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Proc. Nati. Acad. Sci. USAVol. 78, No. 5, pp. 2757-2761, May
1981Biochemistry
Complete amino acid sequence of a-tubulin from porcine
brain(sequence microheterogeneity/homology with muscle
proteins)
H. PONSTINGL, E. KRAUHS, M. LITTLE, AND T. KEMPFInstitute of
Cell and Tumor Biology, German Cancer Research Center, D-6900
Heidelberg, Germany
Communicated by Hans Neurath, January 26, 1981
ABSTRACT The amino acid sequence of a-tubulin from por-cine
brain was determined by automated and manual Edman deg-radation of
eight sets of overlapping peptides. It comprises 450residues plus a
COOH-terminal tyrosine that is present only in15% of the material.
A region of 40 residues at the COOH-ter-minus is highly acidic,
mainly due to 16 glutamyl residues. Thishigh concentration
ofnegative charge suggests a region for bindingcations. At least
six positions, most of them around position 270,are occupied by two
amino acid residues each. Several of theseexchange sites were
assigned to specific peptides by analysis of thepurified
corresponding fragments. These data indicate four a-tu-bulins in
porcine brain. Although a-tubulin on the whole is un-related to
other proteins, there are regions that can be correlatedto
sequences of the myosin head, to actin, to tropomyosin, and
totroponins C and T.
Tubulins occur in all eukaryotic cells as the constituents of
mi-crotubules, which participate in cell division,
intracellulartransport and secretion processes, ciliary and
flagellar move-ment, morphogenesis, and cell orientation. Tubulins
fromwidely differing species and cell types appear to be
remarkablysimilar regarding composition, molecular weight, binding
ofcytostatic and psychopharmacological drugs,
immunologicalcrossreactivity, and capacity to copolymerize. Yet
even withinone cell, there are several types of microtubules that
have dif-fering stabilities and assemble into distinct organelles
at varioustimes. Knowledge of the primary structure should
clarifywhether there is just one tubulin for all functions or
whetherthere exists a family of similar proteins. It will also
facilitatemapping of binding sites for various ligands, production
of an-tibodies to well-defined antigenic sites, matching of
proteinstructure with that of messengers and genes, and
investigationof functionally defective tubulin mutants. Comparison
of thestructure with those of known proteins may give hints for
ex-periments regarding tubulin function.
Tubulin in solution is assumed to exist as a heterodimer oftwo
chains, a and f, each with a molecular weight4of 50,000,and very
similar amino acid compositions. Yet functional dif-ferences have
been reported. For example, only a-tubulin(from blood platelets)
binds cyclic AMP (1) and only /3-tubulinbinds exchangeable GTP (2).
Here we present the sequence ofthe a-chain from porcine brain and
report on the general strat-egy used.
MATERIALS AND METHODSWe have purified tubulin from porcine brain
by a modificationof the methods used by Eipper (3) and by Luduena
et al. (4).The 100,000 X g brain supernatant in 0.05 M sodium
pyro-phosphate buffer (pH 7.0) was incubated with 0.1 mM
colchi-cine for 15 min at 37°C before chromatography on
DEAE-cel-
lulose with a linear gradient of 0.1-0.3 M sodium
chloride.Tubulin was identified by the fluorescence of its complex
withcolchicine (5). The preparation was reduced, alkylated with
io-doacetic acid, and assayed for protein impurities by disc
gelelectrophoresis in the system of Yang and Criddle (6) using
8%polyacrylamide gels. The gels were stained with Coomassie blueand
scanned in a Vernon scanner. Only tubulin of more than95% purity
was processed further.
For separation of a- and f-chains, the protein was
chroma-tographed on hydroxyapatite in 0.1% NaDodSO4with a
lineargradient of 0.2-0.4 M sodium phosphate (7). Fractions
wereassayed for purity by gel electrophoresis as above. Only
a-chainof at least 95% purity was used for sequence determination
asdescribed (7, 8).
