scholarly journals Plasma-membrane location of phosphatidylinositol hydrolysis in rabbit neutrophils stimulated with formylmethionyl-leucylphenylalanine

1982 ◽  
Vol 208 (3) ◽  
pp. 801-808 ◽  
Author(s):  
J P Bennett ◽  
S Cockcroft ◽  
A H Caswell ◽  
B D Gomperts
Keyword(s):  
Plasma Membrane ◽  
High Speed ◽  
Plasma Membranes ◽  
Diazonium Salt ◽  
Gradient Centrifugation ◽  
Particulate Fraction ◽  
Intact Cells ◽  
Hydrolysis Of ◽  
High Speed Supernatant ◽  
Sucrose Step Gradient

Rabbit peritoneal neutrophils, disrupted by sonication, were separated into three subcellular fractions by sucrose-step-gradient centrifugation and these were analysed with respect to biochemical markers. They comprised a high-speed supernatant containing the cytosol, a light particulate fraction enriched in Golgi and plasma membranes and a heavy particulate fraction enriched in granules and nuclei. The light particulate fraction was further separated into its components, which were identified as Golgi membranes (galactosyltransferase activity) and plasma membranes ((radioactivity derived from labelling intact cells with [125I]di-iodosulphanilic acid diazonium salt and [3H]formylmethionyl-leucylphenylalanine ([3H]fMet-Leu-Phe) binding)). In cells prelabelled with [3H]glycerol, the hydrolysis of phosphatidylinositol due to cell stimulation with fMet-Leu-Phe (10 nM) was shown to occur in the light particulate fraction. The [32P]Pi-labelling of phosphatidate, which is an early consequence of phosphatidylinositol hydrolysis, also occurred in this fraction. Analytical sucrose-gradient centrifugation of the light particulate fraction showed that the stimulated increment in [32P]phosphatidate (and thus by implication the initial phosphatidylinositol breakdown) was localized in the 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.

scholarly journals Fluorescent, short-chain C6-NBD-sphingomyelin, but not C6-NBD-glucosylceramide, is subject to extensive degradation in the plasma membrane: implications for signal transduction related to cell differentiation

1995 ◽  
Vol 309 (3) ◽  
pp. 905-912 ◽  
Author(s):  
J W Kok ◽  
T Babia ◽  
K Klappe ◽  
D Hoekstra
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
Cell Differentiation ◽  
Plasma Membranes ◽  
Cell Types ◽  
Short Chain ◽  
Synthetic Activity ◽  
Intact Cells ◽  
Ht29 Cells ◽  
Extensive Degradation

The involvement of the plasma membrane in the metabolism of the sphingolipids sphingomyelin (SM) and glucosylceramide (GlcCer) was studied, employing fluorescent short-chain analogues of these lipids, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]hexanoylsphingosylphosphorylcholine (C6-NBD-SM), C6-NBD-GlcCer and their common biosynthetic precursor C6-NBD-ceramide (C6-NBD-Cer). Although these fluorescent short-chain analogues are metabolically active, some caution is to be taken in view of potential changes in biophysical/biochemical properties of the lipid compared with its natural counterpart. However, these short-chain analogues offer the advantage of studying the lipid metabolic enzymes in their natural environment, since detergent solubilization is not necessary for measuring their activity. These studies were carried out with several cell types, including two phenotypes (differing in state of differentiation) of HT29 cells. Degradation and biosynthesis of C6-NBD-SM and C6-NBD-GlcCer were determined in intact cells, in their isolated plasma membranes, and in plasma membranes isolated from rat liver tissue. C6-NBD-SM was found to be subject to extensive degradation in the plasma membrane, due to neutral sphingomyelinase (N-SMase) activity. The extent of C6-NBD-SM hydrolysis showed a general cell-type dependence and turned out to be dependent on the state of cell differentiation, as revealed for HT29 cells. In undifferentiated HT29 cells N-SMase activity was at least threefold higher than in its differentiated counterpart. In contrast, in all cell types studied, very little if any biosynthesis of C6-NBD-SM from the precursor C6-NBD-Cer occurred. Moreover, in the case of C6-NBD-GlcCer, neither hydrolytic nor synthetic activity was found to be associated with the plasma membrane. These results are discussed in the context of the involvement of the sphingolipids SM and GlcCer in signal transduction pathways in the plasma membrane.

