The Complete Amino Acid Sequence of the Ca2+-dependent Modulator

Vol. 255, No. 3, Issue of February 10, pp. 962-975, 1980 Printed in U.S.A.

The Complete Amino Protein (Calmodulin)

D. Martin Watterson,$

Acid Sequence of the Ca2+-dependent Modulator of Bovine Brain*

(Received for publication, July 12, 1979, and in revised form, September 26, 1979)

Farida Sharief, and Thomas C. Vanaman8

From the Department of Microbiology and Immunology, Duke University Medical Center, Durham, North Carolina 27710

We present the data required to establish the com- plete amino acid sequence of bovine brain modulator protein, the multifunctional calcium-dependent regu- latory protein. Bovine brain modulator protein con- tains 148 amino acid residues and has a molecular mass of 16,680 daltons. The protein commences with an acet- ylated alanyl residue in accord with the previous report that its NH2 terminus was blocked. The single residues of histidine and trimethyllysine occur at positions 107 and 115, respectively, in a region of the linear sequence implicated by other studies as important for calcium- dependent modulator protein-enzyme interactions. The sequence of bovine brain modulator protein demon- strated here is closely related to those of muscle tro- ponin Cs, as originally suggested from considerations of the similarities in calcium binding and functional and physicochemical properties of these proteins (Wat- terson, D. M., Harrelson, W. G., Jr., Keller, P. M., Shar- ief, F., and Vanaman, T. C. (1976) J Biol. Chem 251, 4501-4513). The linear amino acid sequence of bovine brain modulator protein is composed of four internally homologous sequences or domains, each of which con- tains the appropriate amino acids arranged so as to form a helix-loop-helix, calcium-binding structure. The high level of internal homology of bovine brain modu- lator protein and its relationship to the other members of the calcium-binding protein superfamily provide convincing evidence that 1) it arose early in the evolu- tion of these related proteins and 2) it was formed by two successive tandem duplications of a gene encoding a small, single domain ancestral precursor. Comparison with the nearly complete sequences of the bovine uterus and rat testis modulator proteins reported by other laboratories indicates that this ubiquitous cal- cium-dependent regulatory protein does not occur in tissue-specific forms, commensurate with the proposed function of modulator protein as a mediator of calcium- second messenger function in eukaryotic cells.

At least two major classes of s m d molecules act as gener- alized regulatory signals for stimulus-linked cellular responses in eukaryotic cells, cyclic nucleotides, and Ca”. It is now clear that these two types of signals are of equal importance as

* This work was supported by National Institutes of Health Grants NS 10123 and by 5T32-CA09111. D.M.W. acknowledges the support of NIH postdoctoral fellowship, number NS 05132. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate thiq fact.

$ Supported by National Institutes of Health Postdoctoral Fellow- ship NS 05132. Current address, The Rockefeller University, 1230 York Ave., New York, N. Y. 10021.

§ To whom reprint requests should be addressed.

physiological regulators. In many cases they act in concert to provide fine control of numerous cellular events (see Ref. 1 for review). The only documented biochemical action of cyclic nucleotides in eukaryotes is the activation of cyclic nucleotide- dependent protein kinases of broad protein substrate specific- ity (for review, see Ref. 2). Conversely, Ca2+ directly stimulates a variety of enzyme systems and indirectly regulates others through activation of a number of highly specific protein kinases. Studies from a number of laboratories have shown that many of’these regulatory activities of Ca2+ are mediated by a single small acidic calcium-binding protein which will be referred to as modulator protein‘ in this paper. To date, modulator protein has been shown to be a calcium-dependent activator of enzymes of cyclic nucleotide metabolism (3-6), plasma membrane (Ca2+-Mg”)-ATPases associated with Ca2’

efflux (7-lo), myosin light chain kinase in a number of animal tissues (11-14), NAD kinase in plants (15), muscle phospho- rylase kinase (16) and of protein kinase activities in synapto- somal preparations (17, 18).

We have previously reported (19, 20) that bovine brain modulator protein possesses physical, chemical and functional properties similar to those of the calcium-binding subunit of striated muscle troponin, TnC. Stevens et al. (21) made similar conclusions based on studies of the bovine heart protein and in addition showed that, although bovine brain and heart

modulator proteins had indistinguishable tryptic peptide maps, those maps showed little or no resemblance to the tryptic peptide map obtained with rabbit skeletal muscle TnC

.’