To remove NaDodSO4 the protein was extensively dialyzedagainst 1
mM ammonium bicarbonate; the solution was thenconcentrated by
vacuum evaporation, brought to pH 5.5 withacetic acid, and treated
with 9 vol of ice-cold acetone. The su-pernatant was discarded
after 2 hr at -20TC, and the precipitatewas dissolved in dilute
ammonium hydroxide and dialyzedagainst 0.01 M ammonium bicarbonate
for enzymatic digestion,which, in all cases, was done at pH 8.0
with 1-4 mg a-tubulinper ml and, usually, an enzyme/substrate ratio
of 1:100 at 370C.a-Tubulin (50-100 mg) was digested with either
thrombin(Sigma); affinity-purified trypsin (a gift from K.-D. Jany,
Stutt-gart) (9); chymotrypsin (Merck); or protease from
Staphylococ-cus aureus (Miles) (EC 3.4.21.19), from Astacus
leptodactylusEsch. (EC 3.4.99.6), donated by R. Zwilling
(Heidelberg) (10),from Pseudomonasfragi (EC 3.4.24) (a gift from G.
Drapeau,Montreal) (11), or from mouse submaxillary glands (EC
3.4.21)(Boehringer Mannheim). Cleavage times and exceptions fromthe
general schedule were chymotrypsin, 3 hr; trypsin, 7 hr;thrombin, 7
hr; submaxillary protease, 24 hr; staphylococcalprotease/0.2 M
ammonium bicarbonate at an enzyme/sub-strate ratio of 1:50, 24 hr;
Astacus protease/0. 1 M ammoniumbicarbonate at 20'C and an
enzyme/substrate ratio of 1:50, 2hr; protease ofa Pseudomonasfragi
mutant/0.01 M ammoniumbicarbonate/2 M urea, 24 hr. For cleavage
with cyanogen bro-mide (Serva, Heidelberg) the acetone precipitate
was evapo-rated under reduced pressure, and the residue was
dissolvedin pure formic acid, diluted to 70%, and cleaved with a
150-foldexcess of CNBr over methionyl residues for 24 hr in the
dark.The product was lyophilized.
The digests were fractionated on Sephadex G-50 and G-100in 8 M
urea/0. 1 M ammonium bicarbonate, and the fractionswere desalted on
Sephadex G-10. Peptides were further sep-arated by chromatography
on DEAE-cellulose, Dowex 1 x 2and 50 x 2, cellulose thin layers,
and, more recently, by re-versed-phase high-pressure liquid
chromatography with a DuPont 850 liquid chromatograph on a Zorbax
C-8 column, using0.05 M ammonium bicarbonate brought to pH 7.5 with
aceticacid and 0-60% acetonitrile gradients at 400C.Amino acid
analyses were performed on a Durrum D-500
analyzer. Automated Edman degradations used the Beckman2757
The publication costs ofthis article were defrayed in part by
page chargepayment. This article must therefore be hereby marked
"advertise-ment" in accordance with 18 U. S. C. §1734 solely to
indicate this fact.
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2758 Biochemistry: Ponstingl et al.
890 C sequencer with 0. 1 M quadrol as buffer and a single
cleav-age program adapted from Brauer et al. (12). To reduce
peptidelosses by extraction, 3 mg of Polybrene and 200 jig of
glycyl-glycine were applied to the cup and subjected to three
cyclesof degradation prior to analyzing the sample (13).
Phenylthio-hydantoin derivatives of amino acids were identified by
high-pressure liquid chromatography (14) and, in some cases, by
ad-ditional thin-layer chromatography (15); both methods alsoserved
for the assignment of amides. Manual Edman degra-dation plus
dansylation was performed as described (16).
