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Amino acid analysis and peptide mapping of bovine carotid actin
BIOCHIMICA ET BIOPHYSICA ACTA
165
BBA 35318 AMINO ACID ANALYSIS AND PEPTIDE MAPPING OF BOVINE CAROTID ACFIN
CECILE GOSSELIN-REY*, CHARLES GERDAY', ANNIE GASPAR-GODFROID* ANn MARY E. CARSTEN "~ *Laboratory of General Biology, Faculty of Sciences, Universi@ of Li@e, Li@e (Belgium) and ""Mary E. Carsten Research Laboratory, Department of Obsletrics and Gynecology, U.C.L.A. School o[ Medicine, University of California, Los A ngeles, Calif. (U.S.A .)
(Received August 26th, t968)
SUMMARY A c t i n from bovine c a r o t i d is found to be essentially similar to actins e x t r a c t e d from other m u s c u l a r tissues, as d e m o n s t r a t e d b y amino acid analysis a n d p e p t i d e m a p p i n g . So far no difference in m o l e c u l a r s t r u c t u r e has been d e t e c t e d which could a c c o u n t for t h e u n u s u a l i n c o m p l e t e d e p o l y m e r i z a t i o n of this actin. 3-Methylhistidine is present at a r a t i o of I per 8 histidines, similar to t h a t f o u n d in r a b b i t actin. This r a t i o t e n d s to i n d i c a t e t h a t the molecular weight of actin m o n o m e r from s m o o t h a n d s t r i a t e d muscle is near 48 ooo, i n s t e a d of t h e p r e v i o u s l y a c c e p t e d value of 6o ooo.
INTRODUCTION I s o l a t i o n of pure actin from s m o o t h nmscle has m e t with difficulties not enc o u n t e r e d in the p r e p a r a t i o n of s t r i a t e d muscle actin a n d necessitates modified procedures l & A d e t a i l e d c o m p a r i s o n of various m e t h o d s of p r e p a r a t i o n has been carried out in the case of bovine c a r o t i d actin a. T r o p o m y o s i n B was found in all p r e p a r a t i o n s in higher a m o u n t s t h a n those r e p o r t e d for actin p r e p a r a t i o n s from skeletal muscle a n d from uterus. The u l t r a c e n t r i f u g a l b e h a v i o r of bovine c a r o t i d actin was at v a r i a n c e with t h a t d i s p l a y e d b y s t r i a t e d muscle actin< '~ a n d s m o o t h muscle actin, such as u t e r u s actin 1. F r o m its higher r a t e of s e d i m e n t a t i o n , it would a p p e a r t h a t the depolym e r i z a t i o n of c a r o t i d actin d i d not proceed up to the m o n o m e r i c s t a t e a. The size of t h e aggregates a p p e a r e d d e p e n d e n t on the p r o c e d u r e used for the p r e p a r a t i o n ; these aggregates differed, however, from inactive G-actin dimers a l r e a d y described 6-9, since t h e y were found able to polymerize a n d combine with m y o s i n a n d to a c t i v a t e t h e Mg2+-ATPase to t h e s a m e e x t e n t as s t r i a t e d muscle F - a c t i n . To f u r t h e r characterize this p a r t i c u l a r smooth muscle actin, its amino acid c o m p o s i t i o n was i n v e s t i g a t e d after e l i m i n a t i o n of t r o p o m y o s i n B b y a m m o n i u m sulfate fractionation. The c o n t e n t in 3 - m e t h y l h i s t i d i n e a l r e a d y e s t a b l i s h e d in s t r i a t e d Abbreviation: SCM-, S-carboxymethyl. Biovhim. Biophys. Acta, T75 (I969) 165 I73
I0()
C. GOS.SEL1N-REY e t a ] .
muscle actin ] 0,11 w a s determined. Peptide mapping was performed on filter paper and on thin-layer cellulose, and the maps were compared with those obtained with rabbit skeletal actin. The results reported in this paper, compared with those previously established for actin from various other sources1,4,12, fail to reveal any differences greater than would normally be expected from experimental errors and variations, and thus further support the conclusions arrived at by CARSTEN AND KATZ4 that the molecular structure of actin is very stable in all the different muscular tissues of the animal kingdom. The faster rate of sedimentation of bovine carotid G-actin cannot yet be correlated with a difference in primary structure. METHODS
Preparation of actin Actin from the muscular layer of bovine carotid was extracted and purified according to the procedure of CARSTEN A.~D MOMMAERTS 13, including two polymerization and depolymerization cycles as described previously 3. The pellets were suspended in 0.2 mM ATP-o.2 mM ascorbate (pH 7.5), using a tissue homogenizer (Thomas Co.). The yield varied from 20 to 12o mg per IOO g of tissue (media and intima).
Removal of tropomyosin I3 Actin was first polymerized by adding KC1 to o.I M, leaving it for I h at 20 °, and then dialyzed against phosphate buffer, I 0.35 (pH 7.1). To remove tropomyosin B, the solution was brought to 20% saturation (0.76 M) by slow addition with constant stirring of a neutral saturated ammonium sulfate solution. After 5 min additional stirring, the precipitated F-actin was centrifuged down at 14 ooo rev./min for 2o min and suspended in 2o% -saturated ammonium sulfate solution, (pH 7.1). After centrifugation, the pellets were resuspended in deionized water and dialyzed. At this stage, depolymerized actin was no longer soluble at neutral pH. It could be dissolved at pH I I , and the pH could be brought back to about pH 8.5 without any flocculation oceuring. To evaluate the tropomyosin B content, fractionation of the supernatant was carried out. The negligible precipitate occurring between 20 and 400/0 saturation was discarded; the fraction salted out in the range of 4o-65% saturation with ammonium sulfate corresponded to tropomyosin B and sedimented as a single ultracentrifugal boundary. The content of this contaminant amounted to 20% at most.
Ultracentrifugation and starch-gel electrophoresis Sedimentation runs were conducted at 20 ° in the Spinco model E ultracentrifuge in I mM Tris-nitrate 0.2 mM ATP, at pH 7.6 and pH IO, before and after removal of tropomyosin B. Starch-gel electrophoresis according to the method of SMITHIES14 was performed in veronal buffer (pH 7.5, I - o.o12) to which 0. 4 mM ATP and 0.2 mM ascorbic acid were added just before use la.
Alkylation This was carried out according to two different procedures: (a) Actin solution brought to 6 M urea was reacted with iodoacetate as described by CARSTEN 15, Urea Biochim. Biophys. Acta, t75 (I909) I65 173
BOVINE CAROTID ACTIN
167
and iodoacetate were removed by isoelectric precipitation of S-carboxymethylactin (SCM-actin) at pH 5.0 and repeated washing of the precipitatO. SCM-actin was lyophilized. (b) The detailed procedure described by CRESTFIELD, MOORE AND STEINTM was followed. Actin (usually 4 ° mg in 15 ml) dissolved in 8 M urea was reduced with flmercaptoethanol for 5 h at room temperature under N 2 and reacted with iodoacetate for 15 rain. The reaction was stopped by addition offl-mercaptoethanol. After removal of the bulk of the urea by overnight dialysis against deionized water, isoelectric precipitation and washing of SCM-actin was carried out as described above.
