Peripheral proteins and smooth membrane from erythrocyte ghosts. Segregation of ATP-utilizing enzymes into smooth membrane.

Hayashi, Hideo; Jarrett, Harry W.; Penniston, John T.

doi:10.1083/jcb.76.1.105

Cited by 16 publications

(8 citation statements)

References 22 publications

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“…Membranes were pelleted at 47 000 g for 20 min at 4°C. To enrich the membrane fraction for the NR protein, the obtained ghosts were deprived of most of the cytoskeletal proteins as described elsewhere (23). Protein concentration in the obtained smooth membrane samples was determined by using BCA Protein Assay (Pierce; Rockford, Ill) with BSA as standard.…”

Section: Methodsmentioning

confidence: 99%

Functional NMDA receptors in rat erythrocytes

Makhro

Wang

Vogel

et al. 2010

American Journal of Physiology-Cell Physiology

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N-methyl-d-aspartate (NMDA) receptors are ligand-gated nonselective cation channels mediating fast neuronal transmission and long-term potentiation in the central nervous system. These channels have a 10-fold higher permeability for Ca(2+) compared with Na(+) or K(+) and binding of the agonists (glutamate, homocysteine, homocysteic acid, NMDA) triggers Ca(2+) uptake. The present study demonstrates the presence of NMDA receptors in rat erythrocytes. The receptors are most abundant in both erythroid precursor cells and immature red blood cells, reticulocytes. Treatment of erythrocytes with NMDA receptor agonists leads to a rapid increase in intracellular Ca(2+) resulting in a transient shrinkage via Gardos channel activation. Additionally, the exposure of erythrocytes to NMDA receptor agonists causes activation of the nitric oxide (NO) synthase facilitating either NO production in l-arginine-containing medium or superoxide anion (O(2)(.-)) generation in the absence of l-arginine. Conversely, treatment with an NMDA receptor antagonist MK-80, or the removal of Ca(2+) from the incubation medium causes suppression of Ca(2+) accumulation and prevents attendant changes in cell volume and NO/O(2)(.-) production. These results suggest that the NMDA receptor activity in circulating erythrocytes is regulated by the plasma concentrations of homocysteine and homocysteic acid. Moreover, receptor hyperactivation may contribute to an increased incidence of thrombosis during hyperhomocysteinemia.

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Section: Methodsmentioning

confidence: 99%

Functional NMDA receptors in rat erythrocytes

Makhro

Wang

Vogel

et al. 2010

American Journal of Physiology-Cell Physiology

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“…7 displays results of an experiment designed to measure the extent of dephosphorylation during membrane preparation and during incubation in isotonic shape change media containing Tris, 43 6 THE JOURNAL OF CELL BIOLOGY " VOLUME 88, 1981 phosphate, and HEPES . It can be seen that no significant loss of radioactivity occurs after hemolysis and that the decline in specific activity during 37°C incubation in all three buffers is 30 min. ++ = Rapid shape change ; initial rate >75%/10 min.…”

Section: Spectrin Dephosphorylation Under Shapechange Assay Conditionsmentioning

confidence: 87%

“…It has been observed that interconversions of polyphosphoinositides, phosphatidylinositol, 1,2-diacylglycerol, and phosphatidic acid in the erythrocyte membrane are associated with vesiculation and shape change under various conditions (1,2) . The rapid transbilayer diffusion of 1,2-diacylglycerol provides a potential mechanism for changing the relative areas of the two membrane leaflets (1), and biosynthesis of phosphatidate and polyphosphoinositides requires ATP (2,30). It will be of interest to determine the relationship of these reactions to the phenomena we have described.…”

Section: Mechanism Of Shape Changementioning

confidence: 97%

“…++ = Rapid shape change ; initial rate >75%/10 min. § + = 30-40% stomatocytes after [30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45] min, barely perceptible . Accordingly, the buffer effects described above cannot be attributed to differential effects on spectrin dephosphorylation .…”

Section: Spectrin Dephosphorylation Under Shapechange Assay Conditionsmentioning

confidence: 99%

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Spectrin phosphorylation and shape change of human erythrocyte ghosts.

