature on the membrane lipid environment of the psychrophilic bacterium Micrococcus cryophilus

The relationship between the delta 9-desaturase activity of the psychrophilic bacterium Micrococcus cryophilus grown at different temperatures and the physical state of its membrane lipids as measured by ESR spectroscopy has been studied. Arrhenius plots of desaturase activity were biphasic with a discontinuity at a temperature which depended upon the bacterial growth temperature. Changes in the desaturase activation energy, which increased as the growth temperature was lowered, are discussed in the context of membrane lipid fluidity adaptation to changing environmental temperature. The fluidity of membranes and isolated lipids was measured using nitroxide-labeled fatty acids. The spectra of 2-(10-carboxydecyl)-2-hexyl-4,4-dimethyl-3-oxazolidinoxyl in membranes indicated that there were two lipid environments within the membrane whose relative proportions were dependent both on temperature of measurement and on bacterial growth temperature. In contrast, 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinoxyl spectra showed a single lipid environment and plots of log order parameter (S3) vs 1/T were biphasic with inflexion temperatures which were closely related to the bacterial growth temperature. As with membranes, plots of log S3 vs 1/T for total lipids, phosphatidylglycerol and cardiolipin, but not phosphatidylethanolamine, were biphasic and showed inflexions which correlated well with bacterial growth temperature. These results are interpreted as being consistent with a location for the desaturase within the bulk lipid of the membrane rather than in association with specific lipid types.     

The effect of diabetes on hepatocyte plasma membrane fluidity and concanavalin A-induced agglutination

The techniques of fluorescence polarization and lectin-induced agglutination have been utilized to investigate the effects of the diabetic state on some of the dynamic properties of cell membranes. Hepatocyte plasma membranes from streptozotocin-induced diabetic rats exhibited a significant decrease in cholesterol and sialic acid with no alteration in phospholipid content. This membrane system also exhibited a decrease in fluorescence polarization, using the fluorescent probe, 1,6-di-phenyl-1, 3,5-hexatriene, suggesting an increase in membrane fluidity over the value observed in normal hepatocytes. When normal hepatocytes were incubated in the presence of the lectin, concanavalin A (ConA), no significant agglutination was observed. In contrast, hepatocytes from diabetic rats which exhibited a slightly decreased lectin-binding capacity underwent extensive agglutination. In addition, normal hepatocytes which were pretreated with 0.1 mM tetracaine also underwent extensive agglutination with no measurable increase in lectin-binding capacity. These results suggest that altered membrane lipid fluidity and/or cytoskeletal organization may have a profound effect on cell surface dynamics and could result in the uncoupling of the insulin receptor complex from the membrane-associated effector system(s), a defect which may play a role in the problem of insulin resistance observed in some forms of diabetes.     

Comparative lipid analysis of purified plasma membranes and shed extracellular membrane vesicles from normal murine thymocytes and leukemic GRSL cells

The lipid fluidity in purified plasma membranes (PM) of murine leukemic GRSL cells, as measured by fluorescence polarization, is much higher than in PM of normal thymocytes. This was found to be due to relatively low contents of cholesterol and sphingomyelin and a high amount of unsaturated fatty acyl chains, especially linoleic acid, in the phospholipids. PM from GRSL cells contain markedly more phosphatidylethanolamine than those from thymocytes. For both GRSL cells and thymocytes the detailed lipid composition of isolated PM was compared with that of the corresponding shed extracellular membranes (ECM), which were isolated from the ascites fluid and from thymus cell suspensions, respectively. The somewhat decreased lipid fluidity of thymocyte ECM as compared to their PM, can be ascribed to the increased cholesterol/phospholipid molar ratio (0.88 vs. 0.74). No other major differences were found between the lipid composition of these membranes. In contrast, significant differences were found between PM and ECM from GRSL cells. In this system a much lower lipid fluidity of the shed ECM was found, due to the much increased cholesterol/phospholipid molar ratio (3.5-fold) and sphingomyelin (9-fold) content, as compared to the PM. Further, the ECM contain relatively more lysophosphatidylethanolamine and less phosphatidylcholine and -inositol. ECM contain a higher amount of polyunsaturated fatty acids, especially in the phosphatidylethanolamine and lysophosphatidylethanolamine classes. On the other hand, the fatty acids of phosphatidylcholine and lysophosphatidylcholine are more saturated than in PM. In particular, ECM of GRSL cells contain less oleic and linoleic acid residues and more arachidonic acid and 22:polyunsaturated fatty acid residues than PM. The possible relevance of these differences with respect to the mechanism of shedding of vesicles from the cell surface, is discussed.     

Perturbation of lipid metabolism in L-M cultured cells by elaidic acid supplementation: formation of fatty alcohols

Supplementation of culture medium with elaidic acid (400 μg/flask) in L-M cells results in the formation of an otherwise undetected lipid component. We have identified this lipid component to be a mixture of free fatty alcohols containing primarily elaidyl alcohol with cetyl, stearoyl, and oleoyl alcohols as minor constituents. Formation of fatty alcohols by fatty acid supplementation seems to be specific with $\text{trans}$ fatty acids (i.e., elaidate, $\text{trans}$ vaccenate, and linolelaidate); addition of stearate and oleate to the L-M cells does not produce fatty alcohols. The fatty alcohols accumulated by the $\text{trans}$ fatty acid supplementation are associated with both the particulate and supernatant fractions of the cells.     

