Proc. Nat. Acad. Sci. USA Vol. 72, No. 4, pp. 1309-1313, April 1975
Amino-Acid Sequence of Parvalbumin from Rabbit Skeletal Muscle (calcium-binding protein)
DAVID L. ENFIELD, LOWELL H. ERICSSON, HUBERT E. BLUM*, EDMOND H. FISCHER, AND HANS NEURATH Department of Biochemistry, University of Washington, Seattle, Wash. 98195
Contributed by Edmond H. Fischer, January 8, 1975 Determination of the complete aminoABSTRACT acid sequence of rabbit skeletal muscle parvalbumin is described. The sequence of 86 of the 109 total residues was determined automatically by sequenator analyses of peptides obtained after cleavage with CNBr or with trypsin. The positions of the remaining 23 residues were determined by subtractive Edman degradation of tryptic and chymotryptic peptides. The protein has an acetylated amino terminus. Comparison of the rabbit parvalbumin with those from carp, hake, and pike and with the calcium-binding subunit of rabbit muscle troponin indicates that these proteins are homologous. Among the parvalbumins a high degree of identity is observed, especially of residues involved in the binding of calcium or in the formation of the hydrophobic core.
The parvalbumins are low molecular weight, acidic, watersoluble, calcium-binding proteins long believed to occur exclusively in the white muscle of lower vertebrates and for which no physiological function has been determined as yet (1-11). Amino-acid sequences indicate that they are homologous (10, 12, 13) and that this homology extends to other muscle calcium-binding proteins, such as troponin C and one of the light chains of myosin (14). The tertiary structure of carp parvalbumin III has been elucidated (15, 16). Recently, two independent reports have described the isolation of parvalbumins from the skeletal muscle of higher vertebrates, including turtle, chicken, rabbit, and man (17, 18). The retention of these proteins throughout vertebrate evolution suggests that they might be of basic physiological importance. As a first step toward defining their structural and functional relationship to each other and to other muscle proteins, the amino-acid sequence of rabbit parvalbumin is described. METHODS Parvalbumin was isolated from rabbit skeletal muscle (17) by Dr. Pavel Lehky. The protein was fragmented and analyzed as shown diagrammatically in Fig. 1. Cleavages with CNBr and with iL-tosylamido-2-phenylethyl chloromethyl ketonetrypsin were performed as described (10, 19). The CNBr fragments were fractionated by gel filtration (Fig. 2). Enzymatic digests were subjected to ion-exchange chromatography (Spinco AA-15, Bio-Rad AG1-X2, and AG50W-X2 resins), with gradients of pyridine-acetate buffers. Specific cleavage Abbreviations: CB, cyanogen bromide fragment; PhS-hydantoin, phenylthiohydantoin; Tp, tryptic peptide; C, chymotryptic peptide; TN-C, the calcium-binding protein of the myofibrillar troponin complex. * Present address: University of Freiburg, Department of Medicine, Freiburg im Breisgau, West Germany.
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adjacent to the arginyl residue was accomplished after succinylation of the e-amino groups (20). The digest was fractionated on Sephadex G-50 "superfine". Automatic sequence analysis was performed with the Beckman Sequencer (model 890B), and the silylated phenylthiohydantoin (PhS-hydantoin)-amino acids were identified by gas-liquid chromatography (21). The sequence of the remaining residues was determined by subtractive Edman degradation (22) of tryptic and chymotryptic peptides. The amino-terminal acetyl group was identified by the procedure of Schmer and Kreil (23). Amino-acid analyses were performed on Beckman (model 120C) or Durrum (model D-500) amino-acid analyzers. Homoserine lactone was converted to homoserine before analysis (24). Residues containing amide sidechains were identified by gas-liquid chromatography (21), by high voltage electrophoresis at pH 6.5, or by amino-acid analysis after digestion with aminopeptidase M (25). RESULTS
As observed for most other parvalbumins (10, 12, 13), the amino terminus of the rabbit protein is blocked. The entire sequence was, therefore, derived by analysis of peptides produced by specific cleavages of the polypeptide chain. The amino-acid compositions of the protein and the four major CNBr (CB) fragments are given in Table 1. Digestion of the intact protein with trypsin yielded 17 major peptides. Their amino-acid compositions and positions in the molecule are listed in Table 2. For clarity in presentation of the proof of the structure, residues will be numbered according to the complete amino-acid sequence given in Fig. 3. Isolation and Sequenator Analyses of CNBr Fragments. The mixture of CNBr fragments was fractionated into five fractions, CB I to CB V (Fig. 2). By sequence analysis all were homogeneous, except CB V, which yielded no sequence. Extended sequenator analyses were carried out on CB II through CB IV (Fig. 1). The amino-terminal sequence of CB II begins with Val3rGly-Leu and was determined through Phe7o, except for residues 55, 60-62, and 68, which were identified independently (see below). Sequenator analysis of CB III yielded 16 residues beginning with Thrs-Glu-Leu and extending to Phe18. Analysis of CB IV provided 20 residues from Ala87-Ala-Gly to Valio6; Ser,03 was identified by conventional analysis (see below). Alignment of CNBr Fragments. Since the dipeptide CB V was blocked, it was placed at the amino terminus of the molecule. Tryptic peptides Tp I and Tp III were also blocked; an amino-terminal acetyl group was identified on Tp I. The
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Biochemistry: Enfield et al.
Proc. Nat. Acad. Sci. USA 72
L"e
me
L_ ,_Pt
1 2
12
13
Lp
is
LYe
.Lpns
27
32134
26
L"
,
Lys
Arg
.
LYS Met
Lye
SO 6
75
1
63 66
96
(1975)
:19
SOURCES OF FRAGMENTS I Cleavage ot methionines with CNBr CBn
can gSS~~~~~~~~~~~mE -
0
Em~~~~~~~~~~~~~~~~
com
SUBDIGESTS:
CHT
CO 3rr
C HT
mC-2
|CHT 33 C-4
I C-I9
El
I C-Il
11 C-2 EJM.
I...
II Cleavage at arginine after succinylation RI m Tryptic digest TpX-3
Tpl
I
I
TP
Tp
1omm _.....
Tpm
T
Tp X-5 .....
