Association between microtubules and Golgi vesicles isolated from rat parotid glands.
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 micrograms of microtubule protein bound to 500 micrograms of GV fraction. The half-saturation of the GV sites was obtained with a microtubule concentration of 20 micrograms/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.
Coffe
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Laboratoire de Structure et Réactivité des Protéines, CNRS URA 1131, Université Paris XI, Orsay, France.
Raymond
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MN
D000470:Alkaloids / Q000378:metabolism; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic; D001921:Brain / Q000166:cytology / Q000378:metabolism; D002458:Cell Fractionation; D004591:Electrophoresis, Polyacrylamide Gel; D006056:Golgi Apparatus / Q000378:metabolism*; D015151:Immunoblotting; D008297:Male; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D017239:Paclitaxel; D010306:Parotid Gland / Q000166:cytology* / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013552:Swine
D000470:Alkaloids; D000972:Antineoplastic Agents, Phytogenic; D008869:Microtubule-Associated Proteins; D017239:Paclitaxel