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The Complete Amino Acid Sequence of Actins from Bovine Aorta, Bovine Heart, Bovine Fast Skeletal Muscle, and Rabbit, Slow Skeletal Muscle
Differentiation
Dnerentiation 14, 123-133 (1979)
0 Springer-Verlag 1979
Original A rticles The Complete Amino Acid Sequence of Actins born Bovine Aorta, Bovine Heart, Bovine Fast Skeletal Muscle, and Rabbit ,Slow Skeletal Muscle A Protein-Chemical Analysis of Muscle Actin Differentiation JOEL VANDEKERCKHOVEI and KLAUS WEBER2 Max-Planck-Institute for Biophysical Chemistry, Postfach 968, D-3400 Goettingen, Federal Republic of Germany
Complete amino acid sequencesfor four mammalian muscle actins are reported: bovine skeletal muscle actin, bovine cardiac actin, the major component of bovine aorta actin, and rabbit slow skeletal muscle actin. The number of different actins in a higher mammal for which full amino acid sequences are now available is therefore increased from two to five. Screeningof different smooth muscle tissuesrevealed in addition to the aorta type actin a secondsmooth muscle actin,which appearsvery similarifnot identicalto chicken gizzard actin. Since the sequence of chicken gizzard actin is known, six different actins are presently characterized in a higher mammal. The two smooth muscle actins - bovine aorta actin and chicken gizzard actin - differ by only three amino acid substitutions,all located in the amino-terminalend. In the rest of their sequencesboth smooth muscle actins share the same four amino acid substitutions,which distinguishthem from skeletal muscle actin. Cardiac muscle actin differs from skeletal muscle actin by only four amino acid exchanges. No amino acid substitutions were found when actins from rabbit fast and slow skeletal muscle were compared. In addition we summarizethe amino acid substitutionpatterns of the six different mammalian actins and discuss their tissue specificity. The results show a very close relationship between the four muscle actins in comparison to the nonmuscle actins. The amino substitution patterns indicate that skeletal muscle actin is the highest differentiated actin form,whereas smooth muscle actins show a noticeably closer relation tononmuscleactins. Bythesecriteriacardiacmuscleactinliesbetweenskeletalmuscleactin and smooth muscle actins. Introduction
Primary sequence information on apparently similar polypeptides from different sources can establish differences which may give important clues to how genetic programs are regulated during development. It has become increasingly clear in the last few years that, although actin is an ubiquitous protein present in both muscle and nonmuscle tissues, actins from different sources are heterogeneous [I-91. This heterogeneity is 1 Present address: Laboratoriurn voor Histdogie en Genetika, Rijksuniversiteit Gent, Ledeganckstraat 35, B-9000, Ghent, Bel-
Bium
2 To whom reprint requests should be addressed: Dr.Klaus Weber (address see above)
revealed to a limited extent by isoelectric focussing [3,5, 101, but unambiguous identification of the particular actin type, as well as a true estimate of the number of different actins expressed in a eukaryotic organism, demands a direct protein chemical approach. Currently complete amino acid sequence data are available only for rabbit skeletal muscle actin [11-131, for the two mammalian cytoplasmic actins [131,and for the major smooth muscle actin from chicken gizzard [141. In addition, protein chemical analysis of the amino-terminal tryptic peptide of actins from different tissues has shown that at least six different actins are expressed in a higher mammal, i.e., two nonmuscle actins, two smooth muscle actins, one cardiac muscle actin, and one fast skeletal muscle actin [81. These results support the idea that
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higher vertebrates express different actins in a tissuespecific manner, but clearly additional complete actin sequences are necessary, both to establish the extent of actin divergence in tissues of higher vertebrates, and to identify those substitutions that are typical of the different actin types. Here we report the complete amino acid sequences of three actins derived from different bovine muscle types (skeletal muscle, cardiac muscle, and aorta tissue), as well as the sequence of rabbit slow skeletal muscle actin. The similarities and differences revealed in different actin sequences are discussed. Methods Actin Preparations
The following bovine tissues were used: skeletal muscle, cardiac muscle, uterus, rumen wall, complete aorta, and the dissected tunica intima and tunica media respectively. In addition the following chicken tissues were used: gizzard and complete aorta. Dissected slow skeletal muscle tissue from the rabbit soleus was a generous gift of Dr. R. Whalen, Institute Pasteur, Paris, France. Tissues were blended for 30 s in five volumes of acetone (-100 C).ARer low speed centrifugation the acetone was removed and the tissue material subjected to three further acetone extractions. The resulting material was dried for two hours at room temperature (‘acetone powder’) and stored at -200C. Actins were solubilized from acetone powders by two extractions for 1 h each at 4’ C using low salt buffer (2 mM Tris-HC1,0.2 mM ATP, 0.5mM 2-mercaptoethanol, 0.2 mM CaCI,; fmal pH 7.8) [151. Actin was purified from the combined extracts by affinity chromatography on immobilized pancreatic DNAase-I [161. Actin was precipitated from the 3 M guanidineHC1 eluate by the addition of four volumes of ethanol at -200 c. Amino Acid Sequence Determlnation
The amino acid sequence of residues five to 375 is based on a rapid and convenient screening procedure, which requires only 5mg of purified actin. This procedure is an improvement over the one previously described by us [131. Briefly, actin was subjected to performic acid oxidation followed by trypsin digestion. The resulting soluble tryptic peptides were further digested with the protease of Staphylococcus aureus. The insoluble tryptic peptides were cleaved using themolysin digestion. Both secondary digests yielded fully soluble peptides with an average length of 6-7 amino acid residues. Both peptide mixtures were separated on Whatman 3MM paper into individual peptides using a two-dimensional preparative ‘fingerprint’ system in which electrophoresis at either pH 6.5 or pH 3.3 is combined with descending chromatography 1131. Peptides were detected with dilute fluorescamine (171 and recovered. An aliquot was used for amino acid analysis. The amino acid compositions of the peptides were compared with the corresponding rabbit skeletal muscle actin peptides serving as a standard. When an amino acid exchange was indicated by this comparison the peptide was subjected to detailed amino acid sequence analysis in order to locate the exchange. In most cases the direct micro dansyl-Edman procedure [I81 was
J. Vandekerckhove and K. Weber: Different Muscle Actins suEcient for this purpose. Occasionally with longer peptides further enzymatic cleavage by chymotrypsin was necessary. The resulting secondary peptides were separated, detected, and recovered as described above. The secondary peptides were studied by amino acid composition and dansyl-Edman degradation in order to localize the amino acid exchange. Carboxy-terminal sequences when necessary were obtained by a time course digestion using carboxypeptidase A. Amide assignments were obtained either by digestion with leucine amino peptidase or by the use of the Offord plot 1191. The positioning of the peptides in the actin polypeptide chain was made by analogy with the published sequence of rabbit skeletal muscle actin [ l 11 taking into account the recently proposed minor corrections [12, 13, 201. The procedure described above covers the full amino acid sequence of the actin polypeptide with the exception of the very acidic amino terminal tetra peptide [131. The amino acid sequence of these residues is easily studied in the isolated amino terminal tryptic peptide covering residues one to IS. This procedure has been documented [81. The amino terminal sequence of most of the actins for which we provide now the full amino acid sequence has been reported by a detailed analysis of their amino-terminal tryptic peptide
MI. Characterization of Actins by Their Amino-Terminal Ttyptic Peptide
‘he procedure is based on our method for characterization of the amino-terminal tryptic peptide from performic acid oxidized actins by two-dimensional fmgerprints 161. Since all actins studied SO far [8, 9, 11-13, 201 show an unproportionally high relative number of amino acid exchanges in their amino-terminal tryptic peptide, the method can be used to assess possible actin divergence even if a full amino acid sequence is not available. Given the fact that all aminoterminal actin peptides contain at least one cysteine residue MI it is possible to increase the sensitivity of the technique by using actins carboxymethylated by C14-iodoa~eti~ acid. The C*4-labeledaminoterminal tryptic peptides were isolated, stabilized by performic acid oxidation, and fmally separated by a two-dimensional paper electrophoretic system. Peptides were detected by autoradiography using Kodirex Kodak film. Peptides were identified in the two-dimensional fmgerprint system by comparing their position with the corresponding CI4-labeled reference peptides taken from actins with known amino-terminal sequences (skeletal muscle, bovine aorta, chicken gizzard, and the p and y-cytoplasmic actins from bovine brain)
MI. Miscellaneous Procedures
Purfied actins were characterized in 9M urea by isoelectric focussing analysis [211. Polyacrylamidegels w m stained and destained as described [221. The isolated actins showed a purity of more than 95% when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.
Results
Isoelectric Focussing Analysis of Bovine Aorta Actin Isoelectric focussing analysis revealed three! components in total aorta actin (Fig. 1). The major component be-
125
J. Vandekerckhove and K. Weber: Merent Muscle Actins
haved very similarly to sarcomeric a-actin provided by skeletal muscle or cardiac muscle actin. In addition the analysis showed the presence of p-like and y-like actins using bovine brain nonmuscle actin as standard (for actin nomenclature in isoelectric focussing analysis see [3, 5, 6, 131. The ratio of the three isoelectric species
a Py I II
Fig. 1. Isoelectric focussing analysis of bovine aorta actins. Sample 2 is total aorta actin. Samples I , 3, and 4 contain aorta actin mixed with actin from bovine heart (I),bovine brain (3), or chicken gizzard (4). The positions of sarcomeric a-actin and p- and y-nonmuscle actins are indicated
was calculated from a densitometric scan of the gel: alike 72%, p-like 796, and y-like 21%. This ratio agrees with a protein chemical analysis based on the different amino-terminal tryptic peptides of bovine aorta actin [81. In addition use of radioactive labeling using C"iodoacetate and separation of the carboxy-methylated amino-terminal peptides yielded a similar ratio (see below and Fig. 3B). These results identified the d i k e actin as the major smooth muscle actin in aorta tissue. The p-band corresponds to the p-like actin, which is most likely p-nonmuscle adin [81. The y-band corresponds to the sum of y-non-muscle actin and y-like smooth muscle actin of the stomach tissue type (see below and references [8, 141). By these criteria, the major aorta smooth muscle actin (a-like) accounts for close to 75% of the total aorta actin. Since there is currently no preparative procedure for separating the different actin species, we used the unfractionated aorta actin in order to obtain an amino acid sequence of the a-like smooth muscle actin, without finding interference by the three other minor actin species.
