Characterization of D-[3H]cis-diltiazem binding to membrane fractions and specific binding of calcium channel blockers to isolated flagellar membranes of Chlamydomonas reinhardtii

1988 ◽  
Vol 90 (3) ◽  
pp. 457-463
Author(s):  
R. DOLLE ◽  
W. NULTSCH
Keyword(s):  
Plasma Membrane ◽  
Chlamydomonas Reinhardtii ◽  
Binding Site ◽  
Binding Sites ◽  
Membrane Fraction ◽  
Plasma Membranes ◽  
Dissociation Constants ◽  
Intracellular Membrane ◽  
Binding Studies ◽  
Plasma Membrane Fraction

Plasma membranes were separated from the intracellular membranes by using an aqueous two polymerphase system. D-[3H]cis-diltiazem was employed to characterize benzothiazepine-selective receptors in these different membrane fractions of Chlamydomonas reinhardtii. The separation revealed that one type of binding site with higher affinity (KD = 33 nm) can be attributed to the intracellular membrane fraction and a second type with lower affinity (KD = 313nm) to the plasma membrane fraction. The apparent dissociation constants determined from the association and dissociation rate constants in kinetic experiments are comparable to those determined by saturation experiments. The maximum numbers of binding sites of the intracellular membrane fraction and the plasma membrane fraction are Bmax = 6.4 pmol mg−1 protein and Bmax = 19 pmol mg−1 protein, respectively. D-[3H]cis-diltiazem binding is inhibited by (±)verapamil and calcium chloride in both fractions. Moreover, nifedipine stimulates D-[3H]cis-diltiazem diltiazem binding by the intracellular membrane fraction, but shows no effect on the plasma membrane fraction. Ligand displacement binding studies with isolated flagella revealed the occurrence of a d-cisdiltiazem binding site with about the same affinity to this drug (KD = 400 nm) as the one found inthe plasma membrane fraction. The maximum number of binding sites is 4.5 pmol mg−1 protein. The apparent dissociation constants for specific[3H]nimodipine and [3H]verapamil binding to the flagella were calculated to be 8 nm and 38 nm, respectively. The corresponding Bmax values are 345f mol mg−1 protein and 1.3 pmol mg−1 protein, respectively.

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 PREPARATION AND CHARACTERIZATION OF A PLASMA MEMBRANE FRACTION FROM ISOLATED FAT CELLS

1970 ◽  
Vol 44 (2) ◽  
pp. 417-432 ◽  
Author(s):  
Daniel W. McKeel ◽  
Leonard Jarett
Keyword(s):  
Plasma Membrane ◽  
Cytochrome C ◽  
Membrane Fraction ◽  
Plasma Membranes ◽  
Reductase Activity ◽  
Fat Cells ◽  
Isolated Fat Cells ◽  
Adenyl Cyclase Activity ◽  
Cytochrome C Reductase ◽  
Plasma Membrane Fraction

A rapid method of preparing plasma membranes from isolated fat cells is described. After homogenization of the cells, various fractions were isolated by differential centrifugation and linear gradients. Ficoll gradients were preferred because total preparation time was under 3 hr. The density of the plasma membranes was 1.14 in sucrose. The plasma membrane fraction was virtually uncontaminated by nuclei but contained 10% of the mitochondrial succinic dehydrogenase activity and 25–30% of the RNA and reduced nicotinamide adenine dinucleotide cytochrome c reductase activity of the microsomal fraction. Part of the RNA and NADH-cytochrome c reductase activity was believed to be native to the plasma membrane or to the attached endoplasmic reticulum membranes demonstrated by electron microscopy. The adenyl cyclase activity of the plasma membrane fraction was five times that of Rodbell's "ghost" preparation and retained sensitivity to epinephrine. The plasma membrane ATPase activity was five times that of the homogenate and microsomal fractions. Electron microscopic evidence suggested contamination of the plasma membrane fraction by other subcellular components to be less than the biochemical data indicated.

