Partial purification of presynaptic plasma membrane by immunoadsorption

G. P. Miljanich, A. R. Brasier, R. B. Kelly

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

During transmitter release, synaptic vesicle membrane is specifically inserted into the nerve terminal plasma membrane only at specialized sites or 'active zones'. In an attempt to obtain a membrane fraction enriched in active zones, we have utilized the electric organ of the marine ray. From this organ, a fraction enriched in nerve terminals (synaptosomes) was prepared by conventional means. These synaptosomes were bound to microscopic beads by an antiserum to purified electric organ synaptic vesicles (anti-SV). The success of this immunoadsorption procedure was demonstrated by increased specific activities of bead-bound nerve terminal cytoplasmic markers and decreased specific activities of markers for contaminating membranes. To obtain a presynaptic plasma membrane (PSPM) fraction, we lysed the bead-bound synaptosomes by hypoosmotic shock and sonication, resulting in complete release of cytoplasmic markers. When the synaptosomal fraction was surface-labeled with iodine before immunoadsorption, 10% of this label remained bead-bound after lysis, compared with 2% of the total protein, indicating an approximately fivefold enrichment of bead-bound plasma membrane. Concomitantly, the specific activity of bead-bound anti-SV increased ~30-fold, indicating an enrichment of plasma membrane which contained inserted synaptic vesicle components. This PSPM preparation is not simply synaptic vesicle membrane since two-dimensional electrophoresis revealed that the polypeptides of the surface-iodinated PSPM preparation include both vesicle and numerous nonvesicle components. Secondly, antiserum to the PSPM fraction is markedly different from anti-SV and binds to external, nonvesicle, nerve terminal components.

Original languageEnglish (US)
Pages (from-to)88-96
Number of pages9
JournalJournal of Cell Biology
Volume94
Issue number1
StatePublished - 1982
Externally publishedYes

Fingerprint

Cell Membrane
Synaptic Vesicles
Synaptosomes
Electric Organ
Synaptic Membranes
Immune Sera
Sonication
Membranes
Iodine
Electrophoresis
Shock
Catalytic Domain
Peptides
Proteins

ASJC Scopus subject areas

  • Cell Biology

Cite this

Miljanich, G. P., Brasier, A. R., & Kelly, R. B. (1982). Partial purification of presynaptic plasma membrane by immunoadsorption. Journal of Cell Biology, 94(1), 88-96.

Partial purification of presynaptic plasma membrane by immunoadsorption. / Miljanich, G. P.; Brasier, A. R.; Kelly, R. B.

In: Journal of Cell Biology, Vol. 94, No. 1, 1982, p. 88-96.

Research output: Contribution to journalArticle

Miljanich, GP, Brasier, AR & Kelly, RB 1982, 'Partial purification of presynaptic plasma membrane by immunoadsorption', Journal of Cell Biology, vol. 94, no. 1, pp. 88-96.
Miljanich, G. P. ; Brasier, A. R. ; Kelly, R. B. / Partial purification of presynaptic plasma membrane by immunoadsorption. In: Journal of Cell Biology. 1982 ; Vol. 94, No. 1. pp. 88-96.
@article{b14281d8140d488993a4ffe729706e9e,
title = "Partial purification of presynaptic plasma membrane by immunoadsorption",
abstract = "During transmitter release, synaptic vesicle membrane is specifically inserted into the nerve terminal plasma membrane only at specialized sites or 'active zones'. In an attempt to obtain a membrane fraction enriched in active zones, we have utilized the electric organ of the marine ray. From this organ, a fraction enriched in nerve terminals (synaptosomes) was prepared by conventional means. These synaptosomes were bound to microscopic beads by an antiserum to purified electric organ synaptic vesicles (anti-SV). The success of this immunoadsorption procedure was demonstrated by increased specific activities of bead-bound nerve terminal cytoplasmic markers and decreased specific activities of markers for contaminating membranes. To obtain a presynaptic plasma membrane (PSPM) fraction, we lysed the bead-bound synaptosomes by hypoosmotic shock and sonication, resulting in complete release of cytoplasmic markers. When the synaptosomal fraction was surface-labeled with iodine before immunoadsorption, 10{\%} of this label remained bead-bound after lysis, compared with 2{\%} of the total protein, indicating an approximately fivefold enrichment of bead-bound plasma membrane. Concomitantly, the specific activity of bead-bound anti-SV increased ~30-fold, indicating an enrichment of plasma membrane which contained inserted synaptic vesicle components. This PSPM preparation is not simply synaptic vesicle membrane since two-dimensional electrophoresis revealed that the polypeptides of the surface-iodinated PSPM preparation include both vesicle and numerous nonvesicle components. Secondly, antiserum to the PSPM fraction is markedly different from anti-SV and binds to external, nonvesicle, nerve terminal components.",
author = "Miljanich, {G. P.} and Brasier, {A. R.} and Kelly, {R. B.}",
year = "1982",
language = "English (US)",
volume = "94",
pages = "88--96",
journal = "Journal of Cell Biology",
issn = "0021-9525",
publisher = "Rockefeller University Press",
number = "1",