This communication reports the determination of the com- plete amino acid sequence of bovine brain modulator protein.

These studies clearly show its relatedness to the TnCs, the modulator protein sequence being equally homologous to that of either skeletal muscle (22) or cardiac (23) TnC. Unlike the TnCs whose sequences are highly related but tissue-specific, the sequence of bovine brain modulator protein reported here is virtually identical to the nearly complete sequence previ- ously reported for the bovine uterine smooth muscle protein (24). The significance of a number of minor differences be- tween these sequences and the partial sequence previously reported (25) for the rat testis protein are considered. Corre- lation of the sequence data presented here with studies of modulator proteins from diverse organisms and with studies

’

The abbreviations used are: TnC, the calcium-binding subunit of the troponin complex; dansyl, 5-dimethylaminonaphthalene-I-sul- fonyl; EGTA, ethylene glycol bis(fl-aminoethy1 ether)N,N,N’,N’-tet- raacetic acid.

962

This is an Open Access article under the CC BY license.

Complete Sequence of Calmodulin 963 of modulator protein interaction sites indicate that this pro-

tein has remained highly conserved in structure and function throughout eukaryotic evolution and arose as an early ances- tor of the entire super-family of four-domain calcium-binding proteins (26). A preliminary report of some of the conclusions of these studies has been made (27).

EXPERIMENTAL PROCEDURES Materials

Sequanal grade dansyl-chloride, damyl-amino acid standards, L-

norleucine, a-amino-P-guanidinopropionic acid hydrochloride, stan- nous chloride, ninhydrin, hydrindantin, and trifluoroacetic acid were products of Pierce Chemical Co. Glass-distilled benzene, ethylacetate, and I-chlorobutane were obtained from Burdick and Jackson Labo- ratories. Heptafluorobutyric acid, phenylisothiocyanate, ethylacetate, heptane, and Quadrol/t&luoroacetic acid buffer were obtained from Beckman. N"-Methylhistidine, N'-methylhistidine, and W-mono- methyllysine were used as supplied by Cyclo Chemical Co. N"Tri- methyllysine was a generous gift of Dr. R. Adelstein (National Insti- tutes of Health). Pyridine (J. T. Baker) was redistilled following reaction with ninhydrin under reflux conditions (28). Dithiothreitol and aminopeptidase M were obtained from Boehringer Mannheim Biochemicals. a-Chymotrypsin, L-1-tosylamido-2-phenylethyl chlo- romethyl ketone-treated trypsin and phenylmethylsulfonyl fluoride- treated carboxypeptidases A and B were obtained from Worthington.

Formic acid and 2-mercaptoethanol were obtained from Eastman Chemicals. Thermolysin was obtained from Calbiochem. Sephadex chromatography media were prepared for use as described by Phar- macia. All other chemicals were reagent grade and utilized without further purification. Modulator protein was prepared from bovine brain as previously described (20).

Methods

Disjunction of Peptides from Modulator Protein

Trypsin digestion was performed as previously described (20) both on bovine brain modulator protein which had been performic acid- oxidized by the method of Hirs (29) and on the untreated protein in the presence of 1 m~ EGTA. Modulator protein was cleaved with cyanogen bromide (30), digested with chymotrypsin (31), digested with thermolysin (30), and modified with citraconic anhydride (32) using previously described procedures. The resulting peptides were purified by a combination of ion exchange chromatography and gel filtration using volatile buffer systems as described under "Results."

Peptides were detected in column effluents using a modified Techni- con Autoanalyzer system as previously described (33) or by monitor- ing absorbance of column effluents at 220 nm. The appropriate fractions were pooled, the solution was evaporated to dryness, and the residues were dissolved in 50% (v/v) acetic acid and stored at -20°C. Purity of peptides was assessed by ascending chromatography on thin layer cellulose plates (20 X 20 cm, Cel Plate 22, Brinkmann) with pyridine/butanol/acetic acid/water (1015:3:12). Peptides were detected by spraying with ninhydrin spray reagent (Sigma), then developing color at l0O'C for 5 min.

Amino Acid Analysis

Protein samples were hydrolyzed and composition analyses were done exactly as previously described (20). W-Trimethyllysine was determined by amino acid analysis using the following four systems on a Beckman model 121 amino acid analyzer.

System A-Analyses were performed on a column (0.9 X 10 cm) using PA-35 Beckman resin operated at a 35°C column temperature with 0.35 N sodium citrate buffer, pH 5.26. Trimethyllysine eluted 45 min after sample injection.