TJ I
Tp =
TpAX
Detail of peptide sequences and alignment Residue 14-34 15 20 25 30 Ala Ile Gly Ala Phe Ala Ala Ala Glu Ser Phe Asp His Lys Lys Phe Phe Gin Met Val Gly came
TP
HC-2
TPZ-3
Byrd~M
con
z
Residues 58-78 60 65 Ile Glu Glu Glu Glu Leu Gly Phe Ile Leu
70
75
Lys Gly Phe Ser Pro Asp Ala Arg Asp Leu Ser
can
TPN~~~~~Tp
_
_
nc-it
Residues 100- 109 105 100 Asp Glu Phe Ser Thr Leu con
_
acm*
Val
109 Ser Glu Ser
_ EC-2
FIG. 1. Summary of the determination of the amino-acid sequence of rabbit parvalbumin. The top bar represents the intact protein, where specific cleavage points are indicated. Only peptides discussed in the text are shown. Regions enclosed by parentheses and indicated by asterisks are detailed in the lower part of the diagram. Residue numbers correspond to the sequence in Fig. 3. Crosshatching or I, automated degradation; or -, manual degradation; -, unidentified residues. ...
- -
composition of Tp I corresponds to the sum of CB V and the amino-terminal 11 residues of CB III. CB IV contained no homoserine and was thus placed at the carboxyl terminus of the molecule. CB II, therefore, must be located between CB III and CB IV. Since CB II contains the single arginyl residue, an argininespecific tryptic cleavage was performed on the intact protein. Sequenator analysis of fragment R I so obtained (Fig. 1) provided the sequence beginning with Asp76-Leu-Ser and extending through MetweAla-Ala-Gly, thus establishing the overlap between CB II and CB IV. Total Sequence. Sequence analysis of selected tryptic and chymotryptic peptides provided the sequence of the remaining residues of the parvalbumin molecule, as summarized in Fig. 1. The amino-acid compositions of the chymotryptic peptides used are given in Table 3. The sequence of the region of the molecule between Pheis and Met32-Val-Gly was determined by analysis of peptides Tp X-3, Tp XV, III C-2, and Tp IX and confirmed the alignment of fragments CB III and
CB II. Peptides Tp V and Tp XV resulted from cleavage of the Pheig-Ala peptide bond. The high yields of these peptides relative to that of Tp X-3, the expected tryptic peptide, suggests that the hydrolysis of this bond may represent an unusual tryptic cleavage. Residues 55-75 were determined by analysis of peptides Tp IV, II C-19, Tp XI, and II C-11. Sequenator analysis of Tp IV yielded the four glutamyl residues at positions 59-62. Ser55 was identified only at very low levels in three separate sequenator analyses. However, the solution of the PhShydantoin-amino acid after the conversion step was deep pink, which is indicative of the presence of PhS-hydantoinserine (26). The seryl residue was confirmed by subtractive Edman degradation of Tp IV. Furthermore, complete digestion of Tp IV by aminopeptidase M (Rohm and Haas) yielded unmodified serine in 77% yield and confirmed the amino-acid composition. Manual degradation of the arginine-containing chymotryptic peptide II C-11 confirmed that fragment R I had resulted from cleavage at the arginyl residue. The sequence of the carboxyl terminus of the parvalbumin
Proc. Nat. Acad. Sci. USA 72
(1975)
Sequence of Rabbit Parvalbumin
1311
TABLE 1. Amino-acid compositions of CNBr fragments from rabbit parvalbumin* Amino acid CB II
E
0.8-
C
CB III
CB IV
4.0(4) 0.9(1)
2.0(2)
3.0(3) 1. 0(1) 1. 0(1)
4.0(4) 1. 0(1) 2.7(3)
0
I.-
Lys His Arg Asp Thr Ser Hse Glu Pro Gly Ala Val Ile Leu Phe Total
j0.6z 4
Cr
0.4-
0
0.2-
con 20
60
40
csN
CB
100
80
120
140
FRACTION NUMBER
FIG. 2. Gel filtration of the CN&r digest of rabbit parvalbumin. The lyophilized digest (60 mg) was applied to a 2.5 X 115 cm column of Sephadex G-50 "superfine" and developed at 20 ml/hr with 9% formic acid. Fractions were collected every 15 min and monitored by ninhydrin analysis of 50-,.l aliquots after alkaline hydrolysis. Fractions were pooled as indicated and
9.7(10) 0.9(1) 1.0(1) 4.8(5) 2.9(3) 3.6(4) 0.9(1) 5.8(6) 1.0(1) 4.0(4) 1.3(1) 4.2(4) 2.9(3) 5.7(6) 3.9(4) 54
1.5(1)1 3.8(4)
2.0(2)
1.1(1)
3.9(4)
6.8(6)t 3.2(3) 2.0(2) 1.9(2) 4.0(4) 30
1.1(1) 1.0(1) 1.0(1)
1.0(1) 23
Total in seCB V quence 16 2 1 12 5 8 3§ 0.9(1) 12 1 9 11 1.0(1) 5 6 9 9 2 109
Rabbit
parvalbumint 16.1 2.0 1.2 12.2 5.2 8.0 2.8§ 12.7 0.8 9.4 11.3 5.5 6.0 9.0 9.1
* Residues per molecule. Integral values determined by sequence analysis are given in parentheses. t Taken from ref. 9. The composition was normalized to two residues of histidine. The protein contains no cysteine, tryptophan, or tyrosine. $ CB III appears to contain equal quantities of two fragments; one includes residues 3-32, the other the blocked sequence 1-32 (see text). No methionine was observed. § Methionine values.
peptides recovered by lyophilization. was completed by degradation of chymotryptic peptides IV CA4 and IV C-2 derived from the carboxylterminal CNBr fragment CB IV. Residues 97-109 thus correspond in composition to the carboxyl-terminal tryptic peptide Tp II.
molecule
DISCUSSION
Digestion of parvalbumin with trypsin followed the expected enzymatic specificity except for the unusual cleavage in high yield of the PheirAla bond. If the position of this bond (17). The compositions of fragments CB II through CB V in the tertiary structure is the same as that of the analogous correspond to that calculated from the sequence. The high values of alanine and homoserine in pooled fraction CB III Cys18-Ala bond in carp parvalbumin, the bond is internal and hydrolysis could not occur without denaturation or prior (Table 1) are due to the presence in this fraction of fragment CB V-III, which resisted cleavage of the Met2-Thr bond. release of the tryptic peptide, Tp X-3. TABLE 2. Amino-acid compositions of tryptic peptides from rabbit parvalbumin* The amino-acid sequence of rabbit parvalbumin given in Fig. 3 agrees closely with the composition reported by Lehky et al.
