Transmembrane linkage between surface glycoproteins and components of the cytoplasm in neutrophil leukocytes

An experimental approach is described that enables the analysis of interactions between exogenous surface ligands and components of the cytoplasm in neutrophil leukocytes. Neutrophils treated with the nonionic detergent Lubrol PX, under controlled conditions, yield intact detergent-insoluble ghosts. Morphological analysis of neutrophil ghosts shows that they retain the original dimensions of the cell and consist almost entirely of a peripheral filamentous network, representing the submembranous cortical web, concentric to nuclear remnants. All intracellular membrane-bounded organelles, plasma membrane, and background cytoplasmic electron density are absent. Biochemical analysis of the ghosts shows that less than 10% of enzyme markers for the soluble and granule fractions remain, and that greater than 90% of total cell phospholipid is removed during detergent extraction. The major proteins remaining in the ghosts comigrate, on polyacrylamide gels in the presence of SDS, with chicken gizzard actin, myosin, filamin, and a 110-kdalton protein. Patches and caps induced on neutrophils with either fluorescein isothiocyanate-concanavalin A or ferritin-concanavalin A retain their original location and morphology on ghosts after lysis, as determined by both fluorescence and electron microscopy. In similar experiments, but using 125I-labeled lectins, 37% of total cell bound concanavalin A (Con A) and 25% succinylated Con A remain attached to the ghosts. A major 125I-labeled membrane glycoprotein (80 kdaltons) is associated with ghosts prepared from intact neutrophils iodinated in the presence of exogenous lactoperoxidase. Further 125I-labeled membrane glycoproteins (217, 170, and 147 kdaltons) become associated with ghosts prepared from iodinated cells treated before lysis with Con A, but not with succinylated Con A. These data taken together suggest that linkages exist in neutrophils between proteins exposed on the outer surface of the plasma membrane and the peripheral filamentous network independent of the presence of lipid bilayer. The implications of these findings for surface motile phenomena will be discussed.     

Low or undetectable levels of surface high affinity cholera toxin receptors on normal hemopoietic growth factor-dependent cells.

The membrane monosialoganglioside GM1, the high affinity receptor for cholera toxin, is generally considered ubiquitous on normal cells. It was found to be abundant both on normal mature hemopoietic cells and on leukemic cells. By contrast, the normal factor-dependent cell lines, which achieve indefinite proliferation in the presence of the multilineage hemopoietic growth factor apparently displayed the unique character of having low or undetectable levels of surface membrane and cytoplasmic cholera toxin receptors. These results were obtained by the Scatchard analysis of 125iodinated toxin binding, immunofluorescence studies and gel electrophoresis autoradiography. This corroborated the fact that these cells were highly resistant to growth inhibition by cholera toxin (microM to fM) while normal mature cells and leukemic cells of similar phenotype were sensitive.     

Evidence for transmembrane orientation of acylated simian virus 40 large T antigen.

In mKSA cells (a simian virus 40-transformed BALB/c mouse tumor cell line), plasma membrane-associated large T antigen (large T) is found in two subfractions of the plasma membrane; a minor amount of large T is recovered from the Nonidet P-40 (NP-40)-soluble plasma membrane fraction, whereas the majority is tightly bound to a substructure of the plasma membrane, the plasma membrane lamina (PML). Only PML-associated large T is fatty acid acylated (U. Klockmann and W. Deppert, EMBO J. 2:1151-1157, 1983). We have analyzed whether these two forms of plasma membrane-associated large T might differ in features like cell surface expression or metabolic stability. In addition, we have asked whether one of the two large Ts might represent the hypothetic, large T-related protein T* (D. F. Mark and P. Berg, Cold Spring Harbor Symp. Quant. Biol. 44:55-62, 1979). We show that in mKSA cells grown in suspension culture, large T associated with the PML is also exposed on the cell surface. This form of large T, therefore, exhibits properties of a transmembrane protein. Large T in the NP-40-soluble plasma membrane fraction could not be labeled with radioiodine on the cell surface and, for this reason, does not seem to be oriented towards the cell surface. In contrast, when mKSA cells were grown on substratum (culture dish), we found that in these cells both NP-40-soluble large T as well as large T anchored in the PML could be cell surface iodinated. We also have analyzed the plasma membrane association of surface T antigen in mKSA cells grown in a mouse as ascites tumor. In tumor cells, only PML-bound large T is cell surface associated. We conclude that differences in extractibility of cell surface-associated large T most likely depend on cell shape and are not an artifact of cell culture. Both NP-40-soluble and PML-bound large Ts are associated with the plasma membrane in a metabolically stable fashion. Neither of the two large Ts represents T*.     