System B-Analyses were performed on a column (0.9 X 55 cm) of Beckman resin AA-15 using the standard Beckman single-column methodology for acid hydrolysates at 55°C. Trimethyllysine eluted 231 min after sample injection.

System C-This system is a modification of System B of Kuehl and Adelstein (34). Analyses were performed exactly as described except Beckman resin AA-15 and a column temperature of 31.5"C were used. Trimethyllysine eluted 198 min after sample injection.

System D-Analyses were done exactly as described for System A of Kuehl and Adelstein (34) except a column temperature of 52°C was used. Tnmethyllysine eluted 195 min after sample injection.

NH2-terminal Sequence Analysis

Manual sequence analyses of peptides were performed using a modification of the dansyl-Edman procedure of Gray (35), as previ- ously described (20), or using subtractive Edman degradation essen- tially as described by Konigsberg and Hill (36). Automated Edman degradations were performed on a Beckman model 890B Sequencer using the Beckman 0.1 M Quadrol program with combined SI and S?

wash (37). Anilinothlazolinone derivatives were converted to the corresponding phenylthiohydantoin-derivatives of amino acids as de- scribed by Niall (38) and subsequently identified and quantified by gas chromatography (39) and by amino acid analyses after back hydrolysis of the phenylthiohydantoin-derivatives of amino acids (40).

Acetyl groups were detected as the dansyl-hydrazide as described by Schmer and Kreil (41).

Digestion with Exopeptidases

A and carboxypeptidase B exactly as previously described (30).

Nomenclature of Peptides

The nomenclature of peptides is as follows. A set of letters indicates the method of cleavage of the intact protein or peptide (T = trypsin;

Ch = chymotrypsin; CNBr = cyanogen bromide; Th = thermolysin;

and Tc = trypsin cleavage of citraconylated protein.) The initial cleavage method used is given fmt followed by a number (and a letter in some instances), indicating the order of emergence of a particular peptide during fractionation by the technique employed as described in the accompanying figures. Peptides isolated following secondary cleavage of a peptide are indicated by continuing letter and number codes. For example, the peptide T-IC, isolated following trypsin digestion, was further digested with thermolysin, yielding a set of peptides given the names T-IC-Thl, T-IC-Th2, etc.

Minipprint Supplement3

Peptides were digested with aminopeptidase M, carboxypeptidase

Data for much of the peptide isolation, characterization, and se- quence determination obtained in the studies described under "Re- sults" appear in a miniprint supplement immediately following the main text. Tables and figures are numbered in the order of their citation in the main text. Those which are presented in miniprint supplement are labeled in addition by a superscript asterisk.

RESULTS

The complete amino acid sequence of bovine brain modu- lator protein was deduced primarily from analyses of tryptic and cyanogen bromide peptides prepared from the intact protein and encompassing its complete sequence. The blocked NH2 terminus of modulator protein precluded direct deter- mination of its NHz-terminal sequence by automated Edman degradation. Complete or nearly complete sequences were established for each pure tryptic peptide and their NH2- to COOH-terminal order established by studies of cyanogen bro- mide fragments which also provided additional sequence as- signments. Assignments of NHn-terminal (blocked) and COOH-terminal peptides were confirmed by studies of these regions of the intact protein. Remaining sequence assignments and confirmation of tryptic peptide ordering were obtained from studies of peptides isolated from trypsin digests of the citraconylated protein and chymotrypsin digests of the per- formic acid-oxidized protein. The minimal peptides required to establish the complete amino acid sequence of bovine brain modulator protein are shown in Fig. 1.

Sequences of Tryptic Peptides

We have previously reported (20) the isolation and partial characterization of the tryptic peptides of bovine brain mod- Portions of this paper (including Figs. 9' to 15* and Tables IV' to XII*) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, Md. 20014. Request Document No.

79M-1397, cite author(s), and include a check or money order for

$4.65 per set of photocopies.

964

ulator protein used in the current study (see Figs. 10 and 11 in Ref. 20). Preliminary sequence analyses of these peptides performed using the dansyl procedure also reported in that work (20) have been confirmed, extended, and, in some cases, corrected in the studies presented here. A detailed discussion of the data establishing the sequence of each tryptic peptide is given in the miniprint supplement. In addition, these data are completely summarized in Fig. 2.

