A monoclonal antibody to α-tubulin:: Purification of functionally active α-tubulin isoforms

被引:12
作者
Banerjee, A [1 ]
机构
[1] Univ Texas, Hlth Sci Ctr, Dept Biochem, San Antonio, TX 78284 USA
关键词
D O I
10.1021/bi981572n
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Both alpha- and beta-tubulin exist as numerous isotypic forms that originate from different primary sequences as well as a variety of posttranslational modifications. Recent studies allow that tubulin dimers differing in the beta-subunit differ significantly in their subcellular distribution as well as in their functional properties such as assembly, dynamics, conformation, and interaction with antimitotic drugs; however, very little is known about the functional significance of the different alpha-tubulin isoforms and their posttranslational modifications. In an effort to get a better understanding about the alpha-tubulin isoforms, a monoclonal antibody, AYN.6D10, was prepared against the mammalian alpha-tubulin C-terminal sequence Glu-Glu-Gly-Glu-Glu-Tyr. Using an immunoaffinity column, bovine brain tubulin was fractionated into three functionally active alpha beta heterodimers which were identified by immunoblotting with alpha-tubulin-specific antibodies and sequence analysis. Assembly studies in the presence of glycerol and Mg2+ show that one of the fractions, that contains mainly the tyrosinated form of alpha 1/2, assembled poorly, while the nontyrosinated form assembled normally. The results indicate that tubulin dimers differing in their alpha-tubulin may differ in their functional properties. Future studies with the isoforms may yield valuable information regarding the role of alpha-tubulin and its posttranslational modifications in regulating microtubule assembly and function in vivo.
引用
收藏
页码:5438 / 5446
页数:9
相关论文
共 45 条
[11]   PROMOTION OF FLUORESCENCE UPON BINDING OF COLCHICINE TO TUBULIN [J].
BHATTACH.B ;
WOLFF, J .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1974, 71 (07) :2627-2631
[12]  
BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
[13]  
CRESTFIELD AM, 1963, J BIOL CHEM, V238, P622
[14]  
Dustin P, 1982, MICROTUBULES
[15]   POSTTRANSLATIONAL GLUTAMYLATION OF ALPHA-TUBULIN [J].
EDDE, B ;
ROSSIER, J ;
LECAER, JP ;
DESBRUYERES, E ;
GROS, F ;
DENOULET, P .
SCIENCE, 1990, 247 (4938) :83-85
[16]  
EIPPER BA, 1974, J BIOL CHEM, V249, P1407
[17]   MICROTUBULE ASSEMBLY INVITRO - PURIFICATION OF ASSEMBLY-PROMOTING FACTORS [J].
FELLOUS, A ;
FRANCON, J ;
LENNON, AM ;
NUNEZ, J .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1977, 78 (01) :167-174
[18]   A POLYMER-DEPENDENT INCREASE IN PHOSPHORYLATION OF BETA-TUBULIN ACCOMPANIES DIFFERENTIATION OF A MOUSE NEURO-BLASTOMA CELL-LINE [J].
GARD, DL ;
KIRSCHNER, MW .
JOURNAL OF CELL BIOLOGY, 1985, 100 (03) :764-774
[19]   LOCALIZATION OF A HIGHLY DIVERGENT MAMMALIAN TESTICULAR ALPHA-TUBULIN THAT IS NOT DETECTABLE IN BRAIN [J].
HECHT, NB ;
DISTEL, RJ ;
YELICK, PC ;
TANHAUSER, SM ;
DRISCOLL, CE ;
GOLDBERG, E ;
TUNG, KSK .
MOLECULAR AND CELLULAR BIOLOGY, 1988, 8 (02) :996-1000
[20]   CLEAVAGE OF STRUCTURAL PROTEINS DURING ASSEMBLY OF HEAD OF BACTERIOPHAGE-T4 [J].
LAEMMLI, UK .
NATURE, 1970, 227 (5259) :680-+