- Asp - Asn - Gly - S e r - Cly - Leu -m- Lys - Ala - Gly - m-Glu - m-m- - Thr - A l e - Leu - Val - Cys 30 40 Phe - A l a - Gly - Asp - Asp - A l a - Pro - Arg - A h - Val - Phe - Pro - S e t - Ile - V a l - Cly - Arg - Pro - Arg - His 60 -Gin - Gly - Val - Met - Val - Gly - Met - Gly - Gln - Lys - Asp - S e r - Tyr - Val - Gly - Asp - Glu - Ala - Gln - Ser 70 80 Lys - Arg - Gly - Ile - Leu - Thr - Leu - Lys - Tyr - Pro - I l e - Glu - Hi:% Gly - I l e - I l e - Thr - Asn - Trp - Asp 90 100 Asp - Met - Glu - Lys - I l e - Trp - His - His -mPhe - Tyr - Asn - Glu - Leu - Arg - Val - A l a - Pro - Glu - Glu 110 I20 His - Pro - Thr - Leu - Leu - Thr - Glu - A l a - Pro - Leu - Asn - Pro - Lys - A l a - Asn - Arg - Glu - Lys - M e t - Thr I30 140 Gln - Ile - Met - Phe - Glu - Thr - Phe - Asn - Val - Pro - Ala - Met - Tyr - V a l - Ala - Ile - Gln - A l a - Val - Leu I50 160 Ser - Leu - Tyr - A l a - S e r - Gly - Arg - Thr - Thr - Gly - I l e - Val - Leu - Asp - Ser - Gly - Asp - Gly - Val - Thr I 70 I80 His - Asn - Val - Pro - Ile - Tyr - Glu - Gly - Tyr - Ala - Leu - Pro - His - A l a - I l e - M e t - Arg - Leu - Asp - Leu 190 200 Ala - Gly - Arg - Asp - Leu - Thr - Asp - Tyr - Leu - Met - Lys - I l e - Leu - Thr - Glu - Arg - Gly - Tyr - Ser - Phe 2 10 220 V a l - Thr - Thr - A l a - Glu - Arg - Glu - I l e - Val - Arg - Asp - I l e - Lys - Glu - Lys - Leu - Cys - Tyr - V a l - A l a 230 234a Leu - Asp - Phe - Glu - Asn - Glu - Met - Ala - Thr - Ala - Ala - Ser - S e r - S e r - Ser - Leu - Glu - Lys - Ser - Tyr 240 250 Glu - Leu - Pro - Asp - Gly - Gln - Val - I l e - Thr - I l e - Cly - Asn - Glu - Arg - Phe - Arg - Cys - Pro - Glu - Thr 2 70 _260 __ Leu - Phe - Gln - Pro - S e r - Phe - I l e - Gly - Met - Glu - S e r - Ala - Glp - I l e - R i a - Glu - Thr - Thr - Tyr - Asn 280 2 90 S e r - I l e - M e t - Lys - Cys - Asp - I l e - Asp - I l e - Arg - Lys - Asp - Leu - Tyr - A l a - Asn - Asn - Val -mSer 300 310 Gly - Gly - Thr - Thr - Met - Tyr - Pro - Gly - I l e - Ala - Asp - Arg - Met - Gln - Lys - Glu - I l e - Thr - A l a - Leu 320 330 A l a - Pro - S e r - Thr - M e t - Lys - I l e - Lys - Ile - Ile - Ala - Pro - Pro - Clu - Arg - Lys - Tyr - Ser - V a l - Trp 340 350 I l e - Gly - Gly - Ser - Ile - Leu - Ala - Ser - Leu - S e r - Thr - Phe - Gln - Gln - Met - Trp - Ile -uLys - Gln 370 374 _360 __ Glu - Tyr - Asp - Glu - A l a - Gly - Pro - Ser - Ile - Val - His - Arg - Lys - Cys - Phe 10
X
20
_.
50
-
Fig. 2. Amino acid sequence of the major actin species of bovine aorta. X indicates a blocking group which is most likely the acetyl group found in other actins [30]. HisrMcis Nr-methylhistidint.The peptides of aorta actin were aligned in analogy with the amino acid sequence of rabbit skeletal muscle actin [ 111 using the minor corrections proposed later [ 12,131. Residues in boxes indicate amino acid exchanges by comparisonto rabbit skeletal muscle actin. For residue 234a see Discussion
126
J. Vandekerckhove and K. Weber: Different Muscle Actins
Table 1. Peptides of bovine aorta actin (A) differingin amino acid compositionfrom the corresponding peptides of bovine cardiac actin (C), rabbit slow skeletal muscle actin (S) and bovine fast skeletal muscle (F). Values given correspond to 22 h hydrolysates;no corrections were made for incomplete hydrolysis or partial destruction
Peptide 85-91
Peptide 1-18 A Cysteic acid Aspartic acid Methionine sulfone Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan
C
S
F
A
C
Peptide 29 1-3 11 S
F
1.9 1.0 1.0 I-0 3.1A4.12 0.1 0.8 1.9 2.8
1.8 1.8 1.8 02 0.8 - 1.0 0 . 9
2.0 1.0
1.9 1.0
0 . 9
0.9
0.l
0.l
03
2.2 2.0 - 2.1 1.0
2.2 1.0
02.91.91.9 0.8 2.0
1.9
2.0
0.8
1.0
0.8
0.8
A
C
S
F
4.2 1.1 1.9 1.0 0.2 0.9 3.3 2.0 1.0 1.1
4.1 1.1 -
4.3 1.9 1.9 1.1 0.2 1.2 3.3 2.0 1.1 1.0
4.0 1.9 -
1.8 0.9 0.2 0.9 3.2 2.0 0.9 1.0
A
C
S
F
2.0 0.9
1.0
I.0
1.0
1.0
1.1 3.1 2.0 1.0 0.9
0.9
0.8
0.9
0.9
0.9
0.9
0.8
0.9
m u L l 0 . 9
1.8 0.9
0.9
0.8
Peptide 356-358
0.9 1.0
0.7
1.0
1.0
0.9 1.0
1.8
1.7
1.7
1.8
+
+
+
+
0.9
The Complete Amino Acid Sequence of the Mqior Bovine Aorta Actin The 79 different peptides (including some overlapping peptides) which cover the complete amino acid sequence of the actin polypeptide chain (see Methods) were screened by amino acid analysis. Only four of these pep tides showed a difference when their amino acid composition was compared with that of the corresponding peptides of rabbit skeletal muscle actin. These peptides are listed in Table 1. The exact location of the amino acid exchanges was determined as follows:
Peptide 1-18. The complete amino acid sequence of this peptide has been documented [81. It showed the following five amino acid replacements in comparison to rabbit muscle actin: position 1 glutamic acid rather than aspartic acid; position 3 glutamic acid rather than aspartic acid; position 4 aspartic acid rather than glutamic acid; position 5 serine rather than threonine; position 17 cysteine rather than valine (for a summary see Table 2). Peptide 85-91. This peptide originates by a chymotryptic-like cleavage at tyrosine 91 as shown previously for
1.8 0.1 0.1
1.7
1.0
1.1
1.8
1.1
1.8
1.1
gizzard smooth muscle actin [14]. From its amino acid composition (Ser 0.8; Ile 0.8; Tyr 0.9; Phe 1.0; His 1.8 and the presence of tryptophan) a threonine to serine exchange is predicted in comparison to skeletal muscle actin (Table 1). Using a time course digestion of the peptide with carboxypeptidase A (1 h: Tyr 0.7, Phe 0.5, Ser 0.1; 4 h: Tyr 0.9, Phe 0.6, Ser 0.3, His 0.1) this amino acid exchange was localized at position 89.
Peptide 291-31 1. From the composition of this peptide (Asp 4.2; methionine sulfone 1.1; Thr 1.9; Ser 1.0; Pro 0.9; Gly 3.3; Ala 2.0; Val 1.0; Ile 1.1; Leu 2.0; Tyr 1.8; Arg 1.0) a methionine (rabbit muscle actin) to leucine exchange (aorta actin) is predicted (Table 1). Localization of this exchange was possible in a secondary chymotryptic peptide, i.e., residues 294-298 (Asp 1.9; Ala 1.0; Val 0.9; Leu 0.9) by a time course digestion with carboxypeptidase A (after 10 min: Leu 0.6, Val 0.1; after 45 min: Leu 0.8, Val 0.6, Asn 0.1). Thus position 298 in aorta actin is leucine rather than the methionine residue found in skeletal muscle actin. Peptide 356-358. Amino acid composition of this tripeptide (Ser 1.0; Ile 0.9; Lys 0.9) revealed an amino acid exchange from threonine (skeletal muscle actin) to serine
127
J. Vandekerckhove and K. Weber: Different Muscle Actins
Tabk 2. Positions in the amino acid sequences at which exchanges have been detected between four different muscle actins and the two different nonmuscle actins. Positioning of the amino acids in the actin sequences is made in analogy to rabbit skeletal muscle actin 111-131. Amino acid residues in which the four muscle actins differ among themselves are underlined. The data for nonmuscleactins are from Reference 13 and those for stomach smooth muscle actin (chicken gizzard actin) are from Reference 14. Residue number 1 is absent in stomach smooth muscle actin and nonmuscle actin. All actins carry a blocked amheterminus, which is most likely the acetyl group found in skeletal muscle actin [301
Residue number
Actin types Skeletal muscle (fast, slow)
1 2 3 4 5 6 10 16 17 76 89 103 129 153 162 176 20 1 225 259 266 27 1 278 286 296 298 357 364
ASP Glu & P Glu
Thr Thr CYS
Leu Ile .
Thr Val Leu Asn Met Val Am
Cardiac muscle
ASP ASP Glu Glu Thr Thr
_ .
Smooth muscle (aorta)
& G
Glu Glu -
&G
ALP
Glu Thr
Ser
Thr
Thr
CYS Leu Val Ile Thr Thr Val
CYS
Ile
CYS Leu cys Ile
Leu
cys Ser
ser -
Thr
Thr
Val
Val
Leu
Leu
Leu
Asn
Asn Met Val Asn Thr Ile Ala
Asn Met Val Asn Thr Ile Ala
Thr
Thr
Ile Asn
Ile Asn
Met Val Ans
Thr
Thr
Ile Ala Thr Ile Asn
Ile Ala Thr
Met -
Leu Ser -
Ala
Smooth muscle (stomach)
IlC
Asn
Ala
Leu Ser Ala
(aorta actin). Direct dansyl-Wan degradation provided the amino acid sequence Ile-Ser-Lys thus localizing the amino acid exchange at position 357. Figure 2 shows the complete amino acid sequence of the major aorta actin. The order of the peptides covering the full polypeptide chain (see Methods) is based on homology with the rabbit skeletal muscle actin sequence [ l l l . The polypeptide chain contains 375 residues although the numbering system indicates only 3 74. This is because a serine residue following residue 234 has probably been overlooked in the original rabbit skeletal mus-
Non-muscle P-type
v-type
-
-
ASP ASP ASP
Glu Glu Glu Ile Ala
Val
Ile Met CYS Val Thr Val Thr Met Thr
Leu Thr Gln Ala
Leu CYS Phe Phe Thr
Ser
Leu
Leu Sa
Ala
Ser
cle actin sequence [12]. To avoid renumbering of the previously documented amino acid exchanges past this residue in other actins, we agree with the proposal [12, 201 to indicate this residue as serine 234a. Aorta smooth muscle actin differs by eight amino acid exchanges from skeletal muscle actin (Tables 2 and 3). These substitutions occur at positions 1,3,4, 5, 17,89, 298, and 357. Comparison with the chicken gizzard actin sequence 1141 reveals only three substitutions, all of which are localized in the amino-terminal end (positions 1,4, and 5; Table 2). We show below that an actin similar, if not
128
J. Vandekerckhove and K. Weber: Different Muscle Actins
Table 3. Evolutionary relationship of the four different muscle actins from higher vertebrates. Distances are given by the number of amino acid exchanges. For details see Table 2 Skele- Cardiac Stomach Aorta tal Skeletal muscle
-
Cardiac muscle
4
'Stomach' smooth muscle 'Aorta' smooth muscle
4
6
8
6
6
4
4 -
8
6
3
-
/3
25
23
23
23
y
24
22
20
22
-
3
Cytoplasmic (mammalian)
identical with chicken gizzard actin, is also the major smooth muscle actin in bovine stomach tissue.
