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The isolation and characterization of right-side-out plasma membrane vesicles from barley aleurone cells

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Lipids

Abstract

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-as-sociated 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.

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Abbreviations

ATPase:

ATP phosphohydrolase

GPI:

glycerophosphoinositol

GPIP:

glycerophosphoinositolmonophosphate

GPIP2 :

glycerophosphoinositolbisphosphate

HVE:

high-voltage paper electrophoresis

[32Pi]:

32P-labeled phosphoric acid

PI:

phosphatidylinositol

PIP:

phosphatidylinositolmonophosphate

PIP2 :

phosphatidylinositolbisphosphate

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Author notes

  1. Pushpalatha P. N. Murthy

    Present address: Department of Chemistry, Michigan Technological University, 49931, Houghton, Michigan

  2. Kim M. Robbins

    Present address: Math/Science Teacher, Dollar Bay High School, 123 Maple St., 49922, Dollar Bay, MI

  3. Gay Pliska-Matyshak

    Present address: Department of Surgery, Children's Hospital of Michigan, 901 Beaubien Blvd, 48201, Detroit, MI

Authors and Affiliations

  1. Department of Chemistry, Michigan Technological University, 49931, Houghton, Michigan

    Kim M. Robbins, Narasimhan Bhuvarahamurthy & Gay Pliska-Matyshak

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  1. Kim M. Robbins
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  2. Narasimhan Bhuvarahamurthy
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Robbins, K.M., Bhuvarahamurthy, N., Pliska-Matyshak, G. et al. The isolation and characterization of right-side-out plasma membrane vesicles from barley aleurone cells. Lipids 34, 75–82 (1999). https://doi.org/10.1007/s11745-999-340-5

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  • DOI: https://doi.org/10.1007/s11745-999-340-5

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