Establishing the Order of Tryptic Peptides

Peptides encompassing the whole sequence of modulator protein were isolated from a cyanogen bromide digest of modulator protein using a combination of gel filtration and ion exchange chromatography as shown in Figs. 3, 4, and 5.

The initial fractionation of cyanogen bromide-treated modu- lator protein on Sephadex G-75, shown in Fig. 3, yielded four pools containing pure peptides; CNBr-2, -4, -5, and -6. Pools CNBr-1 and CNBr-3 required further separation.

Fig. 4 shows the isolation of pure peptides CNBr-1A and -lB. Ion exchange chromatography of pool CNBr-1 (Fig. 3) on DEAE-Sephadex A-25 following preincubation at pH 8.5 (see legend to Fig. 4) gave the elution profile shown in Fig.

44. Peptide CNBr-1A was isolated as a pure peptide when fractions were pooled as indicated in the figure. Fractionation of CNBr-1 on Sephadex G-150 (Fig. 4B) yielded the unique peptide CNBr-1B which contained the single trimethyllysyl residue in modulator protein.

Pool 3, obtained from the separation shown in Fig. 3, was further resolved into three distinct components as shown in Fig. 5. Ion exchange chromatography on Dowex 50 resin (Beckman AA-15) yielded three peaks of ninhydrin-positive material as shown in Fig. 5A using the conditions described in the legend to that figure. The fist peak corresponded to cysteic acid added to the sample as a marker. The material present in the peak labeled CNBr-3A was a single, unique CNBr cleavage fragment. The pool indicated as CNBr-3B was further resolved into two pure components by gel filtration on Sephadex G-25 in 50% acetic acid as shown in Fig. 5B. The higher peaks of ninhydrin-reactive material present in this elution profile as compared to that obtained for the original pool (CNBr-3B in Fig. 5A) resulted from the larger volume fraction taken for automatic analysis during the separation shown in Fig. 5B.

The amino acid compositions and per cent recoveries of the CNBr fragments isolated as described above are given in Table I. The sum of these compositions accounts well for that

1 10

of the intact protein. As is clear from the analyses described below, these peptides account for the entire sequence of bovine brain modulator protein, allowing the NH2- to COOH- terminal order of the tryptic peptide sequences shown in Fig.

2 to be established.

Peptide CNBr-2 (Residues 1 to 36)"The amino acid com- position of CNBr-2 (Table I) was exactly that expected collec- tively for peptides T-lC, T-3, and T-4 minus an arginyl residue. CNBr-2 was the only cyanogen bromide fragment isolated for which no NH2 terminus was detected by either manual or automated procedures, indicating that it was de- rived from the NH2 terminus of the intact protein. This assignment was confirmed as discussed below.

Subdigestion of CNBr-2 with trypsin yielded three major peptides (Fig. 6; Table 11). Peptide CNBr-2-TZ contained homoserine and was, therefore, placed at the COOH terminus of CNBr-2. The composition of CNBr-2-T2 agreed with that expected for T-4 minus an arginyl residue. Since peptide T-4 had been shown to contain a COOH-terminal methionyl-ar- ginyl bond (see Fig. 2), the cyanogen bromide peptide derived from this sequence should have lacked the COOH-terminal arginine residue.

Peptide CNBr-2-Tl had an amino acid composition identi- cal to peptide T-1C and was resistant to Edman degradation or aminopeptidase M digestion. It was, therefore, placed at the NH2 terminus of CNBr-2. Digestion of CNBr-2-T1 with thermolysin (data not shown) yielded peptides that had amino acid compositions identical to those of thermolysin peptides from T-1C (Table V*; Fig. l l * ) , one of which (T-IC-Th2) was shown to have an acetylated NH2 terminus.

Peptide CNBr-2-T3 had an amino acid composition identi- cal to peptide T-3 (Table IV*) whose sequence, therefore, was placed between peptides CNBr-2-T1 (T-1C) and CNBr-2-T2 (T-4) in the sequence of CNBr-2. The order of tryptic peptides was taken to be T-1C followed by T-3 and T-4. This ordering of tryptic peptides in CNBr-2 was further supported by the isolation from citraconylated modulator protein of a tryptic peptide, Tc-1, whose amino acid composition (Table VIII*) was exactly the sum of T-lC, T-3 and T-4. The NH2-terminal sequence consisting of residues 1 to 37 was deduced to be: Ac-

Ala-Asp-Gln-Leu-Thr-Glu-Glu-Gln-Ile-Ala-Glu-Phe-Lys- Glu-Ala-Phe-Ser-Leu-Phe-Asp-Lys-Asp-Gly-Asn-Gly-Thr- Ile-Thr-Thr-Lys-Glu-Leu-Gly-Thr-Val-Met-Arg.