Ribonucleotide reductase activity in intact mammalian cells: stimulation of enzyme activity by MgCl2, dithiothreitol, and several nucleotides

An intact cell assay system based on Tween-80 permeabilization was used to investigate ribonucleotide reductase activity in Chinese hamster ovary cells. Dithiothreitol, a reducing agent, is required for optimum activity. Analysis of dithiothreitol stimulation of CDP and ADP reductions indicated that in both cases the reducing agent served only to increase the reaction rate without altering the affinity of the enzyme for substrates. Magnesium chloride significantly stimulated the reduction of CDP but not ADP; this elevation in CDP reduction was due to an increase in both the affinity of the enzyme for substrate and the Vmax. In addition to ATP and dGTP, well-known activators of CDP and ADP reductase activities, it was found that dCTP and GTP were also able to activate CDP and ADP reductase activities, respectively. For the dCTP-activated reaction the Vmax was 0.158 nmol dCDP formed 5 X 10(6) cells-1 h-1 and the Km was 0.033 mM CDP, while for the GTP-activated reduction a Vmax of 0.667 nmol dADP formed 5 X 10(6) cells(-1) h-1 and Km of 0.20 mM ADP were observed. Kinetic analysis revealed that dCTP, dGTP, and GTP stimulate ribonucleotide reduction solely by increasing the affinity of the enzyme for substrate without affecting the Vmax of the respective reactions. ATP behaves in a different manner as it stimulates CDP reduction by altering both the affinity of the enzyme for substrate and the Vmax. Cellular concentrations of ribo- and deoxyribonucleoside di- and triphosphate pools were measured to help evaluate the relative physiological importance of the nucleotide activators. These determinations, along with the reaction kinetic studies, strongly imply that ATP is a much more important regulator of CDP reduction that dCTP, whereas GTP may serve as well or better than dGTP as the in vivo activator of ADP reduction.     

Phosphorylation of glycophorin A in membranes of intact human erythrocytes

The qualitative and quantitative contribution of glycophorin A phosphorylation to the general and specific pattern of membrane protein phosphorylation in intact erythrocytes pre-incubated with 32Pi was examined. Intense 32P-labeled bands at 88,000 and 38,000 Mr were identified as phosphorylated glycophorin A dimer and monomer respectively on the basis of several criteria. Quantitatively, phosphorylated glycophorin A dimer accounted for about 70% of 32P in the band 3 region. This value is at least three times that previously reported. The results of ancillary experiments involving selective extraction of ghosts in acidified chloroform/methanol solvents and electrophoresis in the presence of detergents make it unlikely that the 32P associated with glycophorin A was due to bound polyphosphoinositides.     

Conversion of glycerate to serine in intact spinach leaf peroxisomes

Intact spinach (Spinacia oleracea L.) leaf peroxisomes converted glycerate to serine in the presence of NAD and alanine. The reaction proceeded optimally at pH9. Addition of oxaloacetate or alpha-ketoglutarate plus aspartate enhanced the conversion about three-fold. Alteration of the concentration of one of the reaction components, consisting of 2 mM glycerate, 0.2 mM NAD, 0.5 mM oxaloacetate, and 2 mM alanine, revealed half-saturation constants of 0.45 mM for glycerate, 0.06 mM for NAD, 0.02 mM for oxaloacetate, and 0.33 mM for alanine. The conversion proceeded with the formation of hydroxypyruvate followed by serine; hydroxypyruvate did not accumulate to a high amount in the presence or absence of alanine. The amino group donor could be alanine (half-saturation constant, 0.33 mM), glycine (0.45 mM), or asparagine (0.67 mM); the three amino acids produced roughly similar Vmax values. The results indicate that, in the conversion of glycerate to serine, the transamination is catalyzed by a hydroxypyruvate aminotransferase with characteristics unknown among all other studied leaf peroxisomal aminotransferases. The peroxisomal membrane is sparsely permeable to NAD/NADH, and the participation of the peroxisomal malate dehydrogenase in an electron shuttle system across the membrane in the regeneration of NAD/NADH is suggested.     

Lateral mobility of plasma membrane lipids in Xenopus eggs: Regional differences related to animal/vegetal polarity become extreme upon fertilization

Regional differences in the lateral mobility properties of plasma membrane lipids have been studied in unfertilized and fertilized Xenopus eggs by fluorescence photobleaching recovery (FPR) measurements. Out of a variety of commonly used lipid probes only the aminofluorescein-labeled fatty acids HEDAF (5-(N-hexadecanoyl)-aminofluorescein) and TEDAF (5-(N-tetradecanoyl)-aminofluorescein) appear to partition into the plasma membrane. Under all experimental conditions used these molecules show partial recovery upon photobleaching indicating the existence of lipidic microdomains. In the unfertilized egg the mobile fraction of plasma membrane lipids (～50%) has a fivefold smaller lateral diffusion coefficient (D = 1.5 × 10^?8 cm²/sec) in the animal than in the vegetal plasma membrane (D = 7.6 × 10^?8 cm²/sec). This demonstrates the presence of an animal/vegetal polarity within the Xenopus egg plasma membrane. Upon fertilization this polarity is strongly (>100×) enhanced leading to the formation of two distinct macrodomains within the plasma membrane. At the animal side of the egg lipids are completely immobilized on the time scale of FPR measurements (D ? 10^?10 cm²/sec), whereas at the vegetal side D is only slightly reduced (D = 4.4 × 10^?8 cm²/sec). The immobilization of animal plasma membrane lipids, which could play a role in the polyspermy block, probably arises by the fusion of cortical granules which are more numerous here. The transition between the animal and the vegetal domain is sharp and coincides with the boundary between the presumptive ecto- and endoderm. The role of regional differences in the plasma membrane is discussed in relation to cell diversification in early development.