Mechanisms underlying the confined diffusion of cholera toxin B-subunit in intact cell membranes

Multivalent glycolipid binding toxins such as cholera toxin have the capacity to cluster glycolipids, a process thought to be important for their functional uptake into cells. In contrast to the highly dynamic properties of lipid probes and many lipid-anchored proteins, the B-subunit of cholera toxin (CTxB) diffuses extremely slowly when bound to its glycolipid receptor GM(1) in the plasma membrane of living cells. In the current study, we used confocal FRAP to examine the origins of this slow diffusion of the CTxB/GM(1) complex at the cell surface, relative to the behavior of a representative GPI-anchored protein, transmembrane protein, and fluorescent lipid analog. We show that the diffusion of CTxB is impeded by actin- and ATP-dependent processes, but is unaffected by caveolae. At physiological temperature, the diffusion of several cell surface markers is unchanged in the presence of CTxB, suggesting that binding of CTxB to membranes does not alter the organization of the plasma membrane in a way that influences the diffusion of other molecules. Furthermore, diffusion of the B-subunit of another glycolipid-binding toxin, Shiga toxin, is significantly faster than that of CTxB, indicating that the confined diffusion of CTxB is not a simple function of its ability to cluster glycolipids. By identifying underlying mechanisms that control CTxB dynamics at the cell surface, these findings help to delineate the fundamental properties of toxin-receptor complexes in intact cell membranes.     

Cholesterol sensitivity of detergent resistance: a rapid flow cytometric test for detecting constitutive or induced raft association of membrane proteins.

BACKGROUND: Lipid rafts are cholesterol- and glycosphingolipid-rich microdomains in the cellular plasma membranes that play critical roles in compartmentalization (concentration, coupling, and isolation) of receptors and signal molecules. Therefore, detecting constitutive or induced raft associations of such proteins is of central interest in cell biology. This has mostly been done with time- and cell-consuming immunobiochemical techniques affected by several sources of artifacts. A flow cytometric analysis of immunocytochemical staining under differential circumstances of detergent treatment offers a new alternative to this method. METHODS: Membrane microdomains are resistant to nonionic detergents due to extensive, strong interactions between their molecular constituents. We used this feature to develop a rapid flow cytometric assay of differential detergent resistance based on immunocytochemical labeling of extracellular domain epitopes in membrane proteins. Data evaluation is based on comparative detection of their detergent solubility without and with cholesterol depletion of cell membranes, resolved by moderate concentrations of nonionic detergents. RESULTS: Nonionic detergents Triton X-100 and Nonidet-40 (0.05-0.1%) in cold or Brij-98 (0.1-0.5%) at 37 degrees C efficiently resolved detergent solubility or resistance of many lymphocyte cell surface proteins. Kinetic data revealed that a short (5-10 min) detergent treatment is sufficient for this assay. Comparison of detergent solubility in untreated and cholesterol-depleted cells differentiated membrane proteins associated with or excluded from raft microdomains, respectively. Confocal microscopy showed that this mild detergent treatment leaves the cytoskeleton of the cells intact, with a detectable expression of raft marker detergent-resistant proteins attached to it. An induced association with rafts of immunoglobulin E receptors upon antigen cross-linking was also easily detectable in rat mast cells by this approach. CONCLUSIONS: A protocol is proposed for a rapid (5-10 min) test of detergent resistance of membrane proteins in cells. The approach requires only a small amount of cells (10(4)/sample) and offers a good resolution of detergent solubility or resistance of membrane proteins, also in terms of the underlying mechanisms, with an advantage of applicability for all conventional bench-top flow cytometers.     