RESULTS AND DISCUSSIONThe sequence of the 450 amino acid
residues of porcine braina-tubulin (Mr =50,000, depending on the
variant), is given inFig. 1. It is consistent with the amino acid
composition and wasestablished from the eight sets of peptides
generated by cyan-ogen bromide, trypsin, chymotrypsin,
staphylococcal protease,the less-frequently used thrombin and mouse
submaxillaris pro-tease, and by two enzymes that may not have been
used beforein sequence studies, one recently isolated from a mutant
ofPseudomonasfragi, which specifically cleaves at the NH2-ter-minal
side of aspartyl groups, and the other a protease from thedigestive
tract of the crayfish Astacus leptodactylus Esch.,cleaving
preferentially at the NH2-terminal side of alanine, gly-cine,
threonine, and serine.
Tubulin peptides strongly aggregate in Solution, as does
theparent molecule; hence it was necessary to include 8 M urea
in all peptide separations. This limited the types of
separationmethods that could be used, resulted in loss of insoluble
andsmall peptides on desalting by gel filtration, and led to
partialblockage of a- and E-amino groups by cyanate from
decompos-ing urea, producing heterogeneous fragments in low yields
inany further digestion or purification. Therefore, we
abandonedsubdigestions of peptides and chose to work with a larger
num-ber of overlapping primary fragments. A summary of the
frag-ments generated for sequence analysis is given in Fig. 2.A
striking feature of the sequence is the COOH-terminal
region, which we already have discussed in detail (17). The
last66 residues are entirely devoid of asparagine, glutamine,
thre-onine, cysteine, proline, and isoleucine, and the last 40
posi-tions have 47% acidic side chains, 16 glutamic and three
as-partic, rendering this segment one of the most acidic known.Its
high content of glutamyl residues suggests that it may
beresponsible for binding cations, for instance Ca2 , or for
thebasic microtubule-associated proteins, which play
antagonisticroles in microtubule assembly in vitro (for review, see
ref. 18).This part is predicted to have a helical structure, quite
differentfrom the rest of the chain.
In agreement with x-ray data, no indications were found fora
sterical organization in domains-e.g., there are no major se-quence
repeats and the 12 cysteines are spaced unevenly, fourcysteinyl
together with two methionyl residues forming a prom-inent "sulfur"
cluster at residues 295-316. Some other aminoacids also show a
highly asymmetric distribution: although po-sitions 55-135 and
288-378 are devoid of serine with the ex-
25MET-ARG-GLU-CYS-ILE-SER-ILE-HIS-VAL-GLY-GLN-ALA-GLY-VAL-GLN-ILE-GLY-ASN-ALA-CYS-TRP-GLU-LEU-TYR-CYS-
50LEU-GLU-HIS-GLY-ILE-GLN-PRO-ASP-GLY-GLN-MET-PRO-SER-ASP-LYS-THR-ILE-GLY-GLY-GLY-ASP-ASP-SER-PHE-ASN-
