Freeze-thaw and high-voltage discharge allow macromolecule uptake into ileal brush-border vesicles
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.
- Research Organization:
- Tufts Univ. School of Medicine, Boston, MA (USA)
- OSTI ID:
- 6862468
- Journal Information:
- Am. J. Physiol.; (United States), Vol. 252:6
- Country of Publication:
- United States
- Language:
- English
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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