The isolation of plasma membrane from protoplasts of soybean suspension cultures

1977 ◽  
Vol 24 (1) ◽  
pp. 295-310
Author(s):  
D.W. Galbraith ◽  
D.H. Northcote
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Membrane Fraction ◽  
Sucrose Gradient ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Membrane Systems ◽  
Enzymic Digestion ◽  
Nadh Cytochrome

A procedure for the isolation of plasma membranes from protoplasts of suspension-cultured soybean is described. Protoplasts were prepared by enzymic digestion of the cell wall and the plasma membrane was labelled with radioactive diazotized sulphanilic acid. The membrane systems from broken protoplasts were separated by continuous isopycnic sucrose gradient centrifugation. Radioactivity was localized in a band possessing a buoyant density of 1–14 g ml-1. The activities of NADPH- and NADH-cytochrome c reductase, fumarase, Mg2+-ATPase, IDPase and acid phosphodiesterase in the various regions of the density gradient were determined. A plasma membrane fraction was selected which was relatively uncontaminated with membranes derived from endoplasmic reticulum, tonoplasts and mitochondria. The results indicated that Mg2+-ATPase and possibly acid phosphodiesterase were associated with the plasma membrane.

Subcellular location of phage infection protein (Pip) inLactococcus lactis

2006 ◽  
Vol 52 (7) ◽  
pp. 664-672 ◽  
Author(s):  
Duane T Mooney ◽  
Monica Jann ◽  
Bruce L Geller
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Lactococcus Lactis ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Subcellular Location ◽  
Recognition Site ◽  
Phage Infection ◽  
Sucrose Density Gradient Centrifugation ◽  
Membrane Spanning

The amino acid sequence of the phage infection protein (Pip) of Lactococcus lactis predicts a multiple-membrane-spanning region, suggesting that Pip may be anchored to the plasma membrane. However, a near-consensus sortase recognition site and a cell wall anchoring motif may also be present near the carboxy terminus. If functional, this recognition site could lead to covalent linkage of Pip to the cell wall. Pip was detected in both plasma membranes and envelopes (plasma membrane plus peptidoglycan) isolated from the wild-type Pip strain LM2301. Pip was firmly attached to membrane and envelope preparations and was solubilized only by treatment with detergent. Three mutant Pip proteins were separately made in which the multiple-membrane-spanning region was deleted (Pip-Δmmsr), the sortase recognition site was converted to the consensus (Pip-H841G), or the sortase recognition site was deleted (Pip-Δ6). All three mutant Pip proteins co-purified with membranes and could not be solubilized except with detergent. When membranes containing Pip-Δmmsr were sonicated and re-isolated by sucrose density gradient centrifugation, Pip-Δmmsr remained associated with the membranes. Strains that expressed Pip-H841G or Pip-Δ6 formed plaques with near unit efficiency, whereas the strain that expressed Pip-Δmmsr did not form plaques of phage c2. Both membranes and cell-free culture supernatant from the strain expressing Pip-Δmmsr inactivated phage c2. These results suggest that Pip is an integral membrane protein that is not anchored to the cell wall and that the multiple-membrane-spanning region is required for productive phage infection but not phage inactivation.Key words: phage infection protein, Pip, Lactococcus lactis, subcellular location.

Downregulation of surface sodium pumps by endocytosis during meiotic maturation of Xenopus laevis oocytes

1990 ◽  
Vol 258 (1) ◽  
pp. C179-C184 ◽  
Author(s):  
G. Schmalzing ◽  
P. Eckard ◽  
S. Kroner ◽  
H. Passow
Keyword(s):  
Plasma Membrane ◽  
Xenopus Laevis ◽  
Sucrose Gradient ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sodium Dodecyl ◽  
Meiotic Maturation ◽  
Xenopus Laevis Oocytes ◽  
Sodium Pumps