75THR-PHE-PHE-SER-GLU-THR-GLY-ALA-GLY-LYS-HIS-VAL-PRO-AXG-ALA-VAL-PHE-VAL-ASP-LEU-GLU-PRO-THR-VAL-ILE-
100ASP-GLU-VAL-ARG-THR-GLY-THR-TYR-ARG-GLN-LEU-PHE-HIS-PRO-GLU-GLN-LEU-ILE-THR-GLY-LYS-GLU-ASP-ALA-ALA-
125ASN-ASN-TYR-ALA-ARG-GLY-HIS-TYR-THR-ILE-GLY-LYS-GLU-ILE-ILE-ASP-LEU-VAL-LEU-ASP-ARG-ILE-ARG-LYS-LEU-
150ALA-ASP-GLN-CYS-THR-GLY-LEU-GLN-GLY-PHE-SER-VAL-PHE-HIS-SER-PHE-GLY-GLY-GLY-THR-GLY-SER-GLY-PHE-THR-
175SER-LEU-LEU-MET-GLU-ARG-LEU-SER-VAL-ASP-TYR-GLY-LYS-LYS-SER-LYS-LEU-GLU-PHE-SER-ILE-TYR-PRO-ALA-PRO-
200GLN-VAL-SER-THR-ALA-VAL-VAL-GLU-PRO-TYR-ASN-SER-ILE-LEU-THR-THR-HIS-THR-THR-LEU-GLU-HIS-SER-ASP-CYS-
225ALA-PHE-MET-VAL-ASP-ASN-GLU-ALA-ILE-TYR-ASP-ILE-CYS-ARG-ARG-ASN-LEU-ASP-ILE-GLU-ARG-PRO-THR-TYR-THR-
250ASN-LEU-ASN-ARG-LEU-ILE-GLY-GLN-ILE-VAL-SER-SER-ILE-THR-ALA-SER-LEU-ARG-PHE-ASP-GLY-ALA-LEU-ASN-VAL-
ILE HIS TH'Y-L-
275ASP-LEU-THR-GLU-PHE-GLN-THR-ASN-LEU-VAL-PRO-TYR-PRO-ARG-ALA- IE
PHE-PRO-LEU-ALA- HR-TYR ALA.PRO-VAL-GLYILE ~~ARG PHE ASXGLY
~~~~~~~~~300
ILE-SER-ALA-GLU-LYS-ALA-TYR-HIS-GLU-GLN-LEU-SER-VAL-ALA-GLU-ILE-THR-ASN-ALA-CYS-PHE-GLU-PRO-ALA-ASN-325
GLN-MET-VAL-LYS-CYS-ASP-PRO-ARG-HIS-GLY-LYS-TYR-MET-ALA-CYS-CYS-LEU-LEU-TYR-ARG-GLY-ASP-VAL-VAL-PRO-350
LYS-ASP-VAL-ASN-ALA-ALA-ILE-ALA-THR-ILE-LYS-THR-LYS-ARG-
ILE-GLN-PHE-VAL-ASP-TRP-CYS-PRO-THR-GLY-SER375
PHE-LYS-VAL-GLY-ILE-ASN-TYR-GLU-PRO-PRO-THR-VAL-VAL-PRO-GLY-GLY-ASP-LEU-ALA-LYS-VAL-GLN-ARG-ALA-VAL-400
CYS-MET-LEU-SER-ASN-THR-THR-ALA-ILE-ALA-GLU-ALA-TRP-ALA-ARG-LEU-ASP-HIS-LYS-PHE-ASP-LEU-MET-TYR-ALA-425
LYS-ARG-ALA-PHE-VAL-HIS-TRP-TYR-VAL-GLY-GLU-GLY-MET-GLU-GLU-GLY-GLU-PHE-SER-GLU-ALA-ARG-GLU-ASP-MET-450
ALA-ALA-LEU-GLU-LYS-ASP-TYR-GLU-GLU-VAL-GLY-VAL-ASP-SER-VAL-GLU-GLY-GLU-GLY-GLU-GLU-GLU-GLY-GLU-GLU-(TYR)
FIG. 1. Amino acid sequence of a-tubulin from porcine brain.
Positions 265, 266, 271-273, and 340 are heterogeneous. The
COOH-terminaltyrosine is present in only 15% of the material.
Proc. Natl. Acad. Sci. USA 78 (1981)
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Proc. Natl. Acad. Sci. USA 78 (1981) 2759
RESIDUE NUMBER 100 200
T a 0 W
B E
V L- Iu Vi///IILz D
300
m miffyOMMMM/ FMS //F/ //g N//
m~ em 6EI::]
D_ ,,,,,,, I n Th.V~~~ ~ ~ ~~~~~ ~ ~ ~
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~If VZX^, ]
l u MI
E/7/Y1//1, INTACT CHAIN
FIG. 2. Summary of fragments generated for the sequence analysis
of a-tubulin. The hatched section of each bar indicates the portion
of thesequence determined. Peptides were generated by trypsin (T);
cyanogen bromide (B); protease from Staphylococcus aureus V8 (V),
from mouse sub-maxillary glands (S), chymotrypsin (CH), protease
from Astacus (A), thrombin (TO), and a protease from a mutant
ofPseudomonas fragi (F).
ception of a single residue in a variant, 10 serine residues
arerather regularly spaced between positions 136 and 198.