Patel¹,

Fairbanks²

1981

The Journal of Cell Biology

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Human erythrocyte membranes in isotonic medium change shape from crenated spheres to biconcave disks and cup-forms when incubated at 37°C in the presence of MgATP (M . P. Sheetz and S. J . Singer, 1977, J. Cell Biol . 73 :638-646) . The postulated relationship between spectrin phosphorylation and shape change (W . Birchmeier and S. J . Singer, 1977, J. Cell Biol . 73 :647-659) is examined in this report .Salt extraction of white ghosts reduced spectrin phosphorylation during shape change by 85-95%. Salt extraction did not alter crenation, rate of MgATP-dependent shape change, or the fraction (>80%) ultimately converted to disks and cup-forms after 1 h. Spectrin was partially dephosphorylated in intact cells by subjection to metabolic depletion in vitro. Membranes from depleted cells exhibited normal shape-change behavior .Shape-change behavior was influenced by the hemolysis buffer and temperature and by the time required for membrane preparation. Tris and phosphate ghosts lost the capacity to change shape after standing for 1-2 h at 0°C. Hemolysis in HEPES or N-tris(hydroxymethyl)methyl -2-aminoethanesulfonic acid yielded ghosts that were converted rapidly to disks in the absence of ATP and did not undergo further conversion to cup-forms. These effects could not be attributed to differential dephsphorylation of spectrin, because dephosphorylation during ghost preparation and incubation was negligible .These results suggest that spectrin phosphorylation is not required for MgATP-dependent shape change . It is proposed that other biochemical events induce membrane curvature changes and that the role of spectrin is passive.The relationship of cell membrane properties to the biconcave shape of normal human erythrocytes has for many years engaged the attention of investigators from several disciplines . Recently, theoretical treatments (11, 16) and "macro" modeling (13) of the membrane have given satisfactory predictions of observed erythrocyte shapes and simple shape transformations . However, no acceptable explanation for these phenomena at the level of membrane molecular organization has yet been advanced . One of the central issues is the role of intracellular ATP. Nakao et al . (41) first described reversible shape transitions associated with changes in erythrocyte ATP content. Weed et al . (59) suggested that the physical basis for the metabolic dependence of both erythrocyte shape and deformability might be ATP-dependent sot-gel transformations at the inner surface of the membrane. The spectrin-actin meshwork beneath the membrane was found to have some properties of an actomyosin-like system (33,34,49,53) and spectrin polypeptide 2 was shown to be phosphorylated (4,29,42,45,46). These findings raised the prospect that actomyosin-like ATPase 430 activity or protein kinase-mediated phosphorylation would prove to be a link between energy metabolism and control of shape in normal erythrocytes (21,30,33,37,43,44,50,53). Isolated erythrocyte membranes also undergo shape transformations induced by ATP (4...

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“…The ATP requirement for the maintenance of the discoid shape on both erythrocytes and ghosts is well known (21)(22)(23). Hypotheses of a role of spectrin (24) or lipid (25) phosphorylation in mediating this effect have been recently dismissed (26)(27)(28).…”

Section: Discussionmentioning

confidence: 99%

ATP-dependent asymmetric distribution of spin-labeled phospholipids in the erythrocyte membrane: relation to shape changes.

Seigneuret¹,

Devaux²

1984

Proc. Natl. Acad. Sci. U.S.A.

648

432

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Spin-labeled analogs of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine have been used to study phospholipid transverse diffusion and asymmetry in the human erythrocyte membrane. Ascorbate reduction was used to assess the transbilayer distribution of the labels. All three spin-labeled phospholipids initially incorporated into the outer leaflet of the membrane. On fresh erythrocytes at 50C, the phosphatidylcholine label remained mainly in the outer leaflet. In contrast, the phosphatidylserine and phosphatidylethanolamine labels underwent rapid transverse diffusion that led to their asymmetric distribution in favor of the inner leaflet. The latter effect was reversibly inhibited after ATP depletion of the erythrocytes and could be reproduced on resealed erythrocyte ghosts only if hydrolyzable Mg-ATP was included in the internal medium. It is suggested that an ATPdriven transport of amino phospholipids toward the inner leaflet could be the major cause of the phospholipid asymmetry in the erythrocyte membrane. It is also proposed that the same mechanism could explain the ATP requirement of the maintenance of the erythrocyte membrane discoid shape. that this method can be used successfully in erythrocytes, and the transverse diffusion of phosphatidylcholine has been measured. In the present study, the following phospholipid spin labels have been used.These phospholipids possess an unmodified polar head group R that can be either choline, serine, or ethanolamine and areAn asymmetric distribution of phospholipids exists between the two halves of many biological membranes (for review, see ref. 1). The human erythrocyte membrane appears to be the best characterized in this regard. Numerous studies using chemical labeling (2, 3) and phospholipases (4-6) indicate that, in this membrane, phosphatidylcholine and sphingomyelin are distributed in favor of the outer leaflet, while phosphatidylethanolamine and phosphatidylserine are mainly located in the inner leaflet.These observations raise the problem of how this asymmetry is maintained during the 120-day life span of the erythrocyte. The asymmetric distribution could in principle be maintained if transbilayer diffusion of phospholipids was negligible. However, for phosphatidylcholine, transbilayer "flip-flop" has been shown to occur with a half-time of 8-15 hr in the native membrane (7,8), although no such measurements exist for the three other major phospholipid species. A possible role of cytoskeletal proteins in maintaining the asymmetry in spite of transbilayer diffusion has been proposed by Haest et al. (9,10), but no direct evidence for such a process has been observed in the intact native membrane.In the present study, the formation and maintenance of phospholipid asymmetry in the erythrocyte membrane has been addressed by measuring the transverse diffusion of spin-labeled phospholipids bearing different polar head groups. The method, introduced by Kornberg and McConnell (11), is based on the accessibility of membrane-associated spin labe...

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Peripheral proteins and smooth membrane from erythrocyte ghosts. Segregation of ATP-utilizing enzymes into smooth membrane.

Cited by 16 publications

References 22 publications

Functional NMDA receptors in rat erythrocytes

Functional NMDA receptors in rat erythrocytes

Spectrin phosphorylation and shape change of human erythrocyte ghosts.

ATP-dependent asymmetric distribution of spin-labeled phospholipids in the erythrocyte membrane: relation to shape changes.

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