Lymphocyte plasma membranes. III. Composition of lymphocyte plasma membranes from normal and immunized rats

Isolated plasma membranes of thymic and splenic lymphocytes from unimmunized and immunized rats of the inbred ACI and F344 strains were analyzed for chemical and enzymatic composition, for membrane protein patterns by polyacrylamide gel electrophoresis and for membrane-associated immunoglobulins. After immunization, the thymic and splenic lymphocyte membranes from F344 rat contained less carbohydrate and higher phospholipid contents than control animals. In both ACI and F344 inbred rat strains the membrane phospholipid to cholesterol weight ratio increased significantly after immunization. The electrophoretic patterns of solubilized membrane proteins and of iodinated external membrane proteins were similar in unimmunized and immunized animals.When thymic and splenic lymphocytes of normal or immunized animals were surface radioiodinated, solubilized in Triton X-100, NP-40 or 10 M urea in 1.5 M acetic acid and analyzed by immunoprecipitation, labeled IgM immunoglobulin was recovered from thymic lymphocytes but both labeled IgG and IgM were recovered from splenic lymphocytes. However, when unlabeled isolated plasma membranes were solubilized in 1% Triton X-100 and analyzed by immunodiffusion in agarose gels, both IgG and IgM were identified in thymic and splenic cells.     

Interactions of cholera toxin with isolated hepatocytes. Effects of low pH, chloroquine and monensin on toxin internalization, processing and action.

The major steps in cholera-toxin action, i.e. binding, internalization, generation of A1 peptide and activation of adenylate cyclase, were examined in isolated hepatocytes. The binding of toxin involves a single class of high-affinity sites (KD congruent to 0.1 nM; Bmax. congruent to 10(7) sites/cell). At 37 degrees C, cell-associated toxin is progressively internalized, as judged by the loss of its accessibility to antibodies against whole toxin, A and B subunits (about 50, 75 and 30% of initially bound toxin after 40 min respectively). Two distinct pathways are involved in this process: endocytosis of the whole toxin, and selective penetration of the A subunit into the plasma membrane. Exposure of hepatocytes to an acidic medium (pH 5) results in a rapid and marked disappearance of the A subunit from the cell surface. Generation of A1 peptide and activation of adenylate cyclase by the toxin occur after a lag phase (10 min at 37 degrees C), and increase with time in a parallel manner up to 2-3% A1 peptide generated; they are unaffected by exposure of cells to an acidic medium. Chloroquine and monensin, which elevate the pH in acidic organelles, inhibit by 2-4-fold both the generation of A1 peptide and the activation of adenylate cyclase. Unexpectedly, these drugs also inhibit the internalization of the toxin. These results suggest that an acidic pH facilitates the penetration of A subunit into the plasma membrane and presumably the endosomal membrane as well, and that endocytosis of cholera toxin is required for generation of A1 peptide and activation of adenylate cyclase.     

Membrane receptors as general markers for plasma membrane isolation procedures. The use of 125-I-labeled wheat germ agglutinin, insulin, and cholera toxin.

Specific cell surface membrane receptors, labeled by forming a complex with low concentrations (about 10--9 M to 10--10 M) of a highly radioactive (125-I, carrier-free) ligand, can serve as simple, reliable, sensitive, and quantitative markers for plasma membranes in fractionation procedures. 125-I-Labeled insulin, cholera toxin and the plant lictins, wheat germ agglutinin (WGA), and concanavalin A are the receptor ligands used for labeling plasma membranes. Plasma membranes are labeled before homogenization by incubating intact cells briefly at 24 degrees or 4 degrees, or by very brief in situ perfusion of the organ, with the 125-I-Labeled marker. After removing the free 125-I-labeled ligand from the medium by washing (at 4 degrees), the membrane-marker complex remains intact over prolonged (days) periods of time at 4 degrees. Labeling occurs nearly exclusively on the cell surface, the specificity of this plasma membrane reaction is maintained through homogenization and fractionation, and little dissociation of the complex, detectable exchange of label, or aggregation occur even upon prolonged incubation of the homogenates. When desired, the complex can be dissociated deliberately by manipulating experimental conditions such as temperature or by adding specific simple sugars. The most generally suitable marker appears to be WGA. At least in certain tissues (e. g. fat cells) labeling of the plasma membrane with 125-I-WGA and 125-I-isnulin can be performed equally well and selectively in homogenates as in the intact cell. 125-I-Cholera toxin cannot be used in homogenates because of significant binding to nuclei. The use of 125-I-labeled WGA as a specific plasma membrane marker is illustrated in following the course of fractionations, and in quantitating the yield and purity, of plasma membranes from fat cells, lymphocytes, and liver. The results are compared with simultaneous measurements of the plasma membrane enzyme "markers," ATPase, 5-nucleotidase, and basal as well as hormone-stimulated adenylate cyclase activities. The fractionation of liver plasma membranes by aqueous dextran-polyethylene glycol two-phase polymer systems and by conventional differential centrifugation procedures arealso quantitated with the marker, 125I-WGA. Substantial quantities of plasma membrane material are no recovered in the interphase of the two-phase polymer system. Conventional liver fractionation procedures which retain, for further purification, only the readily sedimented pellet (2000 times g, 15 min) discard a very large (at least 70%) questenal hy     