Amino acid analysis Analyses were performed with the Spinco model 12o B automatic amino acid analyzer, according to the method of SPACKMAN, STEIN AND MOORE17. Samples of SCM-actin were hydrolyzed in 6 M HC1 (Mallinckrodt) at lO7 :~ I ° in evacuated sealed tubes for 24, 48 and 72 h. Corrections for decomposition of serine and threonine and incomplete liberation of isoleucine were obtained graphically and found to be the same as those stated by CARSTEN15. Kjeldahl nitrogen was determined by the procedure of BANGTM. Recoveries calculated on the basis of the nitrogen content amounted to IOO ~ 5%. Tryptophan was determined spectrophotometrically according to the method of BEAVEN AND HOLIDAY 19.
The determinations of 3-methylhistidine were carried out as described by JOHNSON, HARRIS AND PERRY1° with the physiological fluids column of the amino
acid analyzer. The column (0. 9 cm × 50 cm) was filled with Beckman 5 ° A resin, and the elution performed with 0.38 M sodium citrate (pH 4.26).
Tryptic digestion SCM-actin was dissolved in 6 M urea at pH II, left for 3 ° min at 20 °, then diluted to 2 M urea and the pH adjusted to 8.0. After equilibration at 38° under N, in a pH stat, trypsin (Worthington salt free, twice crystallised) was added at a concentration of 2% (by wt.), three additions being made at o, 3 ° and 9 ° rain. The digest was filtered through a column of Dowex 5o-X2 (H +) (ref. 12).
Peptide maps Peptide maps were obtained either on filter paper according to the procedure of KATZ AND CARSTEN12 or on cellulose thin layers (5oo-/* layer of Macherey-Nagel MN 3oo cellulose) using technical conditions very similar to those described by BURNS AND TURNER 2°.
In both cases, chromatography was performed in butanol-acetic acid-water (4:1:5, by vol.), followed by electrophoresis in pyridine acetic acid-water (I :1o:289, by vol.) (pH 3.7)- The amount of peptides required per map was 3 mg on filter paper and o.I mg on thin layer. Peptide maps were stained with o.5% ninhydrin in acetone and with a ninhydrin-acetic acid-ethanol-2,4,4-collidine reagent (see ref. 2I), respectively.
Biochim. Biophys. Acta, 175 (1969) i65 I73
c. (;OSSEI.IN REY et al.
I()~
a
ii,ili
b
¢
i!ii!!!
mm~
I
Fig. i. Sedimentation diagram of depolymerized bovine carotid actin prepared by the m e t h o d of CARSTEN AND MOMMAERTS 13, after 48 min at 59 78o rev./min, at a protein concentration of 6 mg/ ml in i mM Tris-nitrate 0.2 mM A T P and ascorbate (pH 7.6); fast component, actin; slow component, t r o p o m y o s i n B. Fig. 2. Tracing of starch-gel electrophoresis p a t t e r n in veronal bufli, r, / = o.ot2 (pH 7.0), c o n taining o. 4 mM A T P and 0.2 mM ascorbic acid. (a) G-actin from bovine carotid, prepared b y the m e t h o d of CARSTEN AND MOMMAERTS13; (b) the same preparation, after precipitation b e t w e e n o and 2o% a m m o n i u m sulfate s a t u r a t i o n ; (c) t r o p o m y o s i n B f r o m bovine carotid, isolated from actin p r e p a r a t i o n s by a m m o n i u m sulfate precipitation between 4 ° and 6 5 % saturation.
RESULTS
Control of purity of actin preparations The presence of tropomyosin B in bovine carotid G-actin prepared by the procedure of CARSTEN AND MOMMAERTS13 is easily detected in view of the unusually high rate of sedimentation of 8-IO S at a concentration of 2-4 mg/ml of this aetin 3 (cf. Fig. I). Aetin solutions further purified by precipitation in the range of o-2O°'o ammonium sulfate saturation were found to be essentially free of tropomyosin B, as shown b y starch-gel electrophoresis. Comparison of the o 20% fraction with tropomyosin B salted out between 40-65% saturation, clearly showed that, although the aggregates of depolymerized aetin did migrate on the starch gel as heterogeneous and trailing material, the o 2O~o fraction was devoid of material migrating at the same rate as tropomyosin B (Fig. 2). The depolymerized 0-20% fraction sedhnented at pH IO as heterogeneous material of 3.5 S at 4.5 mg/ml (Fig. 3a), while reduction followed by alkylation converted this aggregated aetin to a more homogeneous prcduet sedimenting at a rate of 1.5 S at 4.6 mg/ml (Fig. 3b). A similar low rate of sedimentation has been observed with reduced carboxymethylated rabbit skeletal actin and was attributed to large charge effects involved at the very low ionic strength used and to changes in conformation resulting from the new charges introduced on the actin molecule 22. Amino acid analyses and peptide maps of carotid aetin were thus performed on reduced carboxymethylated 0-2o% fraction.
Amino acid composition San:ples of five different actin preparations were analyzed, two of them after Biochim. BiopLys. Acta, t75 (19(i9) 165 173
169
BOVINE CAROTID ACTIN
_/-._ J a
b
Fig. 3. S e d i m e n t a t i o n d i a g r a m s . (a) Depolynlerized bovine carotid actin p r e p a r e d b y t h e m e t h o d CARSTEN A N D } I O M M A E R T S TM after s a l t i n g o u t b e t w e e n o a n d 2 0 % a m m o n i u m sulfate s a t u r a tion ; (b) t h e s a m e p r e p a r a t i o n , r e d u c e d a n d alkylated. P i c t u r e s t a k e n a f t e r 48 rain a t 59 780 rev. / rnin, protein c o n c e n t r a t i o n of 4.5 m g / m l (a) a n d 4.6 m g / m l (b) in i m M T r i s - n i t r a t e (pH lO.O). of
24, 4 8 a n d 72 11, t h e o t h e r s composition of carotid actin
in duplicate after a 24-h hydrolysis. The amino acid ( T a b l e I) w a s f o u n d t o b e q u i t e s i m i l a r t o t h o s e e s t a b -
lished for actins
from other
muscle
small, occurring
mostly
in amino
tissuesa,4,12, ~5. T i l e d i s c r e p a n c i e s
acids whose amounts
are known
observed
were
to be affected by
TABI,E 1 CONIPARISON OF A M I N O ACID C O M P O S I T I O N OF ACT1N P R E P A R A T I O N S
A mino acid
N u m b e r of residues* per 60 ooo g of protein Bovine carotid actin
Lys His*** Arg SCM-Cys Asp Thr~ Ser§ Glu Pro Gly Ala Val Met lie§§ Leu Tyr Phe Trp}§§ Total
Rabbit skeletal actin a~
27.0:4~ o.8"* 25.2 Io.9 .£ 0.2 to.l 25.6 = 0. 7 24. 4 6.4 ± 0.5 6.7 46.9 ~ 1.6 46.9 33.7 ± °-4 37 .8 34.0 +_ i,o 32.3 59.8 ~= o,7 54.7 26.2 2 : 0 . 7 25.2 39.7 ± i.o 37.4 41.4 ~ o.5 40.5 20.I ± 1. 4 24. 9 ~9.o ~ 0.3 21.8 39.8 ~: 1.3 36.7 38.7 ± 0.6 34.5 2o.o ~ o. 7 21. 5 16. 4 ± 0. 4 15. 7 5.o k o.4 5.1 516.5 5Ol.4
Sheep uterus aclin 1
26. 7 1o. 5 26.0 7.3 46.4 36.4 37-7 ,55.9 25.9 30.8 41.9 25.8 16. 3 37.6 38.6 i9.6 16. 9 5.4 515 .
