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Title: A method of resuspending small vesicles separated from suspension by protamine aggregation and centrifugation

Abstract

The technique of separating small vesicles from suspension by aggregation with protamine or polylysine, followed by either filtration or centrifugation has proved a very useful one. This is because the minimum times necessary to separate vesicles from the suspending medium are quite short (15 to 30 s) and the separation is almost complete (over 98% in the cases tested). The primary limitation of this technique, until now, has been that it could only be used for a terminal assay, because the aggregates produced could not be easily disaggregated. It has been found that after aggregation of vesicles by protamine and their separation from suspension by low-speed centrifugation, the addition of an excess of heparin over protamine, followed by vortexing or homogenizing the pellet in fresh medium, satisfactorily disaggregates the vesicles. This process has been tested with phospholipid vesicles and proteoliposomes and with vesicles from inner mitochondrial membrane, sacroplasmic reticulum, and red blood cell membrane, producing satisfactory resuspensions in all cases tested. Leakage of (/sup 14/C)sucrose or /sup 86/Rb/sup +/ from phospholipid vesicles was not found to be significantly more rapid from protamine-heparin-treated vesicles, than from similar vesicles treated with protamine alone or from untreated control vesicles. Good separation of themore » resuspended vesicles from the remaining protamine and heparin was obtained with phospholipid vesicles and proteoliposomes using a gel filtration technique. Most of the protamine and heparin could be removed from the vesicles made of natural biological membrane using a combination of ion exchange chromatography and gel filtration.« less

Authors:
; ; ;
Publication Date:
Research Org.:
Univ. of Rochester, NY
OSTI Identifier:
6437791
DOE Contract Number:  
AC02-76EV03490
Resource Type:
Journal Article
Journal Name:
Anal. Biochem.; (United States)
Additional Journal Information:
Journal Volume: 120
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ORGANOIDS; PARTICLE RESUSPENSION; AGGLOMERATION; CARBON 14 COMPOUNDS; CENTRIFUGATION; FILTRATION; HEPARIN; LABELLED COMPOUNDS; LYSINE; PHOSPHOLIPIDS; PROTAMINES; RUBIDIUM 86; SUSPENSIONS; TRACER TECHNIQUES; ALKALI METAL ISOTOPES; AMINES; AMINO ACIDS; ANTICOAGULANTS; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; CARBOHYDRATES; CARBOXYLIC ACIDS; CELL CONSTITUENTS; COAGULANTS; DAYS LIVING RADIOISOTOPES; DISPERSIONS; DRUGS; ESTERS; HEMATOLOGIC AGENTS; HEPARIN ANTAGONISTS; INTERMEDIATE MASS NUCLEI; ISOMERIC TRANSITION ISOTOPES; ISOTOPE APPLICATIONS; ISOTOPES; LIPIDS; MINUTES LIVING RADIOISOTOPES; MUCOPOLYSACCHARIDES; NUCLEI; ODD-ODD NUCLEI; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC PHOSPHORUS COMPOUNDS; ORGANIC SULFUR COMPOUNDS; POLYSACCHARIDES; PROTEINS; RADIOISOTOPES; RUBIDIUM ISOTOPES; SACCHARIDES; SEPARATION PROCESSES; 550301* - Cytology- Tracer Techniques

Citation Formats

Gunter, K K, Gunter, T E, Jarkowski, A, and Rosier, R N. A method of resuspending small vesicles separated from suspension by protamine aggregation and centrifugation. United States: N. p., 1982. Web. doi:10.1016/0003-2697(82)90326-8.
Gunter, K K, Gunter, T E, Jarkowski, A, & Rosier, R N. A method of resuspending small vesicles separated from suspension by protamine aggregation and centrifugation. United States. doi:10.1016/0003-2697(82)90326-8.
Gunter, K K, Gunter, T E, Jarkowski, A, and Rosier, R N. Fri . "A method of resuspending small vesicles separated from suspension by protamine aggregation and centrifugation". United States. doi:10.1016/0003-2697(82)90326-8.
@article{osti_6437791,
title = {A method of resuspending small vesicles separated from suspension by protamine aggregation and centrifugation},
author = {Gunter, K K and Gunter, T E and Jarkowski, A and Rosier, R N},
abstractNote = {The technique of separating small vesicles from suspension by aggregation with protamine or polylysine, followed by either filtration or centrifugation has proved a very useful one. This is because the minimum times necessary to separate vesicles from the suspending medium are quite short (15 to 30 s) and the separation is almost complete (over 98% in the cases tested). The primary limitation of this technique, until now, has been that it could only be used for a terminal assay, because the aggregates produced could not be easily disaggregated. It has been found that after aggregation of vesicles by protamine and their separation from suspension by low-speed centrifugation, the addition of an excess of heparin over protamine, followed by vortexing or homogenizing the pellet in fresh medium, satisfactorily disaggregates the vesicles. This process has been tested with phospholipid vesicles and proteoliposomes and with vesicles from inner mitochondrial membrane, sacroplasmic reticulum, and red blood cell membrane, producing satisfactory resuspensions in all cases tested. Leakage of (/sup 14/C)sucrose or /sup 86/Rb/sup +/ from phospholipid vesicles was not found to be significantly more rapid from protamine-heparin-treated vesicles, than from similar vesicles treated with protamine alone or from untreated control vesicles. Good separation of the resuspended vesicles from the remaining protamine and heparin was obtained with phospholipid vesicles and proteoliposomes using a gel filtration technique. Most of the protamine and heparin could be removed from the vesicles made of natural biological membrane using a combination of ion exchange chromatography and gel filtration.},
doi = {10.1016/0003-2697(82)90326-8},
journal = {Anal. Biochem.; (United States)},
number = ,
volume = 120,
place = {United States},
year = {1982},
month = {1}
}