Peptides CNBr-3B2, CNBr-3A, CNBr-6, and CNBr-4 (Res- idues 37 to 77)"Residues 38 to 77 in bovine brain modulator protein were contained within one large tryptic peptide, T-lA,

20 30

Complete Sequence of Calmodulin 965

Ftocilon No

Ftc. 3. Purification of cyanogen bromide peptides. Cyanogen bromide cleavage of 1 pmol of bovine brain modulator protein was performed as described under "Methods." The elution profile shown was obtained when the digest was fractionated on a column (2.5 X 85 cm) of Sephadex G-75 in 50% (v/v) acetic acid. The column flow rate was 30 ml/h. A portion of the column effluent (1 d / h ) was continu- ously removed and monitored by reaction with ninhydrin following alkaline hydrolysis (33). e-Dansyllysine (2 pmol) was added to the sample as a marker. Fractions were collected at 6.0-min intervals.

Pools CNBr-1 and CNBr-3 were further fractionated as described in Figs. 4 and 5.

F

E O 50 IO0

FIG. 4. Purification of peptides from CNBr-1. A , CNBr-I (500 nmol) was evaporated to dryness, dissolved in 1 ml of 0.1 M NHdHCOa, incubated for 2 h at room temperature to open the lactone ring, and then freeze-dried. The resulting material was dissolved in 2.0 ml of 0.02 M Tris.HC1, pH 8.2, and applied at room temperature to a column (0.9 X 25 cm) of DEAE-Sephadex A-25 in the same buffer.

The column was eluted at 10 ml/h with a linear gradient consisting of 50 ml each of 0.02 M Tris.HC1, pH 8.2, as starting buffer and 0.02 M

Tris-HCI, 0.3 M NaCl, pH 8.2, as limit buffer. Fractions (1.3 ml) were monitored for absorbance at 210 nm as shown in the elution profde.

B, the pool indicated as CNBr-1 in Fig. 3 was applied to a column (3.0

X 80 cm) of Sephadex G-150 in 50% (v/v) acetic acid. The column was operated with a flow rate of 20 ml/h at 25°C. Fractions were collected at 6.0-min intervals. The elution profile shown was obtained by continuous monitoring of a portion of the column effluent as described in the legend to Fig. 3. The pure peptide CNBr-1B was pooled as indicated.

isolated from the oxidized protein and peptides derived from the sequence Lys-Met-Lys (see Fig. 2). As discussed in the miniprint supplement, trypsin digestion of unoxidized modu- lator protein resulted in an unusual trypsin cleavage of the methionyt-methionine bond contained within T-1A (residues 71 and 72), yielding a large peptide, T-IA', lacking the COOH- terminal tripeptide Met-Ala-Arg. An additional peptide, Met-

05 0 4

-

03

-

02

-

-

g 0.1

E 0 20 ^{1 0 0} I60

-

0

a

i

Frochon No

FIG. 5. Purification of peptides from cyanogen bromide di- gest pool CNBr-3. A , elution profile obtained when CNBr-3 was subjected to ion exchange chromatography on a column (0.9 X 30 cm) of Beckman AA-15 resin operated at 45 ml/h. Consecutive linear gradients were used. First gradient 250 ml each of 0.01 M pyridine/

acetic acid, pH 2.0, and 0.2 M pyridine/acetic acid, pH 3.1. Second gradient: 250 ml each of 0.2 M pyridine/acetic acid, pH 3.1, and 2.0 M pyridine/acetic acid, pH 5.0. B, peptide fraction CNBr-3B was further resolved by chromatography on a column (2.0 X 130 cm) of Sephadex G-25 in 50% (v/v) acetic acid. The flow rate was 20 d / h . Fractions were collected every 6.0 min. A portion of the effluent (1 ml/h) was taken continuously for monitoring as described in Fig. 3.

Ala-Arg-Lys, was isolated from these digests accounting for this sequence. Confirmation of the nature of these various trypsin fragments and their placement within the linear se- quence of the protein was obtained from studies of four of the cyanogen bromide cleavage fragments isolated from intact modulator protein.

