An apparent paradox in the occurrence, and the in vivo turnover, of C-terminal tyrosine in membrane-bound tubulin of brain.
Tubulin tyrosine ligase catalyzes the reversible addition of tyrosine to the C-terminus of tubulin alpha chains. By using ligase and carboxypeptidase A in conjunction, we have previously shown that brain cytoplasmic tubulin exists in three forms: 15-40% already has C-terminal tyrosine, another 10-30% can accept additional tyrosine, and about one-half is an uncharacterized species which is not a ligase substrate. A membrane-bound fraction of brain tubulin, purified by vinblastine precipitation from a detergent extract, has been found to differ by the complete absence of preexisting tyrosine. The membrane fraction from which tubulin was extracted also contained masked forms of both ligase and a distinct detyrosylating enzyme, which can be released by detergent extraction. The turnover of alpha-chain C-terminal tyrosine in vivo was studied by incubating brain mince with labeled tyrosine, or injecting it intracerebrally, under conditions where protein synthesis was inhibited. Tyrosine appeared to turn over to about the same extent in membrane-bound, as in soluble, tubulin. This apparently paradoxical result was not due to ATPase in the membrane fraction, which might have allowed ligase-catalyzed exchange between free and fixed tyrosine. Authentic [14C]tyrosylated tubulin added to the brain membrane fraction was not detyrosylated or subject to endoprotease digestion during subsequent procedures to isolate tubulin. The unexpected finding that tubulin tyrosylated at the C-terminal in vivo appears to be in the "non-substrate" fraction points toward a possible resolution of the paradox.
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002268:Carboxypeptidases / Q000378:metabolism; D043422:Carboxypeptidases A; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D008025:Ligases / Q000378:metabolism; D008297:Male; D008566:Membranes / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D014404:Tubulin; D014443:Tyrosine; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D008025:Ligases