E. Ferber

Isolation and characterization of lymphocyte plasma membranes

We describe a method for the isolation of plasma membranes and other organelles from the lymphocytes of pig mesenteric lymph nodes, as well as from calf mediastinal nodes. The cells were disrupted by nitrogen cavitation leaving the nuclei intact. Nuclei, the large granule fraction and microsomes consisting of vesicles derived from plasma membrane and endoplasmic reticulum were separated by differential centrifugation. Plasma membranes were separated from the fragmented endoplasmic reticulum by equilibrium density ultracentrifugation in buffered dextran solutions. All fractions were characterized using marker enzymes and by their chemical composition. The microsomal fraction seemed to be free of cell constituents other than plasma membranes and endoplasmic reticulum. The yield of plasma membranes was 1.7 % (protein content relative to whole cell homogenate). Specific activities of 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5), a widely used plasma membrane marker, were 125 nmoles·(mg protein)−1·min−1 being enriched by a factor of 25 as compared with whole cell homogenate. The cholesterol content of plasma membranes was 751 nmoles/mg protein (homogenate = 104 nmoles/mg protein) and the phospholipid content 727 nmoles/mg protein (homogenate = 90.3 nmoles/mg protein). The cholesterol: phospholipid molar ratio was 1.03. Lysolecithin acyltransferase (acyl-CoA: 1-acyl-sn-glycero-3-phosphorylcholine-O-acyltransferase, EC 2.3.1.?.) showed specific activities between 10 to 12 nmoles·(mg protein)−1·min−1 in the plasma membrane compared to 3–4 nmoles·(mg protein)−1·min−1 in endoplasmic reticulum and thus appears to be a plasma membrane component in lymphocytes.

Phospholipid metabolism of stimulated lymphocytes: composition of phospholipid fatty acids

Lymph node lymphocytes and thymocytes from different species were isolated. Rabbit and calf thymocytes were stimulated in vitro with concanavalin A. Phospholipid fatty acids of these cells were analyzed and their positional distribution was determined. When compared with liver, phosphatidylcholine of unstimulated lymphocytes was found to contain relatively high amounts of palmitic acid in position 2 and oleic acid in position 1. After stimulation of rabbit thymus cells, the content of polyunsaturated fatty acids (linoleic and arachidonic acid) increased. Thus the ratio of polyenoic acids (18:2 + 20:4) to saturated fatty acids was doubled when compared to control cells. Similar results were obtained after in vivo stimualtion with Mycobacterium Calmette Guerin. The correlation of these findings with the activation of acyl-CoA:lysolecithin acyltransferase, and their relevance for changes of membrane fluidity during lymphocyte stimulation is discussed.

Phospholipid metabolism of stimulated lymphocytes: activation of acyl-CoA:lysolecithin acyltransferases in microsomal membranes.

Abstract Rabbit lymphocytes were stimulated with phytohemagglutinin. After incubation, mitochondria and microsomes were isolated. Microsomes were subfractionated into plasma membranes and endoplasmic reticulum. In these cell fractions the activities of lysolecithin acyltransferases (macyl-CoA:monoacyl-sn-glycero-3-phosphorylcholine acyltransferase) were determined using labeled substrates: acyl-CoAs of different chain lengths and degrees ofunsaturation, and both isomers ofmonoacyl-sn-glycero-3-phosphorylcholine as acceptors. In addition, the lysophospholipase activity was followed. Lysolecithin acyltransferase was found to be activated 2–3 times in the microsomal membranes of stimulated lymphocytes whereas the mitochondrial acyltransferase activity remained unchanged. Separation ofmicrosomal membranes into plasma membranes and endoplasmic reticulum showed that the activation of this enzyme exclusively occurred in the plasma membrane fraction. Km values for acyl-CoAs as well as for lysophosphatides did not change during the stimulation. Time-course experiments indicated that the enzyme activation occurred directly after binding of the stimulant. The activation process did not depend on energy metabolism or on protein synthesis, since it occurred at 0 °C and was not affected by puromycin. The lysophospholipase (1-acyl-sn-glycero-3-phosphorylcholine acylhydrolase, EC 3.1.1.5) activity in mitochondria and microsomes of stimulated lymphocytes was slightly lower than that in control cells. The relevance of these findings for the mechanism of lymphocyte activation is discussed.