Amino acid Lys His
TpI 1.0(1)
TpII
TpIII 2.1(2)
TpIV 1.2(1)
TpV
TpIX 1.0(1)
TpX-2 TpX-3 TpX-5t TpX-6 TpXI 1.0(1) 1.0(1) 2.0(2) 2.0(2) 1. 0(1)
Arg
Asp Thr Ser
Glu Pro
2.0(2) 1.0(1) 2.2(2)
1. 1(1)
0.9(1) 2.7(3) 2.0(2)
1. 1(1)
0.8(1) 3.9(4)
0.9(1) 1.0(1)
1.0(1)
3.0(3) 1.0(1)
1.0(1) 1.0(1)
1. 1(1)
0.9(1) 1.0(1) 1.0(1)
1. 0(1) 1. 1(1)
TpXX TpXXII
1.2(1) 1. 0(1)
1.1(1)
1.1(1)
1.1(1)
1.0(1)
1 .0(1)
1. 1(1)
1. 0(1)
2.1(2) 0.9(1)
1 .0(1)
2.1(2)
1.0(1)
1.0(1)
0.8(1) 1.0(1) 1.1 (1)
0. 9(1) 1.0(1) 1.1 (1)
1.0(1)
0.9(1)
0.9(1) 1.0(1)
1.0(1)
0.9(1)
Gly 2.0(2)
AMet
0.9(1)
Ile Lett
1.0(1)
1. 0(1)
1.9(2)
1.0(1)
no.
2.0(2)
1. 0(1)
Ala Val
Phe Total % Yield Residue
2.0(2) 0.9(1)
TpXV TpXVII TpXVIII TpXIX
1.0(1) 0.8(1)
2.0(2) 1.8(2)
1. 0(1)
1.1 (1)
1.) 1. 0.9(1) 0.9(1) 2.0(2)
1. 0(1)
1.8(2) 2.0(2) 2.0(2)
5
13 100
34
14 36
1-12
97-109
1-13
55-68
2.8(3) 2.0(2)
1.1(1)
0.9(1) 1. 0(1)
12 52
13
1. 0(1) 4.7(5)
2.1(2)
1.1 (1)
1.0(1)
2.8(3)
0.9(1)
0.9(1) 1. 0(1)
1.0(1) 1.8(2)
1.0(1)
1.0(1)
1.0(1)
5 44
14 19
13 29
7
7
37
8 68
36
86
9 46
3 48
2 22
1 164
7 7
9 43
14-18
29-36
76-80
14-27
84-96
39-45
69-75
19-27
81-83
53-54
13, 28 37, 38
46-52
46-54
1. 0(1)
1.0(1) 1.9(2)
molecule. Integral values determined by sequence analysis are given in parentheses. The residue numbers correspond given in Fig. 3. t From a digest of one preparation of parvalbumin, both TpX-5 and a similar peptide, containing phenylalanine instead of threonine at its amino terminus (position 84), were isolated at a molar ratio of 2:1. * Residues per
to the sequence
Biochemistry: Enfield et al.
1312
Proc. Nat. Acad. Sci. USA 72 (1975) 1
16 Glu Ala Asp
5 Val Ile
10 Asp Ala Asp Ala Asp Asp Ile
15 Ala Ala Leu Ala Thr Ala Leu Lys Ala Lys Leu Leu Ac-ALA MET-THR-GLU-LEU-LEU-ASN-ALA-GLU-ASP- ILE - LYS -LYS - ALA-ILE-
Carp Hake Pike Rabbit
Phe Ala Gly Phe Ala Gly Lys Asp Lou
25
Cys Cys Val
Lys Lys Lys
Asp Gly Gly
Glu
Glu
35
Lys Asn
Gly
Ala
Ala
Lys
Glu
Thr
Lys
Ala
Ala
Lys
Ile
GLY-ALA-PHE-ALA-ALA-ALA-GLU-SER-PHE-ASP-HIS-LYS-LYS-PHE-PHE-GLN-MET-VAL-GLY-LEU36 45 Thr Ser Ala Asp Ala Ala lie GIn Gly Ala Ala Gly GIn Ile Ile Ala Met Ala Asn Ala Lys Ala Ile LYS-LYS LYS-SER-THR -GLU -ASP-VAL-LYS- LYS -VAL- PHE-HIS- ILE- LEUQ-R-LYS
55
Ala LYS
-
56
Asp
Val
Asp
Lys
65 Leu
Asp
Lys
Leu
GLYPE -ILE -lGLUGLU -GLU 76 Ala Ala
Thr Asp Gly Thr Asp Ala Thr Asp Ala ASP-LEU -SER -VAL-LYS
-
GLU
LEU-GLY
Gin GIn
Asn Asn Ser
Ala
Lys
Ala
Ala
PHE -ILE -LEU-LYS -GLY-PHE -SER
85 Phe Lou Lys Ser Phe Leu Lys Ser Ala Phe Lou Lys Alq - GLU -THR-LYS -THR - LEU -MET-ALA -ALA-GLY -AP-LYS
Ala -
Gly
Gly
PRO-ASP -ALA-ARG 95
Ala
96
105
-D-GLY-[AS-GLY-
109
Thr Ala Lys Ala Ala Ala Met Lys Gly Ile Glu His Ala ILE -GLY-ALA-ASP-fIE -PHE-SER-THR-LEU- VAL-SER-GLU-SER Val Val
LYs
Phe Phe
Val
Lys
ER
75
Glu
FIG. 3. Amino-acid sequence of rabbit skeletal muscle parvalbumin. The sequence is compared to those of carp III (10), hake (12), and pike III (13) parvalbumins. The sequence of the rabbit parvalbumin is given in capital letters; of the other parvalbumins, only residues that differ from those of the rabbit are shown. Residues of the rabbit parvalbumin corresponding to those involved in calcium binding of carp III (15) are enclosed; residues corresponding to those forming the hydrophobic core of carp III (15) are underlined. The parvalbumins from carp and hake contain 108 residues, whereas those from pike and rabbit have an additional residue at the carboxyl terminus.