Isolation and characterization of plasma membranes from bovine carotid arteries

1986 ◽  
Vol 250 (1) ◽  
pp. C65-C75 ◽  
Author(s):  
R. V. Sharma ◽  
R. C. Bhalla
Keyword(s):  
Plasma Membrane ◽  
Cytochrome C ◽  
Membrane Fraction ◽  
Plasma Membranes ◽  
Carotid Arteries ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Cytochrome C Reductase ◽  
Plasma Membrane Fraction

A plasma membrane fraction from bovine carotid arteries has been isolated by extraction of a crude microsomal fraction with a low-ionic-strength buffer containing ATP and Ca2+. This step was followed by sucrose-density-gradient centrifugation in the presence of 0.6 M KCl. The plasma membrane vesicles were enriched 60- to 80-fold in Na+-K+-adenosinetriphosphatase, 5'-nucleotidase, and phosphodiesterase I activities. The final yields of these marker enzymes were 12-18% of the total activities in the postnuclear supernatant, and the protein yield was 100-120 micrograms/g wet wt of carotid arteries. Contamination of the plasma membrane fraction by mitochondria and sarcoplasmic reticulum was low as judged by low activities of succinate--cytochrome-c reductase and NADPH--cytochrome-c reductase, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoprecipitation with smooth muscle-specific actin antibodies showed that the plasma membrane fraction was substantially free from myosin and actin contamination. The plasma membrane vesicles accumulated Ca2+ in the presence of ATP, and the accumulation was increased by calmodulin. Ca2+ accumulated in the presence or absence of calmodulin could be released almost completely from the vesicles by the addition of the Ca2+ ionophore A23187 but not by ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid, indicating that Ca2+ uptake in the presence of ATP is intravesicular. The effects of phosphate and oxalate on Ca2+ uptake in the plasma membranes were different from one another. Phosphate increased Ca2+ uptake in a concentration- and time-dependent manner, and the increase in Ca2+ uptake could be observed as early as 1 min. On the other hand, oxalate at concentrations up to 5 mM did not increase Ca2+ uptake significantly during the 30-min incubation. These plasma membranes can prove useful for the study of ion transport across plasma membranes, hormone binding, characterization of calcium channels, and preparation of antibodies against plasma membrane proteins.

scholarly journals The Ca2+-activated polyphosphoinositide phosphodiesterase of human and rabbit neutrophil membranes

1984 ◽  
Vol 221 (2) ◽  
pp. 477-482 ◽  
Author(s):  
S Cockcroft ◽  
J M Baldwin ◽  
D Allan
Keyword(s):  
Fatty Acid Composition ◽  
Plasma Membrane ◽  
Fatty Acid ◽  
Phospholipase C ◽  
Acid Composition ◽  
Membrane Fraction ◽  
Plasma Membranes ◽  
Degradation Products ◽  
Diacylglycerol Kinase ◽  
Plasma Membrane Fraction

Addition of Ca2+ to a plasma-membrane fraction derived from human or rabbit neutrophils led to the specific breakdown of polyphosphoinositides. The degradation products were identified as diacylglycerol and inositol bis- and tris-phosphate, thus demonstrating the presence of a Ca2+-activated phospholipase C. The newly generated diacylglycerol resembled the polyphosphoinositides in its fatty acid composition, and in the presence of MgATP2- it was converted into phosphatidate. These results therefore demonstrate the presence in neutrophil plasma membranes not only of polyphosphoinositide phosphodiesterase but also of diacylglycerol kinase.

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.

scholarly journals Characterization and isolation of a high-density-lipoprotein-binding protein from bovine corpus luteum plasma membrane

1992 ◽  
Vol 287 (3) ◽  
pp. 841-848 ◽  
Author(s):  
K Ferreri ◽  
K M J Menon
Keyword(s):  
Plasma Membrane ◽  
Membrane Fraction ◽  
Plasma Membranes ◽  
Binding Protein ◽  
High Density ◽  
High Density Lipoproteins ◽  
Plasma Membrane Fraction ◽  
High Affinity ◽  
High Concentrations ◽  
Single Polypeptide