Reactive oxygen species are involved in the activation of cellular phospholipase A2.

Vanadate (V) potentiated (4‐ to 10‐fold) the activation of cellular phospholipase A2 (PLA2) induced by H2O2 (H), a phorbol ester (T), a Ca2+‐ionophore (A) and opsonized zymosan in macrophages. V+H induced in intact cells the activation and translocation of PLA2 and protein kinase C(PKC) to the plasma membrane. V+H and V+T+A induced strong chemiluminescence (CL) which was abrogated by a specific NADPH oxidase inhibitor diphenylene iodonium (DPI). DPI markedly suppressed the stimulation of PLA2 by V+T+A and V+OZ. The results suggest that the formation of endogenous reactive oxygen species (ROS) is important for PLA2 activation.

The study of lipid-protein interactions in membranes by fluorescent probes

Abstract 1. 1. We report the fluorescence of 1-anilinonaphthalene-8-sulfonate from 220 mμ up in various solvents, in association with lecithin and lysolecithin and bound to membranes of human erythrocytes. 2. 2. The membrane spectra indicate that there is transfer of electronic excitation energy from some of the tryptophans of membrane-bound 1-anilinonaphthalene-8-sulfonate. 3. 3. Our findings raise the possibility that some or all of the membrane-bound 1-anilinonaphthalene-8-sulfonate is associated with membrane proteins; the dye can therefore not be assumed to act solely as a probe for possible lipid regions in membranes.

Analysis of the proteins in thymocyte plasma membrane and smooth endoplasmic reticulum by sodium dodecylsulfate-gel electrophoresis

Abstract We have purified the plasma membranes and membranes of endoplasmic reticulum from calf and rabbit thymocytes and from calf mediastinal lymph node lymphocytes. We disrupted the cells by the “nitrogen cavitation method” and prepared a microsomal isolate by differential centrifugation. We fractionated this by isopycnic ultracentrifugation in dextran gradients into membrane vesicles, PM 1 and PM 2 , most likely derived from plasma membrane and a fraction, ER, most likely originating from endoplasmic reticulum. More than 80% of the microsomal 5′-nucleotidase and acid p -nitrophenylphosphatase concentrates in the PM 1 and PM 2 fractions; alkaline p -nitrophenylphosphatase, another presumptive PM marker, is concentrated in the PM 1 fraction. These data are confirmed by the lacroperoxidase radioiodination of intact rabbit thymocytes followed by subcellular fractionation. The specific content of phospholipids (822 nmoles/mg protein) and cholesterol (1032 nmoles/mg protein) is highest in PM 1 and PM 2 plasma membrane fractions. NADH-oxidoreductase, our endoplasmic reticulum marker, is clearly enriched in gradient pellet. The membrane proteins were separated by electrophoretic molecular sieving in sodium dodecylsulfate-polyacrylamide gel electrophoresis, containing dithiothreitol (sodium dodecylsulfate-polyacrylamide gel electrophoresis). We numbered the 10 major protein components of the “microsomal fraction” (apparent molecular weights between 280000 and 15000) from 1–10 according to their decreasing molecular weights. Of these proteins, those with higher molecular weight, predominantly glycoproteins, appear in the PM 1 fraction, while the endoplasmic reticulum fraction contains mainly low molecular weight components.