* Corrected to a 95 % recovery- of nitrogen. ** V a r i a t i o n expressed as t h e a v e r a g e of t h e d e v i a t i o n s f r o m t h e m e a n . *** I n c l u d i n g 3 - m e t h y l h i s t i d i n e . Corrected for losses d u r i n g hydrolysis. §.~ Corrected for i n c o m p l e t e hydrolysis. ~ B y u l t r a v i o l e t absorption. Biochim. Biophys. Acta, 175 (i969) 165-173
c. GOSSELIN-REYet al.
17o
tile time of hydrolysis; thus they do not appear to be significant. The glutamic acid value was slightly higher compared to that of other actins. Cysteine residues. These residues were estimated as S-carboxymethylcysteine. When alkylation of the carotid actin was performed as described by CARSTEN (see METHODS), only approx. 2 cysteine residues were found to be alkylated, and 4 residues were recovered as half cystine. However, when the actin preparations were reduced with fl-mercaptoethanol prior to alkylation, 6 7 S-carboxymethylcysteines were recovered in 6o ooo g of actin. Results were in good agreement with data on rabbit skeletal actin 15,22,23. Cysteic acid, amounting to approx. I residue, was occasionally observed (Table II). Control experiments carried out in the same laboratory showed TABLE II STATE OF C Y S T E I N E R E S I D U E S RECOVERED ON AMINO ACID ANALYSIS OF CAROTID ACTIN
Treatment of actin preparations Cystine (half)
SCMcysteine
Cysteic acid
3.4 4.8
o o
--* --
4.4 3.4 4.5
2.3 1.6 1.4
-~. i 0-4
o o o o o
7.3 5.8 6.o O, i 7.0
-o 1.9 0.9 o
No alkylation Preparation Preparation
t 2
d lhylation without reduction Preparation Preparation Preparation
6 IO 13
Reduction before alkvlation Preparation Preparation Preparation Preparation Preparation
8 9 i I 12 t3
* Not estimated,
that rabbit skeletal actin could not be fully alkylated either unless reduction was performed first, and that the partially alkylated actin yielded cystine on amino acid analysis. Occurrence of disulfide bridges has been suggested by KRANS, VAN E I J K AND WESTENBRINK22, while CARSTEN15,~4 fully alkylated rabbit skeletal actin without reduction and found no cystine on amino acid analysis. Thus the discrepancies observed seem related to experimental conditions of preparation and/or of alkylation peculiar to the laboratories concerned. 3-Methylhistidine. This amino acid, known to be present in actin from striated muscle, was also found in bovine carotid actin. On the small basic amino acids column, the histidine peak appeared to be symmetrical and the presence of 3-methylhistidine was not indicated; on the physiological fluid column, however, these 2 amino acids were perfectly separated, and peak areas could be accurately calculated. In 6 determinations of 2 different preparations after 24 and 4 8 h of hydrolysis, 1.22 ± o.o3 (standard deviation) residues of 3-methylhistidine were found in 6o ooo g of actin. This value is in agreement with the finding of JOHNSON, HARRIS AND PERRY1° who found I residue of 3-methylhistidine for 7.6 residues of histidine; our corresponding value is I for 7-95. Biochim. Biophys. ,4cta,
I75 (I969)
i65-173
BOVINE CAROTID ACTIN
171
Peptide maps E x p e r i m e n t a l conditions of t r y p t i c digestion h a d to be a d j u s t e d before reproducible p e p t i d e m a p s could be o b t a i n e d for different samples of bovine c a r o t i d actin. F o r t r y p t i c digestion, the SCM-actin m u s t be c o m p l e t e l y dissolved a n d digestion allowed to proceed for a considerable t i m e after cessation of alkali u p t a k e . Complete dissolution of SCM-actin was o b t a i n e d in 6 M u r e a b y raising the p H to a b o u t I I " the m i x t u r e was t h e n a d j u s t e d to 2 M urea a n d p H 8.o, a n d the t r y p s i n added. Most of the digestion a p p e a r e d to occur within the first 3 ° m i n ; the alkali u p t a k e curve leveled off within t h e n e x t 3 ° min. W h e n t h e digestion was s t o p p e d after 9 ° min, a v a r i a b l e a m o u n t of p e p t i d e was found to be insoluble in w a t e r a n d to r e m a i n at the origin on the p e p t i d e maps. The p a t t e r n s observed were found to v a r y in some maps. In most cases, the 3o m a j o r spots d e t e c t e d on filter p a p e r m a p s were easily m a t c h e d with those o b s e r v e d for s t r i a t e d 4, c a r d i a c ~2 a n d s m o o t h muscle actin I (Fig. 4), no
Fig. 4. Peptide map on filter paper of tryptie digest of partially alkylated actin from bovine ca rorid (a~ and of alkvlated actin from rabbit skeletal muscle (b). spot being found which h a d n o t been occasionally observed previously. W h e n digestion was carried out for 18o min, p e p t i d e s o b t a i n e d were soluble in w a t e r a n d m a p s were reproducible a n d essentially similar to those of r a b b i t skeletal muscle actin. W h e n c o m p a r i n g t h i n - l a y e r cellulose a n d filter p a p e r m a p s of the same sample of actin peptides, the general p a t t e r n s were o b v i o u s l y quite similar. In b o t h cases, a b o u t 3o m a j o r spots were found. Nevertheless, a few spots a p p e a r e d to be s o m e w h a t at v a r i a n c e or not as h e a v i l y s t a i n e d on b o t h t y p e s of m a p , a n d for this reason we did not a t t e m p t to m a t c h the spots. I t was concluded t h a t the p a t t e r n s o b s e r v e d all agreed well within the limits of v a r i a b i l i t y of the m e t h o d s , a n d t h a t bovine c a r o t i d actin d i s p l a y s no peculiar c h a r a c t e r i s t i c s of p e p t i d e composition. DISCUSSION N u m e r o u s d a t a a c c u m u l a t e d so far d e m o n s t r a t e the l i m i t e d v a r i a b i l i t y of the m o l e c u l a r s t r u c t u r e of aetin a m o n g the different m u s c u l a r tissues of a wide range of
Biochim. Biophvs. 14cta, 175 (1969) t6S I73
I72
(;. GOSSELIN-REYel al.
