The isolation and characterization of right-side-out plasma membrane vesicles from barley aleurone cells.
Examination of organelle- and membrane-specific processes such as signal transduction necessitates the use of plasma membrane vesicles with cytoplasmic side-in orientation. We are interested in the structural identity and subcellular localization of in vivo [32P]phosphoric acid ([32Pi])-labeled phosphoinositides, including the recently discovered phosphatidyl-scyllo-inositol, for signal transduction studies. In the first part of this investigation, plasma membrane vesicles from barley aleurone cells were isolated employing the aqueous polymer (Dextran and polyethylene glycol) two-phase partition method. The membrane vesicles that partitioned into the upper and lower phases of the aqueous polymer two-phase system were characterized and the purity of the vesicles ascertained by assaying for two marker enzymes, K+-stimulated, Mg2+-dependent adenosine triphosphatase (EC 3.6.1.3, ATPase), localized in the plasma membranes, and cytochrome c oxidase, localized in the mitochondria. Inhibitors for ATPases such as azide, molybdate, and vanadate were used to distinguish between plasma membrane-associated and intracellular membrane-associated ATPases. These inhibitor studies suggest that the plasma membrane preparation contained about 7% of intracellular membrane vesicles and the intracellular membrane fraction contained about 6% of plasma membrane vesicles. Orientation of the plasma membrane vesicles was ascertained by measuring the latent ATPase activity. These latency studies suggest that about 95% of the plasma membrane vesicles were of cytoplasmic side-in orientation. In the second part of this investigation, intracellular distribution and in vivo [32Pi] labeling of phosphoinositides in the plasma membranes and intracellular membranes were investigated. Preferential accumulation of [32Pi]-labeled phosphatidyl-myo-inositol monophosphate (myo-PIP) and phosphatidyl-myo-inositol bisphosphate (myo-PIP2) was observed in the plasma membrane. However, scyllo-phosphatidylinositol (scyllo-PI) was detected in both the plasma membrane and the intracellular membranes. The cellular concentration of myo-phosphoinositides was determined, and, after 24 h of labeling with [32Pi], the ratio of radiolabel in myo-PI, PIP, and PIP2 paralleled the relative concentrations in aleurone cells.
Robbins
K M
KM
Department of Chemistry, Michigan Technological University, Houghton 49931, USA.
Bhuvarahamurthy
N
N
Pliska-Matyshak
G
G
Murthy
P P
PP
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry*; D003902:Detergents / Q000494:pharmacology; D003576:Electron Transport Complex IV / Q000378:metabolism; D005615:Freezing; D001467:Hordeum / Q000166:cytology / Q000648:ultrastructure*; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000737:chemistry; D008967:Molecular Biology / Q000379:methods*; D017830:Octoxynol / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000737:chemistry / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D003902:Detergents; D010716:Phosphatidylinositols; D010743:Phospholipids; D010761:Phosphorus Radioisotopes; D014638:Vanadates; D017830:Octoxynol; D003576:Electron Transport Complex IV; D000251:Adenosine Triphosphatases