Freeze-thaw and high-voltage discharge allow macromolecule uptake into ileal brush-border vesicles

Donowitz, M; Emmer, E; McCullen, J; Reinlib, L; Cohen, M E; Rood, R P; Madara, J; Sharp, G W.G.; Murer, H; Malmstrom, K

Title: Freeze-thaw and high-voltage discharge allow macromolecule uptake into ileal brush-border vesicles

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Abstract

High-voltage discharge or one cycle of freeze-thawing are shown to transiently permeabilize rabbit ileal brush-border membrane vesicles to macromolecules. Uptake of the radiolabeled macromolecule dextran, mol wt 70,000, used as a marker for vesicle permeability, was determined by a rapid filtration technique, with uptake defined as substrate associated with the vesicle and releasable after incubation of vesicles with 0.1% saponin. Dextran added immediately after electric shock (2000 V) or at the beginning of one cycle of freeze-thawing was taken up approximately eightfold compared with control. ATP also was taken up into freeze-thawed vesicles, whereas there was no significant uptake into control vesicles. The increase in vesicle permeability was reversible, based on Na-dependent D-glucose uptake being decreased when studied 5 but not 15 min after electric shock, and was not significantly decreased after completion of one cycle of freeze-thawing. In addition, adenosine 3',5'-cyclic monophosphate and Ca/sup 2 +/-calmodulin-dependent protein kinase activity were similar in control vesicles and vesicles exposed to high-voltage discharge or freeze-thawing. Also, vesicles freeze-thawed with (/sup 32/P)ATP demonstrated increased phosphorylation compared with nonfrozen vesicles, while freeze-thawing did not alter vesicle protein as judged by Coomassie blue staining. These techniques should allow intestinal membrane vesicles to be used formore »« less

Authors:: Donowitz, M; Emmer, E; McCullen, J; Reinlib, L; Cohen, M E; Rood, R P; Madara, J; Sharp, G W.G.; Murer, H; Malmstrom, K

Publication Date:: Mon Jun 01 00:00:00 EDT 1987

Research Org.:: Tufts Univ. School of Medicine, Boston, MA (USA)

OSTI Identifier:: 6862468

Resource Type:: Journal Article

Journal Name:: Am. J. Physiol.; (United States)

Additional Journal Information:: Journal Volume: 252:6

Country of Publication:: United States

Language:: English

Subject:: 59 BASIC BIOLOGICAL SCIENCES; 62 RADIOLOGY AND NUCLEAR MEDICINE; CARBON 14 COMPOUNDS; UPTAKE; DEXTRAN; ELECTRIC SHOCK; BIOLOGICAL EFFECTS; MEMBRANES; AUTORADIOGRAPHY; FREEZING; THAWING; AMP; ATP; CALCIUM COMPOUNDS; CATIONS; GLUCOSE; PERMEABILITY; PHOSPHORUS 32; RABBITS; SMALL INTESTINE; TRITIUM COMPOUNDS; ALDEHYDES; ALKALINE EARTH METAL COMPOUNDS; ANIMALS; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; BLOOD SUBSTITUTES; BODY; CARBOHYDRATES; CHARGED PARTICLES; DAYS LIVING RADIOISOTOPES; DIGESTIVE SYSTEM; DRUGS; GASTROINTESTINAL TRACT; HEMATOLOGIC AGENTS; HEXOSES; INTESTINES; IONS; ISOTOPES; LABELLED COMPOUNDS; LIGHT NUCLEI; MAMMALS; MONOSACCHARIDES; NUCLEI; NUCLEOTIDES; ODD-ODD NUCLEI; ORGANIC COMPOUNDS; ORGANS; PHOSPHORUS ISOTOPES; POLYSACCHARIDES; RADIOISOTOPES; SACCHARIDES; VERTEBRATES; 551001* - Physiological Systems- Tracer Techniques; 550601 - Medicine- Unsealed Radionuclides in Diagnostics

Citation Formats


                    Donowitz, M, Emmer, E, McCullen, J, Reinlib, L, Cohen, M E, Rood, R P, Madara, J, Sharp, G W.G., Murer, H, and Malmstrom, K. Freeze-thaw and high-voltage discharge allow macromolecule uptake into ileal brush-border vesicles.  United States: N. p., 1987. 
        Web.

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                    Donowitz, M, Emmer, E, McCullen, J, Reinlib, L, Cohen, M E, Rood, R P, Madara, J, Sharp, G W.G., Murer, H, & Malmstrom, K. Freeze-thaw and high-voltage discharge allow macromolecule uptake into ileal brush-border vesicles.  United States.

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                    Donowitz, M, Emmer, E, McCullen, J, Reinlib, L, Cohen, M E, Rood, R P, Madara, J, Sharp, G W.G., Murer, H, and Malmstrom, K. 1987.  
        "Freeze-thaw and high-voltage discharge allow macromolecule uptake into ileal brush-border vesicles".  United States.

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@article{osti_6862468,

  title        = {Freeze-thaw and high-voltage discharge allow macromolecule uptake into ileal brush-border vesicles},

  author       = {Donowitz, M and Emmer, E and McCullen, J and Reinlib, L and Cohen, M E and Rood, R P and Madara, J and Sharp, G W.G. and Murer, H and Malmstrom, K},

  abstractNote = {High-voltage discharge or one cycle of freeze-thawing are shown to transiently permeabilize rabbit ileal brush-border membrane vesicles to macromolecules. Uptake of the radiolabeled macromolecule dextran, mol wt 70,000, used as a marker for vesicle permeability, was determined by a rapid filtration technique, with uptake defined as substrate associated with the vesicle and releasable after incubation of vesicles with 0.1% saponin. Dextran added immediately after electric shock (2000 V) or at the beginning of one cycle of freeze-thawing was taken up approximately eightfold compared with control. ATP also was taken up into freeze-thawed vesicles, whereas there was no significant uptake into control vesicles. The increase in vesicle permeability was reversible, based on Na-dependent D-glucose uptake being decreased when studied 5 but not 15 min after electric shock, and was not significantly decreased after completion of one cycle of freeze-thawing. In addition, adenosine 3',5'-cyclic monophosphate and Ca/sup 2 +/-calmodulin-dependent protein kinase activity were similar in control vesicles and vesicles exposed to high-voltage discharge or freeze-thawing. Also, vesicles freeze-thawed with (/sup 32/P)ATP demonstrated increased phosphorylation compared with nonfrozen vesicles, while freeze-thawing did not alter vesicle protein as judged by Coomassie blue staining. These techniques should allow intestinal membrane vesicles to be used for studies of intracellular control of transport processes, for instance, studies of protein kinase regulation of transport.},

  doi          = {},

  url          = {https://www.osti.gov/biblio/6862468},
  journal      = {Am. J. Physiol.; (United States)},
number       = ,

  volume       = 252:6,

  place        = {United States},

  year         = {Mon Jun 01 00:00:00 EDT 1987},

  month        = {Mon Jun 01 00:00:00 EDT 1987}

}

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