animal species. Bovine carotid actin appears to be no exception in this respect. No difference in amino acid composition was detected. A somewhat high glutamic acid content was observed, which might correspond to 3.5-5% contamination with tropomyosin B if one assumes that the amino acid compositions of tropomyosin B of different smooth muscles are similar 2a. Such low amounts might not be detected on starch-gel electrophoresis. No obvious difference was demonstrated between the patterns of peptide maps originating from fully digested bovine carotid and from other sources of actins. The resistance of actin to trypsin digestion was encountered previously1°, 23 and in that respect it nfight be pointed out that under our conditions, the fact that the pH no longer drifted after 6o min digestion was misleading as an indication of whether the reaction was complete. However, in agreement with the findings of JonxsoN, HARRIS AND P E R R Y TM, it was observed that the full complement of spots was found on the peptide map when roughly 6o to 7o% digestion had occurred, as estimated on the basis of nitrogen recovery. 3o maior spots were numbered on filter paper as well as on cellulose thin-layer maps. Additional faint spots were detected, especially on thin-layer maps, so that the number mentioned above should be taken as the minimum estimate. The significance of the total number of spots detected by peptide mapping of actin has already been discussed, and it has been suggested that if two subunits are present in the actin molecule, they are not identical -04. The detection of 4 I - 4 2 peptides in the actin molecule 2a and the presence of one 3-methylhistidine residue per mole in striated muscle TM and bovine carotid actin are compatible with the existence of either 2 nonidentical polypeptide chains, as discussed above, or one polypeptide chain, as recently suggested by REES AND YOUNG9. Furthermore, the finding of a ratio of one 3-methylhistidine per approx. 8 histidine residues in bovine carotid actin, as already determined in rabbit skeletal actin, indicates a lower minimum molecular weight than the value of 6o ooo accepted up to now, and perfectly agrees with the proposed molecular weight of actin of 4 8 ooo which has been derived from ultracentrifugation data 9. The number and behavior of thiol groups of actin have been extensively studied (see refs. 23, 24, 26, 27). The great sensitivity of these groups to oxidation is well exemplified by the variations in the data published and by the discrepancies met with in rabbit skeletal actin solutions prepared in our two laboratories using the same procedure. From our results, it would appear that, out of 7 cysteine residues found when carboxymethylation is preceded by reduction, four would exhibit a marked tendency to form disulfide bridges. These four did not react with iodoacetate in 6 M urea when reduction was omitted and were recovered as cystine on amino acid analysis. It has been suggested that intramoleeular disulfide bridges occur in actin preparations 22, while on the other hand, 4 SH groups were found to be remarkably resistent to oxidation 24. In spite of a careful examination of the maps, no difference could be detected between digests originating from partially and fully alkylated actin, or between those from bovine carotid and rabbit skeletal muscle as would be expected if disulfide bridges did occur between parts of the molecule corresponding to different peptides. However it is possible that experimental conditions might fail to reveal existing differences. The unusual sedimentation behavior of bovine carotid G-actin cannot be correlated with any particular feature in its molecular structure. One might speculate that Biochim. Biophys...tcta, ~75 (I9()9) 165 I73
B O V I N E CAROTID ACTIN
173
it is related to the sluggishness of polymerization and depolymerization pertaining to smooth muscle actins. Further work will show whether these peculiar properties relate to differences in molecular structure which could not be detected by the methods used in this study, or are due to interference of minor intra- or extracellular components associated with these G-actins. ACKNOWLEDGEMENTS
This work was supported in part by grant No. HD-ooolo from the National Institute of Child Health and Human Development and by a research career development award 5-K3-GM-4547 from the U.S. Public Health Service to M.E.C. REFERENCES I 2 3 4 5 6 7 8 9 lo 11 12 13 14 15 16 17 18 19 20 2t 22 23 24 25 26 27
~{. E. CARSTEN, Biochemistry, 4 ( 1 9 6 5 ) I O 4 9 . J. C. RUEGG, E. STRASSNER AND R. H. SCHIRMER, Biochem. Z., 243 (1965) 7 ° . A. GASPAR-GODFROID, G. HAMOIR AND L. LASZT, Angiologica, 4 (1967) 323 . M. E. CARSTEN, AND A. M. KATZ, Biochim. Biophys. Acta, 90 (1964) 534. A. M. [~ATZ AND E. J. HALL, Circul. Res., 13 (1963) 187. }(. LAKI, 1(. ~IARUYAMA AND D. R. I~OMINZ, Arch. Biochem., 98 (t962) 323 . A. M. KATZ, Biochim. Biophys. Acta, 78 (1963) "226. M. S. LEWIS, I(. MARUYAMA, ~V. ]~. CARROLL, D. R. I•OMINZ AND K. LAKI, Biochemislry, 2 (1963) 34. M. K. REES AND M. YOONG, J. Biol. Chem., 242 (1967) 4449P. JOHNSON, C. I. HARRIS AND S. V. PERRY, Biochem. J., IO 5 (I967) 361. A. M. ASATOOR AND M. D. ARMSTRONG, Bioehem. Biophys. Res. Commun., 26 (I967) I68. A. M. KATZ AND M. E. CARSTEN, Circul. Res., 13 (I963) 474. M. E. CARSTEN AND W. F. H. M. MOMMAERTS, Biochemistry, 2 (I963) 28. O. SMITHIES, Biochem. J., 6I (1955) 629. M. E. CARSTEN, Biochemistry, 2 (1963) 32. A. M. CRESTFIELD, S. MOORE AND W . H. STEIN, J. Biol. Chem., 238 (1963) ()22. D. H. SPACKMAN, XV. S. STEIN AND S. MOORE, Anal. Chem., 3 ° (I958) II9O. 1. BANG, Methoden zur Mikrobestimmung einiger Blutbestandteile, J. F. Bergmann, Wiesbaden, i916. G. 1f. BEAVEN AND E. R. HOLIDAY, Adv. Prot. Chem., 7 (1952) 3~9 . D. J. \V. BURNS AND N. A. TURNER, J. Chromatog., 3 ° (1967) 469. b~. RANDERATH, Thin Layer Chromatography, 2nd ed. V e r l a g C h e m i e , A c a d e m i c Press, N e w Y o r k , 1966. H. ]~¢I. J. I~RANS, H. G. VAN E I J K AND H. G. K. \VESTENBRINK, Biochim. Biophys. Acla, Ioo (1965) i93. A. )¢IARTONOSI, Arch. Biochem. Biophys., 123 (1968) 29. ),~. E. CARSTEN,Biochemistry, .5 (1966) 297. ~ . E. CARSTEN, Biochemistry, 7 (1968) 960. VV. \~.r. KIELLEY, A/4n. Rev. Bioehem., 33 (1964) 403 • \V. DRABIKOWSKY AND .q. BITNY-SLACHTO, Acta Bioehim. Polon., i i (1964) 421.