During meiotic maturation, plasma membranes of Xenopus laevis oocytes completely lose the capacity to transport Na and K and to bind ouabain. To explore whether the downregulation might be due to an internalization of the sodium pump molecules, the intracellular binding of ouabain was determined. Selective permeabilization of the plasma membrane of mature oocytes (eggs) by digitonin almost failed to disclose ouabain binding sites. However, when the eggs were additionally treated with 0.02% sodium dodecyl sulfate (SDS) to permeabilize inner membranes, all sodium pumps present before maturation were recovered. Phosphorylation by [gamma-32P]ATP combined with SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography showed that sodium pumps were greatly reduced in isolated plasma membranes of eggs. According to sucrose gradient fractionation, maturation induced a shift of sodium pumps from the plasma membrane fraction to membranes of lower buoyant density with a protein composition different from that of the plasma membrane. Endocytosed sodium pumps identified on the sucrose gradient from [3H]ouabain bound to the cell surface before maturation could be phosphorylated with inorganic [32P]phosphate. The findings suggest that downregulation of sodium pumps during maturation is brought about by translocation of surface sodium pumps to an intracellular compartment, presumably endosomes. This contrasts the mechanism of downregulation of Na-dependent cotransport systems, the activities of which are reduced as a consequence of a maturation-induced depolarization of the membrane without a removal of the corresponding transporter from the plasma membrane.

Enrichment of sulfogalactosylalkylacylglycerol in a plasma membrane fraction from adult rat testis

1980 ◽  
Vol 58 (10) ◽  
pp. 1230-1239 ◽  
Author(s):  
Margaret A. Shirley ◽  
Harry Schachter
Keyword(s):  
Plasma Membrane ◽  
Membrane Fraction ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Membrane Material ◽  
Radioactive Label ◽  
Rat Testis ◽  
Plasma Membrane Fraction ◽  
Adult Rat ◽  
Dna And Rna

Adult rat testis homogenates were fractionated by differential centrifugation followed by two discontinuous gradient centrifugation steps under identical conditions except for the absence of digitonin in the first gradient and the presence of 0.03% digitonin in the second gradient. The first gradient centrifugation yielded a membrane fraction enriched 28.8-fold in 5′-nucleotidase, 21.5-fold in UDP-Gal:GlcNAc galactosyltransferase and 18.6-fold in UDP-GlcNAc:α-D-mannoside N-acetylglucosaminyltransferase. Repeat centrifugation of this membrane fraction in the presence of digitonin resulted in the sedimentation of most of the membrane material to a denser level of the gradient; this material was enriched 32.1-fold in 5′-nucleotidase but only 1.9-fold in galactosyltransferase and 8.4-fold in N-acetylglucosaminyltransferase. The plasma membrane fraction was shown to be free of glucose-6-phosphatase, succinate dehydrogenase, β-N-acetylglucosaminidase, DNA, and RNA. The fraction therefore appears to be enriched in plasma membrane but relatively free of Golgi membrane contamination, as indicated by the relatively low levels of glycosyltransferases, and of contamination by other organelles. The testicular cells which contribute plasma membrane to this fraction have not yet been definitively identified; the contribution by Sertoli cells is particularly difficult to assess since these cells have been reported to be enriched in 5′-nucleotidase. However, sulfogalactosylalkylacylglycerol (SGG), a lipid previously shown to be present primarily in primary spermatocytes, spermatids, and spermatozoa, was enriched 33.1-fold in the plasma membrane fraction; this finding as well as experiments with [36S]sulfate-labeled sulfogalactosylalkylacylglycerol at various times after injection of radioactive label have indicated that both spermatocytes and spermatids were contributing SGG-rich membrane material to our plasma membrane preparation. This membrane material is most probably derived from the plasma membranes of the spermatocytes and spermatids.