Screening of the Two Smooth Muscle Actins in Different Tissues Although the bovine aorta actin (a-like) and chicken gizzard (y-like) smooth muscle actins are separated by isoelectric focussing, the technique does not readily resolve the a-like actin from sarcomeric a-actin and the ylike actin from y-nonmuscle actin (Fig. 1). The two smooth muscle actins are however readily identified in the two-dimensional fingerprint analysis of the amino-terminal tryptic peptides derived from performic acid oxidized actins [81. We have improved this technique by using carboxymethylation with C 14-iodoacetate rather than performic acid to modify the cysteine residues. This approach offered two advantages. First, detection of the amino-terminal peptides is now based on autoradiography, which is much more sensitive than the peptide stains previously used. Second, because much smaller amounts could be loaded on the paper, the amino-terminal peptide of y-nonmuscle actin no longer smeared during the pH 3.3 and pH 4.7 electrophoresis 181. Thus we can now detect the presence of traces of ynonmuscle actin as minor component in smooth muscle tissues. In Fig. 3 use of this characterization of C"-labeled amino-terminal tryptic peptides is made. Figure 3H shows with a reconstituted mixture the separation pattern of the peptides from the following five actins commonly found in higher vertebrates: a-like smooth muscle actin, y-like smooth muscle, a-skeletal muscle actin, and the two nonmuscle actins p and y. Note the clear
separation of the two smooth muscle actin peptides. Actins from different tissues containing smooth muscle cells were screened by this procedure (Fig. 3A-G). Chicken aorta actins (Fig. 3A) showed a pattern of amino terminal peptides very similar to that of bovine aorta actins (Fig. 3B). In both cases the a-like smooth muscle actin was the major actin species, although a small amount of y-like smooth muscle actin is clearly detected. In addition both actin samples contained a small amount of the nonmuscle actins p and y with p expressed preferentially. The spot labeled by an asterisk has also been noted in a previous study using performic acid oxidized actins and is most likely due to an isomerization at aspartic acid residue 4 in aorta a-like actin and should therefore be counted as a derivative of the major aorta actin 181. Currently we do not know if this conversion occurs only in vitro or already in vivo. We also have no explanation why this derivative is apparently missing in chicken aorta actin. The presence of two smooth muscle actins in aorta prompted us to study their relative ratio in dissected tunica intima (innermost coat) and tunica media. No significant difference was detected (Figs. 3C and 3D). The major component of chicken gizzard (7-like) actin (Fig. 3E) was also detected as the major component in the actin from the muscular wall of bovine rumen (Fig. 3F), although the latter actin also contained in addition a considerable amount of a like smooth muscle actin. In bovine uterus both smooth muscle actins are present in equal amounts (Fig. 3G). It should be noted that all smooth muscle tissues so far characterized revealed as minor components the p-nonmuscle actin and frequently the y-nonmuscle actin with the former expressed at a higher level than the latter. In addition there are a few minor spots, which so far have not been identified.
The Complete Amino Acid Sequence of Bovine Cardiac Actin A comparative study on the sequences in the aminoterminal tryptic peptides (residues 1-18) of various bovine actins [81 revealed two amino acid exchanges when bovine cardiac actin was compared with rabbit and bovine skeletal muscle actin (see below). These exchanges involved residue 2, aspartic acid to glutamic acid, and residue 3, glutamic acid to aspartic acid, with the first residue being typical of the skeletal muscle actins. Performic acid oxidized bovine cardiac actin was fragmented as described under Methods. The peptides purified from the various digests cover residues 5-375 of the actin polypeptide chain. Only two main peptides,
J. Vandekerckhove and K. Weber: Different Muscle Actins
129
Flg. 3. Two-dimensional separation pattern of the C"-carboxymethylated actin amino-terminaltryptic peptides. Horizontal separation is by electrophoresis at pH 3.3, and vertical separation is by electrophoresisat pH 6.5. Distances in cm on both axes were measured from the origin. The amino-tmninal peptides are from actins of the following tissues:chicken aorta (A),bovine aorta (B), bovine aorta tunicu intimu (C), bovine aorta tunicu medfa @), chicken gizzard (E), bovine rumen (P),and bovine uterus (G). (H)shows a reconstituted mixture of a-like smooth muscle actin of bovine aorta (I),y-like smooth muscle actin of chicken gizzard (2),/3-nonmuscle actin (3), y-nonmuscleactin (4, and a-skeletal muscle actin (5).For an explantation of the spot labeled by an asterisk see Results
130
carrying an amino acid exchange in comparison to rabbit skeletal muscle actin, were detected (peptide 291-31 1, and peptide 356-358) (Table 1; see also reference [91). These peptides were processed as described above for the corresponding aorta actin peptides. The results proved conclusively a methionine to leucine (heart) exchange at position 298 and a threonine to serine (heart) exchange at position 357. The latter amino acid substitution was previously repotted by Elzinga et al. in a study on human and bovine cardiac actin 121. The four amino acid substitutions of bovine cardiac actin in relation to skeletal muscle actin are listed in Table 2. It should be emphasized that the bovine cardiac actin peptide containing residue 89 is identical with the corresponding skeletal muscle actin (Table 1). Thus the amino acid substitution at position 89 in aorta and gizzard actins (see above) is typical only of smooth muscle actins. The Complete Amino Acid Sequence of Bovine Skeletal Muscle Actin
In order to detect whether some of the amino acid exchanges observed in various bovine actins [7-9, 12,131 in comparison to rabbit skeletal muscle actin reflect a species specificity rather than tissue specificity, we determined the sequence of bovine skeletal muscle. This actin was already studied in 19% of its total sequence using automated procedures on selected cyanogen bromide fragments [71. In addition a previous screening of the amino acid compositionsof tryptic peptides covering 7 1% of the total sequence did not reveal any amino acid difference between skeletal muscle actins from rabbit and beef [91. Using the procedures given in Methods we have covered the complete sequence of bovine skeletal muscle actin and were unable to detect any variation from the standard reference sequence of rabbit skeletal muscle actin [ll-131. The Complete Sequence of Rabbit Slow Skeletal Muscle Actin The complete sequence of the amino-terminal -tic peptide (residues 1-18) of rabbit slow skeletal muscle actin was determined by the procedures described elsewhere [81. The sequence of this peptide was found to be identical with the sequence of the amino-terminal pep tide of rabbit back muscle [ l 1,131. The remainder of the actin polypeptide was screened by analysis of the 79 peptides which cover residues 5-375 (see Methods). No
J. Vandekerckhove and K. Weber: Different Muscle Actins
amino acid exchanges were discovered in comparison to rabbit fast skeletal muscle actin (back muscle). As an illustration for the results, we have listed in Table 1 the amino acid composition of those slow muscle actin peptides whose counterparts in bovine aorta contain an amino acid exchange in relation to rabbit back muscle actin.