Selective iodination and polypeptide composition of pinocytic vesicles

We describe a method for the specific radioiodination of pinocytic vesicles (PVs) based upon the simultaneous endocytosis of lactoperoxidase (LPO) and glucose oxidase (GO). Initial experiments indicated that LPO was interiorized by the macrophage cell line J774 by fluid phase pinocytosis and without detectable binding to the plasma membrane (PM). Interiorization varied linearly with enzyme concentration and exposure time, was temperature dependent, and was undetectable at 4 degrees C. Employing EM cytochemistry, LPO activity was restricted to PVs after a 3- to 5-min pulse at 37 degrees C. These results formed the basis of the method for iodinating the luminal surface of PVs: 5-min exposure to both LPO and GO at 37 degrees C followed by washes and iodination (addition of 125I and glucose) at 4 degrees C. Enzyme-dependent incorporation of iodide into the polypeptides of both PV membrane and contents occurred. Several lines of evidence indicated that there was selective labeling of PV as opposed to PM. Iodination did not occur if the pinocytic uptake of LPO ad GO was inhibited by low temperature. EM autoradiography showed a cytoplasmic localization of grains, whereas a clear PM association was evident with surface labeling. LPO was iodinated only after PV labeling and was present within organelles demonstrating latency. After PV iodination, > 75% of several labeled membrane antigens could be immunoprecipitated by monoclonal antibodies only after cell lysis. In contrast, all labeled antigens were accessible to antibody on intact cells after surface labeling. The polypeptide compositions of PM and PV membrane were compared by SDS polyacrylamide gel electrophoresis and by quantitative immune precipitation using a panel of anti-J774 monoclonal antibodies. The electrophoretic profiles of iodinated proteins (15-20 bands) were strikingly similar in NP-40 lysates of both PV and PM iodinated cells. In addition, eight membrane antigens examined by immune precipitation, including the trypsin-resistant immunoglobulin (Fc) receptor and the H-2Dd histocompatibility antigen, were found to be iodinated to the same relative extents by both labeling procedures. We conclude that PV membrane is formed from a representative sample of PM polypeptide components.     

Asymmetric distribution of gangliosides in rat renal brush-border and basolateral membranes

Highly enriched brush-border and basolateral membranes isolated from rat renal cortex were used to study the distribution of endogenous gangliosides in the two distinct plasma membrane domains of epithelial cells. These two membrane domains differed in their glycolipid composition. The basolateral membranes contained more of both neutral and acidic glycolipids, expressed on a protein basis. In both membranes, the neutral glycolipids corresponding to mono-, di-, tri- and tetraglycosylceramides were present. The basolateral membranes contained more diglycosylceramide than the brush-border membranes. The major gangliosides found were GM4, GM3, and GD3 with minor amounts of GM1 and GD1a. The latter were identified and quantified by sensitive iodinated cholera toxin binding assays. When the distribution of individual gangliosides was calculated as a percent of total gangliosides, the brush-border membranes were enriched with GM3, GM1 and GD1a compared to the basolateral membranes, which were enriched with GD3 and GM4. The observation of a distinct distribution of glycolipids between brush-border and basolateral membranes of the same epithelial cell suggests that there may be a specific sorting and insertion process for epithelial plasma membrane glycolipids. In turn, asymmetric glycolipid biogenesis may reflect differences in glycolipid function between the two domains of the epithelial plasma membrane.     