Posi-tions 43-45 and 142-148 carry clusters of glycines,
suggestingthat these areas may be flexible regions, while positions
163-231and 247-309 are devoid of this frequent amino acid in
tubulin,whose abundance may be responsible for the low amount
ofsecondary structure.
Although tubulin has been reported to be present in orclosely
associated with membranes (19, 20), there are no regionsof the
sequence that are predominantly hydrophobic. Also tu-bulin, its
isolated a-chain, and most of the peptides obtainedfrom digests
were fairly soluble in aqueous solution at pH =7.5.Thus, the
tendency for tubulin and its fragments to aggregateand the
interaction of tubulin with membranes may be due toionic forces.One
possible way ofregulating tubulin assembly is posttrans-
lational modification of side chains. So far, however, we
havenot detected any modified amino acids. An additional
COOH-terminal tyrosine is present in 15% of our material (17)
and,recently, a ligase has been isolated from porcine brain
(21),which specifically adds this residue to the
COOH-terminalglutamate.
There is no evidence for a carbohydrate moiety, nor for
y-carboxyglutamic acid. Also, not having any radioactive label
inour material, we did not detect any phosphorylated residues.
Microheterogeneity. The establishment ofthe sequence wasimpeded
by microheterogeneity in several positions. Althoughthe
electrophoretic homogeneity of the starting material madethe
presence ofimpurities unlikely, it was nevertheless possiblethat
the preparation contained similar peptides derived fromdifferent
regions of the protein or that incomplete degradationhad resulted
in the presence of more than one residue in a po-sition. The first
possibility could be excluded by extensive over-lapping and the
second by separating variant peptides by high-pressure liquid
chromatography and analyzing the homogene-ous fractions. A total of
at least six positions carry amino acidexchanges (Fig. 3) and most
of them are concentrated in a "hotspot" around position 270.
Analyses of homogeneous fractionsof these variant peptides allowed
unambiguous identificationof the residues at several exchange
sites. Other peptides in thesame area and around residue 160,
however, were found to yieldheterogeneous degradation products at
one position. Discus-sion of them is omitted from this paper. As a
rule, we found a
mixture of two amino acid residues in a given position.
How-ever, position 265 appears to have three-isoleucine,
glycine,and alanine-in four different linkage groups. Hence at
leastfour different a-chains may be present in our preparation.
Most of these exchanges can be explained by a single
basesubstitution in the codon except that the isoleucine to
glycine,and isoleucine to alanine at 265 and isoleucine to
histidine at266 each require two base changes.
Although this heterogeneity might be due to alleles, it mayalso
reflect the presence of different tubulins in different celltypes
of the brain-e.g., nerve and glia cells. An
organ-specific/3-tubulin has already been described in Drosophila
(22). Al-ternatively, more than one a-tubulin may be required
evenwithin one cell.
Secondary Structure Prediction. We have tried to predictthe
secondary structure of a-tubulin according to Chou andFasman (23).
a-Tubulin appears to be rather irregularly folded:only 26% of the
chain is predicted to be helical and 33% is pre-dicted to have a
,3-sheet conformation, which is similar to resultsofearlier
circular dichroism studies with native a- and ,3-tubulinfrom pig
(22% a, 30% /3) (24) and calfbrain (26% a, 47% ,3) (25).
All major helix potentials reside in the COOH-terminal halfof
the chain around residues 275-291 and the three helices atthe
COOH-terminus already reported in an earlier paper (17),residues
383-403, 413-435, and 440-450. Major /8-sheet re-gions are expected
at positions 49-94 (five strands), 169-195(three strands), and
223-239 and 340-378 (four strands). A seriesof overlapping turns
are predicted at positions 31-49 and139-149.
In these regions, there is only one position with well-docu-
270ILE-HIS-PHE-PRO-LEU-ALA-THR-TYR-ALA
GLY-HIS-PHE-PRO-LEU-ALA-THR-TYR
GLY-ILE-PHE-PRO-LEU-ALA-ARG-PHE-ASX
ALA-HIS-PHE-PRO-LEU-ALA- X -PHE-ASX
FIG. 3. Assignment ofsequence variants around position 270.