Discussion
The complete amino acid sequences of the actins from bovine aorta, bovine cardiac muscle, bovine fast skeletal muscle, and rabbit slow skeletal muscle provide further aspects to our understanding of actin divergence during muscle differentiation in higher vertebrates. This study brings the number of different actin polypeptides fully characterized in the same mammalian species from two (p- and y-nonmuscle actins) 1131 to five by the results provided for bovine skeletal muscle actin, bovine cardiac actin, and smooth muscle actin from bovine aorta. In addition our results confirm the concept that a sixth actin, which has been sequenced in the case of chicken gizzard [141 is also the predominant form of bovine rumen smooth muscle tissue. In our sequence studies we have used a convenient preparative fingerprint technique in which peptides covering the whole actin polypeptide are compared with the corresponding rabbit skeletal muscle actin peptides by chromatographic and electrophoretic behaviour as well as by amino acid composition. Although this approach is very fast and especially suitable for comparative studies, we cannot rigorously exclude the possibility of some double compensating amino acid substitution within some of the peptides so far studied. Although this possibility has been encountered once in cardiac muscle actin, in general it should be a rare event because of the following reasons. First, the extremely conservative nature of the amino acid sequence of various actins has been documented in previous studies [7-9, 11-14,201. Second, the average size of the peptides encountered is six to seven residues thus lowering statistically the chance of double compensating amino acid exchanges. Third, the majority of the amino acid exchanges typically found in mammalian nonmuscle actins by direct sequence analysis of similar peptides [7, 9, 12, 131 are clearly excluded in the various muscle actins studied here. Fourth, in those cases where partial amino acid sequences were obtained by automated direct analysis of some cyanogen bromide fragments the results [71 are in full agreement with our data. For example all amino acid exchanges, which we document in bovine cardiac actin in addition to that at position 357 171 occur in
J. Vandekerckhove and K. Weber: Different Muscle Actins
regions of this actin polypeptide chain, which have not been previously subjected to direct automated analysis. Recently Lu and Elzinga [12lproposed in a study of bovine brain actin that a serine residue following residue 234 was probably overlooked in the original rabbit skeletal muscle actin sequence 11 11. We confmed this extra residue in three different mammalian nonmuscle actins [131,in chicken gizzard actin [141and in the actin from Physarum polycephalum [20l.We also found this additional serine residue in all four muscle actins studied in this report. To avoid renumbering of the previously documented amino acid exchanges past this residue, we agree with the proposal [121 to indicate this residue as serine 234a (see also Fig. 2). Our results on the major actin species of bovine aorta provide the second full amino acid sequence of a smooth muscle actin. Aorta actin diffms by only three amino acid exchanges from the smooth muscle actin expressed in the chicken stomach tissue (chicken gizzard actin) [141. All amino acid exchanges occur in the amino-terminal tryptic peptide (residues 1, 4, and 5 ) again emphasizing that this region of the actin polypep tide contains a disproportionally high number of changes [8, 91. The presence of an additional aspartic acid residue in aorta actin (position 4) explains the lower isoelectric point of this actin (a-like) in comparison to the more basic gizzard actin (y-like). In the remainder of their sequence both smooth muscle actins share four amino acid exchanges in comparison to rabbit skeletal muscle actin (Table 2). Of these positions 17, 298, and 357 involve amino acid residues, which are also typical of nonmuscle actin [7,9, 12, 131. Position 89 is especially interesting because this amino acid substitution is typical of smooth muscle actins and is missing in other muscle actins as well as in nonmuscle actins [9, 13, 141. The three amino acid differences between bovine aorta actin and chicken gizzard actin do not reflect species divergence but are clearly due to tissue specificity because of the following results. First, bovine aorta contains a minor actin, which has an amino-terminal tryptic peptide identical in amino acid sequence with the corresponding gizzard actin peptide [81.Second, actins from bovine and chicken aorta give an extremely similar ftngerprint pattern of their amino-terminalpeptides labeled by C14-carboxymethylation.In both cases the peptide correspondingto the a-like actin, detected by isoelectric focussing analysis, is the major product accounting for close to 7096 of the total actin of the tissues. Third, a similar analysis of cow rumen tissue indicates in agreement with previous findings on chicken gizzard that the
131
y-like smooth muscle actin is the major component of stomach tissue. Fourth, in the case of bovine uterus both actin components are present in equal amounts. There are several aspects of actin expression in smooth muscle tissues, which we currently do not understand. Thus we do not know if the minor actin species, which most likely corresponds to p and y-nonmuscle actins, are derived from contaminating nonmuscle cells especially rich in p-actin or if they are inherent components in the smooth muscle cells of the tissue. Since we have characterized these species only by their amino-terminal tryptic peptide, we cannot yet say whether they really reflect nonmuscle actins in the rest of their sequence. In addition it is not clear if in those smooth muscle tissues like aorta and uterus the two different smooth muscle actins (a-like and y-like) are present in the same cell or are expressed in different cells. In an attempt to answer this question aorta tissue was separated into the two histologically well defined parts, i.e., tunica intima and tunica media, but the same ratio of different actins was found as in the undissected tissue. Clearly further experiments are necessary to understand the complexity of actin expression in smooth muscle tissues. The complete amino acid sequence of bovine cardiac actin revealed only four amino acid exchanges in comparison to rabbit skeletal muscle actin (positions 2, 3, 298,and 357). Two of these (positions 298 and 357) are also typical of the two nonmuscle actins 17, 9, 12, 131 and the two smooth muscle actins. The other two changes (positions 2,3)although involving charged residues reflect only a permutation. Thus the overall net charge of cardiac actin and skeletal muscle actin must be the same, in agreement with isoelectric focussing studies, which classified both actins as a-type [6, 231. Since the amino acid exchanges in four different bovine actins - two nonmuscle actins [ 131;aorta actin and cardiac muscle actin - have been expressed in reference to the standard rabbit muscle actin sequence [ 11, 131, we also completed the sequence of bovine skeletal muscle actin in order to avoid a potential species specificity of actin. No amino acid differences were found between the two skeletal muscle actins in agreement with previous studies indicating lack of species specificity in the case of nonmuscle actins from various mammals [9, 131. Although troponins, tropomyosins, a-actinins, and myosins of fast and slow skeletal muscle can be generally distinguished immunologically as well as by protein chemical data [24-291 we were unable to detect any amino acid exchanges between the actins prepared from
132
the two different rabbit muscles. This result could indicate that both actins are coded by the same gene. Alternatively in view of the extremely conserved amino acid sequence of actins in general by contrast to other muscle proteins (see for instance [241) it remains possible that although the actin gene products are the same in slow and fast skeletal muscle they are coded by different genes. This possibility would stay in line with the current concept of different contractile proteins in various muscles. Table 2 summarizes the various amino acid exchanges, which distinguish the four different muscle actins so far characterized by full amino acid sequence analysis. In addition it shows the amino acid substitution pattern for the two mammalian nonmuscle actins [131. The combined data reveal a distinct evolutionary development between nonmuscle actins and the various muscle actins. Clearly nonmuscle actins have to be considered as the archetype actin, since they occur in all nonmuscle cells including the lower eukaryotic unicellular organisms as for instance in amoebas. In line with this interpretation is the fact that even mammalian nonmuscle actins differ by fewer amino acid exchanges from the actin of Physarum polycephalurn than from mammalian muscle actins [20l.The typical amino acid exchange patterns at positions 17, 298, and 357 indicate that skeletal muscle actin is the highest differentiated actin species. Closest to skeletal muscle actin is cardiac actin (Tables 2 and 3) which shows only four replacements. The smooth muscle actins and also probably the aorta type actin show a somewhat closer relation to nonmuscle actins than skeletal muscle actin, although they still differ by some 23 or 22 residues in a comparison of p- and y-nonmuscle actin and aorta actin. The results emphasize again that residues 1 to 17 comprise a relatively variable region of the actin polypeptide chain 19, 131. Since full amino acid sequences are now available for six different mammalian actins it will be interesting to see, if some of the amino acid exchanges can be correlated with functional properties of actin in vivo or in vitro. Acknowledgements:J. V. is bevoegdverklaard nawrser at the Belgian National Fund for Scientific Research (N.F.W.O.). We thank M. Puype for excelllent technical assistance. We thank Dr. R. Whalen for a sample of rabbit slow skeletal muscle tissue. This work was supported in part by a research grant from the Belgian National Fund for Scientific Research (N.F.W.O.) (Krediet aan Navorsers to J.V.).
References 1. Gruenstein. E., Rich, A.: Non-identity of muscle and non-muscle actins. Biochem. Biophys. Res. Commun. 64, 472 (1975)
J. Vandekerckhove and K. Weber: Dflerent Muscle Actins
S.: Human heart and platelet actins are products of different genes. Science 191, 94
2. Elzinga, M., Maron, B. J., Adelstein, R.
(1976) 3. Whalen, R.G., Butler-Browne, G. S., Gros, F.: Protein synthesis
and actin heterogeneity in calf muscle cells in culture. Proc. Natl. Acad. Sci. USA 73, 2018 (1976) 4. Storti, R. V., Rich, A.: Chick cytoplasmic actin and muscle actin have different structural genes. Proc. Natl. Acad. Sci. USA 73, 2346 (1976) 5. Garrels, J., Gibson, W.: Identification and characterization of multiple forms of actin. Cell 9, 793 (1976) 6. Rubenstein, P., Spudich, J.: Actin microheterogeneity in chick embryo fibroblasts. Proc. Natl. Acad. Sci. USA 74, 120 (1977) 7. Elzinga, M., Lu, R.:Comparative amino acid sequence studies
on actin. In: Contractile systems in nonmuscle tissues. Perry, S. V., Margreth, A., Adelstein, R. (eds.), p. 29. Amsterdam: North Holland Biomedical Press 1976 8. Vandekwckhove,J., Weber, K.: At least six different actins are expressed in a higher mammal: an analysis based on the amino acid sequence of the amino terminal tryptic peptide. J. Mol.Biol. 126, 783 (1978) 9. Vandekerckhove, J., Weber, K.: Mammalian cytoplasmic actins
are the products of at least two genes and differ in primary structure in at least 25 identified positions from skeletal muscle actins. Proc. Natl. Acad. Sci. USA 75, 1106 (1978) 10. Zechel, K., Weber, K.: Actins from mammals, bird, fish and slime mold characterized by isoelectric focussing in polyacrylamide gels. Eur. J. Biochem. 89, 105 (1978) 11. Collins, J., Elzinga, M.: The primary structure of actin from rabbit skeletal muscle. J. Biol. Chem. 250, 5915 (1975) 12. Lu, R., Elzinga, M.: Partial amino acid sequence of brain actin and its homology with muscle actin. Biochemistry 16, 5801 (1977) 13. Vandekerckhove, J., Weber, K.: Actin amino acid sequences.
Comparison of actins from calf thymus, bovine brain and SV40transformed mouse 3T3 cells with rabbit skeletal muscle actin. Eur. J. Biochem. 90, 451 (1978) 14. Vandekerckhove, J., Weber, K.: The amino acid sequence of actin from two different chicken muscles: skeletal muscle actin and gizzard smooth muscle actin. FEES Lett. 102, 219 (1979) 15. Spudich, J., Watt, S.: The regulation of rabbit skeletal muscle contraction. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J. Biol. Chem. 246, 4866 (1971) 16. Lazarides, E., Lindberg, U.: Actin is the naturally occurring inhibitor of deoxyribonucleaseI. Proc. Natl. Acad. Sci. USA 71, 4742 (1974) 17. Vandekerckhove, J., Van Montagu,
M.:Sequence analysis of fluorescamine-stainedpeptides and proteins purified on a nanomole scale. Application to proteins of bacteriophage MS2. Eur. J. Biochem. 44, 279 (1974) 18. Bruton, C. J., Hartley, B. S.: Chemical studies on methionyl-tRNA synthetase from Escherichia colf. J. Mol. Biol. 52, 165 (1970) 19. Offord,
R. E.: Electrophoretic mobilities of peptides on paper and their use in the determination of amide groups. Nature 211,
591 (1966) 20. Vandekerckhove, J., Weber, K.: The amino acid sequence of Physarum actin. Nature 276, 720 (1978) 21. O’Farrell, P. H.: High resolution two-dimensional elcctrophoresis of proteins. J. Biol. Chem. 250, 4007 (1975)
133
J. Vandekerckhove and K. Weber: M e r e n t Muscle Actins 22. Zechel, K.: On the resolution of polypeptides by isoelectric focussing in polyacryltunidegels in the presence of urea and nonidet - P40. Anal. Biochem. 83, 240 (1977) 23. Izant, J. G., Lazarides, E.: Invariance and heterogeneity in the major structural and regulatory protehs of chick muscle cells revealed by two-dimensional gel electrophoresis. Proc. Natl. Acad. Sci. USA 74, 1450 (1977) 24. Willrinson, J. M.,Grand, R. J. A,: Comparison of amino acid sequence of troponin I from Werent striated muscles. Nature 271, 31 (1978) 25. Wilkinson, J. M.: The components of troponin from chicken fast skeletal muscle. A comparison of troponin T and troponin I from breast and leg muscle. Biochem. J. 169, 229 (1978) 26. Cummins, P., Perry, S. V.: The subunits and biological activity
27. 28. 29. 30.