The sequence of rabbit parvalbumin is compared in Fig. 3 to those of carp, hake, and pike to demonstrate the homologous relationship among these proteins. All of the residues serving as calcium ligands in carp parvalbumin (15) occupy identical positions in the rabbit protein. Residues neighboring these ligand sites are also highly conserved. Likewise, Arg75 and Glu81, which form the internal salt bridge and play an TABLE 3. Amino-acid compositions of chymotryptic peptides from CNBr fragments of rabbit parvalbumin* Amino acid Lys His Arg Asp Thr Ser Glu Pro Gly Ala Val Leu Phe
IIC-1 I IC-19
IIIC-2
1.1(1)
2.1(2) 1.0(1)
1.0(1) 2.0(2)
IVC-2
IVC-4
1.0(1)
1.0(1)
1.8(2) 1.0(1)
1.0(1)
1.0(1)
1.0(1)
essential role in determining the tertiary structure of the molecule, are conserved, as are all of the residues whose sidechains are involved in the formation of hydrogen bonds in the region of this salt bridge (15), except for the substitution of glutamic acid for aspartic acid at position 22. Finally, residues composing the hydrophobic core of carp parvalbumin (15) are either conserved in the rabbit protein or replaced by other hydrophobic residues. Comparison of the amino-acid sequence of rabbit parvalbumin with that of rabbit TN-C (14) confirms (a) that the two proteins are structurally similar, supporting earlier suggestions (14, 27, 28) that they might have evolved from a common ancestor, and (b) that the parvalbumins are not formed simply by limited proteolysis of the TN-C molecule (27, 29). Alignment of the sequences of rabbit parvalbumin and TN-C reveals a degree of identity similar to that found in the comparison of the parvalbumins of carp, hake, and pike with rabbit TN-C (14). Again, maximum identities between the two proteins are observed in the regions implicated in the binding of calcium, especially the carboxyl-terminal segments.
1.0(1) 1.1(1)
1.0(1)
1.0(1)
1.0(1) 1.0(1)
1.1(1)
Total % Yield Residue
7 28
3 30
5 10
no.
71-77
68-70
25-29
4
40
3 34
106-109 103-103
* Residues per molecule. Integral values determined by sequence analysis are given in parentheses. The residue numbers correspond to the sequence given in Fig. 3.
The fact that parvalbumins are not restricted to aquatic lower vertebrates but have been conserved through close to 500 million years to evolution suggests that they must serve some essential physiological function, possibly related to the contractile process. Analysis of the sequence of rabbit parvalbumin is being carried out independently in the laboratory of Dr. J.-F. Pechere, I)epartment of MIacromolecular Biochemistry, CNRS, Montpellier, France. We acknowledge the excellent technical assistance of Brita Moody, Dr. Albert Boosman, Richard Granberg, and Richard Olsgaard. We thank Dr. P. Lehky for providing the rabbit
Proc. Nat. Acad. Sci. USA 72
(1975)
parvalbumin used in this investigation. We also thank Drs. K. A. Walsh and K. Titani for valuable discussion. This work was supported in part by the National Institutes of Health (GM 15731 and AM 07902), the National Science Foundation (GB 20482), the Muscular Dystrophy Association of America, and the American Cancer Society (BC91P). D.L.E. is an Associate Investigator of the Howard Hughes Medical Institute. 1. Deuticke, H. J. (1934) Hoppe-Seyler's Z. Physiol. Chem. 224, 216-228. 2. Focant, B. & Pechbre, J.-F. (1965) Arch. Irt. Physiol. Biochim. 73, 334-354. 3. Hamoir, G. & Konosu, S. (1965) Biochem. J. 96, 85-97. 4. Konosu, S., Hamoir, G. & Pechere, J.-F. (1965) Biochem. J. 96, 98-112. 5. Pechbre, J.-F., Demaille, J. & Capony, J.-P. (1971) Biochim. Biophys. Acta 236, 391-408. 6. Benzonana, G., Capony, J.-P. & Pechbre, J.-F. (1972) Biochim. Biophys. Acta 278, 110-116. 7. Gerday, C. & Teuwis, J.-C. (1972) Biochim. Biophys. Acta 271, 320-331. 8. Pechbre, J.-F., Capony, J.-P. & Demaille, J. (1973) Syst. Zool. 22, 533-548. 9. Bushana Rao, K. S. P. & Gerday, C. (1973) Comp. Biochem. Physiol. B 44, 1113-1125. 10. Coffee, C. J. & Bradshaw, R. A. (1973) J. Biol. Chem. 248, 3305-3312. 11. Pechere, J.-F., Capony, J.-P. & Ryden, L. (1971) Eur. J. Biochem. 23, 421-428. 12. Capony, J.-P., Ryden, L., Demaille, J. & Pechere, J.-F. (1973) Eur. J. Biochem. 32, 97-108.
Sequence of Rabbit Parvalbumin
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13. Frankenne, F., Joassin, L. & Gerday, C. (1973) FEBS Lett. 35, 145-147. 14. Collins, J. H. (1974) Biochem. Biophys. Res. Commun. 58, 301-308. 15. Kretsinger, R. H. & Nockolds, C. E. (1973) J. Biol. Chem. 248, 3313-3326. 16. Hendrickson, W. A. & Karle, J. (1973) J. Biol. Chem. 248, 3327-3334. 17. Lehky, P., Blum, H. E., Stein, E. A. & Fischer, E. H. (1974) J. Biol. Chem. 249, 4332-4334. 18. Pechere, J.-F. (1974) C.R. Acad. Sci. 278, 2577-2579. 19. Titani, K., Hermodson, M. A., Fujikawa, K., Ericsson, L. H., Walsh, K. A., Neurath, H. & Davie, E. W. (1972) Biochemistry 11, 4899-4903. 20. Hermodson, M. A., Ericsson, L. H., Neurath, H. & Walsh, K. A. (1973) Biochemistry 12, 3146-3153. 21. Hermodson, M. A., Ericsson, L. H., Titani, K., Neurath, H. & Walsh, K. A. (1972) Biochemistry 11, 4493-4502. 22. Shearer, W. T., Bradshaw, R. A., Gurd, F. R. N. & Peters, T., Jr. (1967) J. Biol, Chem. 242, 5451-5459. 23. Schmer, G. & Kreil, G. (1969) Anal. Biochem. 29, 186-
192 24. Ambler, R. P. (1965) Biochem. J. 96, 32P. 25. Jackson, R. L. & Hirs, C. H. W. (1970) J. Biol. Chem. 245, 624-636. 26. Ingram, V. M. (1953) J. Chem. Soc., 3717-3718. 27. Malencik, D. A., Heizmann, C. W. & Fischer, E. H. (1975) Biochemistry, in press. 28. Demaille, J., Dutruge, E., Eisenberg, E., Capony, J.-P. & Pechere, J.-F. (1974) FEBS Lett. 42, 173-178. 29. Heizmann, C. W., Malencik, D. A. & Fischer, E. H. (1974) Biochem. Biophys. Res. Commun. 57, 162-168.