Plasma membranes from Physarum polycephalum Plasmodia: purification, characterization, and comparison with amoebae plasma membranes

1984 ◽  
Vol 62 (9) ◽  
pp. 831-836 ◽  
Author(s):  
Dominick Pallotta ◽  
Anne Barden ◽  
Rémi Martel ◽  
Josée Kirouac-Brunet ◽  
François Bernier ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Gel Electrophoresis ◽  
Membrane Fraction ◽  
Physarum Polycephalum ◽  
Sucrose Gradient ◽  
Plasma Membranes ◽  
Two Dimensional ◽  
Two Phase ◽  
Crude Membrane Fraction

Two methods are described for preparing plasma membranes from Physarum polycephalum plasmodia. In the first method Plasmodia were broken in homogenizing medium (250 mM sucrose, 1 mM ZnCl2, 10 mM Tris (pH 8.0)), and the nuclei were removed by low speed centrifugation. A crude membrane fraction was collected and the membranes were purified by centrifugation in a linear 30–65% (w/w) sucrose gradient. The membranes sedimented in a single band at a density of 1.26 g/cm3. In the second method the plasmodia were broken in the same homogenizing medium and a crude membrane fraction was prepared by differential centrifugation. The membranes were purified by the Dextran – polyethylene glycol aqueous two-phase system. Membranes from both preparations were found by enzymatic assay and by electron microscopy to be virtually free of contaminating cell organelles. Analyses by one- and two-dimensional polyacrylamide gel electrophoresis showed a great similarity in the proteins from two-phase and sucrose gradient prepared membranes, indicating that either technique can be used to prepare membranes for protein studies. An analysis by two-dimensional gel electrophoresis of plasma membrane proteins from amoebae and plasmodia showed differences in some of the major proteins, indicating that changes occur in plasma membrane proteins during differentiation.

scholarly journals Purification and characterization of two plasma membrane domains from ejaculated bull spermatozoa.

1986 ◽  
Vol 102 (5) ◽  
pp. 1813-1825 ◽  
Author(s):  
I de Curtis ◽  
G Fumagalli ◽  
N Borgese
Keyword(s):  
Plasma Membrane ◽  
Membrane Fraction ◽  
Sucrose Gradient ◽  
Plasma Membranes ◽  
Polyclonal Antibodies ◽  
Binding Activity ◽  
Equilibrium Density ◽  
Anterior Portion ◽  
Plasma Membrane Fraction ◽  
Scattering Material

Plasma membranes were detached from ejaculated bull spermatozoa by a brief sonication in a moderately hypotonic medium, and the released plasma membranes were partially purified by differential centrifugation. The resulting fraction was enriched 8- and 15-fold in alkaline phosphatase and 5' nucleotidase activities, respectively, compared with the starting sonicated spermatozoa. This total plasma membrane fraction was separated into two distinct fractions by equilibrium density centrifugation on a continuous linear sucrose gradient. Two peaks of light scattering material were formed at densities of 1.117 and 1.148 g/ml. The denser peak contained most of the protein of the plasma membrane fraction, whereas nearly all the concanavalin A binding activity was found in the lighter peak. The two bands had distinctly different polypeptide compositions when analyzed by SDS PAGE. Polyclonal antibodies were raised in rabbits against a major integral membrane glycoprotein of each fraction (Mr of 92,000 in the light peak and 98,000 in the dense peak). The two antigens were detected on the surface of intact spermatozoa by indirect immunofluorescence microscopy. The 92-kD protein (present in the lighter band) was detected only on the plasma membrane of the acrosomal and anterior postacrosomal regions of the head. The 98-kD antigen, present in the heavier band, was localized to the surface of the postacrosomal region of the head, to the principal piece of the tail, and to the connecting piece between the head and tail. The exclusive localization of the 92-kD polypeptide to the surface of the anterior portion of the head was confirmed by immunoelectron microscopy. These data show that the two fractions isolated on the sucrose gradient originate from different regions of the sperm cell plasma membrane.