Tryptic peptide T-1A was placed COOH-terminal to T-4 in the linear sequence by studies of peptide CNBr-3B2. The amino acid composition of this peptide shown in Table I is identical to that expected for the NH2-terminal 14 residues of T-1A’ established by automated Edman degradation (Table XII*) plus a single residue of arginine. Subtractive Edman degradation of CNBr-3B2 (Table XI*) established its NH2- terminal sequence as Arg-Ser-Leu-Gly-Glx. The NH~terminal

Froctm No.

FIG. 6. Purification of peptides from a trypsin digest of CNBr-2. Cyanogen bromide peptide CNBr-2 (500 nmol) was digested with trypsin as described under “Methods.” Peptides were resolved by chromatography on a column (0.9 X 30 cm) of Beckman AA-15 resin eluted at 30 d / h with a linear gradient of 200 ml each of 0.02

M pyridine/acetic acid, pH 2.20, and 0.5 M pyridine/acetic acid, pH 2.65, as the starting and limit buffers, respectively. After completion of the gradient the column was step-eluted with 2.0 M pyridine/acetic acid, pH 5.0. The elution profile was obtained exactly as described in Fig. 3.

TABLE I

Cyanogen bromide peptides from bovine brain modulator protein

Peptides were isolated from a cyanogen bromide digest of modulator protein as described in the text and Figs. 3, 4, and 5. Amino acid compositions were determined as described in text. Values in parentheses are assumed residues/mol.

Amino acid CNBr-1A” CNBr-1B“ CNBr-2 CNBr-3Ab CNBr-3Blh CNBr-3B2’ CNBr-4 CNBr-5

Lysine 2.1 (2) 2.8 (3) 1.3 (1) 1.0 (1)

Histidine 0.8 (1)

Trimethylly- 0.9 (1)

Arginine 2.6 (3) 0.9 (1) 1.1 (1) 1.0 (1)

Aspartic acid 4.7 (5) 3.0 (3) 4.4 (4) 5.1 (5) 4.1 (4) 1.8 (2)

Threonine 1.1 (1) 1.6 (2) 4.8 (5) 1.9 (2) 1.0 (1) 0.7 (1)

Serine 1.7 (2) 1.1 (1) 1.0 (1)

Glutamic acid 5.2 (5) 3.8 (4) 7.0 (7) 2.3 (2) 5.4 (5) 4.0 (4)

Proline 1.0 (1) 1.0 (1)

CNBr-6

sine

Glycine Alanine Valine Homoserine‘

Isoleucine Leucine Tyrosine Phenylalanine

2.3 (2) 1.1 (1) 2.6 (3)

1.8 (2) 1.0 (1)

0.6 (1) 0.6 (1)

1.7 (2) 0.5 (1) 1.7 (2)

1.2 (1) 2.0 (2)

3.3 (3) 2.0 (2) 2.2 (2) 1.1 (1) 1.2 (1) 1.0 (1) 1.9 (2)

3.1 (3) 1.0 (1) 1.1 (1) 1.0 (1) 1.3 0.6 (1) 0.5 (1) 0.5 (1) 0.5 (1) 0.5 2.0 (2) 1.6 (2) 1.6 (2)

3.2 (3) 1.0 (1) 2.1 (2)

0.9 (1) 3.0 (3) 1.7 (2) 0.9 ( 1 )

(1) 0.8 (1)

(1) Free homo-

serine

Total assumed 33 15 36 20 20 15 4 3

residues

1

% yield 33 15 52 50 24 52 51 54 59

Pool 1 of Fig. 3 resolved into two peptides as described in Fig. 4.

Pool 3 of Fig. 3 resolved into one peptide and a mixture of two peptides as described in Fig. 4 A . The peptide mixture (CNBr-3B) was Determined as homoserine and homoserine lactone.

resolved into two peptides as described in Fig. 4B.

Complete Sequent

TABLE I1

Amino acid compositions ofpeptides isolated from a trypsin digest of CNBr-2

Peptides were isolated as described in Fig. 6. Compositions were determined as described in the text. Values in parentheses are as- sumed residues/mol.