Amino-Acid Sequence of Parvalbumin from Rabbit Skeletal Muscle (calcium-binding protein)
DAVID L. ENFIELD, LOWELL H. ERICSSON, HUBERT E. BLUM*, EDMOND H. FISCHER, AND HANS NEURATH Department of Biochemistry, University of Washington, Seattle, Wash. 98195
Contributed by Edmond H. Fischer, January 8, 1975 Determination of the complete aminoABSTRACT acid sequence of rabbit skeletal muscle parvalbumin is described. The sequence of 86 of the 109 total residues was determined automatically by sequenator analyses of peptides obtained after cleavage with CNBr or with trypsin. The positions of the remaining 23 residues were determined by subtractive Edman degradation of tryptic and chymotryptic peptides. The protein has an acetylated amino terminus. Comparison of the rabbit parvalbumin with those from carp, hake, and pike and with the calcium-binding subunit of rabbit muscle troponin indicates that these proteins are homologous. Among the parvalbumins a high degree of identity is observed, especially of residues involved in the binding of calcium or in the formation of the hydrophobic core.
The parvalbumins are low molecular weight, acidic, watersoluble, calcium-binding proteins long believed to occur exclusively in the white muscle of lower vertebrates and for which no physiological function has been determined as yet (1-11). Amino-acid sequences indicate that they are homologous (10, 12, 13) and that this homology extends to other muscle calcium-binding proteins, such as troponin C and one of the light chains of myosin (14). The tertiary structure of carp parvalbumin III has been elucidated (15, 16). Recently, two independent reports have described the isolation of parvalbumins from the skeletal muscle of higher vertebrates, including turtle, chicken, rabbit, and man (17, 18). The retention of these proteins throughout vertebrate evolution suggests that they might be of basic physiological importance. As a first step toward defining their structural and functional relationship to each other and to other muscle proteins, the amino-acid sequence of rabbit parvalbumin is described. METHODS Parvalbumin was isolated from rabbit skeletal muscle (17) by Dr. Pavel Lehky. The protein was fragmented and analyzed as shown diagrammatically in Fig. 1. Cleavages with CNBr and with iL-tosylamido-2-phenylethyl chloromethyl ketonetrypsin were performed as described (10, 19). The CNBr fragments were fractionated by gel filtration (Fig. 2). Enzymatic digests were subjected to ion-exchange chromatography (Spinco AA-15, Bio-Rad AG1-X2, and AG50W-X2 resins), with gradients of pyridine-acetate buffers. Specific cleavage Abbreviations: CB, cyanogen bromide fragment; PhS-hydantoin, phenylthiohydantoin; Tp, tryptic peptide; C, chymotryptic peptide; TN-C, the calcium-binding protein of the myofibrillar troponin complex. * Present address: University of Freiburg, Department of Medicine, Freiburg im Breisgau, West Germany.
1309
adjacent to the arginyl residue was accomplished after succinylation of the e-amino groups (20). The digest was fractionated on Sephadex G-50 "superfine". Automatic sequence analysis was performed with the Beckman Sequencer (model 890B), and the silylated phenylthiohydantoin (PhS-hydantoin)-amino acids were identified by gas-liquid chromatography (21). The sequence of the remaining residues was determined by subtractive Edman degradation (22) of tryptic and chymotryptic peptides. The amino-terminal acetyl group was identified by the procedure of Schmer and Kreil (23). Amino-acid analyses were performed on Beckman (model 120C) or Durrum (model D-500) amino-acid analyzers. Homoserine lactone was converted to homoserine before analysis (24). Residues containing amide sidechains were identified by gas-liquid chromatography (21), by high voltage electrophoresis at pH 6.5, or by amino-acid analysis after digestion with aminopeptidase M (25). RESULTS
As observed for most other parvalbumins (10, 12, 13), the amino terminus of the rabbit protein is blocked. The entire sequence was, therefore, derived by analysis of peptides produced by specific cleavages of the polypeptide chain. The amino-acid compositions of the protein and the four major CNBr (CB) fragments are given in Table 1. Digestion of the intact protein with trypsin yielded 17 major peptides. Their amino-acid compositions and positions in the molecule are listed in Table 2. For clarity in presentation of the proof of the structure, residues will be numbered according to the complete amino-acid sequence given in Fig. 3. Isolation and Sequenator Analyses of CNBr Fragments. The mixture of CNBr fragments was fractionated into five fractions, CB I to CB V (Fig. 2). By sequence analysis all were homogeneous, except CB V, which yielded no sequence. Extended sequenator analyses were carried out on CB II through CB IV (Fig. 1). The amino-terminal sequence of CB II begins with Val3rGly-Leu and was determined through Phe7o, except for residues 55, 60-62, and 68, which were identified independently (see below). Sequenator analysis of CB III yielded 16 residues beginning with Thrs-Glu-Leu and extending to Phe18. Analysis of CB IV provided 20 residues from Ala87-Ala-Gly to Valio6; Ser,03 was identified by conventional analysis (see below). Alignment of CNBr Fragments. Since the dipeptide CB V was blocked, it was placed at the amino terminus of the molecule. Tryptic peptides Tp I and Tp III were also blocked; an amino-terminal acetyl group was identified on Tp I. The
1310
Biochemistry: Enfield et al.
Proc. Nat. Acad. Sci. USA 72
L"e
me
L_ ,_Pt
1 2
12
13
Lp
is
LYe
.Lpns
27
32134
26
L"
,
Lys
Arg
.
LYS Met
Lye
SO 6
75
1
63 66
96
(1975)
:19
SOURCES OF FRAGMENTS I Cleavage ot methionines with CNBr CBn
can gSS~~~~~~~~~~~mE -
0
Em~~~~~~~~~~~~~~~~
com
SUBDIGESTS:
CHT
CO 3rr
C HT
mC-2
|CHT 33 C-4
I C-I9
El
I C-Il
11 C-2 EJM.
I...
II Cleavage at arginine after succinylation RI m Tryptic digest TpX-3
Tpl
I
I
TP
Tp
1omm _.....
Tpm
T
Tp X-5 .....