scholarly journals Immunocytochemical localization and biochemical characterization of a novel plasma membrane-associated, neutral pH optimum α-l-fucosidase from rat testis and epididymal spermatozoa

1996 ◽  
Vol 318 (3) ◽  
pp. 821-831 ◽  
Author(s):  
Manuel AVILÉS ◽  
Irene ABASCAL ◽  
José Angel MARTÍNEZ-MENÁRGUEZ ◽  
María Teresa CASTELLS ◽  
Sheri R. SKALABAN ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Gradient Centrifugation ◽  
Light And Electron Microscopy ◽  
Enzymic Activity ◽  
Epididymal Sperm ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Neutral Ph

1. Immunocytochemical and biochemical techniques have been used to localize and characterize a novel plasma membrane-associated, neutral-pH-optimum α-l-fucosidase from rat spermatozoa. Light and electron microscopy specifically localized the fucosidase on the plasma membrane of the convex region of the principal segment of testicular and cauda epididymal sperm heads. Immunoreactivity for α-l-fucosidase was also detected in the Golgi apparatus of spermatocytes and spermatids but no immunoreactivity was observed in the acrosome. 2. Fractionation of epididymal sperm homogenates indicated that over 90% of the α-l-fucosidase activity was associated with the 48000 g pellet. This pellet-associated activity could be solubilized with 0.5 M NaCl but not with 0.5% Triton X-100, suggesting that fucosidase is peripherally associated with membranes. Sucrose-density-gradient centrifugation of sperm homogenates indicated that fucosidase was enriched in the plasma membrane-enriched fraction. Analysis of α-l-fucosidase on intact epididymal sperm indicated that the enzyme was active, displayed linear kinetics and had a pH–activity curve (with an optimum near 7) which was comparable to that of fucosidase from epididymal sperm extracts. These results further suggest that fucosidase is associated with plasma membranes, and that its active site is accessible to fucoconjugates. Evidence that most of the fucosidase is associated with the exterior of the plasma membrane came from studies in which intact sperm had fucosidase activity comparable to that of sperm sonicates, and from studies in which approx. 90% of the fucosidase activity on intact sperm could be released from the sperm by gentle shaking with 0.5 M NaCl. Isoelectric focusing indicated that the NaCl-solubilized epididymal sperm fucosidase appears to have one major and one minor isoform with pIs near 7.2 and 5.2, respectively. SDS/PAGE and Western blotting indicated that the NaCl-solubilized extract of epididymal sperm contains two protein bands of 54 and 50 kDa which were highly immunoreactive with the IgG fraction of anti-fucosidase antibodies. Although the function of the novel sperm fucosidase is not known, its specific localization to the plasma membrane of the region of the rat sperm head involved in sperm–egg binding and its high enzymic activity at neutral pH on intact sperm suggest that this enzyme may have a role in sperm–egg interactions.

Reduction of Selenite and Detoxification of Elemental Selenium by the Phototrophic BacteriumRhodospirillum rubrum

1999 ◽  
Vol 65 (11) ◽  
pp. 4734-4740 ◽  
Author(s):  
J. Kessi ◽  
M. Ramuz ◽  
E. Wehrli ◽  
M. Spycher ◽  
R. Bachofen
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Cell Density ◽  
Culture Medium ◽  
Rhodospirillum Rubrum ◽  
Buoyant Density ◽  
Elemental Selenium ◽  
Selenite Reduction ◽  
Final Cell Density ◽  
Cell Densities