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.

scholarly journals Uridine diphosphate glucose-sterol glucosyltransferase and nucleoside diphosphatase activities in etiolated pea seedlings

1978 ◽  
Vol 169 (2) ◽  
pp. 297-303 ◽  
Author(s):  
M J Staver ◽  
K Glick ◽  
D J Baisted
Keyword(s):  
Density Gradient ◽  
High Speed ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Particulate Fraction ◽  
Partial Recovery ◽  
Sucrose Density Gradient Centrifugation ◽  
Freeze Thaw ◽  
Pea Seedlings ◽  
Nucleoside Diphosphatase

1. UDP-glucose-sterol glucosyltransferase and nucleoside diphosphatases were isolated in a particulate fraction from 7-day-old etiolated pea seedlings. The glucosyltransferase and UDPase (uridine diphosphatase) are stimulated by Ca2+ cation, less so by Mg2+ cation, and inhibited by Zn2+. 2. Each activity has a pH optimum near 8. 3. The glucosyltransferase is specific for UDP-glucose as the glucosyl donor and is inhibited by UDP. Partial recovery from UDP inhibition is effected by preincubation of the enzyme. 4. Freeze-thaw treatment and subsequent sucrose-density-gradient centrifugation of the particulate fraction shows the glucosyltransferase to be widely distributed among cell fractions but to be most active in particles with a density of 1.15 g/ml. UDPase is most active in particulate material with a density of over 1.18 g/ml but an activity peak also appears at 1.15 g/ml. Of several nucleoside diphosphatase activities, UDPase activity is most enhanced by the freeze-thaw and sucrose-density-gradient-fractionation procedures. 5. Detergent treatment with 0.1% sodium deoxycholate allows the partial solubilization of the glucosyltransferase and UDPase. The two activities are similarly distributed between pellet and supernatant after high-speed centrifugation for two different time intervals. 6. A role for UDPase in the functioning of glucosylation reactions is discussed.

Composition, biophysical properties, and morphometry of plasma membranes in pulmonary interstitial edema

2002 ◽  
Vol 282 (6) ◽  
pp. L1382-L1390 ◽  
Author(s):  
Paola Palestini ◽  
Chiara Calvi ◽  
Elena Conforti ◽  
Laura Botto ◽  
Carla Fenoglio ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Plasma Membranes ◽  
Cell Activation ◽  
Membrane Lipids ◽  
Gradient Centrifugation ◽  
Mechanical Stimuli ◽  
Biophysical Properties ◽  
Interstitial Edema ◽  
Vascular Perfusion

We evaluated the changes in plasma membrane composition, biophysical properties, and morphology of pulmonary endothelial cells in anesthetized rabbits receiving 0.5 ml · kg−1 · min−1 saline infusion for 180 min, causing mild interstitial edema. Plasma membrane fractions were obtained from lung homogenates with gradient centrifugation, allowing a sixfold enrichment in caveolin-1. In edematous lungs, cholesterol content and phospholipidic phosphorus increased by 15 and 40%, respectively. These data correlated with morphometric analysis of lungs fixed in situ by vascular perfusion with 2.5% glutaraldehyde, suggesting a relative increase in surface of luminal to interstitial front of the capillary endothelial cells, due to a convoluted luminal profile. In edematous lungs, the fraction of double-bound fatty acids increased in membrane lipids; moreover, the phosphatidylcholine/phosphatidylethanolamine and the cholesterol/phospholipid ratios decreased. These changes were consistent with the increase in fluorescence anisotropy of plasma membrane, indicating an increase in its fluidity. Data suggest that mechanical stimuli elicited by a modest (∼4%) increase in extravascular water cause marked changes in plasma membranes that may be of relevance in signal transduction and endothelial cell activation.

scholarly journals Localization of ferrochelatase and of newly synthesized haem in membrane fractions from Rhodopseudomonas spheroides

1978 ◽  
Vol 174 (1) ◽  
pp. 277-281 ◽  
Author(s):  
J Barrett ◽  
O T G Jones
Keyword(s):  
Cell Envelope ◽  
Gradient Centrifugation ◽  
Specific Radioactivity ◽  
Sucrose Density Gradient ◽  
Particulate Fraction ◽  
Outer Face ◽  
Sucrose Density Gradient Centrifugation ◽  
Intact Cells ◽  
Rhodopseudomonas Spheroides ◽  
Cytochrome Content

Cells of Rhodopseudomonas spheroides, strains R-26 or GVP, were grown photosynthetically, disrupted and two particulate fractions separated by sucrose-density-gradient centrifugation. The upper particulate fraction, enriched in bacteriochlorophyll, was identified as containing the chromatophores; the lower particulate fraction had the characteristics of the cell envelope. The two fractions differed in cytochrome content and cytochrome spectra. Ferrochelatase was found almost exclusively in the chromatophore fraction and was located on the outer face of the chromatophores, i.e. in contact with the cytosol in intact cells. The addition of 59FeCl3 to cells growing in low-iron media resulted in labelling of the protohaem fraction (probably arising from cytochrome b) of the membranes. The specific radioactivity of the haem of the chromatophores rose more rapidly than that of the envelope fraction and then after 2 h declined to approximately the same value, suggesting that haems of the chromatophore may act as precursors of haem of the envelope.

scholarly journals Membrane-associated phosphoproteins in Plasmodium berghei-infected murine erythrocytes.