Characterization of the receptor for cholera toxin and Escherichia coli heat-labile toxin in rabbit intestinal brush borders.

125I-labelled heat-labile toxin (from Escherichia coli) and 125I-labelled cholera toxin bound to immobilized ganglioside GM1 and Balb/c 3T3 cell membranes with identical specificities, i.e. each toxin inhibited binding of the other. Binding of both toxins to Balb/c 3T3 cell membranes was saturable, with 50% of maximal binding occurring at 0.3 nM for cholera toxin and 1.1 nM for heat-labile toxin, and the number of sites for each toxin was similar. The results suggest that both toxins recognize the same receptor, namely ganglioside GM1. In contrast, binding of 125I-heat-labile toxin to rabbit intestinal brush borders at 0 degree C was not inhibited by cholera toxin, although heat-labile toxin inhibited 125I-cholera toxin binding. In addition, there were 3-10-fold more binding sites for heat-labile toxin than for cholera toxin. At 37 degrees C cholera toxin, but more particularly its B-subunit, did significantly inhibit 125I-heat-labile toxin binding. Binding of 125I-cholera toxin was saturable, with 50% maximal of binding occurring at 1-2 nM, and was quantitatively inhibited by 10(-8) M unlabelled toxin or B-subunit. By contrast, binding of 125I-heat-labile toxin was non-saturable (up to 5 nM), and 2 X 10(-7) M unlabelled B-subunit was required to quantitatively inhibit binding. Neuraminidase treatment of brush borders increased 125I-cholera toxin but not heat-labile toxin binding. Extensive digestion of membranes with Streptomyces griseus proteinase or papain did not decrease the binding of either toxin. The additional binding sites for heat-labile toxin are not gangliosides. Thin-layer chromatograms of gangliosides which were overlayed with 125I-labelled toxins showed that binding of both toxins was largely restricted to ganglioside GM1. However, 125I-heat-labile toxin was able to bind to brush-border galactoproteins resolved by SDS/polyacrylamide-gel electrophoresis and transferred to nitrocellulose.     

LACTOPEROXIDASE-COUPLED IODINATION OF BOVINE CHROMAFFIN GRANULES

Abstract— Chromaffin granules were iodinated with lactoperoxidase at either their external or internal membrane surfaces. When iodination of internal soluble granule proteins and membrane phospholipids was minimized, the majority of the membrane proteins, including the 83,000 component, were iodinated. Components with molecular weights 63,000, 61,000, 51,000, 44,000, 32,000, 26,000 and 19,000 had a higher ¹²⁵I specific activity than did the other membrane components, suggesting they were more accessible at the outer membrane surface than were the other components. In the presence of detergent, the iodination of all membrane components was increased more than 10-fold; the incorporation of ¹²⁵I was now similar to their Coomassie Blue staining intensity in disc gels, indicating that all components were equally accessible to lactoperoxidase. In the presence of detergent, iodine incorporation into the MW 83,000 and 16,000 components was stimulated approx 100-fold.
The MW 83,000, 63,000, 61,000 and 37,000 components incorporated significant amounts of ¹²⁵I when granule membranes were iodinated from their internal surface, suggesting these components have a portion of their polypeptide chain accessible at the inner membrane surface. Thus the MW 83,000 component, which we identified as dopamine β hydroxylase, and the MW 63,000/61,000 components, which are part of the membrane ATPase, can be iodinated from both membrane surfaces. This would suggest that these are transmembrane proteins. However, the major portion of all the proteins in this membrane were inaccessible to lactoperoxidase at either membrane surface.     

Isolation and characterization of the plasma membrane of L-1210 cells. Iodination as a marker for the plasma membrane.