of polymorphic forms of tropomyosin. Biochem. J. 133, 765 (1973) Suzuki, A., ell,D. E., Stromer, M.H., Singh, I., Temple, J.: aActinin from red and white porcine muscle. Biochim. Biophys. Acta 295, 188 (1973) Lorvey, S., Risby, D.: Light chains from fast and slow muscle myosins. Nature 234, 81 (1971) Hoh, J. F. Y., McGrath, P. A., White, R. I.: Electrophoretic analysis of multiple forms of myosin in fast-twith and slowtwitch muscles of the chick. Biochem. J. 157, 87 (1976) Alving, R. E., Laki, K.:N-terminal sequence of actin. Biochemistry 5, 2597 (1966)
Received June 1979/Accepted July 1979
Dnerentiation 14, 123-133 (1979)
0 Springer-Verlag 1979
Original A rticles The Complete Amino Acid Sequence of Actins born Bovine Aorta, Bovine Heart, Bovine Fast Skeletal Muscle, and Rabbit ,Slow Skeletal Muscle A Protein-Chemical Analysis of Muscle Actin Differentiation JOEL VANDEKERCKHOVEI and KLAUS WEBER2 Max-Planck-Institute for Biophysical Chemistry, Postfach 968, D-3400 Goettingen, Federal Republic of Germany
Complete amino acid sequencesfor four mammalian muscle actins are reported: bovine skeletal muscle actin, bovine cardiac actin, the major component of bovine aorta actin, and rabbit slow skeletal muscle actin. The number of different actins in a higher mammal for which full amino acid sequences are now available is therefore increased from two to five. Screeningof different smooth muscle tissuesrevealed in addition to the aorta type actin a secondsmooth muscle actin,which appearsvery similarifnot identicalto chicken gizzard actin. Since the sequence of chicken gizzard actin is known, six different actins are presently characterized in a higher mammal. The two smooth muscle actins - bovine aorta actin and chicken gizzard actin - differ by only three amino acid substitutions,all located in the amino-terminalend. In the rest of their sequencesboth smooth muscle actins share the same four amino acid substitutions,which distinguishthem from skeletal muscle actin. Cardiac muscle actin differs from skeletal muscle actin by only four amino acid exchanges. No amino acid substitutions were found when actins from rabbit fast and slow skeletal muscle were compared. In addition we summarizethe amino acid substitutionpatterns of the six different mammalian actins and discuss their tissue specificity. The results show a very close relationship between the four muscle actins in comparison to the nonmuscle actins. The amino substitution patterns indicate that skeletal muscle actin is the highest differentiated actin form,whereas smooth muscle actins show a noticeably closer relation tononmuscleactins. Bythesecriteriacardiacmuscleactinliesbetweenskeletalmuscleactin and smooth muscle actins. Introduction
Primary sequence information on apparently similar polypeptides from different sources can establish differences which may give important clues to how genetic programs are regulated during development. It has become increasingly clear in the last few years that, although actin is an ubiquitous protein present in both muscle and nonmuscle tissues, actins from different sources are heterogeneous [I-91. This heterogeneity is 1 Present address: Laboratoriurn voor Histdogie en Genetika, Rijksuniversiteit Gent, Ledeganckstraat 35, B-9000, Ghent, Bel-
Bium
2 To whom reprint requests should be addressed: Dr.Klaus Weber (address see above)
revealed to a limited extent by isoelectric focussing [3,5, 101, but unambiguous identification of the particular actin type, as well as a true estimate of the number of different actins expressed in a eukaryotic organism, demands a direct protein chemical approach. Currently complete amino acid sequence data are available only for rabbit skeletal muscle actin [11-131, for the two mammalian cytoplasmic actins [131,and for the major smooth muscle actin from chicken gizzard [141. In addition, protein chemical analysis of the amino-terminal tryptic peptide of actins from different tissues has shown that at least six different actins are expressed in a higher mammal, i.e., two nonmuscle actins, two smooth muscle actins, one cardiac muscle actin, and one fast skeletal muscle actin [81. These results support the idea that
0301-4681/79/0014/0123/$02.20
124
higher vertebrates express different actins in a tissuespecific manner, but clearly additional complete actin sequences are necessary, both to establish the extent of actin divergence in tissues of higher vertebrates, and to identify those substitutions that are typical of the different actin types. Here we report the complete amino acid sequences of three actins derived from different bovine muscle types (skeletal muscle, cardiac muscle, and aorta tissue), as well as the sequence of rabbit slow skeletal muscle actin. The similarities and differences revealed in different actin sequences are discussed. Methods Actin Preparations
The following bovine tissues were used: skeletal muscle, cardiac muscle, uterus, rumen wall, complete aorta, and the dissected tunica intima and tunica media respectively. In addition the following chicken tissues were used: gizzard and complete aorta. Dissected slow skeletal muscle tissue from the rabbit soleus was a generous gift of Dr. R. Whalen, Institute Pasteur, Paris, France. Tissues were blended for 30 s in five volumes of acetone (-100 C).ARer low speed centrifugation the acetone was removed and the tissue material subjected to three further acetone extractions. The resulting material was dried for two hours at room temperature (‘acetone powder’) and stored at -200C. Actins were solubilized from acetone powders by two extractions for 1 h each at 4’ C using low salt buffer (2 mM Tris-HC1,0.2 mM ATP, 0.5mM 2-mercaptoethanol, 0.2 mM CaCI,; fmal pH 7.8) [151. Actin was purified from the combined extracts by affinity chromatography on immobilized pancreatic DNAase-I [161. Actin was precipitated from the 3 M guanidineHC1 eluate by the addition of four volumes of ethanol at -200 c. Amino Acid Sequence Determlnation
The amino acid sequence of residues five to 375 is based on a rapid and convenient screening procedure, which requires only 5mg of purified actin. This procedure is an improvement over the one previously described by us [131. Briefly, actin was subjected to performic acid oxidation followed by trypsin digestion. The resulting soluble tryptic peptides were further digested with the protease of Staphylococcus aureus. The insoluble tryptic peptides were cleaved using themolysin digestion. Both secondary digests yielded fully soluble peptides with an average length of 6-7 amino acid residues. Both peptide mixtures were separated on Whatman 3MM paper into individual peptides using a two-dimensional preparative ‘fingerprint’ system in which electrophoresis at either pH 6.5 or pH 3.3 is combined with descending chromatography 1131. Peptides were detected with dilute fluorescamine (171 and recovered. An aliquot was used for amino acid analysis. The amino acid compositions of the peptides were compared with the corresponding rabbit skeletal muscle actin peptides serving as a standard. When an amino acid exchange was indicated by this comparison the peptide was subjected to detailed amino acid sequence analysis in order to locate the exchange. In most cases the direct micro dansyl-Edman procedure [I81 was
J. Vandekerckhove and K. Weber: Different Muscle Actins suEcient for this purpose. Occasionally with longer peptides further enzymatic cleavage by chymotrypsin was necessary. The resulting secondary peptides were separated, detected, and recovered as described above. The secondary peptides were studied by amino acid composition and dansyl-Edman degradation in order to localize the amino acid exchange. Carboxy-terminal sequences when necessary were obtained by a time course digestion using carboxypeptidase A. Amide assignments were obtained either by digestion with leucine amino peptidase or by the use of the Offord plot 1191. The positioning of the peptides in the actin polypeptide chain was made by analogy with the published sequence of rabbit skeletal muscle actin [ l 11 taking into account the recently proposed minor corrections [12, 13, 201. The procedure described above covers the full amino acid sequence of the actin polypeptide with the exception of the very acidic amino terminal tetra peptide [131. The amino acid sequence of these residues is easily studied in the isolated amino terminal tryptic peptide covering residues one to IS. This procedure has been documented [81. The amino terminal sequence of most of the actins for which we provide now the full amino acid sequence has been reported by a detailed analysis of their amino-terminal tryptic peptide
MI. Characterization of Actins by Their Amino-Terminal Ttyptic Peptide
‘he procedure is based on our method for characterization of the amino-terminal tryptic peptide from performic acid oxidized actins by two-dimensional fmgerprints 161. Since all actins studied SO far [8, 9, 11-13, 201 show an unproportionally high relative number of amino acid exchanges in their amino-terminal tryptic peptide, the method can be used to assess possible actin divergence even if a full amino acid sequence is not available. Given the fact that all aminoterminal actin peptides contain at least one cysteine residue MI it is possible to increase the sensitivity of the technique by using actins carboxymethylated by C14-iodoa~eti~ acid. The C*4-labeledaminoterminal tryptic peptides were isolated, stabilized by performic acid oxidation, and fmally separated by a two-dimensional paper electrophoretic system. Peptides were detected by autoradiography using Kodirex Kodak film. Peptides were identified in the two-dimensional fmgerprint system by comparing their position with the corresponding CI4-labeled reference peptides taken from actins with known amino-terminal sequences (skeletal muscle, bovine aorta, chicken gizzard, and the p and y-cytoplasmic actins from bovine brain)
MI. Miscellaneous Procedures
Purfied actins were characterized in 9M urea by isoelectric focussing analysis [211. Polyacrylamidegels w m stained and destained as described [221. The isolated actins showed a purity of more than 95% when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.
Results
Isoelectric Focussing Analysis of Bovine Aorta Actin Isoelectric focussing analysis revealed three! components in total aorta actin (Fig. 1). The major component be-
125
J. Vandekerckhove and K. Weber: Merent Muscle Actins
haved very similarly to sarcomeric a-actin provided by skeletal muscle or cardiac muscle actin. In addition the analysis showed the presence of p-like and y-like actins using bovine brain nonmuscle actin as standard (for actin nomenclature in isoelectric focussing analysis see [3, 5, 6, 131. The ratio of the three isoelectric species
a Py I II
Fig. 1. Isoelectric focussing analysis of bovine aorta actins. Sample 2 is total aorta actin. Samples I , 3, and 4 contain aorta actin mixed with actin from bovine heart (I),bovine brain (3), or chicken gizzard (4). The positions of sarcomeric a-actin and p- and y-nonmuscle actins are indicated
was calculated from a densitometric scan of the gel: alike 72%, p-like 796, and y-like 21%. This ratio agrees with a protein chemical analysis based on the different amino-terminal tryptic peptides of bovine aorta actin [81. In addition use of radioactive labeling using C"iodoacetate and separation of the carboxy-methylated amino-terminal peptides yielded a similar ratio (see below and Fig. 3B). These results identified the d i k e actin as the major smooth muscle actin in aorta tissue. The p-band corresponds to the p-like actin, which is most likely p-nonmuscle adin [81. The y-band corresponds to the sum of y-non-muscle actin and y-like smooth muscle actin of the stomach tissue type (see below and references [8, 141). By these criteria, the major aorta smooth muscle actin (a-like) accounts for close to 75% of the total aorta actin. Since there is currently no preparative procedure for separating the different actin species, we used the unfractionated aorta actin in order to obtain an amino acid sequence of the a-like smooth muscle actin, without finding interference by the three other minor actin species.