Sep-arate peptide fractions were degraded and yielded
homogeneoussequences.
S
CH
400
A E
TO-
F
V/v ml I I
Q 0 mm, E0I --I
F//7//7///7//-
Biochemistry: Ponstingl et al.
OR ROMP mmff-_. 0 0rl- rg ra 0m 0m
El EM 0 gm 0 M E:::]V/Z//- M-/l EJ 0 % rg
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2760 Biochemistry: Ponstingl et al.
mented microheterogeneity-the threonine to serine exchangeat
position 340-presumably at the beginning of a strand of f3-sheet
that is not likely to be greatly influenced by this substi-tution.
The other amino acid substitutions are located in areaswith less
clearcut structural potentials; thus, their effect on therespective
structures would be difficult to predict.
The known lability of the tubulin molecule, as measured byits
capacity to polymerize and to bind colchicine, may be ex-plained by
its low level of secondary structures, as predictedby this
model.
Tubulin Peptides from Other Sources. Some sequence in-formation
for tubulin peptides from other sources has been re-ported:
Comparison with previous data on the NH2-terminal25 residues
ofchicken brain a-tubulin (26) shows six differences.Residues 10,
13, and 17 have been identified as threonine inchicken brain
a-tubulin and are glycine in the porcine protein.Cysteine residues
are present in porcine a-tubulin at positions4, 20, and 25 whereas,
in the chicken protein, the residues atthese positions have been
tentatively identified as serine. Res-idues 22-36, 303-313,
389-398, and 414-425 correspond tounlocated fragments and residue
426-450 corresponds to theCOOH-terminal cyanogen bromide peptide
isolated from calfbrain and sequenced by Lu and Elzinga (27).
However, twoother peptides designated a by these authors have no
counter-part in our a-sequence but resemble porcine /3-tubulin
(un-published observations).
Homology to Other Proteins. Several conflicting hypotheseshave
been advanced to explain microtubule function in intra-cellular
transport. It has been suggested that microtubules arepassive
skeletons or pointers for directional movement, providescaffolds to
which force-generating molecules are attached, oreven actively
function as motors ofmovement. Although knowl-
edge of a structure alone does not explain function, it forms
abasis on which to tackle functional problems, and comparisonof
protein structures may suggest further experiments.
a-Tubulin on the whole is unrelated to any other known pro-tein,
but some parts of the sequence appear to be variations ofknown
motifs. Because several regions of a-tubulin resembleareas of
various proteins, we can not assume a genetic relation-ship. More
likely, the similarities indicate that a given func-tion-for
example, binding and hydrolysis of a nucleotide-canbe performed by
a limited number of similar structures. Belowwe give a few examples
(Fig. 4).
Four regions of a-tubulin are similar to actin sequences
(28)and, with one exception, they are in the same order in
bothproteins. Between 32% and 70% ofthe residues in these
regionsare identical, comparable with the similarity of a- and
13-tu-bulin. The first of these segments includes a thrombic
cleavagesite in a-tubulin and in actin.A particularly interesting
relationship exists between a-tu-
bulin positions 192-238 and a fragment from the globular
headofmyosin (29). This segment ofthe myosin heavy chain
includestwo cysteines, whose alkylation modifies the ATPase
activity ofmyosin (31). The head ofmyosin can form a crossbridge
betweenthe thick and thin filaments by attaching to an actin
molecule.The reaction between actin and myosin is cyclical, and
eachcycle includes the hydrolysis of one molecule ofATP.