}

TY - JOUR

T1 - Partial purification of presynaptic plasma membrane by immunoadsorption

AU - Miljanich, G. P.

AU - Brasier, A. R.

AU - Kelly, R. B.

PY - 1982

Y1 - 1982

N2 - During transmitter release, synaptic vesicle membrane is specifically inserted into the nerve terminal plasma membrane only at specialized sites or 'active zones'. In an attempt to obtain a membrane fraction enriched in active zones, we have utilized the electric organ of the marine ray. From this organ, a fraction enriched in nerve terminals (synaptosomes) was prepared by conventional means. These synaptosomes were bound to microscopic beads by an antiserum to purified electric organ synaptic vesicles (anti-SV). The success of this immunoadsorption procedure was demonstrated by increased specific activities of bead-bound nerve terminal cytoplasmic markers and decreased specific activities of markers for contaminating membranes. To obtain a presynaptic plasma membrane (PSPM) fraction, we lysed the bead-bound synaptosomes by hypoosmotic shock and sonication, resulting in complete release of cytoplasmic markers. When the synaptosomal fraction was surface-labeled with iodine before immunoadsorption, 10% of this label remained bead-bound after lysis, compared with 2% of the total protein, indicating an approximately fivefold enrichment of bead-bound plasma membrane. Concomitantly, the specific activity of bead-bound anti-SV increased ~30-fold, indicating an enrichment of plasma membrane which contained inserted synaptic vesicle components. This PSPM preparation is not simply synaptic vesicle membrane since two-dimensional electrophoresis revealed that the polypeptides of the surface-iodinated PSPM preparation include both vesicle and numerous nonvesicle components. Secondly, antiserum to the PSPM fraction is markedly different from anti-SV and binds to external, nonvesicle, nerve terminal components.

AB - During transmitter release, synaptic vesicle membrane is specifically inserted into the nerve terminal plasma membrane only at specialized sites or 'active zones'. In an attempt to obtain a membrane fraction enriched in active zones, we have utilized the electric organ of the marine ray. From this organ, a fraction enriched in nerve terminals (synaptosomes) was prepared by conventional means. These synaptosomes were bound to microscopic beads by an antiserum to purified electric organ synaptic vesicles (anti-SV). The success of this immunoadsorption procedure was demonstrated by increased specific activities of bead-bound nerve terminal cytoplasmic markers and decreased specific activities of markers for contaminating membranes. To obtain a presynaptic plasma membrane (PSPM) fraction, we lysed the bead-bound synaptosomes by hypoosmotic shock and sonication, resulting in complete release of cytoplasmic markers. When the synaptosomal fraction was surface-labeled with iodine before immunoadsorption, 10% of this label remained bead-bound after lysis, compared with 2% of the total protein, indicating an approximately fivefold enrichment of bead-bound plasma membrane. Concomitantly, the specific activity of bead-bound anti-SV increased ~30-fold, indicating an enrichment of plasma membrane which contained inserted synaptic vesicle components. This PSPM preparation is not simply synaptic vesicle membrane since two-dimensional electrophoresis revealed that the polypeptides of the surface-iodinated PSPM preparation include both vesicle and numerous nonvesicle components. Secondly, antiserum to the PSPM fraction is markedly different from anti-SV and binds to external, nonvesicle, nerve terminal components.

UR - http://www.scopus.com/inward/record.url?scp=0020327714&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0020327714&partnerID=8YFLogxK

M3 - Article

C2 - 6749869

AN - SCOPUS:0020327714

VL - 94

SP - 88

EP - 96

JO - Journal of Cell Biology

JF - Journal of Cell Biology

SN - 0021-9525

IS - 1

ER -