- Asp - Asn - Gly - S e r - Cly - Leu -m- Lys - Ala - Gly - m-Glu - m-m- - Thr - A l e - Leu - Val - Cys 30 40 Phe - A l a - Gly - Asp - Asp - A l a - Pro - Arg - A h - Val - Phe - Pro - S e t - Ile - V a l - Cly - Arg - Pro - Arg - His 60 -Gin - Gly - Val - Met - Val - Gly - Met - Gly - Gln - Lys - Asp - S e r - Tyr - Val - Gly - Asp - Glu - Ala - Gln - Ser 70 80 Lys - Arg - Gly - Ile - Leu - Thr - Leu - Lys - Tyr - Pro - I l e - Glu - Hi:% Gly - I l e - I l e - Thr - Asn - Trp - Asp 90 100 Asp - Met - Glu - Lys - I l e - Trp - His - His -mPhe - Tyr - Asn - Glu - Leu - Arg - Val - A l a - Pro - Glu - Glu 110 I20 His - Pro - Thr - Leu - Leu - Thr - Glu - A l a - Pro - Leu - Asn - Pro - Lys - A l a - Asn - Arg - Glu - Lys - M e t - Thr I30 140 Gln - Ile - Met - Phe - Glu - Thr - Phe - Asn - Val - Pro - Ala - Met - Tyr - V a l - Ala - Ile - Gln - A l a - Val - Leu I50 160 Ser - Leu - Tyr - A l a - S e r - Gly - Arg - Thr - Thr - Gly - I l e - Val - Leu - Asp - Ser - Gly - Asp - Gly - Val - Thr I 70 I80 His - Asn - Val - Pro - Ile - Tyr - Glu - Gly - Tyr - Ala - Leu - Pro - His - A l a - I l e - M e t - Arg - Leu - Asp - Leu 190 200 Ala - Gly - Arg - Asp - Leu - Thr - Asp - Tyr - Leu - Met - Lys - I l e - Leu - Thr - Glu - Arg - Gly - Tyr - Ser - Phe 2 10 220 V a l - Thr - Thr - A l a - Glu - Arg - Glu - I l e - Val - Arg - Asp - I l e - Lys - Glu - Lys - Leu - Cys - Tyr - V a l - A l a 230 234a Leu - Asp - Phe - Glu - Asn - Glu - Met - Ala - Thr - Ala - Ala - Ser - S e r - S e r - Ser - Leu - Glu - Lys - Ser - Tyr 240 250 Glu - Leu - Pro - Asp - Gly - Gln - Val - I l e - Thr - I l e - Cly - Asn - Glu - Arg - Phe - Arg - Cys - Pro - Glu - Thr 2 70 _260 __ Leu - Phe - Gln - Pro - S e r - Phe - I l e - Gly - Met - Glu - S e r - Ala - Glp - I l e - R i a - Glu - Thr - Thr - Tyr - Asn 280 2 90 S e r - I l e - M e t - Lys - Cys - Asp - I l e - Asp - I l e - Arg - Lys - Asp - Leu - Tyr - A l a - Asn - Asn - Val -mSer 300 310 Gly - Gly - Thr - Thr - Met - Tyr - Pro - Gly - I l e - Ala - Asp - Arg - Met - Gln - Lys - Glu - I l e - Thr - A l a - Leu 320 330 A l a - Pro - S e r - Thr - M e t - Lys - I l e - Lys - Ile - Ile - Ala - Pro - Pro - Clu - Arg - Lys - Tyr - Ser - V a l - Trp 340 350 I l e - Gly - Gly - Ser - Ile - Leu - Ala - Ser - Leu - S e r - Thr - Phe - Gln - Gln - Met - Trp - Ile -uLys - Gln 370 374 _360 __ Glu - Tyr - Asp - Glu - A l a - Gly - Pro - Ser - Ile - Val - His - Arg - Lys - Cys - Phe 10
X
20
_.
50
-
Fig. 2. Amino acid sequence of the major actin species of bovine aorta. X indicates a blocking group which is most likely the acetyl group found in other actins [30]. HisrMcis Nr-methylhistidint.The peptides of aorta actin were aligned in analogy with the amino acid sequence of rabbit skeletal muscle actin [ 111 using the minor corrections proposed later [ 12,131. Residues in boxes indicate amino acid exchanges by comparisonto rabbit skeletal muscle actin. For residue 234a see Discussion
126
J. Vandekerckhove and K. Weber: Different Muscle Actins
Table 1. Peptides of bovine aorta actin (A) differingin amino acid compositionfrom the corresponding peptides of bovine cardiac actin (C), rabbit slow skeletal muscle actin (S) and bovine fast skeletal muscle (F). Values given correspond to 22 h hydrolysates;no corrections were made for incomplete hydrolysis or partial destruction
Peptide 85-91
Peptide 1-18 A Cysteic acid Aspartic acid Methionine sulfone Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan
C
S
F
A
C
Peptide 29 1-3 11 S
F
1.9 1.0 1.0 I-0 3.1A4.12 0.1 0.8 1.9 2.8
1.8 1.8 1.8 02 0.8 - 1.0 0 . 9
2.0 1.0
1.9 1.0
0 . 9
0.9
0.l
0.l
03
2.2 2.0 - 2.1 1.0
2.2 1.0
02.91.91.9 0.8 2.0
1.9
2.0
0.8
1.0
0.8
0.8
A
C
S
F
4.2 1.1 1.9 1.0 0.2 0.9 3.3 2.0 1.0 1.1
4.1 1.1 -
4.3 1.9 1.9 1.1 0.2 1.2 3.3 2.0 1.1 1.0
4.0 1.9 -
1.8 0.9 0.2 0.9 3.2 2.0 0.9 1.0
A
C
S
F
2.0 0.9
1.0
I.0
1.0
1.0
1.1 3.1 2.0 1.0 0.9
0.9
0.8
0.9
0.9
0.9
0.9
0.8
0.9
m u L l 0 . 9
1.8 0.9
0.9
0.8
Peptide 356-358
0.9 1.0
0.7
1.0
1.0
0.9 1.0
1.8
1.7
1.7
1.8
+
+
+
+
0.9
The Complete Amino Acid Sequence of the Mqior Bovine Aorta Actin The 79 different peptides (including some overlapping peptides) which cover the complete amino acid sequence of the actin polypeptide chain (see Methods) were screened by amino acid analysis. Only four of these pep tides showed a difference when their amino acid composition was compared with that of the corresponding peptides of rabbit skeletal muscle actin. These peptides are listed in Table 1. The exact location of the amino acid exchanges was determined as follows:
Peptide 1-18. The complete amino acid sequence of this peptide has been documented [81. It showed the following five amino acid replacements in comparison to rabbit muscle actin: position 1 glutamic acid rather than aspartic acid; position 3 glutamic acid rather than aspartic acid; position 4 aspartic acid rather than glutamic acid; position 5 serine rather than threonine; position 17 cysteine rather than valine (for a summary see Table 2). Peptide 85-91. This peptide originates by a chymotryptic-like cleavage at tyrosine 91 as shown previously for
1.8 0.1 0.1
1.7
1.0
1.1
1.8
1.1
1.8
1.1
gizzard smooth muscle actin [14]. From its amino acid composition (Ser 0.8; Ile 0.8; Tyr 0.9; Phe 1.0; His 1.8 and the presence of tryptophan) a threonine to serine exchange is predicted in comparison to skeletal muscle actin (Table 1). Using a time course digestion of the peptide with carboxypeptidase A (1 h: Tyr 0.7, Phe 0.5, Ser 0.1; 4 h: Tyr 0.9, Phe 0.6, Ser 0.3, His 0.1) this amino acid exchange was localized at position 89.
Peptide 291-31 1. From the composition of this peptide (Asp 4.2; methionine sulfone 1.1; Thr 1.9; Ser 1.0; Pro 0.9; Gly 3.3; Ala 2.0; Val 1.0; Ile 1.1; Leu 2.0; Tyr 1.8; Arg 1.0) a methionine (rabbit muscle actin) to leucine exchange (aorta actin) is predicted (Table 1). Localization of this exchange was possible in a secondary chymotryptic peptide, i.e., residues 294-298 (Asp 1.9; Ala 1.0; Val 0.9; Leu 0.9) by a time course digestion with carboxypeptidase A (after 10 min: Leu 0.6, Val 0.1; after 45 min: Leu 0.8, Val 0.6, Asn 0.1). Thus position 298 in aorta actin is leucine rather than the methionine residue found in skeletal muscle actin. Peptide 356-358. Amino acid composition of this tripeptide (Ser 1.0; Ile 0.9; Lys 0.9) revealed an amino acid exchange from threonine (skeletal muscle actin) to serine
127
J. Vandekerckhove and K. Weber: Different Muscle Actins
Tabk 2. Positions in the amino acid sequences at which exchanges have been detected between four different muscle actins and the two different nonmuscle actins. Positioning of the amino acids in the actin sequences is made in analogy to rabbit skeletal muscle actin 111-131. Amino acid residues in which the four muscle actins differ among themselves are underlined. The data for nonmuscleactins are from Reference 13 and those for stomach smooth muscle actin (chicken gizzard actin) are from Reference 14. Residue number 1 is absent in stomach smooth muscle actin and nonmuscle actin. All actins carry a blocked amheterminus, which is most likely the acetyl group found in skeletal muscle actin [301
Residue number
Actin types Skeletal muscle (fast, slow)
1 2 3 4 5 6 10 16 17 76 89 103 129 153 162 176 20 1 225 259 266 27 1 278 286 296 298 357 364
ASP Glu & P Glu
Thr Thr CYS
Leu Ile .
Thr Val Leu Asn Met Val Am
Cardiac muscle
ASP ASP Glu Glu Thr Thr
_ .
Smooth muscle (aorta)
& G
Glu Glu -
&G
ALP
Glu Thr
Ser
Thr
Thr
CYS Leu Val Ile Thr Thr Val
CYS
Ile
CYS Leu cys Ile
Leu
cys Ser
ser -
Thr
Thr
Val
Val
Leu
Leu
Leu
Asn
Asn Met Val Asn Thr Ile Ala
Asn Met Val Asn Thr Ile Ala
Thr
Thr
Ile Asn
Ile Asn
Met Val Ans
Thr
Thr
Ile Ala Thr Ile Asn
Ile Ala Thr
Met -
Leu Ser -
Ala
Smooth muscle (stomach)
IlC
Asn
Ala
Leu Ser Ala
(aorta actin). Direct dansyl-Wan degradation provided the amino acid sequence Ile-Ser-Lys thus localizing the amino acid exchange at position 357. Figure 2 shows the complete amino acid sequence of the major aorta actin. The order of the peptides covering the full polypeptide chain (see Methods) is based on homology with the rabbit skeletal muscle actin sequence [ l l l . The polypeptide chain contains 375 residues although the numbering system indicates only 3 74. This is because a serine residue following residue 234 has probably been overlooked in the original rabbit skeletal mus-
Non-muscle P-type
v-type
-
-
ASP ASP ASP
Glu Glu Glu Ile Ala
Val
Ile Met CYS Val Thr Val Thr Met Thr
Leu Thr Gln Ala
Leu CYS Phe Phe Thr
Ser
Leu
Leu Sa
Ala
Ser
cle actin sequence [12]. To avoid renumbering of the previously documented amino acid exchanges past this residue in other actins, we agree with the proposal [12, 201 to indicate this residue as serine 234a. Aorta smooth muscle actin differs by eight amino acid exchanges from skeletal muscle actin (Tables 2 and 3). These substitutions occur at positions 1,3,4, 5, 17,89, 298, and 357. Comparison with the chicken gizzard actin sequence 1141 reveals only three substitutions, all of which are localized in the amino-terminal end (positions 1,4, and 5; Table 2). We show below that an actin similar, if not
128
J. Vandekerckhove and K. Weber: Different Muscle Actins
Table 3. Evolutionary relationship of the four different muscle actins from higher vertebrates. Distances are given by the number of amino acid exchanges. For details see Table 2 Skele- Cardiac Stomach Aorta tal Skeletal muscle
-
Cardiac muscle
4
'Stomach' smooth muscle 'Aorta' smooth muscle
4
6
8
6
6
4
4 -
8
6
3
-
/3
25
23
23
23
y
24
22
20
22
-
3
Cytoplasmic (mammalian)
identical with chicken gizzard actin, is also the major smooth muscle actin in bovine stomach tissue.