Biochim. Biophys. Acta, 175 (1969) I 6 5 173
165
BBA 35318 AMINO ACID ANALYSIS AND PEPTIDE MAPPING OF BOVINE CAROTID ACFIN
CECILE GOSSELIN-REY*, CHARLES GERDAY', ANNIE GASPAR-GODFROID* ANn MARY E. CARSTEN "~ *Laboratory of General Biology, Faculty of Sciences, Universi@ of Li@e, Li@e (Belgium) and ""Mary E. Carsten Research Laboratory, Department of Obsletrics and Gynecology, U.C.L.A. School o[ Medicine, University of California, Los A ngeles, Calif. (U.S.A .)
(Received August 26th, t968)
SUMMARY A c t i n from bovine c a r o t i d is found to be essentially similar to actins e x t r a c t e d from other m u s c u l a r tissues, as d e m o n s t r a t e d b y amino acid analysis a n d p e p t i d e m a p p i n g . So far no difference in m o l e c u l a r s t r u c t u r e has been d e t e c t e d which could a c c o u n t for t h e u n u s u a l i n c o m p l e t e d e p o l y m e r i z a t i o n of this actin. 3-Methylhistidine is present at a r a t i o of I per 8 histidines, similar to t h a t f o u n d in r a b b i t actin. This r a t i o t e n d s to i n d i c a t e t h a t the molecular weight of actin m o n o m e r from s m o o t h a n d s t r i a t e d muscle is near 48 ooo, i n s t e a d of t h e p r e v i o u s l y a c c e p t e d value of 6o ooo.
INTRODUCTION I s o l a t i o n of pure actin from s m o o t h nmscle has m e t with difficulties not enc o u n t e r e d in the p r e p a r a t i o n of s t r i a t e d muscle actin a n d necessitates modified procedures l & A d e t a i l e d c o m p a r i s o n of various m e t h o d s of p r e p a r a t i o n has been carried out in the case of bovine c a r o t i d actin a. T r o p o m y o s i n B was found in all p r e p a r a t i o n s in higher a m o u n t s t h a n those r e p o r t e d for actin p r e p a r a t i o n s from skeletal muscle a n d from uterus. The u l t r a c e n t r i f u g a l b e h a v i o r of bovine c a r o t i d actin was at v a r i a n c e with t h a t d i s p l a y e d b y s t r i a t e d muscle actin< '~ a n d s m o o t h muscle actin, such as u t e r u s actin 1. F r o m its higher r a t e of s e d i m e n t a t i o n , it would a p p e a r t h a t the depolym e r i z a t i o n of c a r o t i d actin d i d not proceed up to the m o n o m e r i c s t a t e a. The size of t h e aggregates a p p e a r e d d e p e n d e n t on the p r o c e d u r e used for the p r e p a r a t i o n ; these aggregates differed, however, from inactive G-actin dimers a l r e a d y described 6-9, since t h e y were found able to polymerize a n d combine with m y o s i n a n d to a c t i v a t e t h e Mg2+-ATPase to t h e s a m e e x t e n t as s t r i a t e d muscle F - a c t i n . To f u r t h e r characterize this p a r t i c u l a r smooth muscle actin, its amino acid c o m p o s i t i o n was i n v e s t i g a t e d after e l i m i n a t i o n of t r o p o m y o s i n B b y a m m o n i u m sulfate fractionation. The c o n t e n t in 3 - m e t h y l h i s t i d i n e a l r e a d y e s t a b l i s h e d in s t r i a t e d Abbreviation: SCM-, S-carboxymethyl. Biovhim. Biophys. Acta, T75 (I969) 165 I73
I0()
C. GOS.SEL1N-REY e t a ] .
muscle actin ] 0,11 w a s determined. Peptide mapping was performed on filter paper and on thin-layer cellulose, and the maps were compared with those obtained with rabbit skeletal actin. The results reported in this paper, compared with those previously established for actin from various other sources1,4,12, fail to reveal any differences greater than would normally be expected from experimental errors and variations, and thus further support the conclusions arrived at by CARSTEN AND KATZ4 that the molecular structure of actin is very stable in all the different muscular tissues of the animal kingdom. The faster rate of sedimentation of bovine carotid G-actin cannot yet be correlated with a difference in primary structure. METHODS
Preparation of actin Actin from the muscular layer of bovine carotid was extracted and purified according to the procedure of CARSTEN A.~D MOMMAERTS 13, including two polymerization and depolymerization cycles as described previously 3. The pellets were suspended in 0.2 mM ATP-o.2 mM ascorbate (pH 7.5), using a tissue homogenizer (Thomas Co.). The yield varied from 20 to 12o mg per IOO g of tissue (media and intima).
Removal of tropomyosin I3 Actin was first polymerized by adding KC1 to o.I M, leaving it for I h at 20 °, and then dialyzed against phosphate buffer, I 0.35 (pH 7.1). To remove tropomyosin B, the solution was brought to 20% saturation (0.76 M) by slow addition with constant stirring of a neutral saturated ammonium sulfate solution. After 5 min additional stirring, the precipitated F-actin was centrifuged down at 14 ooo rev./min for 2o min and suspended in 2o% -saturated ammonium sulfate solution, (pH 7.1). After centrifugation, the pellets were resuspended in deionized water and dialyzed. At this stage, depolymerized actin was no longer soluble at neutral pH. It could be dissolved at pH I I , and the pH could be brought back to about pH 8.5 without any flocculation oceuring. To evaluate the tropomyosin B content, fractionation of the supernatant was carried out. The negligible precipitate occurring between 20 and 400/0 saturation was discarded; the fraction salted out in the range of 4o-65% saturation with ammonium sulfate corresponded to tropomyosin B and sedimented as a single ultracentrifugal boundary. The content of this contaminant amounted to 20% at most.
Ultracentrifugation and starch-gel electrophoresis Sedimentation runs were conducted at 20 ° in the Spinco model E ultracentrifuge in I mM Tris-nitrate 0.2 mM ATP, at pH 7.6 and pH IO, before and after removal of tropomyosin B. Starch-gel electrophoresis according to the method of SMITHIES14 was performed in veronal buffer (pH 7.5, I - o.o12) to which 0. 4 mM ATP and 0.2 mM ascorbic acid were added just before use la.
Alkylation This was carried out according to two different procedures: (a) Actin solution brought to 6 M urea was reacted with iodoacetate as described by CARSTEN 15, Urea Biochim. Biophys. Acta, t75 (I909) I65 173
BOVINE CAROTID ACTIN
167
and iodoacetate were removed by isoelectric precipitation of S-carboxymethylactin (SCM-actin) at pH 5.0 and repeated washing of the precipitatO. SCM-actin was lyophilized. (b) The detailed procedure described by CRESTFIELD, MOORE AND STEINTM was followed. Actin (usually 4 ° mg in 15 ml) dissolved in 8 M urea was reduced with flmercaptoethanol for 5 h at room temperature under N 2 and reacted with iodoacetate for 15 rain. The reaction was stopped by addition offl-mercaptoethanol. After removal of the bulk of the urea by overnight dialysis against deionized water, isoelectric precipitation and washing of SCM-actin was carried out as described above.