TJ I
Tp =
TpAX
Detail of peptide sequences and alignment Residue 14-34 15 20 25 30 Ala Ile Gly Ala Phe Ala Ala Ala Glu Ser Phe Asp His Lys Lys Phe Phe Gin Met Val Gly came
TP
HC-2
TPZ-3
Byrd~M
con
z
Residues 58-78 60 65 Ile Glu Glu Glu Glu Leu Gly Phe Ile Leu
70
75
Lys Gly Phe Ser Pro Asp Ala Arg Asp Leu Ser
can
TPN~~~~~Tp
_
_
nc-it
Residues 100- 109 105 100 Asp Glu Phe Ser Thr Leu con
_
acm*
Val
109 Ser Glu Ser
_ EC-2
FIG. 1. Summary of the determination of the amino-acid sequence of rabbit parvalbumin. The top bar represents the intact protein, where specific cleavage points are indicated. Only peptides discussed in the text are shown. Regions enclosed by parentheses and indicated by asterisks are detailed in the lower part of the diagram. Residue numbers correspond to the sequence in Fig. 3. Crosshatching or I, automated degradation; or -, manual degradation; -, unidentified residues. ...
- -
composition of Tp I corresponds to the sum of CB V and the amino-terminal 11 residues of CB III. CB IV contained no homoserine and was thus placed at the carboxyl terminus of the molecule. CB II, therefore, must be located between CB III and CB IV. Since CB II contains the single arginyl residue, an argininespecific tryptic cleavage was performed on the intact protein. Sequenator analysis of fragment R I so obtained (Fig. 1) provided the sequence beginning with Asp76-Leu-Ser and extending through MetweAla-Ala-Gly, thus establishing the overlap between CB II and CB IV. Total Sequence. Sequence analysis of selected tryptic and chymotryptic peptides provided the sequence of the remaining residues of the parvalbumin molecule, as summarized in Fig. 1. The amino-acid compositions of the chymotryptic peptides used are given in Table 3. The sequence of the region of the molecule between Pheis and Met32-Val-Gly was determined by analysis of peptides Tp X-3, Tp XV, III C-2, and Tp IX and confirmed the alignment of fragments CB III and
CB II. Peptides Tp V and Tp XV resulted from cleavage of the Pheig-Ala peptide bond. The high yields of these peptides relative to that of Tp X-3, the expected tryptic peptide, suggests that the hydrolysis of this bond may represent an unusual tryptic cleavage. Residues 55-75 were determined by analysis of peptides Tp IV, II C-19, Tp XI, and II C-11. Sequenator analysis of Tp IV yielded the four glutamyl residues at positions 59-62. Ser55 was identified only at very low levels in three separate sequenator analyses. However, the solution of the PhShydantoin-amino acid after the conversion step was deep pink, which is indicative of the presence of PhS-hydantoinserine (26). The seryl residue was confirmed by subtractive Edman degradation of Tp IV. Furthermore, complete digestion of Tp IV by aminopeptidase M (Rohm and Haas) yielded unmodified serine in 77% yield and confirmed the amino-acid composition. Manual degradation of the arginine-containing chymotryptic peptide II C-11 confirmed that fragment R I had resulted from cleavage at the arginyl residue. The sequence of the carboxyl terminus of the parvalbumin
Proc. Nat. Acad. Sci. USA 72
(1975)
Sequence of Rabbit Parvalbumin
1311
TABLE 1. Amino-acid compositions of CNBr fragments from rabbit parvalbumin* Amino acid CB II
E
0.8-
C
CB III
CB IV
4.0(4) 0.9(1)
2.0(2)
3.0(3) 1. 0(1) 1. 0(1)
4.0(4) 1. 0(1) 2.7(3)
0
I.-
Lys His Arg Asp Thr Ser Hse Glu Pro Gly Ala Val Ile Leu Phe Total
j0.6z 4
Cr
0.4-
0
0.2-
con 20
60
40
csN
CB
100
80
120
140
FRACTION NUMBER
FIG. 2. Gel filtration of the CN&r digest of rabbit parvalbumin. The lyophilized digest (60 mg) was applied to a 2.5 X 115 cm column of Sephadex G-50 "superfine" and developed at 20 ml/hr with 9% formic acid. Fractions were collected every 15 min and monitored by ninhydrin analysis of 50-,.l aliquots after alkaline hydrolysis. Fractions were pooled as indicated and
9.7(10) 0.9(1) 1.0(1) 4.8(5) 2.9(3) 3.6(4) 0.9(1) 5.8(6) 1.0(1) 4.0(4) 1.3(1) 4.2(4) 2.9(3) 5.7(6) 3.9(4) 54
1.5(1)1 3.8(4)
2.0(2)
1.1(1)
3.9(4)
6.8(6)t 3.2(3) 2.0(2) 1.9(2) 4.0(4) 30
1.1(1) 1.0(1) 1.0(1)
1.0(1) 23
Total in seCB V quence 16 2 1 12 5 8 3§ 0.9(1) 12 1 9 11 1.0(1) 5 6 9 9 2 109
Rabbit
parvalbumint 16.1 2.0 1.2 12.2 5.2 8.0 2.8§ 12.7 0.8 9.4 11.3 5.5 6.0 9.0 9.1
* Residues per molecule. Integral values determined by sequence analysis are given in parentheses. t Taken from ref. 9. The composition was normalized to two residues of histidine. The protein contains no cysteine, tryptophan, or tyrosine. $ CB III appears to contain equal quantities of two fragments; one includes residues 3-32, the other the blocked sequence 1-32 (see text). No methionine was observed. § Methionine values.
peptides recovered by lyophilization. was completed by degradation of chymotryptic peptides IV CA4 and IV C-2 derived from the carboxylterminal CNBr fragment CB IV. Residues 97-109 thus correspond in composition to the carboxyl-terminal tryptic peptide Tp II.
molecule
DISCUSSION
Digestion of parvalbumin with trypsin followed the expected enzymatic specificity except for the unusual cleavage in high yield of the PheirAla bond. If the position of this bond (17). The compositions of fragments CB II through CB V in the tertiary structure is the same as that of the analogous correspond to that calculated from the sequence. The high values of alanine and homoserine in pooled fraction CB III Cys18-Ala bond in carp parvalbumin, the bond is internal and hydrolysis could not occur without denaturation or prior (Table 1) are due to the presence in this fraction of fragment CB V-III, which resisted cleavage of the Met2-Thr bond. release of the tryptic peptide, Tp X-3. TABLE 2. Amino-acid compositions of tryptic peptides from rabbit parvalbumin* The amino-acid sequence of rabbit parvalbumin given in Fig. 3 agrees closely with the composition reported by Lehky et al.