Residues1-46 of this fragment appear to be similar to positions
192-238in a-tubulin. In particular, the cysteine SH(1), which can
bealkylated in the absence of bound nucleotides with the resultthat
the Ca2+-ATPase activity is stimulated, resembles the
cys-teine-213, and the SH(2), which can be alkylated in the
presenceof ADP, occupies a position comparable with cysteine-200
ina-tubulin. Myosin alkylated at both sulfhydryl groups is
devoid
a 57- 68 G A G K H V P R A V F VACTIN 21- 32 F A G D D A P R A V
F P
a 95-127 G K E D A 'A N Y A R G H Y[T I G K E I I D L V L D RIR
K L A DACTIN 289-321 R K D L Y A N N V M S G G T T M Y P G T A D R
M Q K E I T A L A P
a 239-254 T H S L F D IG A L N V D L T EACTIN 173-187 H A I ML L
L A G R - D L T D
a 299-312 A iQ M V K C DlP R H GHKYACTIN 278-291 Y N S I M K C D
I D I R K D
a 191-215 T H T T L E H S - D[ A F M V D Nf A I Y D I C R
RMYOSIN 1- 23 E H E L V L H Q L RU- - N G V LWE GW- RLI CRL
KHEAD
a 216-239 N L D I E R P T T N L-N[ L I G N I V S SHTMYOSIN 24-
47 G F P - S[R I LIJ A D F K Q Y K V L N A S A II PHEAD
a 430-448 K Y|E E VIG V D S VGEG E E E|GTNT 1- 16 s - |E E V - -
E HV E E E AIE E EIA
FIG. 4. Homology of a-tubulin with actin (28), a fragment of the
myosin head (29), and troponin T (30) from rabbit skeletal muscle.
A, Ala; B,Asx; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I,
Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S,
Ser; T, Thr; V, Val; W, Trp; Y,Tyr; Z, Glx.
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Proc. Natl. Acad. Sci. USA 78 (1981) 2761
of ATPase activity. Although direct participation of this
regionin actin or nucleotide binding has not been proven, the
evidencesuggests that they are at or near the catalytic site for
myosinATPase. No secondary structure could be predicted for the
re-gion between SH(2) and SH(1), which is also the case for
thecorresponding tubulin sequence. Sulfhydryls are essential
fortubulin polymerization, and blockage ofas few as one or two
SHgroups inhibits the assembly of microtubules by an as yet
un-determined mechanism (32, 33).The highly acidic COOH-terminal
part of a-tubulin resem-
bles the NH2-terminal sequence of troponin T (see Fig. 4)
(30).This protein, as a component of the troponin complex,
partic-ipates in the Ca2+ regulation ofactin-myosin contacts. One
mayspeculate that these similar structures ofa-tubulin and
troponinT perform analogous physiological functions that, in view
of theclusters of glutamyl residues, could involve cation
binding.
In addition to the sequence similarity to troponin T,
somesimilarities have been observed to the structures of
a-tropomy-osin and troponin C. Also a tripeptide, His-Gly-Lys, that
hasbeen isolated from cat spinal cord and reported to impair
firingof neurones in the dorsal horn (34) is present at
positions309-311 of a-tubulin.
As these proteins are quite unrelated, it is difficult to
explainthe similarities on the basis ofevolutionary relationship.
We feelthat a more useful approach would be to evaluate the
sequencesimilarities strictly on the basis of structure-function
criteria.Thus, one would expect that two unrelated proteins or
regionsof proteins that perform analogous functions should also
havesimilar amino acid sequences regardless of the
evolutionaryrelationship.Note Added in Proof. After we had
communicated this article, the nu-cleotide sequence ofcDNA from
chicken brain tubulin messengers waspublished by Valenzuela et al.
(35). From these data, an amino acidsequence for chicken brain
a-tubulin was deduced, corresponding toresidues 41-451 of our
sequence and differing only in residues 175 (ar-ginine), 295
(tyrosine), and 358 (glutamate) from one of our variants.We wish to
thank Mr. Jurgen Kretschmer, Mrs. Ch. Orlando, and
Miss Herta Scherer for their skillful technical assistance; Dr.
G. Os-terburg for programming the method ofsecondary structure
prediction,and Drs. G. Schulz and R. Woodbury for discussions. This
work wassupported by the Deutsche Forschungsgemeinschaft.1.
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