scholarly journals Calcium-dependent fusion of the plasma membrane fraction from human neutrophils with liposomes

1990 ◽  
Vol 1025 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Joseph W. Francis ◽  
James E. Smolen ◽  
Kenneth J. Balazovich ◽  
Rebecca R. Sandborg ◽  
Laurence A. Boxer
Keyword(s):  
Plasma Membrane ◽  
Membrane Fraction ◽  
Human Neutrophils ◽  
Plasma Membrane Fraction ◽  
Calcium Dependent

scholarly journals Regulation of chemoattractant receptor interaction with transducing proteins by organizational control in the plasma membrane of human neutrophils.

1989 ◽  
Vol 109 (6) ◽  
pp. 2783-2790 ◽  
Author(s):  
A J Jesaitis ◽  
J O Tolley ◽  
G M Bokoch ◽  
R A Allen
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
G Proteins ◽  
Membrane Fraction ◽  
Human Neutrophils ◽  
Membrane Domain ◽  
Plasma Membrane Fraction ◽  
A Minor ◽  
Plasma Membrane Domain ◽  
Gamma S

Isolated purified plasma membrane domains from unstimulated human neutrophils were photoaffinity labeled with F-Met-Leu-Phe-N epsilon-(2-(p-azido-[125I]salicylamido)ethyl- 1,3'-dithiopropionyl)-Lys also referred to as FMLPL-SASD[125I]. Most of the photoaffinity-labeled N-formyl peptide receptors were found in light plasma membrane fraction (PM-L) which has been previously shown to be enriched in guanyl nucleotide binding proteins and the plasma membrane marker alkaline phosphatase (Jesaitis, A. J., G. M. Bokoch, J. O. Tolley, and R. A. Allen. 1988. J. Cell Biol. 107:921-928). Furthermore, the heavy plasma membrane fraction (PM-H), which is enriched in actin and fodrin, was depleted in receptors. Solubilization of PM-L and PM-H in divalent cation-free buffer containing octylglucoside and subsequent sedimentation at 180,000 g in detergent-containing sucrose gradients revealed two receptor forms. The major population, found in PM-L sedimented as a globular protein with an apparent sedimentation coefficient of 6-7S, while a minor fraction found in the PM-H fraction sedimented as a 4S particle. In addition, the 6-7S form could be converted to the 4S form by inclusion of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in the extraction buffer (ED50 = 10-30 nM). ATP was not effective at doses of up to 10 microM. In contrast, isolation and solubilization of receptors from desensitized cells (photoaffinity labeled after a 15 degrees C incubation with FMLPL-SASD[125I]) revealed that the majority of receptors (greater than 60-90%), which are found in PM-H, sedimented as 4S particles. A minor fraction of receptors found in the PM-L sedimented as 6-7S species. The receptors in the PM-H fraction, however, were still capable of interacting with G-proteins, since addition of unlabeled PM-L membrane fraction as a G-protein source reconstituted a more rapidly sedimenting form showing sensitivity to GTP gamma S. These results suggest that receptors in unstimulated human neutrophils have a higher probability of interacting with G-proteins because they are in the light plasma membrane domain. The results also suggest that receptors that have been translocated to the heavy plasma membrane domain during the process of desensitization or response termination have a lower probability of interacting with G-protein. Since the latter receptors are still capable of forming G protein associations, then their lateral segregation would represent a mechanism of controlling of receptor G-protein interactions. This reorganization of the plasma membrane, therefore, may form the molecular basis for response termination or homologous desensitization in human neutrophils.

Association of the hepatic IP3 receptor with the plasma membrane: relevance to mode of action

1993 ◽  
Vol 265 (6) ◽  
pp. C1588-C1596 ◽  
Author(s):  
L. Feng ◽  
N. Kraus-Friedmann
Keyword(s):  
Plasma Membrane ◽  
Membrane Fraction ◽  
Ip3 Receptors ◽  
Ip3 Receptor ◽  
Plasma Membrane Fraction ◽  
T Lymphoma Cells ◽  
T Lymphoma ◽  
Triton X ◽  
Brain Receptor ◽  
The Brain

Studies were carried out to characterize the interaction between inositol 1,4,5-trisphosphate (IP3) receptors and the plasma membrane fraction. Extraction of the membranes with the nonionic detergents Nonidet P-40 and Triton X-100, followed by centrifugation at 100,000 g, resulted in the doubling of the IP3 receptor in the pellets, whereas no detectable binding was found in the supernatants. These data indicate that the detergents did not solubilize the receptor, that it remained associated with membrane particles, and that it is likely to be associated with the cytoskeleton. The cytoskeleton proteins actin, ankyrin, and spectrin were identified in the plasma membrane fraction. However, comparison of the amount of these proteins in different fractions of the detergent, or otherwise treated plasma membrane fractions, showed no direct correlation between the presence of any of these proteins in the plasma membrane fraction and their ability to bind [3H]IP3. This is in contrast to the brain and T-lymphoma cells in which the IP3 receptor is attached to ankyrin (L. Y. W. Bourguigon, H. Jin, N. Iida, N. R. Brandt, and S. H. Zhang. J. Biol. Chem. 268: 6477-6486, 1993; and S. K. Joseph and S. Samanta. J. Biol. Chem 268: 6477-6486, 1993). Thus the hepatic IP3 receptor, which is different from the brain receptor, might attach to the cytoskeleton by anchoring to a different protein. Because cytochalasin D treatment of livers diminishes the ability of IP3 to raise cytosolic free Ca2+ levels, the attachment of the IP3 receptor to the cytoskeleton seems to involve an association with microfilaments.

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.

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