Screening of the Two Smooth Muscle Actins in Different Tissues Although the bovine aorta actin (a-like) and chicken gizzard (y-like) smooth muscle actins are separated by isoelectric focussing, the technique does not readily resolve the a-like actin from sarcomeric a-actin and the ylike actin from y-nonmuscle actin (Fig. 1). The two smooth muscle actins are however readily identified in the two-dimensional fingerprint analysis of the amino-terminal tryptic peptides derived from performic acid oxidized actins [81. We have improved this technique by using carboxymethylation with C 14-iodoacetate rather than performic acid to modify the cysteine residues. This approach offered two advantages. First, detection of the amino-terminal peptides is now based on autoradiography, which is much more sensitive than the peptide stains previously used. Second, because much smaller amounts could be loaded on the paper, the amino-terminal peptide of y-nonmuscle actin no longer smeared during the pH 3.3 and pH 4.7 electrophoresis 181. Thus we can now detect the presence of traces of ynonmuscle actin as minor component in smooth muscle tissues. In Fig. 3 use of this characterization of C"-labeled amino-terminal tryptic peptides is made. Figure 3H shows with a reconstituted mixture the separation pattern of the peptides from the following five actins commonly found in higher vertebrates: a-like smooth muscle actin, y-like smooth muscle, a-skeletal muscle actin, and the two nonmuscle actins p and y. Note the clear
separation of the two smooth muscle actin peptides. Actins from different tissues containing smooth muscle cells were screened by this procedure (Fig. 3A-G). Chicken aorta actins (Fig. 3A) showed a pattern of amino terminal peptides very similar to that of bovine aorta actins (Fig. 3B). In both cases the a-like smooth muscle actin was the major actin species, although a small amount of y-like smooth muscle actin is clearly detected. In addition both actin samples contained a small amount of the nonmuscle actins p and y with p expressed preferentially. The spot labeled by an asterisk has also been noted in a previous study using performic acid oxidized actins and is most likely due to an isomerization at aspartic acid residue 4 in aorta a-like actin and should therefore be counted as a derivative of the major aorta actin 181. Currently we do not know if this conversion occurs only in vitro or already in vivo. We also have no explanation why this derivative is apparently missing in chicken aorta actin. The presence of two smooth muscle actins in aorta prompted us to study their relative ratio in dissected tunica intima (innermost coat) and tunica media. No significant difference was detected (Figs. 3C and 3D). The major component of chicken gizzard (7-like) actin (Fig. 3E) was also detected as the major component in the actin from the muscular wall of bovine rumen (Fig. 3F), although the latter actin also contained in addition a considerable amount of a like smooth muscle actin. In bovine uterus both smooth muscle actins are present in equal amounts (Fig. 3G). It should be noted that all smooth muscle tissues so far characterized revealed as minor components the p-nonmuscle actin and frequently the y-nonmuscle actin with the former expressed at a higher level than the latter. In addition there are a few minor spots, which so far have not been identified.
The Complete Amino Acid Sequence of Bovine Cardiac Actin A comparative study on the sequences in the aminoterminal tryptic peptides (residues 1-18) of various bovine actins [81 revealed two amino acid exchanges when bovine cardiac actin was compared with rabbit and bovine skeletal muscle actin (see below). These exchanges involved residue 2, aspartic acid to glutamic acid, and residue 3, glutamic acid to aspartic acid, with the first residue being typical of the skeletal muscle actins. Performic acid oxidized bovine cardiac actin was fragmented as described under Methods. The peptides purified from the various digests cover residues 5-375 of the actin polypeptide chain. Only two main peptides,
J. Vandekerckhove and K. Weber: Different Muscle Actins
129
Flg. 3. Two-dimensional separation pattern of the C"-carboxymethylated actin amino-terminaltryptic peptides. Horizontal separation is by electrophoresis at pH 3.3, and vertical separation is by electrophoresisat pH 6.5. Distances in cm on both axes were measured from the origin. The amino-tmninal peptides are from actins of the following tissues:chicken aorta (A),bovine aorta (B), bovine aorta tunicu intimu (C), bovine aorta tunicu medfa @), chicken gizzard (E), bovine rumen (P),and bovine uterus (G). (H)shows a reconstituted mixture of a-like smooth muscle actin of bovine aorta (I),y-like smooth muscle actin of chicken gizzard (2),/3-nonmuscle actin (3), y-nonmuscleactin (4, and a-skeletal muscle actin (5).For an explantation of the spot labeled by an asterisk see Results
130
carrying an amino acid exchange in comparison to rabbit skeletal muscle actin, were detected (peptide 291-31 1, and peptide 356-358) (Table 1; see also reference [91). These peptides were processed as described above for the corresponding aorta actin peptides. The results proved conclusively a methionine to leucine (heart) exchange at position 298 and a threonine to serine (heart) exchange at position 357. The latter amino acid substitution was previously repotted by Elzinga et al. in a study on human and bovine cardiac actin 121. The four amino acid substitutions of bovine cardiac actin in relation to skeletal muscle actin are listed in Table 2. It should be emphasized that the bovine cardiac actin peptide containing residue 89 is identical with the corresponding skeletal muscle actin (Table 1). Thus the amino acid substitution at position 89 in aorta and gizzard actins (see above) is typical only of smooth muscle actins. The Complete Amino Acid Sequence of Bovine Skeletal Muscle Actin
In order to detect whether some of the amino acid exchanges observed in various bovine actins [7-9, 12,131 in comparison to rabbit skeletal muscle actin reflect a species specificity rather than tissue specificity, we determined the sequence of bovine skeletal muscle. This actin was already studied in 19% of its total sequence using automated procedures on selected cyanogen bromide fragments [71. In addition a previous screening of the amino acid compositionsof tryptic peptides covering 7 1% of the total sequence did not reveal any amino acid difference between skeletal muscle actins from rabbit and beef [91. Using the procedures given in Methods we have covered the complete sequence of bovine skeletal muscle actin and were unable to detect any variation from the standard reference sequence of rabbit skeletal muscle actin [ll-131. The Complete Sequence of Rabbit Slow Skeletal Muscle Actin The complete sequence of the amino-terminal -tic peptide (residues 1-18) of rabbit slow skeletal muscle actin was determined by the procedures described elsewhere [81. The sequence of this peptide was found to be identical with the sequence of the amino-terminal pep tide of rabbit back muscle [ l 1,131. The remainder of the actin polypeptide was screened by analysis of the 79 peptides which cover residues 5-375 (see Methods). No
J. Vandekerckhove and K. Weber: Different Muscle Actins
amino acid exchanges were discovered in comparison to rabbit fast skeletal muscle actin (back muscle). As an illustration for the results, we have listed in Table 1 the amino acid composition of those slow muscle actin peptides whose counterparts in bovine aorta contain an amino acid exchange in relation to rabbit back muscle actin.