ABSTRACT The effect of selenite on growth kinetics, the ability of cultures to reduce selenite, and the mechanism of detoxification of selenium were investigated by using Rhodospirillum rubrum. Anoxic photosynthetic cultures were able to completely reduce as much as 1.5 mM selenite, whereas in aerobic cultures a 0.5 mM selenite concentration was only reduced to about 0.375 mM. The presence of selenite in the culture medium strongly affected cell division. In the presence of a selenite concentration of 1.5 mM cultures reached final cell densities that were only about 15% of the control final cell density. The cell density remained nearly constant during the stationary phase for all of the selenite concentrations tested, showing that the cells were not severely damaged by the presence of selenite or elemental selenium. Particles containing elemental selenium were observed in the cytoplasm, which led to an increase in the buoyant density of the cells. Interestingly, the change in the buoyant density was reversed after selenite reduction was complete; the buoyant density of the cells returned to the buoyant density of the control cells. This demonstrated that R. rubrum expels elemental selenium across the plasma membrane and the cell wall. Accordingly, electron-dense particles were more numerous in the cells during the reduction phase than after the reduction phase.

scholarly journals Plasma membrane association of Acanthamoeba myosin I.

1989 ◽  
Vol 109 (4) ◽  
pp. 1519-1528 ◽  
Author(s):  
H Miyata ◽  
B Bowers ◽  
E D Korn
Keyword(s):  
Plasma Membrane ◽  
Binding Site ◽  
Heavy Chain ◽  
Membrane Fraction ◽  
Plasma Membranes ◽  
Actin Binding ◽  
Plasma Membrane Fraction ◽  
Myosin I ◽  
Membrane Bound ◽  
Actin Binding Site

Myosin I accounted for approximately 2% of the protein of highly purified plasma membranes, which represents about a tenfold enrichment over its concentration in the total cell homogenate. This localization is consistent with immunofluorescence analysis of cells that shows myosin I at or near the plasma membrane as well as diffusely distributed in the cytoplasm with no apparent association with cytoplasmic organelles or vesicles identifiable at the level of light microscopy. Myosin II was not detected in the purified plasma membrane fraction. Although actin was present in about a tenfold molar excess relative to myosin I, several lines of evidence suggest that the principal linkage of myosin I with the plasma membrane is not through F-actin: (a) KI extracted much more actin than myosin I from the plasma membrane fraction; (b) higher ionic strength was required to solubilize the membrane-bound myosin I than to dissociate a complex of purified myosin I and F-actin; and (c) added purified myosin I bound to KI-extracted plasma membranes in a saturable manner with maximum binding four- to fivefold greater than the actin content and with much greater affinity than for pure F-actin (apparent KD of 30-50 nM vs. 10-40 microM in 0.1 M KCl plus 2 mM MgATP). Thus, neither the MgATP-sensitive actin-binding site in the NH2-terminal end of the myosin I heavy chain nor the MgATP-insensitive actin-binding site in the COOH-terminal end of the heavy chain appeared to be the principal mechanism of binding of myosin I to plasma membranes through F-actin. Furthermore, the MgATP-sensitive actin-binding site of membrane-bound myosin I was still available to bind added F-actin. However, the MgATP-insensitive actin-binding site appeared to be unable to bind added F-actin, suggesting that the membrane-binding site is near enough to this site to block sterically its interaction with actin.

scholarly journals Purification and characterization of the human neutrophil NADH- cytochrome b5 reductase

Blood ◽  
1985 ◽  
Vol 66 (3) ◽  
pp. 673-678 ◽  
Author(s):  
AI Tauber ◽  
J Wright ◽  
FK Higson ◽  
SA Edelman ◽  
DJ Waxman
Keyword(s):  
Plasma Membrane ◽  
Human Neutrophil ◽  
Membrane Fraction ◽  
Cytochrome B5 ◽  
Sodium Dodecyl ◽  
High Specificity ◽  
Biochemical Properties ◽  
Human Neutrophils ◽  
Cytochrome B5 Reductase ◽  
Nadh Cytochrome