Amino acid analysis Analyses were performed with the Spinco model 12o B automatic amino acid analyzer, according to the method of SPACKMAN, STEIN AND MOORE17. Samples of SCM-actin were hydrolyzed in 6 M HC1 (Mallinckrodt) at lO7 :~ I ° in evacuated sealed tubes for 24, 48 and 72 h. Corrections for decomposition of serine and threonine and incomplete liberation of isoleucine were obtained graphically and found to be the same as those stated by CARSTEN15. Kjeldahl nitrogen was determined by the procedure of BANGTM. Recoveries calculated on the basis of the nitrogen content amounted to IOO ~ 5%. Tryptophan was determined spectrophotometrically according to the method of BEAVEN AND HOLIDAY 19.
The determinations of 3-methylhistidine were carried out as described by JOHNSON, HARRIS AND PERRY1° with the physiological fluids column of the amino
acid analyzer. The column (0. 9 cm × 50 cm) was filled with Beckman 5 ° A resin, and the elution performed with 0.38 M sodium citrate (pH 4.26).
Tryptic digestion SCM-actin was dissolved in 6 M urea at pH II, left for 3 ° min at 20 °, then diluted to 2 M urea and the pH adjusted to 8.0. After equilibration at 38° under N, in a pH stat, trypsin (Worthington salt free, twice crystallised) was added at a concentration of 2% (by wt.), three additions being made at o, 3 ° and 9 ° rain. The digest was filtered through a column of Dowex 5o-X2 (H +) (ref. 12).
Peptide maps Peptide maps were obtained either on filter paper according to the procedure of KATZ AND CARSTEN12 or on cellulose thin layers (5oo-/* layer of Macherey-Nagel MN 3oo cellulose) using technical conditions very similar to those described by BURNS AND TURNER 2°.
In both cases, chromatography was performed in butanol-acetic acid-water (4:1:5, by vol.), followed by electrophoresis in pyridine acetic acid-water (I :1o:289, by vol.) (pH 3.7)- The amount of peptides required per map was 3 mg on filter paper and o.I mg on thin layer. Peptide maps were stained with o.5% ninhydrin in acetone and with a ninhydrin-acetic acid-ethanol-2,4,4-collidine reagent (see ref. 2I), respectively.
Biochim. Biophys. Acta, 175 (1969) i65 I73
c. (;OSSEI.IN REY et al.
I()~
a
ii,ili
b
¢
i!ii!!!
mm~
I
Fig. i. Sedimentation diagram of depolymerized bovine carotid actin prepared by the m e t h o d of CARSTEN AND MOMMAERTS 13, after 48 min at 59 78o rev./min, at a protein concentration of 6 mg/ ml in i mM Tris-nitrate 0.2 mM A T P and ascorbate (pH 7.6); fast component, actin; slow component, t r o p o m y o s i n B. Fig. 2. Tracing of starch-gel electrophoresis p a t t e r n in veronal bufli, r, / = o.ot2 (pH 7.0), c o n taining o. 4 mM A T P and 0.2 mM ascorbic acid. (a) G-actin from bovine carotid, prepared b y the m e t h o d of CARSTEN AND MOMMAERTS13; (b) the same preparation, after precipitation b e t w e e n o and 2o% a m m o n i u m sulfate s a t u r a t i o n ; (c) t r o p o m y o s i n B f r o m bovine carotid, isolated from actin p r e p a r a t i o n s by a m m o n i u m sulfate precipitation between 4 ° and 6 5 % saturation.
RESULTS
Control of purity of actin preparations The presence of tropomyosin B in bovine carotid G-actin prepared by the procedure of CARSTEN AND MOMMAERTS13 is easily detected in view of the unusually high rate of sedimentation of 8-IO S at a concentration of 2-4 mg/ml of this aetin 3 (cf. Fig. I). Aetin solutions further purified by precipitation in the range of o-2O°'o ammonium sulfate saturation were found to be essentially free of tropomyosin B, as shown b y starch-gel electrophoresis. Comparison of the o 20% fraction with tropomyosin B salted out between 40-65% saturation, clearly showed that, although the aggregates of depolymerized aetin did migrate on the starch gel as heterogeneous and trailing material, the o 2O~o fraction was devoid of material migrating at the same rate as tropomyosin B (Fig. 2). The depolymerized 0-20% fraction sedhnented at pH IO as heterogeneous material of 3.5 S at 4.5 mg/ml (Fig. 3a), while reduction followed by alkylation converted this aggregated aetin to a more homogeneous prcduet sedimenting at a rate of 1.5 S at 4.6 mg/ml (Fig. 3b). A similar low rate of sedimentation has been observed with reduced carboxymethylated rabbit skeletal actin and was attributed to large charge effects involved at the very low ionic strength used and to changes in conformation resulting from the new charges introduced on the actin molecule 22. Amino acid analyses and peptide maps of carotid aetin were thus performed on reduced carboxymethylated 0-2o% fraction.
Amino acid composition San:ples of five different actin preparations were analyzed, two of them after Biochim. BiopLys. Acta, t75 (19(i9) 165 173
169
BOVINE CAROTID ACTIN
_/-._ J a
b
Fig. 3. S e d i m e n t a t i o n d i a g r a m s . (a) Depolynlerized bovine carotid actin p r e p a r e d b y t h e m e t h o d CARSTEN A N D } I O M M A E R T S TM after s a l t i n g o u t b e t w e e n o a n d 2 0 % a m m o n i u m sulfate s a t u r a tion ; (b) t h e s a m e p r e p a r a t i o n , r e d u c e d a n d alkylated. P i c t u r e s t a k e n a f t e r 48 rain a t 59 780 rev. / rnin, protein c o n c e n t r a t i o n of 4.5 m g / m l (a) a n d 4.6 m g / m l (b) in i m M T r i s - n i t r a t e (pH lO.O). of
24, 4 8 a n d 72 11, t h e o t h e r s composition of carotid actin
in duplicate after a 24-h hydrolysis. The amino acid ( T a b l e I) w a s f o u n d t o b e q u i t e s i m i l a r t o t h o s e e s t a b -
lished for actins
from other
muscle
small, occurring
mostly
in amino
tissuesa,4,12, ~5. T i l e d i s c r e p a n c i e s
acids whose amounts
are known
observed
were
to be affected by
TABI,E 1 CONIPARISON OF A M I N O ACID C O M P O S I T I O N OF ACT1N P R E P A R A T I O N S
A mino acid
N u m b e r of residues* per 60 ooo g of protein Bovine carotid actin
Lys His*** Arg SCM-Cys Asp Thr~ Ser§ Glu Pro Gly Ala Val Met lie§§ Leu Tyr Phe Trp}§§ Total
Rabbit skeletal actin a~
27.0:4~ o.8"* 25.2 Io.9 .£ 0.2 to.l 25.6 = 0. 7 24. 4 6.4 ± 0.5 6.7 46.9 ~ 1.6 46.9 33.7 ± °-4 37 .8 34.0 +_ i,o 32.3 59.8 ~= o,7 54.7 26.2 2 : 0 . 7 25.2 39.7 ± i.o 37.4 41.4 ~ o.5 40.5 20.I ± 1. 4 24. 9 ~9.o ~ 0.3 21.8 39.8 ~: 1.3 36.7 38.7 ± 0.6 34.5 2o.o ~ o. 7 21. 5 16. 4 ± 0. 4 15. 7 5.o k o.4 5.1 516.5 5Ol.4
Sheep uterus aclin 1
26. 7 1o. 5 26.0 7.3 46.4 36.4 37-7 ,55.9 25.9 30.8 41.9 25.8 16. 3 37.6 38.6 i9.6 16. 9 5.4 515 .