Discussion
The complete amino acid sequences of the actins from bovine aorta, bovine cardiac muscle, bovine fast skeletal muscle, and rabbit slow skeletal muscle provide further aspects to our understanding of actin divergence during muscle differentiation in higher vertebrates. This study brings the number of different actin polypeptides fully characterized in the same mammalian species from two (p- and y-nonmuscle actins) 1131 to five by the results provided for bovine skeletal muscle actin, bovine cardiac actin, and smooth muscle actin from bovine aorta. In addition our results confirm the concept that a sixth actin, which has been sequenced in the case of chicken gizzard [141 is also the predominant form of bovine rumen smooth muscle tissue. In our sequence studies we have used a convenient preparative fingerprint technique in which peptides covering the whole actin polypeptide are compared with the corresponding rabbit skeletal muscle actin peptides by chromatographic and electrophoretic behaviour as well as by amino acid composition. Although this approach is very fast and especially suitable for comparative studies, we cannot rigorously exclude the possibility of some double compensating amino acid substitution within some of the peptides so far studied. Although this possibility has been encountered once in cardiac muscle actin, in general it should be a rare event because of the following reasons. First, the extremely conservative nature of the amino acid sequence of various actins has been documented in previous studies [7-9, 11-14,201. Second, the average size of the peptides encountered is six to seven residues thus lowering statistically the chance of double compensating amino acid exchanges. Third, the majority of the amino acid exchanges typically found in mammalian nonmuscle actins by direct sequence analysis of similar peptides [7, 9, 12, 131 are clearly excluded in the various muscle actins studied here. Fourth, in those cases where partial amino acid sequences were obtained by automated direct analysis of some cyanogen bromide fragments the results [71 are in full agreement with our data. For example all amino acid exchanges, which we document in bovine cardiac actin in addition to that at position 357 171 occur in
J. Vandekerckhove and K. Weber: Different Muscle Actins
regions of this actin polypeptide chain, which have not been previously subjected to direct automated analysis. Recently Lu and Elzinga [12lproposed in a study of bovine brain actin that a serine residue following residue 234 was probably overlooked in the original rabbit skeletal muscle actin sequence 11 11. We confmed this extra residue in three different mammalian nonmuscle actins [131,in chicken gizzard actin [141and in the actin from Physarum polycephalum [20l.We also found this additional serine residue in all four muscle actins studied in this report. To avoid renumbering of the previously documented amino acid exchanges past this residue, we agree with the proposal [121 to indicate this residue as serine 234a (see also Fig. 2). Our results on the major actin species of bovine aorta provide the second full amino acid sequence of a smooth muscle actin. Aorta actin diffms by only three amino acid exchanges from the smooth muscle actin expressed in the chicken stomach tissue (chicken gizzard actin) [141. All amino acid exchanges occur in the amino-terminal tryptic peptide (residues 1, 4, and 5 ) again emphasizing that this region of the actin polypep tide contains a disproportionally high number of changes [8, 91. The presence of an additional aspartic acid residue in aorta actin (position 4) explains the lower isoelectric point of this actin (a-like) in comparison to the more basic gizzard actin (y-like). In the remainder of their sequence both smooth muscle actins share four amino acid exchanges in comparison to rabbit skeletal muscle actin (Table 2). Of these positions 17, 298, and 357 involve amino acid residues, which are also typical of nonmuscle actin [7,9, 12, 131. Position 89 is especially interesting because this amino acid substitution is typical of smooth muscle actins and is missing in other muscle actins as well as in nonmuscle actins [9, 13, 141. The three amino acid differences between bovine aorta actin and chicken gizzard actin do not reflect species divergence but are clearly due to tissue specificity because of the following results. First, bovine aorta contains a minor actin, which has an amino-terminal tryptic peptide identical in amino acid sequence with the corresponding gizzard actin peptide [81.Second, actins from bovine and chicken aorta give an extremely similar ftngerprint pattern of their amino-terminalpeptides labeled by C14-carboxymethylation.In both cases the peptide correspondingto the a-like actin, detected by isoelectric focussing analysis, is the major product accounting for close to 7096 of the total actin of the tissues. Third, a similar analysis of cow rumen tissue indicates in agreement with previous findings on chicken gizzard that the
131
y-like smooth muscle actin is the major component of stomach tissue. Fourth, in the case of bovine uterus both actin components are present in equal amounts. There are several aspects of actin expression in smooth muscle tissues, which we currently do not understand. Thus we do not know if the minor actin species, which most likely corresponds to p and y-nonmuscle actins, are derived from contaminating nonmuscle cells especially rich in p-actin or if they are inherent components in the smooth muscle cells of the tissue. Since we have characterized these species only by their amino-terminal tryptic peptide, we cannot yet say whether they really reflect nonmuscle actins in the rest of their sequence. In addition it is not clear if in those smooth muscle tissues like aorta and uterus the two different smooth muscle actins (a-like and y-like) are present in the same cell or are expressed in different cells. In an attempt to answer this question aorta tissue was separated into the two histologically well defined parts, i.e., tunica intima and tunica media, but the same ratio of different actins was found as in the undissected tissue. Clearly further experiments are necessary to understand the complexity of actin expression in smooth muscle tissues. The complete amino acid sequence of bovine cardiac actin revealed only four amino acid exchanges in comparison to rabbit skeletal muscle actin (positions 2, 3, 298,and 357). Two of these (positions 298 and 357) are also typical of the two nonmuscle actins 17, 9, 12, 131 and the two smooth muscle actins. The other two changes (positions 2,3)although involving charged residues reflect only a permutation. Thus the overall net charge of cardiac actin and skeletal muscle actin must be the same, in agreement with isoelectric focussing studies, which classified both actins as a-type [6, 231. Since the amino acid exchanges in four different bovine actins - two nonmuscle actins [ 131;aorta actin and cardiac muscle actin - have been expressed in reference to the standard rabbit muscle actin sequence [ 11, 131, we also completed the sequence of bovine skeletal muscle actin in order to avoid a potential species specificity of actin. No amino acid differences were found between the two skeletal muscle actins in agreement with previous studies indicating lack of species specificity in the case of nonmuscle actins from various mammals [9, 131. Although troponins, tropomyosins, a-actinins, and myosins of fast and slow skeletal muscle can be generally distinguished immunologically as well as by protein chemical data [24-291 we were unable to detect any amino acid exchanges between the actins prepared from
132
the two different rabbit muscles. This result could indicate that both actins are coded by the same gene. Alternatively in view of the extremely conserved amino acid sequence of actins in general by contrast to other muscle proteins (see for instance [241) it remains possible that although the actin gene products are the same in slow and fast skeletal muscle they are coded by different genes. This possibility would stay in line with the current concept of different contractile proteins in various muscles. Table 2 summarizes the various amino acid exchanges, which distinguish the four different muscle actins so far characterized by full amino acid sequence analysis. In addition it shows the amino acid substitution pattern for the two mammalian nonmuscle actins [131. The combined data reveal a distinct evolutionary development between nonmuscle actins and the various muscle actins. Clearly nonmuscle actins have to be considered as the archetype actin, since they occur in all nonmuscle cells including the lower eukaryotic unicellular organisms as for instance in amoebas. In line with this interpretation is the fact that even mammalian nonmuscle actins differ by fewer amino acid exchanges from the actin of Physarum polycephalurn than from mammalian muscle actins [20l.The typical amino acid exchange patterns at positions 17, 298, and 357 indicate that skeletal muscle actin is the highest differentiated actin species. Closest to skeletal muscle actin is cardiac actin (Tables 2 and 3) which shows only four replacements. The smooth muscle actins and also probably the aorta type actin show a somewhat closer relation to nonmuscle actins than skeletal muscle actin, although they still differ by some 23 or 22 residues in a comparison of p- and y-nonmuscle actin and aorta actin. The results emphasize again that residues 1 to 17 comprise a relatively variable region of the actin polypeptide chain 19, 131. Since full amino acid sequences are now available for six different mammalian actins it will be interesting to see, if some of the amino acid exchanges can be correlated with functional properties of actin in vivo or in vitro. Acknowledgements:J. V. is bevoegdverklaard nawrser at the Belgian National Fund for Scientific Research (N.F.W.O.). We thank M. Puype for excelllent technical assistance. We thank Dr. R. Whalen for a sample of rabbit slow skeletal muscle tissue. This work was supported in part by a research grant from the Belgian National Fund for Scientific Research (N.F.W.O.) (Krediet aan Navorsers to J.V.).
References 1. Gruenstein. E., Rich, A.: Non-identity of muscle and non-muscle actins. Biochem. Biophys. Res. Commun. 64, 472 (1975)
J. Vandekerckhove and K. Weber: Dflerent Muscle Actins
S.: Human heart and platelet actins are products of different genes. Science 191, 94
2. Elzinga, M., Maron, B. J., Adelstein, R.
(1976) 3. Whalen, R.G., Butler-Browne, G. S., Gros, F.: Protein synthesis
and actin heterogeneity in calf muscle cells in culture. Proc. Natl. Acad. Sci. USA 73, 2018 (1976) 4. Storti, R. V., Rich, A.: Chick cytoplasmic actin and muscle actin have different structural genes. Proc. Natl. Acad. Sci. USA 73, 2346 (1976) 5. Garrels, J., Gibson, W.: Identification and characterization of multiple forms of actin. Cell 9, 793 (1976) 6. Rubenstein, P., Spudich, J.: Actin microheterogeneity in chick embryo fibroblasts. Proc. Natl. Acad. Sci. USA 74, 120 (1977) 7. Elzinga, M., Lu, R.:Comparative amino acid sequence studies
on actin. In: Contractile systems in nonmuscle tissues. Perry, S. V., Margreth, A., Adelstein, R. (eds.), p. 29. Amsterdam: North Holland Biomedical Press 1976 8. Vandekwckhove,J., Weber, K.: At least six different actins are expressed in a higher mammal: an analysis based on the amino acid sequence of the amino terminal tryptic peptide. J. Mol.Biol. 126, 783 (1978) 9. Vandekerckhove, J., Weber, K.: Mammalian cytoplasmic actins
are the products of at least two genes and differ in primary structure in at least 25 identified positions from skeletal muscle actins. Proc. Natl. Acad. Sci. USA 75, 1106 (1978) 10. Zechel, K., Weber, K.: Actins from mammals, bird, fish and slime mold characterized by isoelectric focussing in polyacrylamide gels. Eur. J. Biochem. 89, 105 (1978) 11. Collins, J., Elzinga, M.: The primary structure of actin from rabbit skeletal muscle. J. Biol. Chem. 250, 5915 (1975) 12. Lu, R., Elzinga, M.: Partial amino acid sequence of brain actin and its homology with muscle actin. Biochemistry 16, 5801 (1977) 13. Vandekerckhove, J., Weber, K.: Actin amino acid sequences.
Comparison of actins from calf thymus, bovine brain and SV40transformed mouse 3T3 cells with rabbit skeletal muscle actin. Eur. J. Biochem. 90, 451 (1978) 14. Vandekerckhove, J., Weber, K.: The amino acid sequence of actin from two different chicken muscles: skeletal muscle actin and gizzard smooth muscle actin. FEES Lett. 102, 219 (1979) 15. Spudich, J., Watt, S.: The regulation of rabbit skeletal muscle contraction. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J. Biol. Chem. 246, 4866 (1971) 16. Lazarides, E., Lindberg, U.: Actin is the naturally occurring inhibitor of deoxyribonucleaseI. Proc. Natl. Acad. Sci. USA 71, 4742 (1974) 17. Vandekerckhove, J., Van Montagu,
M.:Sequence analysis of fluorescamine-stainedpeptides and proteins purified on a nanomole scale. Application to proteins of bacteriophage MS2. Eur. J. Biochem. 44, 279 (1974) 18. Bruton, C. J., Hartley, B. S.: Chemical studies on methionyl-tRNA synthetase from Escherichia colf. J. Mol. Biol. 52, 165 (1970) 19. Offord,
R. E.: Electrophoretic mobilities of peptides on paper and their use in the determination of amide groups. Nature 211,
591 (1966) 20. Vandekerckhove, J., Weber, K.: The amino acid sequence of Physarum actin. Nature 276, 720 (1978) 21. O’Farrell, P. H.: High resolution two-dimensional elcctrophoresis of proteins. J. Biol. Chem. 250, 4007 (1975)
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J. Vandekerckhove and K. Weber: M e r e n t Muscle Actins 22. Zechel, K.: On the resolution of polypeptides by isoelectric focussing in polyacryltunidegels in the presence of urea and nonidet - P40. Anal. Biochem. 83, 240 (1977) 23. Izant, J. G., Lazarides, E.: Invariance and heterogeneity in the major structural and regulatory protehs of chick muscle cells revealed by two-dimensional gel electrophoresis. Proc. Natl. Acad. Sci. USA 74, 1450 (1977) 24. Willrinson, J. M.,Grand, R. J. A,: Comparison of amino acid sequence of troponin I from Werent striated muscles. Nature 271, 31 (1978) 25. Wilkinson, J. M.: The components of troponin from chicken fast skeletal muscle. A comparison of troponin T and troponin I from breast and leg muscle. Biochem. J. 169, 229 (1978) 26. Cummins, P., Perry, S. V.: The subunits and biological activity
27. 28. 29. 30.
of polymorphic forms of tropomyosin. Biochem. J. 133, 765 (1973) Suzuki, A., ell,D. E., Stromer, M.H., Singh, I., Temple, J.: aActinin from red and white porcine muscle. Biochim. Biophys. Acta 295, 188 (1973) Lorvey, S., Risby, D.: Light chains from fast and slow muscle myosins. Nature 234, 81 (1971) Hoh, J. F. Y., McGrath, P. A., White, R. I.: Electrophoretic analysis of multiple forms of myosin in fast-twith and slowtwitch muscles of the chick. Biochem. J. 157, 87 (1976) Alving, R. E., Laki, K.:N-terminal sequence of actin. Biochemistry 5, 2597 (1966)
Received June 1979/Accepted July 1979