Amino acid Lys His
TpI 1.0(1)
TpII
TpIII 2.1(2)
TpIV 1.2(1)
TpV
TpIX 1.0(1)
TpX-2 TpX-3 TpX-5t TpX-6 TpXI 1.0(1) 1.0(1) 2.0(2) 2.0(2) 1. 0(1)
Arg
Asp Thr Ser
Glu Pro
2.0(2) 1.0(1) 2.2(2)
1. 1(1)
0.9(1) 2.7(3) 2.0(2)
1. 1(1)
0.8(1) 3.9(4)
0.9(1) 1.0(1)
1.0(1)
3.0(3) 1.0(1)
1.0(1) 1.0(1)
1. 1(1)
0.9(1) 1.0(1) 1.0(1)
1. 0(1) 1. 1(1)
TpXX TpXXII
1.2(1) 1. 0(1)
1.1(1)
1.1(1)
1.1(1)
1.0(1)
1 .0(1)
1. 1(1)
1. 0(1)
2.1(2) 0.9(1)
1 .0(1)
2.1(2)
1.0(1)
1.0(1)
0.8(1) 1.0(1) 1.1 (1)
0. 9(1) 1.0(1) 1.1 (1)
1.0(1)
0.9(1)
0.9(1) 1.0(1)
1.0(1)
0.9(1)
Gly 2.0(2)
AMet
0.9(1)
Ile Lett
1.0(1)
1. 0(1)
1.9(2)
1.0(1)
no.
2.0(2)
1. 0(1)
Ala Val
Phe Total % Yield Residue
2.0(2) 0.9(1)
TpXV TpXVII TpXVIII TpXIX
1.0(1) 0.8(1)
2.0(2) 1.8(2)
1. 0(1)
1.1 (1)
1.) 1. 0.9(1) 0.9(1) 2.0(2)
1. 0(1)
1.8(2) 2.0(2) 2.0(2)
5
13 100
34
14 36
1-12
97-109
1-13
55-68
2.8(3) 2.0(2)
1.1(1)
0.9(1) 1. 0(1)
12 52
13
1. 0(1) 4.7(5)
2.1(2)
1.1 (1)
1.0(1)
2.8(3)
0.9(1)
0.9(1) 1. 0(1)
1.0(1) 1.8(2)
1.0(1)
1.0(1)
1.0(1)
5 44
14 19
13 29
7
7
37
8 68
36
86
9 46
3 48
2 22
1 164
7 7
9 43
14-18
29-36
76-80
14-27
84-96
39-45
69-75
19-27
81-83
53-54
13, 28 37, 38
46-52
46-54
1. 0(1)
1.0(1) 1.9(2)
molecule. Integral values determined by sequence analysis are given in parentheses. The residue numbers correspond given in Fig. 3. t From a digest of one preparation of parvalbumin, both TpX-5 and a similar peptide, containing phenylalanine instead of threonine at its amino terminus (position 84), were isolated at a molar ratio of 2:1. * Residues per
to the sequence
Biochemistry: Enfield et al.
1312
Proc. Nat. Acad. Sci. USA 72 (1975) 1
16 Glu Ala Asp
5 Val Ile
10 Asp Ala Asp Ala Asp Asp Ile
15 Ala Ala Leu Ala Thr Ala Leu Lys Ala Lys Leu Leu Ac-ALA MET-THR-GLU-LEU-LEU-ASN-ALA-GLU-ASP- ILE - LYS -LYS - ALA-ILE-
Carp Hake Pike Rabbit
Phe Ala Gly Phe Ala Gly Lys Asp Lou
25
Cys Cys Val
Lys Lys Lys
Asp Gly Gly
Glu
Glu
35
Lys Asn
Gly
Ala
Ala
Lys
Glu
Thr
Lys
Ala
Ala
Lys
Ile
GLY-ALA-PHE-ALA-ALA-ALA-GLU-SER-PHE-ASP-HIS-LYS-LYS-PHE-PHE-GLN-MET-VAL-GLY-LEU36 45 Thr Ser Ala Asp Ala Ala lie GIn Gly Ala Ala Gly GIn Ile Ile Ala Met Ala Asn Ala Lys Ala Ile LYS-LYS LYS-SER-THR -GLU -ASP-VAL-LYS- LYS -VAL- PHE-HIS- ILE- LEUQ-R-LYS
55
Ala LYS
-
56
Asp
Val
Asp
Lys
65 Leu
Asp
Lys
Leu
GLYPE -ILE -lGLUGLU -GLU 76 Ala Ala
Thr Asp Gly Thr Asp Ala Thr Asp Ala ASP-LEU -SER -VAL-LYS
-
GLU
LEU-GLY
Gin GIn
Asn Asn Ser
Ala
Lys
Ala
Ala
PHE -ILE -LEU-LYS -GLY-PHE -SER
85 Phe Lou Lys Ser Phe Leu Lys Ser Ala Phe Lou Lys Alq - GLU -THR-LYS -THR - LEU -MET-ALA -ALA-GLY -AP-LYS
Ala -
Gly
Gly
PRO-ASP -ALA-ARG 95
Ala
96
105
-D-GLY-[AS-GLY-
109
Thr Ala Lys Ala Ala Ala Met Lys Gly Ile Glu His Ala ILE -GLY-ALA-ASP-fIE -PHE-SER-THR-LEU- VAL-SER-GLU-SER Val Val
LYs
Phe Phe
Val
Lys
ER
75
Glu
FIG. 3. Amino-acid sequence of rabbit skeletal muscle parvalbumin. The sequence is compared to those of carp III (10), hake (12), and pike III (13) parvalbumins. The sequence of the rabbit parvalbumin is given in capital letters; of the other parvalbumins, only residues that differ from those of the rabbit are shown. Residues of the rabbit parvalbumin corresponding to those involved in calcium binding of carp III (15) are enclosed; residues corresponding to those forming the hydrophobic core of carp III (15) are underlined. The parvalbumins from carp and hake contain 108 residues, whereas those from pike and rabbit have an additional residue at the carboxyl terminus.
