Calcium-induced inactivation of microtubule formation in brain extracts. Presence of a calcium-dependent protease acting on polymerization-stimulating microtubule-associated proteins.
Incubation of brain extracts in the presence of 1 mM CaCl2 results in the permanent loss of tubulin polymerization, even after later addition of ethyleneglycol-bis(beta-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), when assembly conditions are chosen which rely on the presence of microtubule-associated proteins (such as MAP1 and MAP2). Purified microtubular protein, by contrast, recovers readily from calcium inhibition by the later addition of EGTA. Mixing experiments, using purified microtubular protein and brain extract, show that permanent loss of tubulin assembly is always accompanied by proteolysis of high-molecular-weight microtubular-associated proteins. Addition of purified protein MAP2 after chelation of calcium by EGTA, immediately restores microtubule assembly. Furthermore, substitution of guanosine 5'-[alpha, beta-methylene]triphosphate for GTP after EGTA treatment results in the typical tubulin polymerization process, which is independent of the presence of microtubule-associated proteins. Thus, the proteolytic action of a calcium-dependent protease is specific for high-molecular-weight microtubule-associated proteins and not tubulin itself. The protease is soluble and therefore removing during the purification of microtubular protein by cycles of temperature-dependent polymerization and depolymerization. We discuss the potential physiological importance of this calcium-dependent protease.
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology*; D004533:Egtazic Acid / Q000494:pharmacology; D006023:Glycoproteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008870:Microtubules / Q000378:metabolism*; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism*; D051381:Rats; D014020:Tissue Extracts / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism*
D006023:Glycoproteins; D014020:Tissue Extracts; D014404:Tubulin; D004533:Egtazic Acid; D006160:Guanosine Triphosphate; D010447:Peptide Hydrolases; D002118:Calcium