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Association between microtubules and Golgi vesicles isolated from rat parotid glands
Author(s) -
Coffe Gérard,
Raymond MarieNoëlle
Publication year - 1990
Publication title -
biology of the cell
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.543
H-Index - 85
eISSN - 1768-322X
pISSN - 0248-4900
DOI - 10.1016/0248-4900(90)90371-9
Subject(s) - microtubule , colcemid , vesicle , biology , golgi apparatus , tubulin , organelle , astral microtubules , microbiology and biotechnology , biophysics , biochemistry , cell , spindle apparatus , membrane , cell division , endoplasmic reticulum
Summary— We report an isolation procedure of trans‐Golgi vesicles (GVs) from rat parotid glands. Various organelle markers were used, particularly galactosyl transferase as a trans‐Golgi marker, to test the purity of the GV fraction. A quantitative in vitro binding assay between microtubules and GVs is described. The vesicles were incubated with taxol‐induced microtubules, layered between 50% and 43% sucrose cushions and subjected to centrifugation. Unlike free microtubules which were sedimented, the GV‐bound microtubules co‐migrated upward with GVs. Quantification of these bound microtubules was carried out by densitometric scanning of Coomassie blue‐stained gels. The association between microtubules and GVs followed a saturation curve, with a plateau value of 20 μg of microtubule protein bound to 500 μg of GV fraction. The half‐saturation of the GV sites was obtained with a microtubule concentration of 20 μg/ml. Electron microscopy of negatively stained re‐floated material showed numerous microtubule‐vesicle complexes. Coating of microtubules with an excess of brain microtubule‐associated proteins (MAPs) abolished binding. In the absence of exogenous microtubules, we showed that the GV fraction was already interacting with a class of endogenous rat parotid microtubules. This class of colcemid and cold‐stable microtubules represents 10–20% of the total tubulin content of the parotid cell.