The sequence of rabbit parvalbumin is compared in Fig. 3 to those of carp, hake, and pike to demonstrate the homologous relationship among these proteins. All of the residues serving as calcium ligands in carp parvalbumin (15) occupy identical positions in the rabbit protein. Residues neighboring these ligand sites are also highly conserved. Likewise, Arg75 and Glu81, which form the internal salt bridge and play an TABLE 3. Amino-acid compositions of chymotryptic peptides from CNBr fragments of rabbit parvalbumin* Amino acid Lys His Arg Asp Thr Ser Glu Pro Gly Ala Val Leu Phe
IIC-1 I IC-19
IIIC-2
1.1(1)
2.1(2) 1.0(1)
1.0(1) 2.0(2)
IVC-2
IVC-4
1.0(1)
1.0(1)
1.8(2) 1.0(1)
1.0(1)
1.0(1)
1.0(1)
essential role in determining the tertiary structure of the molecule, are conserved, as are all of the residues whose sidechains are involved in the formation of hydrogen bonds in the region of this salt bridge (15), except for the substitution of glutamic acid for aspartic acid at position 22. Finally, residues composing the hydrophobic core of carp parvalbumin (15) are either conserved in the rabbit protein or replaced by other hydrophobic residues. Comparison of the amino-acid sequence of rabbit parvalbumin with that of rabbit TN-C (14) confirms (a) that the two proteins are structurally similar, supporting earlier suggestions (14, 27, 28) that they might have evolved from a common ancestor, and (b) that the parvalbumins are not formed simply by limited proteolysis of the TN-C molecule (27, 29). Alignment of the sequences of rabbit parvalbumin and TN-C reveals a degree of identity similar to that found in the comparison of the parvalbumins of carp, hake, and pike with rabbit TN-C (14). Again, maximum identities between the two proteins are observed in the regions implicated in the binding of calcium, especially the carboxyl-terminal segments.
1.0(1) 1.1(1)
1.0(1)
1.0(1)
1.0(1) 1.0(1)
1.1(1)
Total % Yield Residue
7 28
3 30
5 10
no.
71-77
68-70
25-29
4
40
3 34
106-109 103-103
* Residues per molecule. Integral values determined by sequence analysis are given in parentheses. The residue numbers correspond to the sequence given in Fig. 3.
The fact that parvalbumins are not restricted to aquatic lower vertebrates but have been conserved through close to 500 million years to evolution suggests that they must serve some essential physiological function, possibly related to the contractile process. Analysis of the sequence of rabbit parvalbumin is being carried out independently in the laboratory of Dr. J.-F. Pechere, I)epartment of MIacromolecular Biochemistry, CNRS, Montpellier, France. We acknowledge the excellent technical assistance of Brita Moody, Dr. Albert Boosman, Richard Granberg, and Richard Olsgaard. We thank Dr. P. Lehky for providing the rabbit
Proc. Nat. Acad. Sci. USA 72
(1975)
parvalbumin used in this investigation. We also thank Drs. K. A. Walsh and K. Titani for valuable discussion. This work was supported in part by the National Institutes of Health (GM 15731 and AM 07902), the National Science Foundation (GB 20482), the Muscular Dystrophy Association of America, and the American Cancer Society (BC91P). D.L.E. is an Associate Investigator of the Howard Hughes Medical Institute. 1. Deuticke, H. J. (1934) Hoppe-Seyler's Z. Physiol. Chem. 224, 216-228. 2. Focant, B. & Pechbre, J.-F. (1965) Arch. Irt. Physiol. Biochim. 73, 334-354. 3. Hamoir, G. & Konosu, S. (1965) Biochem. J. 96, 85-97. 4. Konosu, S., Hamoir, G. & Pechere, J.-F. (1965) Biochem. J. 96, 98-112. 5. Pechbre, J.-F., Demaille, J. & Capony, J.-P. (1971) Biochim. Biophys. Acta 236, 391-408. 6. Benzonana, G., Capony, J.-P. & Pechbre, J.-F. (1972) Biochim. Biophys. Acta 278, 110-116. 7. Gerday, C. & Teuwis, J.-C. (1972) Biochim. Biophys. Acta 271, 320-331. 8. Pechbre, J.-F., Capony, J.-P. & Demaille, J. (1973) Syst. Zool. 22, 533-548. 9. Bushana Rao, K. S. P. & Gerday, C. (1973) Comp. Biochem. Physiol. B 44, 1113-1125. 10. Coffee, C. J. & Bradshaw, R. A. (1973) J. Biol. Chem. 248, 3305-3312. 11. Pechere, J.-F., Capony, J.-P. & Ryden, L. (1971) Eur. J. Biochem. 23, 421-428. 12. Capony, J.-P., Ryden, L., Demaille, J. & Pechere, J.-F. (1973) Eur. J. Biochem. 32, 97-108.
Sequence of Rabbit Parvalbumin
1313
13. Frankenne, F., Joassin, L. & Gerday, C. (1973) FEBS Lett. 35, 145-147. 14. Collins, J. H. (1974) Biochem. Biophys. Res. Commun. 58, 301-308. 15. Kretsinger, R. H. & Nockolds, C. E. (1973) J. Biol. Chem. 248, 3313-3326. 16. Hendrickson, W. A. & Karle, J. (1973) J. Biol. Chem. 248, 3327-3334. 17. Lehky, P., Blum, H. E., Stein, E. A. & Fischer, E. H. (1974) J. Biol. Chem. 249, 4332-4334. 18. Pechere, J.-F. (1974) C.R. Acad. Sci. 278, 2577-2579. 19. Titani, K., Hermodson, M. A., Fujikawa, K., Ericsson, L. H., Walsh, K. A., Neurath, H. & Davie, E. W. (1972) Biochemistry 11, 4899-4903. 20. Hermodson, M. A., Ericsson, L. H., Neurath, H. & Walsh, K. A. (1973) Biochemistry 12, 3146-3153. 21. Hermodson, M. A., Ericsson, L. H., Titani, K., Neurath, H. & Walsh, K. A. (1972) Biochemistry 11, 4493-4502. 22. Shearer, W. T., Bradshaw, R. A., Gurd, F. R. N. & Peters, T., Jr. (1967) J. Biol, Chem. 242, 5451-5459. 23. Schmer, G. & Kreil, G. (1969) Anal. Biochem. 29, 186-
192 24. Ambler, R. P. (1965) Biochem. J. 96, 32P. 25. Jackson, R. L. & Hirs, C. H. W. (1970) J. Biol. Chem. 245, 624-636. 26. Ingram, V. M. (1953) J. Chem. Soc., 3717-3718. 27. Malencik, D. A., Heizmann, C. W. & Fischer, E. H. (1975) Biochemistry, in press. 28. Demaille, J., Dutruge, E., Eisenberg, E., Capony, J.-P. & Pechere, J.-F. (1974) FEBS Lett. 42, 173-178. 29. Heizmann, C. W., Malencik, D. A. & Fischer, E. H. (1974) Biochem. Biophys. Res. Commun. 57, 162-168.