Phospholipid metabolism of stimulated lymphocytes: Comparison of the activation of acyl-CoA: Lysolecithin acyltransferase with the binding of concanavalin A to thymocytes

Calf thymocytes were isolated and incubated with concanavalin A. The effect of the mitogen on the enzyme activity of membrane-bound lysolecithin acyltransferase (acyl-CoA:1-acylglycero-3-phosphorylcholine-O-acyltransferase, EC 2.3.1.23) was determined as also the binding of 125I-labelled concanavalin A to intact cells and isolated membranes. The lysolecithin acyltransferase was found to be activated three times in microsomal membranes. The activation occurred directly after binding of concanavalin A and was temperature independent, since similar activities were found in cells treated with concanavalin A at 0 and 37 degrees C. The acyltransferase activation using increasing concentrations of concanavalin A revealed a different behaviour, as compared to the binding of concanavalin A. While the binding of concanavalin A to intact cells expressed a normal hyperbolic saturation function the activation process of the acyltransferase described a sigmoidal relationship. Correspondingly, the interaction coefficients for both functions were different (Sips coefficient for binding = 1.0 and Hill coefficient of the enzyme activation = 1.8). These results indicate that the acyltransferase activation is due to a cooperative interaction between the ligand-receptor complex and the enzyme.

Effect of cellular fatty acid composition on the phospholipase A2 activity of bone marrow-derived macrophages, and their ability to induce lucigenin-dependent chemiluminescence

Mouse bone marrow macrophages were obtained by cultivation in serum-free medium. Addition of specific fatty acids to the medium leads to macrophage populations which differ in their fatty acid composition. The fatty acid composition of the cellular membranes directly modulates functional abilities of the macrophages such as the generation of superoxide anion and phospholipase A2 activity in response to phorbol ester and zymosan. Both capacities were lowest in macrophages cultured serum-free without lipids. Incorporation of unsaturated fatty acids into macrophage phospholipids leads to an increase of O2- production as measured by lucigenin-dependent chemiluminescence and to an increased phospholipase A2 activity after challenge with phorbol ester or zymosan.

Translocation of phospholipase A2 from cytosol to membranes induced by 1-oleoyl-2-acetyl-glycerol in serum-free cultured macrophages

Macrophages exhibit high activities of a phospholipase A2 which preferentially cleaves arachidonic acid (I. Flesch, B. Schmidt, and E. Ferber, Z. Naturforsch. 40c, 356-363, 1985). In unstimulated cells more than 90% of the total activity of this enzyme is localized in the cytosol. Treatment of these cells with 100 microM 1-oleoyl-2-acetyl-glycerol (OAG) for 30 min induced a translocation of phospholipase A2 to cellular membranes. The amount of translocated phospholipase A2 was about 30% of the total activity and correlated with a similar translocation of protein kinase C to membranes. These data suggest that the translocation of phospholipase A2 to membranes is related to the activation process of this enzyme.

CoA-Dependent cleavage of arachidonic acid from phosphatidylcholine and transfer to phosphatidylethanolamine in homogenates of murine thymocytes

Increased turnover of the fatty acyl moieties of membrane phospholipids and in particular the metabolism of arachidonic acid play a central role in the early phase of ligand-receptor-mediated activation in many cell types. Alterations in the membrane lipid phase are thought to initiate changes in the membrane permeability to ions and the activation of membrane-bound enzymes [ 11. The synthesis of arachidonic acid metabolites by the cycle-oxygenase and lipoxygenase systems is also considered to be controlled by the availability of free arachidonic acid; this in turn is determined by the release of arachidonic acid from membrane-bound lipids [2-l]. In mitogen-induced activation of T lymphocytes, one of the early changes reported in activated cells is an increased incorporation of long-chain fatty acids into phosphatidylcholine and phosphatidylethanolamine [5]. In mitogen-activated human T cells an elevated release of arachidonic acid from. membrane phospholipids has been reported [6,7], coupled with a synthesis of arachidonic acid derivatives [8]. There is good evidence that phospholipid synthesising enzymes are activated in stimulated T cells [9] but we have been unable to detect significant phospholipase Aa activity in thymocytes using exogeneous substrate [lo]. The rapid incorporation of fatty acids