The ovary uses the cholesterol from high-density lipoproteins (HDL) as a substrate source for steroid hormone production. It is not clear, however, how ovarian cells acquire the lipoprotein cholesterol. This study describes the characterization and isolation of a high-affinity-binding protein for apolipoprotein E-free HDL from the plasma-membrane fraction of bovine corpora lutea. Plasma membranes were prepared by differential centrifugation with 5-6-fold enrichment of 5′-nucleotidase activity. The binding of 125I-HDL to the plasma membranes was time-dependent, and there appeared to be a single high-affinity site with a Kd of 6.7 micrograms of HDL/ml of assay buffer. The binding was not affected by high concentrations of low-density lipoproteins or the Ca2+ chelator EDTA, nor by changes in pH in the range 6.5-9.0. The binding was affected by the salt concentration in the buffer, with a dose-dependent increase that reached a maximum at 150-250 mM-NaCl. Binding was increased in the presence of high concentrations of KCl and KBr, and most significantly increased by high concentrations of bivalent metal ions. Ligand-blot analysis under reducing conditions revealed that the binding protein was a single polypeptide of about 108 kDa that was associated with the plasma-membrane fraction. This HDL-binding protein was purified to homogeneity by solubilization with Triton X-100, poly(ethylene glycol) precipitation, DEAE-Sephadex chromatography, and preparative SDS/PAGE. The purified binding protein is a single polypeptide of 108 kDa that retains high affinity and specificity for HDL as assayed by ligand blotting.

scholarly journals Identification of dexamethasone-binding sites on male-rat liver plasma membranes by affinity labelling

1989 ◽  
Vol 260 (2) ◽  
pp. 435-441 ◽  
Author(s):  
G M Howell ◽  
C Po ◽  
Y A Lefebvre
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Male Rat ◽  
Binding Studies ◽  
Rat Serum ◽  
Neuraminidase Treatment ◽  
Affinity Labelling ◽  
Protein Components

Binding studies with [3H]dexamethasone identified two binding sites on plasma membranes prepared from the male rat liver, a low-capacity site with a KD of 7.0 nM and a higher-capacity site with a KD of 90.1 nM. Both sites exhibited glucocorticoid responsiveness and specificity for glucocorticoids and progestins. Triamcinolone acetonide, which competes well for the binding of dexamethasone to the cytosolic glucocorticoid receptor, did not compete well for the binding of [3H]dexamethasone to the plasma-membrane binding sites. The binding sites were sensitive to protease and neuraminidase treatment, and resistant to extraction with NaCl, but were extracted with the detergent Triton X-100. As these experiments indicated the presence of plasma-membrane protein components which bind glucocorticoids at physiological concentrations, affinity-labelling experiments with dexamethasone mesylate were conducted. Two peptides were specifically labelled, one at approx. Mr 66,000 and one at Mr 45,000. The Mr-66,000 peptide was not sensitive to glucocorticoids, and was extracted by NaCl, and so did not correspond to either of the sites identified in the dexamethasone-binding studies. The Mr-45,000 entity, on the other hand, resembled the dexamethasone-binding sites in its response to glucocorticoid manipulation of the animal and in its resistance to salt extraction. This peptide was not present in rat serum. Thus we have identified a plasma-membrane peptide which binds dexamethasone. Whether this peptide is involved in transport of the glucocorticoid across the plasma membrane remains to be determined.

scholarly journals HELA CELL PLASMA MEMBRANES

1974 ◽  
Vol 63 (2) ◽  
pp. 357-363 ◽  
Author(s):  
Sven Johnsen ◽  
Torbjørn Stokke ◽  
Hans Prydz
Keyword(s):  
Plasma Membrane ◽  
Hela Cell ◽  
Membrane Fraction ◽  
Phase Contrast ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Balance Sheet ◽  
Marker Enzyme ◽  
Plasma Membrane Fraction ◽  
Cell Plasma

A method for the preparation of HeLa cell plasma membrane ghosts is described. The purity of the plasma membrane fraction was examined by phase contrast and electron microscopy, by chemical analysis, and by assay of marker enzymes. Data on the composition of the plasma membrane fraction are given. It was observed that the distribution pattern of 5'-nucleotidase activity among the subcellular fractions differed from that of ouabain-sensitive ATPase. In addition, the specific activity of 5'-nucleotidase did not follow the distribution of the membrane ghosts. Thus, this enzyme would seem unsuitable as a plasma membrane marker. A complete balance sheet for marker enzyme activities during the fractionation is necessary for the calculation of increase in specific activity because the activities of both 5'-nucleotidase and ouabain-sensitive ATPase might change during the fractionation procedures.

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