Mouse leukemia L-1210 cells were iodinated with 125I; this permitted the development of a method for the isolation of the plasma membranes. These show a 10- to 16-fold increase in the specific activity of 125I over that of the cell homogenate and a 20-fold increase in the specific activities of 5'-nucleotidase and alkaline phosphatase; 20-fold increase in the specific activities of 5'-nucleotidase and alkaline phosphatase; no mitochondrial or microsomal marker enzyme activities were detected. Sodium dodecyl sulfate gel electrophoresis of the plasma membranes shows approx. 40 peptides with molecular weights ranging from 10 000 to over 200 000; a polypeptide (Mr 50 000) predominates. Of 13 iodinated surface membrane proteins, the major radioactive peptide has a molecular weight of 85 000. The importance of the selection of the appropriate gel system for the analysis of membrane proteins is emphasized.     

Relationship between trinitrophenyl and H-2 antigens on trinitrophenyl-modified spleen cells. I. H-2 antigens on cells treated with trinitrobenzene sulfonic acid are derivatized.

Spleen cells were treated with TNBS in order to determine if cell surface H-2 antigens are derivatized with TNP. By labeling the cell membrane of the TNP-modified cells with 125I, followed by detergent lysis and immune precipitation with anti-TNP, it was determined that no H-2 antigenic activity remained in the supernatant. Further, by the use of an antibody-induced antigen redistribution assay it was found that previous exposure to TNP-modified cells to anti-TNP in the absence of complement rendered these cells resistant to lysis by anti-H-2 in the presence of complement. Together these data indicate that at the concentration of TNBS used for modification, H-2 antigens are derivatized with TNP. However, in addition to H-2, other proteins including immunoglobulin were also derivatized with TNP. Anti-TNP cytotoxic effector cells were blocked from their cytotoxic activity by anti-TNP antiserum. These data indicate that TNP directly couples to H-2 antigens on the cell surface of TNP-modified cells and that TNP is associated with the antigenic determinant that the cytotoxic T cell recognizes.     

Alterations in the cell surface proteins of Drosophila during morphogenesis

Summary Unevaginated and evaginated Drosophila imaginal discs were surface-labeled with ¹²⁵I. Relative labeling was greater in eleven peptides and lower in three peptides of evaginated discs compared to unevaginated discs. These results are compared to the effects of 20-hydroxyecdysone (20-HOE) on metabolic labeling of membrane proteins fractionated from imaginal discs, and on cell surface labeling of a hormone-responsive Drosophila tissue culture line. A group of ³⁵S-methionine labeled membrane fraction peptides whose metabolic labeling is 20-HOE dependent have isoelectric points and apparent molecular weights very similar to those of a group of proteins only labeled in iodinated evaginated discs, supporting the conclusion that these are hormone-dependent, cell surface proteins (Rickoll and Fristrom 1983). Based upon two-dimensional gel electrophoretic and immunological criteria three of the proteins showing increased labeling in evaginated discs are related to three proteins induced by 20-HOE in tissue culture cells. Two different subsets of radiolabeled peptides were observed in the imaginal discs based upon detergent solubility. Some of the proteins which are soluble in NP-40 plus urea but insoluble in NP-40 alone may be localized in the basal lamina of the imaginal discs, a structure which labels heavily with ¹²⁵I and is lacking in tissue culture cells. In discs, the majority of hormone-dependent changes in radiolabeled peptides were seen in the fraction solubilized by NP-40 and urea with a sulfhydryl reducing agent, while in tissue culture cells, the majority of differences is seen in the fraction solubilized by NP-40 only. We speculate that these proteins may be involved in similar processes, e.g., cell rearrangement, that occur during both disc morphogenesis and 20-HOE induced aggregation in tissue culture cells.This work was supported by grants CD-205 from the American Cancer Society, RR08132 from NIH to C.A.P. and GM 19937 from NIH to J.W.F.     