Abstract NADH-cytochrome b5 reductase is the predominant NADH-diaphorase found in the human neutrophil (Blood 62:152, 1983). Although this reductase segregates with the light membranes of nitrogen-cavitated neutrophils separated on Percoll gradients (which include the plasma membrane markers alkaline phosphatase and NADPH-oxidase), it is approximately 95% excluded from plasma membrane-enriched phagocytic vacuoles. The reductase constitutes approximately 5% of the light membrane fraction FAD-flavoprotein (14.8 +/- 5.5 pmol/mg protein) and was found in equimolar concentration with a high potential b cytochrome also present in this light membrane fraction and tentatively identified as cytochrome b5. Isolation of the reductase from human neutrophils was accomplished by Triton X-114 solubilization of the light Percoll gradient membranes, followed by temperature-dependent phase separation and then affinity chromatography on AMP-Sepharose. The active preparation contained 1.3 mol FAD/mol protein, migrated on sodium dodecyl sulfate-polyacrylamide gels as a single band corresponding to an apparent mol wt of 45,000 daltons, exhibited a pl of 5.7 on chromatofocusing and was obtained in greater than 70% yield, with an overall purification of almost 900-fold. The purified enzyme was characterized by a high specificity for NADH as electron donor (Km = 6.4 mumol/L v Km greater than 1.6 mmol/L for NADPH) and exhibited a maximal turnover of ca. 30,000 min-1 at 22 degrees C with either ferricyanide or cytochrome b5 (Km = 10 nmol/L) as electron acceptor. Although the physical characterization and biochemical properties described here demonstrate that this neutrophil NADH b5 reductase is similar to the corresponding liver and erythrocyte enzymes, its unique function in the neutrophil has yet to be determined.

scholarly journals Isolation and characterization of monoclonal antibodies directed against plant plasma membrane and cell wall epitopes: identification of a monoclonal antibody that recognizes extensin and analysis of the process of epitope biosynthesis in plant tissues and cell cultures.

1988 ◽  
Vol 107 (1) ◽  
pp. 163-175 ◽  
Author(s):  
D J Meyer ◽  
C L Afonso ◽  
D W Galbraith
Keyword(s):  
Monoclonal Antibody ◽  
Cell Wall ◽  
Monoclonal Antibodies ◽  
Plasma Membrane ◽  
Solid Phase ◽  
Culture Media ◽  
Gradient Centrifugation ◽  
Cell Suspension Cultures ◽  
Isolation And Characterization

Membranes from tobacco cell suspension cultures were used as antigens for the preparation of monoclonal antibodies. Use of solid phase and indirect immunofluorescence assays led to the identification of hybridomas producing antibodies directed against cell surface epitopes. One of these monoclonal antibodies (11.D2) was found to recognize a molecular species which on two-dimensional analysis (using nonequilibrium pH-gradient electrophoresis and SDS-PAGE) was found to have a high and polydisperse molecular mass and a very basic isoelectric point. This component was conspicuously labeled by [3H]proline in vivo. The monoclonal antibody cross-reacted with authentic tomato extensin, but not with potato lectin nor larch arabinogalactan. Use of the monoclonal antibody as an immunoaffinity reagent allowed the purification of a tobacco glycoprotein which was identical in amino acid composition to extensin. Finally, immunocytological analyses revealed tissue-specific patterns of labeling by the monoclonal antibody that were identical to those observed with a polyclonal antibody raised against purified extensin. We have concluded that monoclonal antibody 11.D2 recognizes an epitope that is carried exclusively by extensin. Analysis of cellular homogenates through differential and isopycnic gradient centrifugation revealed that biosynthesis of the extensin epitope was found on or within the membranes of the endoplasmic reticulum, Golgi region and plasma membrane. This result is consistent with the progressive glycosylation of the newly-synthesized extensin polypeptide during its passage through a typical eukaryotic endomembrane pathway of secretion. The 11.D2 epitope was not found in protoplasts freshly isolated from leaf tissues. However, on incubation of these protoplasts in appropriate culture media, biosynthesis of the epitope was initiated. This process was not impeded by the presence of chemicals that are reported to be inhibitors of cell wall production or of proline hydroxylation.

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