ATTACHMENT TO THE LUTEAL PLASMA MEMBRANES: AN EARLY EVENT IN THE ACTION OF LUTEINIZING HORMONE

1973 ◽  
Vol 57 (2) ◽  
pp. 199-NP ◽  
Author(s):  
HANNU RAJANIEMI ◽  
TAPANI VANHA-PERTTULA
Keyword(s):  
Plasma Membrane ◽  
Luteinizing Hormone ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Early Event ◽  
Target Tissue ◽  
Marker Enzyme ◽  
Sucrose Density Gradient Centrifugation ◽  
Initial Event

SUMMARY The distribution of 125I-labelled luteinizing hormone (LH) was studied by autoradiography of the murine pregnant ovary 10 min after intravenous administration. The grains in autoradiograms were localized around the luteal cells. The pregnant ovary showed the highest uptake of 125I-labelled LH 30 min after the injection. No similar accumulation of 125I-labelled follicle-stimulating hormone, 125I-labelled albumin or free 125I was obtained. The ribosomal fraction of the corpus luteum contained a slightly higher level of radioactivity than the other subcellular fractions after injection of 125I-labelled LH. Sucrose density gradient centrifugation of the luteal particle preparation resulted in an enrichment of radioactivity in particles containing Na+,K+-activated ATPase, a marker enzyme of the plasma membrane. These findings support the concept of plasma membrane binding as the initial event in LH action on the target tissue.

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.

Mass spectrometry is a powerful tool for identification of proteins associated with lipid rafts of Jurkat T-cell line

2004 ◽  
Vol 32 (5) ◽  
pp. 777-779
Author(s):  
P. Pompach ◽  
P. Man ◽  
P. Novák ◽  
V. Havlíček ◽  
A. Fišerová ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Extraction Procedure ◽  
Sucrose Density Gradient ◽  
Cellular Adhesion ◽  
Jurkat Cells ◽  
Membrane Microdomains ◽  
Sucrose Density Gradient Centrifugation ◽  
Biologically Relevant ◽  
Jurkat T Cell

Many proteins involved in signal-transduction pathways are concentrated in membrane microdomains enriched in lipids with distinct physical properties. Since these microdomains are insoluble in non-ionic detergents in cold, proteins associated with them could be efficiently purified by techniques such as sucrose-density gradient centrifugation. The complexity of the resulting protein mixture requires powerful MS technique for its analysis. We have found that successful identification of biologically relevant proteins is critically dependent on the enrichment of the starting material (plasma membranes), and on the extraction procedure. Applying these conditions in combination with microHPLC-ESI (electrospray ionization)-MS/MS, we have identified proteins involved in signalling, cytoskeletal association and cellular adhesion in Jurkat cells that are not stimulated by any antibody incubation.

scholarly journals The αβ monomer of the insulin receptor has hormone-responsive tyrosine kinase activity

1991 ◽  
Vol 273 (1) ◽  
pp. 49-56 ◽  
Author(s):  
E R Mortensen ◽  
J G Drachman ◽  
G Guidotti
Keyword(s):  
Plasma Membranes ◽  
Kinase Activity ◽  
Gradient Centrifugation ◽  
Insulin Receptors ◽  
Sucrose Density Gradient ◽  
Erythrocyte Membranes ◽  
Protein Kinase Activity ◽  
Sucrose Density Gradient Centrifugation ◽  
Adipocyte Plasma Membranes ◽  
Receptor Dimers

Insulin receptors from turkey erythrocyte membranes exist as monomers and dimers when membranes are solubilized with detergent. We examined the ability of monomers and dimers to act as protein kinases to effect both autophosphorylation of the receptor and phosphorylation of an exogenous substrate. After separation by sucrose-density-gradient centrifugation, only receptor dimers show significant basal and insulin-stimulated kinase activity, whereas material at the position of receptor monomers is not active. Partial reduction of the membrane-bound receptors with dithiothreitol, however, produces a receptor monomer containing an alpha and a beta chain which has protein kinase activity similar to that of the original dimers. With rat adipocyte plasma membranes, which in the absence of reducing agents only contain receptor dimers, reduction with dithiothreitol also produces monomers with receptor kinase activity. Receptor monomer hormone-dependent kinase activity is insensitive to receptor concentration and shows stimulation after immobilization on an affinity support.

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.

