Inside-out but not right side-out plasma membrane vesicles from soybean enlarge when treated with ATP + 2,4-D as determined by electron microscopy and light scattering: Evidence for involvement of a plasma membrane AAA-ATPase

BioFactors ◽  
2006 ◽  
Vol 28 (2) ◽  
pp. 91-104 ◽  
Author(s):  
Carrie Hicks-Bergera ◽  
D. James Morreb
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Light Scattering ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Aaa Atpase ◽  
Inside Out

scholarly journals Isolation of InsP4 and InsP6 binding proteins from human platelets: InsP4 promotes Ca2+ efflux from inside-out plasma membrane vesicles containing 104 kDa GAP1IP4BP protein

1996 ◽  
Vol 315 (3) ◽  
pp. 1027-1034 ◽  
Author(s):  
Flavia O'ROURKE ◽  
Eileen MATTHEWS ◽  
Maurice B. FEINSTEIN
Keyword(s):  
Plasma Membrane ◽  
Calcium Oxalate ◽  
Binding Sites ◽  
Amino Acid Content ◽  
Membrane Vesicles ◽  
Human Platelets ◽  
Binding Activity ◽  
Plasma Membrane Vesicles ◽  
Internal Peptide ◽  
Inside Out

A low-density membrane fraction from human platelets contained the plasma membrane marker glycoprotein Ib (GpIb) and selective binding sites for InsP4 and InsP6. It was separated from the bulk of InsP3-receptor-containing membranes, but was heterogeneous, probably also containing surface-connected canalicular system and some lighter elements of the internal dense tubule system. After loading with calcium oxalate and re-centrifugation on Percoll gradients, this mixed fraction was subfractionated into light membranes containing all of the GpIb, high-affinity InsP4 binding sites (KD = 18 nM) and phosphate-stimulated Ca2+ transport activity. InsP4 (EC50 0.6 μM), but not InsP3 or InsP6, released up to 35% of the accumulated Ca2+ from these vesicles, which were shown to be inside-out plasma membrane vesicles by a biotinylation labelling technique and selective removal of right-side-out plasma membrane vesicles with streptavidin–agarose. Most of the InsP4, and all of the InsP6, binding was present in the much denser calcium oxalate-loaded subfractions, which were free of GpIb. InsP6 binding activity was chromatographically purified as a 116 kDa protein (KD for InsP6 = 5.9 nM), with an amino acid content and two internal peptide sequences identical to those of 116 kDa vinculin. A 104 kDa InsP4 binding protein (KD for InsP4 = 12 nM), probably identical to GAP1IP4BP described by Cullen, Hsuan, Truong, Letcher, Jackson, Dawson and Irvine [(1995) Nature (London) 376, 527–530], was also isolated. This InsP4 receptor may mediate Ca2+ influx in platelets that occurs subsequent to receptor-stimulated production of InsP3 and unloading of internal Ca2+ stores.

scholarly journals Rat ovarian lutropin receptor is a transmembrane protein. Evidence for an Mr-20,000 cytoplasmic domain

1986 ◽  
Vol 239 (1) ◽  
pp. 83-87 ◽  
Author(s):  
K P Keinänen ◽  
H J Rajaniemi
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Protein ◽  
Membrane Vesicles ◽  
Cytoplasmic Domain ◽  
Proteolytic Digestion ◽  
Plasma Membrane Vesicles ◽  
Human Choriogonadotropin ◽  
Lutropin Receptor ◽  
Membrane Bound ◽  
Inside Out

Membrane topography of the rat ovarian lutropin receptor was studied by two different approaches. Ovarian membrane preparation, labelled with 125I-labelled human choriogonadotropin in vivo, was subjected to extensive chymotryptic digestion. The soluble and membrane-bound radioactive complexes were cross-linked with glutaraldehyde, and analysed by SDS/polyacrylamide-gel electrophoresis and autoradiography. Chymotrypsin solubilized 70-75% of the radioactivity as Mr-96,000, Mr-74,000 and Mr-61,000 complexes, and decreased the size of the membrane-bound 125I-labelled human choriogonadotropin-receptor complex from Mr 130,000 to Mr 110,000. The Mr-110,000 complex was not observed when 0.1% Triton X-100 was present in the proteolytic digestion. Enrichment of inside-out-oriented plasma-membrane vesicles by concanavalin A affinity chromatography increased by 70% the fraction of radioactivity that remained in the membrane fraction after chymotrypsin treatment. Chymotrypsin also diminished the size of the membrane-bound unoccupied receptor from Mr 90,000 to Mr 70,000, as detected by ligand (125I-labelled human choriogonadotropin) blotting. These results suggest that the lutropin receptor is a transmembrane protein with a cytoplasmic domain of Mr 20,000 that is sensitive to proteolytic digestion in the inside-out-oriented plasma-membrane vesicles.

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