1983 ◽  
Vol 97 (1) ◽  
pp. 196-201 ◽  
Author(s):  
M F Wiser ◽  
P A Wood ◽  
J W Eaton ◽  
J R Sheppard
Keyword(s):  
Plasma Membrane ◽  
Plasmodium Berghei ◽  
Plasma Membranes ◽  
Intact Cell ◽  
Erythrocyte Membranes ◽  
Amino Acid Residues ◽  
Two Dimensional Gel Electrophoresis ◽  
Intact Cells ◽  
Membrane Phosphorylation ◽  
Triton X

Normal and Plasmodium berghei (NYU-2 strain)-infected murine erythrocytes display substantially different patterns of plasma membrane phosphoproteins phosphorylation. Intact erythrocytes (normal and parasite infected) incubated with 32Pi and isolated washed erythrocyte plasma membranes incubated with gamma-32P-ATP were analyzed for phosphoproteins by SDS PAGE and autoradiography. Two new phosphoproteins of molecular weight 45,000 (pp45) and 68,000 (pp68), which are absent in normal erythrocyte membranes, are associated with the membranes of infected erythrocytes subjected to both intact-cell and isolated-membrane phosphorylation conditions. Two-dimensional gel electrophoresis indicates that pp45 and pp68 are of parasite origin. Partial or complete proteolytic digestion reveals that pp45 is phosphorylated at similar amino acid residues both in intact cells and in isolated membranes. The pp45 phosphoprotein can be detected at as low as 3% parasitemia and its phosphorylation is not affected by 10 microM cAMP, 1 mM Ca2+, or 5 mM EGTA. Extraction of isolated washed plasma membranes with 0.5% Triton X-100 or 0.1 M NaOH indicates that pp45 is detergent insoluble and only partially extractable with NaOH, suggesting that pp45 is closely associated with the host erythrocyte 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.

scholarly journals Modulation of 5′-nucleotidase activity in plasma membranes and intact cells by the extracellular matrix proteins laminin and fibronectin

1992 ◽  
Vol 282 (1) ◽  
pp. 181-188 ◽  
Author(s):  
N Olmo ◽  
J Turnay ◽  
G Risse ◽  
R Deutzmann ◽  
K von der Mark ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Plasma Membrane ◽  
Inhibitory Activity ◽  
Plasma Membranes ◽  
Extracellular Matrix Proteins ◽  
Cell Receptor ◽  
Inhibitory Effect ◽  
Matrix Proteins ◽  
Nucleotidase Activity ◽  
Intact Cells

Modulation of 5′-nucleotidase activity by the extracellular matrix proteins fibronectin, laminin and their fragments has been studied in plasma membrane preparations as well as in intact BCS-TC2 and Rugli cells. The ectoenzyme on plasma membranes is activated by laminin; fibronectin inhibits the AMPase activity on BCS-TC2 plasma membranes but no inhibitory effect is found in plasma membrane preparations from Rugli cells. These effects are dependent on the preincubation time and protein concentration. When the effect of the extracellular matrix proteins is studied on intact cells, both BCS-TC2 and Rugli cells show similar behaviour. A decrease in the enzyme activity is observed in the presence of fibronectin. The AMPase inhibitory activity is located on its 40 kDa fragment. No inhibitory activity is found in other fibronectin fragments, including the 140 kDa fragment which contains the RGDS cell-adhesion sequence. Laminin and its E1-4 and E8 fragments are able to activate the ecto-5′-nucleotidase activity of both BCS-TC2 and Rugli cells. The effect of the E1-4 fragment on intact cells is greater than that observed for the E8 fragment and uncleaved laminin. Our results suggest a bifunctional role for 5′-nucleotidase as ectoenzyme and cell receptor for extracellular matrix proteins.

Purification of distinct plasma membranes from canine renal medulla

1978 ◽  
Vol 234 (3) ◽  
pp. F247-F254
Author(s):  
R. Iyengar ◽  
D. S. Mailman ◽  
G. Sachs
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Collecting Duct ◽  
Gradient Centrifugation ◽  
Phospholipid Composition ◽  
Renal Medulla ◽  
Similar Distribution ◽  
Mitochondrial Atpase ◽  
Fold Enrichment

Two types of plasma membrane were purified from canine distal renal medulla by the techniques of differential and zonal density-gradient centrifugation followed by free-flow electrophoresis. One group of plasma membranes was identified as basal-laterally derived based on a 30-fold enrichment of Na-K-ATPase, a 20-fold enrichment of vasopressin-stimulated adenylate cyclase, and a 33-fold enrichment of [3H]vasopressin binding sites. The second type of plasma membrane was free of these markers, but had a cholesterol and phospholipid composition similar to them. Alkaline phosphatase also had a similar distribution in the two fractions. This lighter membrane fraction contained a membrane-bound cyclic AMP-dependent protein kinase as well as substrate for this kinase. In addition there was a 26-fold enrichment of specific activity of an anion (SO32-)-activated ATPase which was insensitive to mitochondrial ATPase inhibitor protein, in contrast to the mitochondrial fraction of the tissue. Based on the relative preponderance of collecting duct tissue in the distal medulla and the yield of membrane protein, these membranes are tentatively identified as containing apical membranes of the collecting duct.

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