SPECIFIC INTERACTION OF CORTICOSTEROIDS WITH BINDING SITES IN THE PLASMA MEMBRANES OF THE RAT ANTERIOR PITUITARY GLAND

1978 ◽  
Vol 79 (2) ◽  
pp. 215-222 ◽  
Author(s):  
B. KOCH ◽  
B. LUTZ-BUCHER ◽  
B. BRIAUD ◽  
C. MIALHE
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Specific Interaction ◽  
Sucrose Density Gradient ◽  
Pituitary Cells ◽  
Sucrose Density Gradient Centrifugation ◽  
Binding Characteristics ◽  
Rat Pituitary ◽  
Rat Pituitary Cells

The binding of glucocorticoids to a crude fraction of rat pituitary plasma membranes and to solubilized membrane proteins was measured. The binding characteristics were similar to those exhibited by transcortin: radioactive corticosterone was bound to a greater extent than radioactive dexamethasone and labelled corticosterone, but not labelled dexamethasone, was displaced by unlabelled corticosterone, deoxycorticosterone and progesterone. A Scatchard plot of the binding data revealed the presence of a binding material with a dissociation constant of about 3·2 nmol/l, which sedimented at 4S after sucrose density-gradient centrifugation. It was found that the number of binding sites was inversely related to the concentration of corticosterone in the circulation and was increased after long-term adrenalectomy. These data suggest that a material similar to transcortin is complexed to the plasma membrane of rat pituitary cells.

scholarly journals Plasma Membrane Vesicles from Mouse 15091A Tumor Cells: Agent of Platelet Aggregating Activity

1977 ◽  
Author(s):  
G.J. Gasic ◽  
J.L. Catafalmo ◽  
G.P. Gasic ◽  
S.J. Shattil ◽  
G.J. Stewart
Keyword(s):  
Plasma Membrane ◽  
Tumor Cells ◽  
Cell Transformation ◽  
Gradient Centrifugation ◽  
Amorphous Material ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Plasma Membrane Vesicles ◽  
Sucrose Density Gradient Centrifugation ◽  
Artificial Plasma

We reported that cells from most tumors display platelet aggregating activity (PAA) in heparinized plasma and that this activity contributes to metastasis.Recently, we demonstrated that PAA can be used as a marker of cell transformation in virally infected rat cells. The material responsible for PAA is shed into culture medium. Characterization revealed a material which is particulate and sedimentable at 50,000x g for 60 min.; it contains proteins and lipids with a free cholesterol to phospholipid ratio of 0.556.Delipida-tion as well as complete solubilization abolished PAA.SDS-ME PAGE, 7.5% slab gels, revealed 20 bands.EM studies of 50,000x g pellets shed by 15091A cells indicated they contained numerous vesicles, some solid bodies, numerous free or vesicle associated small particles, and some amorphous material. Discontinuous sucrose density gradient centrifugation of the 50,000x g pellet yielded at the 1.07-1.17 g/cm3 interface a predominantly vesicular fraction which was the most active interfacial material. The vesicles, visible with phase contrast microscopy, resemble those produced by artificial plasma membrane vesiculation in various cell systems, including normal cells. Since PAA is only shown by transformed cells, vesicles from these must be different or much more numerous. Spontaneous vesiculation by tumor cells may be potentially important in understanding cell transformation and tumor metastases.

scholarly journals Solubilization of pig lymphocyte plasma membrane and fractionation of some of the components

1971 ◽  
Vol 123 (5) ◽  
pp. 967-975 ◽  
Author(s):  
D. Allan ◽  
M. J. Crumpton
Keyword(s):  
Plasma Membrane ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Biological Activities ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sucrose Density Gradient ◽  
Similar Distribution ◽  
Sucrose Density Gradient Centrifugation

The degree of solubilization of pig lymphocyte plasma membrane by sodium deoxycholate was determined at a variety of temperatures and detergent concentrations. Approx. 95% of the membrane protein was soluble in 2% deoxycholate at 23°C. Some of the biological activities of the membrane survived this treatment. The leucine β-naphthylamidase activity was more readily soluble than the 5′-nucleotidase and these enzymes could be separated by extraction with 0.5% deoxycholate at 0°C. Membrane solubilized in 2% deoxycholate at 23°C was fractionated by sucrose-density-gradient centrifugation in 1% deoxycholate. The phospholipid was separated from the protein, which formed a fairly symmetrical peak that sedimented slightly slower than ovalbumin; the leucine naphthylamidase and 5′-nucleotidase activities were resolved from each other and from the main protein peak. Similar separations were achieved by elution from Sephadex G-200 and Sepharose 6B in 1% deoxycholate. The main proteins, however, appeared to possess much higher molecular weights than those indicated by sucrose-density-gradient centrifugation. This disparity suggests that many of the membrane proteins have a rod-like shape, especially since the results of experiments with [14C]deoxycholate revealed that the proteins did not bind significant amounts of deoxycholate. In contrast, 5′-nucleotidase and leucine naphthylamidase appeared to be globular. Polyacrylamide-gel electrophoresis of membrane solubilized in sodium dodecyl sulphate gave a similar distribution of protein to that achieved by sucrose-density-gradient centrifugation. Trace amounts only of polypeptides of molecular weight less than 10000 were detected.

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