* Corrected to a 95 % recovery- of nitrogen. ** V a r i a t i o n expressed as t h e a v e r a g e of t h e d e v i a t i o n s f r o m t h e m e a n . *** I n c l u d i n g 3 - m e t h y l h i s t i d i n e . Corrected for losses d u r i n g hydrolysis. §.~ Corrected for i n c o m p l e t e hydrolysis. ~ B y u l t r a v i o l e t absorption. Biochim. Biophys. Acta, 175 (i969) 165-173
c. GOSSELIN-REYet al.
17o
tile time of hydrolysis; thus they do not appear to be significant. The glutamic acid value was slightly higher compared to that of other actins. Cysteine residues. These residues were estimated as S-carboxymethylcysteine. When alkylation of the carotid actin was performed as described by CARSTEN (see METHODS), only approx. 2 cysteine residues were found to be alkylated, and 4 residues were recovered as half cystine. However, when the actin preparations were reduced with fl-mercaptoethanol prior to alkylation, 6 7 S-carboxymethylcysteines were recovered in 6o ooo g of actin. Results were in good agreement with data on rabbit skeletal actin 15,22,23. Cysteic acid, amounting to approx. I residue, was occasionally observed (Table II). Control experiments carried out in the same laboratory showed TABLE II STATE OF C Y S T E I N E R E S I D U E S RECOVERED ON AMINO ACID ANALYSIS OF CAROTID ACTIN
Treatment of actin preparations Cystine (half)
SCMcysteine
Cysteic acid
3.4 4.8
o o
--* --
4.4 3.4 4.5
2.3 1.6 1.4
-~. i 0-4
o o o o o
7.3 5.8 6.o O, i 7.0
-o 1.9 0.9 o
No alkylation Preparation Preparation
t 2
d lhylation without reduction Preparation Preparation Preparation
6 IO 13
Reduction before alkvlation Preparation Preparation Preparation Preparation Preparation
8 9 i I 12 t3
* Not estimated,
that rabbit skeletal actin could not be fully alkylated either unless reduction was performed first, and that the partially alkylated actin yielded cystine on amino acid analysis. Occurrence of disulfide bridges has been suggested by KRANS, VAN E I J K AND WESTENBRINK22, while CARSTEN15,~4 fully alkylated rabbit skeletal actin without reduction and found no cystine on amino acid analysis. Thus the discrepancies observed seem related to experimental conditions of preparation and/or of alkylation peculiar to the laboratories concerned. 3-Methylhistidine. This amino acid, known to be present in actin from striated muscle, was also found in bovine carotid actin. On the small basic amino acids column, the histidine peak appeared to be symmetrical and the presence of 3-methylhistidine was not indicated; on the physiological fluid column, however, these 2 amino acids were perfectly separated, and peak areas could be accurately calculated. In 6 determinations of 2 different preparations after 24 and 4 8 h of hydrolysis, 1.22 ± o.o3 (standard deviation) residues of 3-methylhistidine were found in 6o ooo g of actin. This value is in agreement with the finding of JOHNSON, HARRIS AND PERRY1° who found I residue of 3-methylhistidine for 7.6 residues of histidine; our corresponding value is I for 7-95. Biochim. Biophys. ,4cta,
I75 (I969)
i65-173
BOVINE CAROTID ACTIN
171
Peptide maps E x p e r i m e n t a l conditions of t r y p t i c digestion h a d to be a d j u s t e d before reproducible p e p t i d e m a p s could be o b t a i n e d for different samples of bovine c a r o t i d actin. F o r t r y p t i c digestion, the SCM-actin m u s t be c o m p l e t e l y dissolved a n d digestion allowed to proceed for a considerable t i m e after cessation of alkali u p t a k e . Complete dissolution of SCM-actin was o b t a i n e d in 6 M u r e a b y raising the p H to a b o u t I I " the m i x t u r e was t h e n a d j u s t e d to 2 M urea a n d p H 8.o, a n d the t r y p s i n added. Most of the digestion a p p e a r e d to occur within the first 3 ° m i n ; the alkali u p t a k e curve leveled off within t h e n e x t 3 ° min. W h e n t h e digestion was s t o p p e d after 9 ° min, a v a r i a b l e a m o u n t of p e p t i d e was found to be insoluble in w a t e r a n d to r e m a i n at the origin on the p e p t i d e maps. The p a t t e r n s observed were found to v a r y in some maps. In most cases, the 3o m a j o r spots d e t e c t e d on filter p a p e r m a p s were easily m a t c h e d with those o b s e r v e d for s t r i a t e d 4, c a r d i a c ~2 a n d s m o o t h muscle actin I (Fig. 4), no
Fig. 4. Peptide map on filter paper of tryptie digest of partially alkylated actin from bovine ca rorid (a~ and of alkvlated actin from rabbit skeletal muscle (b). spot being found which h a d n o t been occasionally observed previously. W h e n digestion was carried out for 18o min, p e p t i d e s o b t a i n e d were soluble in w a t e r a n d m a p s were reproducible a n d essentially similar to those of r a b b i t skeletal muscle actin. W h e n c o m p a r i n g t h i n - l a y e r cellulose a n d filter p a p e r m a p s of the same sample of actin peptides, the general p a t t e r n s were o b v i o u s l y quite similar. In b o t h cases, a b o u t 3o m a j o r spots were found. Nevertheless, a few spots a p p e a r e d to be s o m e w h a t at v a r i a n c e or not as h e a v i l y s t a i n e d on b o t h t y p e s of m a p , a n d for this reason we did not a t t e m p t to m a t c h the spots. I t was concluded t h a t the p a t t e r n s o b s e r v e d all agreed well within the limits of v a r i a b i l i t y of the m e t h o d s , a n d t h a t bovine c a r o t i d actin d i s p l a y s no peculiar c h a r a c t e r i s t i c s of p e p t i d e composition. DISCUSSION N u m e r o u s d a t a a c c u m u l a t e d so far d e m o n s t r a t e the l i m i t e d v a r i a b i l i t y of the m o l e c u l a r s t r u c t u r e of aetin a m o n g the different m u s c u l a r tissues of a wide range of
Biochim. Biophvs. 14cta, 175 (1969) t6S I73
I72
(;. GOSSELIN-REYel al.
