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Volume 17, Number 8,
Issue of April 15, 1997
pp. 2722-2727
Copyright ©1997 Society for Neuroscience
Cysteine String Proteins Associated with Secretory Granules of
the Rat Neurohypophysis
Received Oct. 24, 1996; revised Jan. 13, 1997; accepted Feb. 4, 1997.
Sandrine Pupier1,
Christian Leveque1,
Beatrice Marqueze1,
Masakazu Kataoka2,
Masami Takahashi2, and
Michael J. Seagar1
1 Institut National de la Santé et de la
Recherche Médicale U464, Institut Jean Roche, Faculté de
Médecine Secteur Nord, 13916 Marseille Cedex 20, France, and
2 Mitsubishi Kasei Institute of Life Science, Machida, 194 Tokyo, Japan
The properties and subcellular distribution of cysteine string
proteins (csps) were analyzed in peptidergic nerve terminals of the rat
neurohypophysis. Polyclonal antibodies raised against recombinant rat
brain csp recognized a 36 kDa protein in isolated neurosecretosomes
from the post-pituitary. After chemical deacylation, a single 27 kDa
form was detected that displayed identical properties to csps in a
whole-brain synaptosomal fraction. Immunoisolation demonstrated that
synaptophysin and csps were located in the same vesicles. Density
gradient centrifugation of postsynaptosomal supernatants of
neurohypophysial homogenates revealed that csps and VAMP were present
in two distinct vesicle populations. Synaptophysin was only detected in
the slowly migrating population corresponding to small synaptic
vesicles, whereas arginine vasopressin was present in the more rapidly
sedimenting population indicating that it contains large dense core
vesicles (LDCVs). Immobilized antibodies against csp, synaptotagmin, or
VAMP captured vesicular arginine vasopressin confirming the association
of these proteins with LDCVs. Co-immunoprecipitation assays with
proteins solubilized from neurohypophysial or whole-brain nerve
terminals failed to reveal complexes containing csp and
[125I] GVIA receptors. These results indicate that csps
in the CNS are associated with both small synaptic vesicles and LDCVs.
However, they do not provide support for the hypothesis that protein
complexes implicated in exocytosis, which interact with presynaptic
N-type calcium channels, contain csps.
Key words:
cysteine string proteins;
VAMP;
calcium channels;
large dense core vesicles;
synaptic vesicles;
neurohypophysis
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