Amino acid Lysine Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Valine Methionine”

Isoleucine Leucine Phenylalanine

CNBr-2-TI CNBr-2-T2 0.6 (1) 1.5 (1)

1.1 (1) 1.0 (1)

4.7 (5) 0.8 (1)

0.4 1.4 (1)

1.8 (2)

0.2 1.0 (1)

0.9 (1) 1.0 (1)

1.1 ( I ) 1.1 (I) 1.0 (1)

CNBr-2-T3

~~

1.5 (2) 2.6 (3) 2.5 (3) 0.9 (1) 1.4 (1) 2.1 (2) 1.2 (1)

0.8 (1) 1.0 (1) 2.1 (2) Total assumed 13 6 17

residues

% Yield 80 32 50

Residue num- 1-13 31-36 14-30

be9

Determined as homoserine plus homoserine lactone.

’

arginine must have been produced by CNBr cleavage of the only methionyl-arginine bond in modulator protein, that pres- ent at the COOH terminus of T-4. The remaining 4-residue sequence Ser-Leu-Gly-Glx- is identical to that of the NH2 terminus of T-1A. The placement of T-1A adjacent to the COOH terminus of T-4 is thus unequivocal despite the single arginyl residue overlap provided by CNBr-3B2.

One of the 2 prolyl residues in modulator protein, both of which are present in peptide T-1A (and T-lA’), was present in CNBr-3B2 discussed above. The only other proline con- taining CNBr peptide isolated from digests of the intact protein was peptide CNBr-3A. The amino acid composition of this peptide shown in Table I accounted for the remainder of the composition of T-1A except for single residues of methionine, alanine, and arginine. In addition, an identical peptide, T-1A”CNBr-2 (Table VU*) was isolated from a cyan- ogen bromide digest of T-1A’ (see miniprint supplement).

Automated Edman degradation of CNBr-3A (Table XII*) unequivocally placed it within T-1A contiguous to CNBr-3B2 and provided some of the information required to establish this portion of the sequence. The sequence of residues 37 to 71 established by these data as summarized in Figs. 1 and 2,

was: Arg-Ser-Leu-Gly-Gln-Asn-Pro-Thr-Glu-Ala-Glu-Leu- Gln-Asp-Met-Ile-Asn-Glu-Val-Asp-Ala-Asp-Gly-Asn- Gly

-

-

-

-

-

The COOH-terminal3 residues of T-lA, Met-Ala-Arg, were isolated from the cyanogen bromide digest of modulator pro- tein as free homoserine (CNBr-6, Table I) and as part of the tetrapeptide CNBr-4, which contained single residues of ala- nine, arginine, lysine, and homoserine (Table I). Subtractive Edman degradation of CNBr-4 (Table XI*) established its sequence to be Ala-Arg-Lys-Hse. These were the only Ala- Arg and Arg-Lys sequences found in bovine brain modulator protein. As noted above, the methionyl-methionine bond in T-1A was completely susceptible to trypsin cleavage in the unoxidized protein producing the tetrapeptide Met-Ala-Arg- Lys (T-10) in high yields. Peptide T-11 isolated from the oxidized protein (Table IV*) had the sequence Lys-Met-Lys.

These were the only Lys-Met and Met-Lys sequences found

:e of Calmodulin 967

in modulator protein. The sequences of CNBr-4, T-11, and T- 10 together with the information presented above for T-1A unequivocally established the COOH-terminal sequence of T-

1.4 to be Met-Ala-Arg and position the sequence Lys-Met-Lys (T-11) immediately adjacent to its COOH terminus. The sequence for residues 37 to 77 thus was established as: ^Arg- Ser-Leu-Gly-Gln-Asn-Pro-Thr-Glu-Ala-Glu-Leu-Gln- Asp-Met-Ile-Asn-Glu-Val-Asp-Ala-Asp-Gly-Asn-Gly- Thr-Ile-Asp-Phe-Pro-Glu-Phe-Leu-Thr-Met-Met-Ala- Arg-Lys-Met-Lys.

Peptides CNBr-1A and CNBr-IB (Residues 77 to 124)- CNBr-1A was a large peptide whose amino acid composition (Table I) indicated that it contained tryptic peptides T-2, T- 9, and T-5B (Table IV*, Fig. 2), plus 1 additional residue each of lysine, histidine, valine, and homoserine. Automated se- quence analysis of CNBr-1A (Table XII’) demonstrated a partial sequence of Lys-Asp-Thr-Asp-Ser-Glu-Glu-Glu-Ile- Arg-Glu-Ala-Phe-Arg-Val-Phe. The sequence Val-Phe- is unique to the NH2 terminus of the tryptic peptide T-5B. Thus, the order of tryptic peptides within CNBr-1A was established as Lys, T-2, T-9, and T-5B. In addition, peptide T-6, resulting from incomplete trypsin cleavage, was found to have a se- quence of Met-Lys-Asp-Thr-Asp-Ser-Glu-Glu-Glu-Ile-Arg

(see miniprint supplement discussion of T-2 and T-6). There- fore, CNBr-1A was clearly adjacent to the COOH terminus of CNBr-4 discussed above, confuming the placement of peptide T-11 and placing in order the sequences of tryptic peptides T- 2, T-9, and T-5B as summarized in Fig. 1.