animal species. Bovine carotid actin appears to be no exception in this respect. No difference in amino acid composition was detected. A somewhat high glutamic acid content was observed, which might correspond to 3.5-5% contamination with tropomyosin B if one assumes that the amino acid compositions of tropomyosin B of different smooth muscles are similar 2a. Such low amounts might not be detected on starch-gel electrophoresis. No obvious difference was demonstrated between the patterns of peptide maps originating from fully digested bovine carotid and from other sources of actins. The resistance of actin to trypsin digestion was encountered previously1°, 23 and in that respect it nfight be pointed out that under our conditions, the fact that the pH no longer drifted after 6o min digestion was misleading as an indication of whether the reaction was complete. However, in agreement with the findings of JonxsoN, HARRIS AND P E R R Y TM, it was observed that the full complement of spots was found on the peptide map when roughly 6o to 7o% digestion had occurred, as estimated on the basis of nitrogen recovery. 3o maior spots were numbered on filter paper as well as on cellulose thin-layer maps. Additional faint spots were detected, especially on thin-layer maps, so that the number mentioned above should be taken as the minimum estimate. The significance of the total number of spots detected by peptide mapping of actin has already been discussed, and it has been suggested that if two subunits are present in the actin molecule, they are not identical -04. The detection of 4 I - 4 2 peptides in the actin molecule 2a and the presence of one 3-methylhistidine residue per mole in striated muscle TM and bovine carotid actin are compatible with the existence of either 2 nonidentical polypeptide chains, as discussed above, or one polypeptide chain, as recently suggested by REES AND YOUNG9. Furthermore, the finding of a ratio of one 3-methylhistidine per approx. 8 histidine residues in bovine carotid actin, as already determined in rabbit skeletal actin, indicates a lower minimum molecular weight than the value of 6o ooo accepted up to now, and perfectly agrees with the proposed molecular weight of actin of 4 8 ooo which has been derived from ultracentrifugation data 9. The number and behavior of thiol groups of actin have been extensively studied (see refs. 23, 24, 26, 27). The great sensitivity of these groups to oxidation is well exemplified by the variations in the data published and by the discrepancies met with in rabbit skeletal actin solutions prepared in our two laboratories using the same procedure. From our results, it would appear that, out of 7 cysteine residues found when carboxymethylation is preceded by reduction, four would exhibit a marked tendency to form disulfide bridges. These four did not react with iodoacetate in 6 M urea when reduction was omitted and were recovered as cystine on amino acid analysis. It has been suggested that intramoleeular disulfide bridges occur in actin preparations 22, while on the other hand, 4 SH groups were found to be remarkably resistent to oxidation 24. In spite of a careful examination of the maps, no difference could be detected between digests originating from partially and fully alkylated actin, or between those from bovine carotid and rabbit skeletal muscle as would be expected if disulfide bridges did occur between parts of the molecule corresponding to different peptides. However it is possible that experimental conditions might fail to reveal existing differences. The unusual sedimentation behavior of bovine carotid G-actin cannot be correlated with any particular feature in its molecular structure. One might speculate that Biochim. Biophys...tcta, ~75 (I9()9) 165 I73
B O V I N E CAROTID ACTIN
173
it is related to the sluggishness of polymerization and depolymerization pertaining to smooth muscle actins. Further work will show whether these peculiar properties relate to differences in molecular structure which could not be detected by the methods used in this study, or are due to interference of minor intra- or extracellular components associated with these G-actins. ACKNOWLEDGEMENTS
This work was supported in part by grant No. HD-ooolo from the National Institute of Child Health and Human Development and by a research career development award 5-K3-GM-4547 from the U.S. Public Health Service to M.E.C. REFERENCES I 2 3 4 5 6 7 8 9 lo 11 12 13 14 15 16 17 18 19 20 2t 22 23 24 25 26 27
~{. E. CARSTEN, Biochemistry, 4 ( 1 9 6 5 ) I O 4 9 . J. C. RUEGG, E. STRASSNER AND R. H. SCHIRMER, Biochem. Z., 243 (1965) 7 ° . A. GASPAR-GODFROID, G. HAMOIR AND L. LASZT, Angiologica, 4 (1967) 323 . M. E. CARSTEN, AND A. M. KATZ, Biochim. Biophys. Acta, 90 (1964) 534. A. M. [~ATZ AND E. J. HALL, Circul. Res., 13 (1963) 187. }(. LAKI, 1(. ~IARUYAMA AND D. R. I~OMINZ, Arch. Biochem., 98 (t962) 323 . A. M. KATZ, Biochim. Biophys. Acta, 78 (1963) "226. M. S. LEWIS, I(. MARUYAMA, ~V. ]~. CARROLL, D. R. I•OMINZ AND K. LAKI, Biochemislry, 2 (1963) 34. M. K. REES AND M. YOONG, J. Biol. Chem., 242 (1967) 4449P. JOHNSON, C. I. HARRIS AND S. V. PERRY, Biochem. J., IO 5 (I967) 361. A. M. ASATOOR AND M. D. ARMSTRONG, Bioehem. Biophys. Res. Commun., 26 (I967) I68. A. M. KATZ AND M. E. CARSTEN, Circul. Res., 13 (I963) 474. M. E. CARSTEN AND W. F. H. M. MOMMAERTS, Biochemistry, 2 (I963) 28. O. SMITHIES, Biochem. J., 6I (1955) 629. M. E. CARSTEN, Biochemistry, 2 (1963) 32. A. M. CRESTFIELD, S. MOORE AND W . H. STEIN, J. Biol. Chem., 238 (1963) ()22. D. H. SPACKMAN, XV. S. STEIN AND S. MOORE, Anal. Chem., 3 ° (I958) II9O. 1. BANG, Methoden zur Mikrobestimmung einiger Blutbestandteile, J. F. Bergmann, Wiesbaden, i916. G. 1f. BEAVEN AND E. R. HOLIDAY, Adv. Prot. Chem., 7 (1952) 3~9 . D. J. \V. BURNS AND N. A. TURNER, J. Chromatog., 3 ° (1967) 469. b~. RANDERATH, Thin Layer Chromatography, 2nd ed. V e r l a g C h e m i e , A c a d e m i c Press, N e w Y o r k , 1966. H. ]~¢I. J. I~RANS, H. G. VAN E I J K AND H. G. K. \VESTENBRINK, Biochim. Biophys. Acla, Ioo (1965) i93. A. )¢IARTONOSI, Arch. Biochem. Biophys., 123 (1968) 29. ),~. E. CARSTEN,Biochemistry, .5 (1966) 297. ~ . E. CARSTEN, Biochemistry, 7 (1968) 960. VV. \~.r. KIELLEY, A/4n. Rev. Bioehem., 33 (1964) 403 • \V. DRABIKOWSKY AND .q. BITNY-SLACHTO, Acta Bioehim. Polon., i i (1964) 421.
Biochim. Biophys. Acta, 175 (1969) I 6 5 173