Polymerization of Antarctic fish tubulins at low temperatures: role of carboxy-terminal domains.
We have proposed previously that the efficient polymerization of tubulins from Antarctic fishes at low, physiological temperatures (-1.8 to +2 degrees C) may result in part from adaptations (e.g., reductions in acidic residues) located in their carboxy-terminal (C-terminal) tails [Detrich & Overton (1986) J. Biol. Chem. 261, 10922-10930]. To test this hypothesis, we have examined the polymerization of Antarctic fish neural tubulins modified at their C termini by proteolysis or by neutralization of carboxyl groups. Addition of subtilisin to low concentrations of Notothenia coriiceps tubulin induced a biphasic assembly reaction: stage I corresponded to the C-terminal cleavage of beta chains to produce alpha beta s dimers, and stage II coincided with the slower, C-terminal cleavage of alpha chains to yield alpha s beta s. Both stage I and stage II polymers consisted of protofilament sheets and microtubules with attached sheets. The critical concentration for assembly of the stage II polymer was at least 10-fold lower than that of untreated tubulin. Neutralization of Glu and Asp carboxyls in Gobionotothen gibberifrons microtubules by the carbodiimide-catalyzed incorporation of glycine ethyl ester (GEE) moieties produced a tubulin, modified largely in its C termini, that assembled more readily than did control tubulin. When 12 GEE groups were incorporated per dimer, the critical concentrations for assembly of modified tubulin at 5-10 degrees C were 2-3-fold smaller than those for the unmodified protein. Comparably modified bovine tubulin (10 GEE/dimer) assembled at 37 degrees C with a critical concentration 2.6-fold lower than that for the unmodified tubulin.(ABSTRACT TRUNCATED AT 250 WORDS)
Singer
W D
WD
Department of Biochemistry, University of Kansas, Lawrence 66045.
Parker
S K
SK
Himes
R H
RH
Detrich
H W
HW
D000222:Adaptation, Physiological; D000818:Animals; D000864:Antarctic Regions; D001224:Aspartic Acid / Q000737:chemistry; D002234:Carbodiimides / Q000737:chemistry; D003080:Cold Temperature; D005399:Fishes / Q000502:physiology*; D005971:Glutamates / Q000737:chemistry; D008854:Microscopy, Electron; D008870:Microtubules / Q000737:chemistry; D011108:Polymers; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013381:Subtilisins / Q000494:pharmacology; D014404:Tubulin / Q000737:chemistry*
D002234:Carbodiimides; D005971:Glutamates; D011108:Polymers; D014404:Tubulin; D001224:Aspartic Acid; D013381:Subtilisins