The activation of adenylate cyclase from small intestinal epithelium by cholera toxin

ADP-ribosylation of membrane proteins from rabbit small intestinal epithelium was investigated following incubation of membranes with [32P]NAD and cholera toxin. Cholera toxin catalyzes incorporation of 32P into three proteins of 40 kDA, 45 kDa and 47 kDa located in the brush-border membrane. In contrast, basal lateral membranes do not contain any protein which becomes labeled in a toxin-dependent manner when incubated with cholera toxin and [32P]NAD. The modification of membrane proteins from brush border occurred in spite of the virtual absence in these membranes of adenylate cyclase activatable either by cholera toxin, vasoactive intestinal peptide (VIP) or fluoride. The three agents activated adenylate cyclase when crude plasma membrane were used. Cholera toxin activated fivefold at 10 micrograms/ml. Vasoactive intestinal peptide activated at concentrations from 10-300 nM, the maximal stimulation being sixfold. Fluoride activated 10-fold at 10 mM. When basal lateral membranes were assayed for adenylate cyclase it was found that, with respect to the crude membranes, the specific activity of fluoride-activated enzyme was 3.3-fold higher, VIP stimulated enzyme was maintained while cholera-toxin-stimulated enzyme showed half specific activity. Moreover, while fluoride stimulated ninefold and VIP stimulated fivefold, cholera toxin only stimulated twofold at the highest concentration. The results suggest that the activation by cholera toxin of adenylate cyclase located at the basal lateral membrane requires ADPribosylation of proteins in the brush border membrane.     

Analytical approach for the extraction of recombinant membrane viral glycoprotein from stably transfected Drosophila melanogaster cells

Parameters for storage, lysis and concentration of Drosophila melanogaster Schneider 2 (S2AcRVGP) cells expressing the recombinant rabies virus glycoprotein (RVGP) were studied with regard to RVGP quantification by ELISA, for productivity evaluation and future purification. Lysis buffers were formulated with Tris, NaCl, glycerol, EDTA, KCl, Na(2)PO(4), MgCl(2), PMSF and NP-40 or CHAPS. S2AcRVGP cells (10(7) cells at the exponential growth phase) were frozen at -20 degrees C as a dry pellet, suspended in buffer (B) formulations or after treatment with lysis buffer (LB) formulations. They were then thawed as cell pellets or with B formulations or PBS at 4 degrees C or at room temperature and then lysed with LB formulations. For RVGP quantification by ELISA, a protocol was chosen of cell preparation including cell freezing as dry pellet, cell thawing at 4 degrees C with B4 (Tris, NaCl, MgCl(2), PMSF) and cell lysis with the LB4 (B4 + NP-40) since it fulfilled requirements of high RVGP detection, and was easily performed with mixtures freezing quickly, and a cost-saving LB formulation could be used. Using these established conditions, we examined the optimal cell concentration for RVGP quantification by ELISA. Results showed that an increase in the RVGP detection (from 62.5 to 1083 ng/10(7) cells) paralleled a decrease in the cell number (3 x 10(7) - 10(5) cells) used. The NP-40 concentration present in the LB4 was further investigated as a function of the cell number used for sample preparation. Previous results were confirmed indicating that higher NP-40 concentrations led to a decreased detection of RVGP. Altogether our data clearly pointed out the crucial effects of cell freeze, thaw, lysis and concentration on immune detection of recombinant membrane glycoproteins and can be useful as a guideline for sample preparation for this purpose.     

Adenylate cyclase from rabbit small intestine: activation by cholera toxin and interaction with calcium

The stimulation of adenylate cyclase in various fractions of plasma membranes from rabbit small intestinal epithelium has been studied. In crude plasma membranes cholera toxin activated 5-fold at 10 micrograms/ml; vasoactive intestinal peptide (VIP) activated at concentration from 10(-8) to 10(-7) M, the maximal stimulation being 6-fold. Fluoride activated 10-fold at 10 mM. VIP-stimulated enzyme was inhibited by Ca2+ concentrations in the micromolar range. In the presence of calmodulin a biphasic response was obtained. At low Ca2+ concentration (4 x 10(-9)-6 x 10(-8) M) the enzyme was activated. As the Ca2+ concentration was increased the enzyme was concomitantly inhibited. We have investigated the mechanism by which cholera toxin activates intestinal adenylate cyclase. We have found that cholera toxin catalyzed incorporation of 32P into proteins located in the brush-border membrane whose molecular weights are in the range of 40-45kDa. These membranes bind [3H]GTP with a Kd of 1.8 x 10(-7) M. In contrast, basal lateral membranes do not contain any protein which becomes labeled in a toxin-dependent manner when incubated with cholera toxin and [32P]NAD. The modification of brush-border membrane protein occurred in spite of the absence of adenylate cyclase in these membranes. Adenylate cyclase in basal lateral membranes was poorly activated by cholera toxin as compared to crude plasma membranes. On the other hand, the ability of VIP and fluoride to activate the enzyme was enhanced in basal lateral membranes with respect to crude membranes. The results are discussed in relation to the mechanism by which cholera toxin activates adenylate cyclase in intact intestinal cells.     

