Studies on in vitro polymerization of tubulin from renal medullary extracts.

Biochimica Et Biophysica Acta
L D BarnesT P Dousa

Abstract

Previous in vivo studies showed that microtubules are involved in the cellular action of vasopressin. In order to analyze the role of renal medullary microtubules, a system was developed which would allow the study of the assembly of tubulin in renal medulla extracts into microtubules in vitro. The assembly of tubulin into microtubules occurred in renal medullary cytosol (100 000 times g supernatant) under specific conditions which include pre-concentration of cytosol by ultrafiltration, the presence of ethylene glycol bis(2-aminoethyl)ether tetraacetic acid (EGTA) and 4 M glycerol, and warming at 37 degrees C. Formation of microtubules, which sedimented at 100 000 times g, was proved by (a) an increase in the apparent [3H]colchicine-binding activity of depolymerized pellets, (b) appearance of typical microtubules as shown by electron microscopy, and (c) by the increase in the quantity of microtubular protein analyzed by polyacrylamide gel electrophoresis. Vinblastine at a concentrationof 10(-6) M completely blocked formation of microtubules. A slight increase of ionized calcium in the polymerization mixture also prevented microtubule assembly; this inhibitory effect of ionized calcium was present at concentrations as low as 10...Continue Reading

References

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Citations

Jul 1, 1978·The Journal of Cell Biology·J A WeatherbeeR R Weihing
Feb 1, 1982·The American Journal of Medicine·R L Jamison, R E Oliver
Jul 1, 1976·Kidney International·T P Dousa, H Valtin
Oct 1, 1981·Clinical Endocrinology·P H BaylisD A Heath
Nov 23, 1979·Biochimica Et Biophysica Acta·P RayJ Nath
Oct 26, 1977·Biochimica Et Biophysica Acta·E Meier, O S Jorgensen
Jun 2, 1978·Biochimica Et Biophysica Acta·R E Scott, T P Dousa
Sep 1, 1979·Archives of Biochemistry and Biophysics·L D Barnes, G M Roberson

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