Accessibility of plasma membrane antigens

Serological techniques applied to intact cells register only those antigens of the plasma membrane that are exposed at the cell surface and are therefore accessible to antibody. Solubilization of the plasma membrane by detergent, used in the conventional surface-iodination immunoprecipitation technique, renders other plasma membrane antigens accessible. We have shown this by using a modified version of the technique in which lysis with detergent is postponed until after the cells have been reacted with antibody. Comparison of the conventional and modified methods confirms that the plasma membrane glycoprotein gp70 has antigen that is not exposed on the intact cells as well as accessible antigen, for example, G_IX. The modified surface-iodination immunoprecipitation method is useful for distinguishing cell-surface antigens from plasma membrane antigens that normally are not accessible. This is exemplified by the fact that standard anti-TL and anti-X.1 sera identify gp70 antigen in the plasma membrane that is registered by the conventional, but not by the modified method.Abbreviations used in this paper are anti - BALB BALB/c - gp70 MuLV envelope glycoprotein of molecular weight about 70,000 daltons, sometimes referred to as gp69/71 - gs group-specific - ¹²⁵I-imm-pptn surface labeling of viable cells with¹²⁵I followed by immunoprecipitation analysis - Ig immunoglobulin - MuLV murine leukemia virus - NMS normal mouse serum - PAGE polyacrylamide gel electrophoresis - PBS Dulbecco's phosphate-buffered saline, Ca⁺⁺- and Mg⁺⁺-free - SDS sodium dodecyl sulfate - TL thymus leukemia antigen     

Cholera toxin differentially decreases membrane levels of alpha and beta subunits of G proteins in NG108-15 cells

Treatment of NG108-15 neuroblastoma x glioma cells (24 h) with cholera toxin (0.1-10 micrograms/ml) resulted in a concentration-dependent reduction of the membrane levels of subunits of GTP-binding regulatory proteins (G proteins), as determined by quantitative immunoblot procedures. The extent of reduction differed for different types of subunits: the levels of Go alpha and G beta 1 were reduced by 40-50%, whereas those of G alpha common immunoreactivity and Gi2 alpha were only reduced by 10-20% following treatment with 10 micrograms/ml cholera toxin. This effect of the toxin could not be mimicked by incubation with the resolved B oligomer of cholera toxin, nor by exposure of cells to agents able to raise the intracellular levels of cAMP. Basal adenylate cyclase was stimulated in a biphasic manner by cholera toxin, being stimulated at low concentrations (0.01-10 ng/ml) and then decreased at high (0.1-10 micrograms/ml) concentrations. Thus, the down regulation of G-protein subunits produced by cholera toxin requires its (ADP-ribosyl)transferase activity but does not result from a cAMP-mediated mechanism. The toxin-mediated decrease of Go alpha in the membrane was correlated with a diminution of opioid-receptor-mediated stimulation of high-affinity GTPase activity, suggesting that opioid receptors interact with Go in native membranes of NG108-15 cells. Northern-blot analysis of cytoplasmic RNA prepared from cells treated with cholera toxin showed that the levels of mRNA coding for G beta 1 did not change. Thus, the cholera-toxin-induced decrease of G-protein subunits may not result from an alteration in mRNA levels, but may involve a direct effect of the toxin on the process of insertion and/or clearance of G proteins into and/or from the membrane. These data indicate that cholera toxin, besides catalyzing the ADP-ribosylation of Gs and Gi/Go types of G proteins, can also reduce the steady state levels of Go alpha and G beta 1 subunits in the membrane and thus alter by an additional mechanism the function of inhibitory receptor systems.