Peptide CNBr-1A contained the single histidyl residue found in modulator protein. From the amino acid composition of CNBr-1A and the above sequence data, the only residues which were not placed in sequence at the COOH terminus of CNBr-1A were 1 residue each of histidine, valine, and homo- serine. These COOH-terminal 3 residues could have been derived only from T-5A (Table IV*; Fig. 2) whose NHz-ter- minal sequence was established as His-Val-Met-Thr-(see min- iprint supplement). This unequivocally established the se- quential order of tryptic peptides within modulator protein as T-2, T-9, T-5B, and T-5A. Therefore, the COOH-terminal sequence of CNBr-1A must be His-Val-Hse. This established the sequence of residues 77-124 as Lys-Asp-Thr-Asp-Ser- Glu-Glu-Glu-Ile-Arg-Glu-Ala-Phe-Arg-Val-Phe-Asp-Lys- Asp-Gly-Asn-Gly-Tyr-Ile-Ser-Ala-Ala-Glu-Leu-Arg-His- Val-Met-Thr-Asn-Leu-Gly-Glu-Tml-Leu-Thr-Asp-Glu- Glu-Val-Asp-Glu-Met. As further confirmation of this se-

quence, peptides whose amino acid compositions (Table X*) fit those expected of residues 90 to 92 (Ch-12), 93 to 99 (Ch-71, 100 to 105 (Ch-2), and 106 to 109 (Ch-13) were isolated (Fig.

15*) from a chymotryptic digest of modulator protein. Also, peptide CNBr-1B isolated as described above had an amino acid composition (Table I) expected for residues 110 to 124 whose sequence had been established during the studies of peptide T-5A (residues 107 to 126).

Peptides CNBr-3Bl and CNBr-5 (Residues 125-148)”Two remaining unique CNBr fragments, CNBr-3B1 and CNBr-5, were isolated from digests of intact modulator protein in high yields. The sum of their amino acid compositions, shown in Table I, plus 1 residue of free homoserine (CNBr-6) was exactly that expected for the COOH-terminal tryptic peptide T-1B plus additional single residues of isoleucine and arginine.

Peptide CNBr-3B1, a 20-residue peptide, encompassed all but the COOH-terminal4 residues of T-lB, Met-Thr-Ala-Lys.

Automated sequence analysis of CNBr-3B1 (Table XII*) dem- onstrated a partial sequence of Ile-Arg-Glu-Ala-Asn-Ile-Asx- Gly. Since peptide T-1B began with the unique sequence Glu- Ala-Asn-Ile-Asp-Gly- and peptide T-5A ended in the unique sequence Met-Ile-Arg, the sequence shown for CNBr-3B1

represented an overlap in sequence for tryptic peptides T-5A and T-1B.

Peptide CNBr-5 (Thr,Ala,Lys) contained no homoserine or homoserine lactone and, therefore, represented the COOH- terminal 3 residues of modulator protein (residues 146 to 148), confirming the sequence of this region established as described below and in the miniprint supplement. The amino acid sequence of residues 125-148 was established as: Ile-kg-Glu- Ala-Asn-Ile-Asp-Gly-Asp-Gly-Glu-Val-Asn-T~-Glu-Glu- Phe-Val-Gln-Met-Met-Thr-Ala-Lys.

Confirmation of COOH-terminal Sequence Assignment Studies of cyanogen bromide peptide CNBr-5 and tryptic peptide T-1B (see miniprint supplement) indicated that the COOH-terminal sequence of bovine brain modulator protein was Thr-Ala-Lys-COOH. This assignment was confmed by digestion of the intact protein with carboxypeptidases A and B. When 3 mg (180 nmol) of performic acid-oxidized bovine brain modulator protein were treated with 0.25 mg of carbox- ypeptidase A as described under "Methods," no amino acids were detected in aliquots containing 22.5 nmol of substrate polypeptide in significantly greater amounts than were present in the same aliquot prior to incubation (at zero time). Addition of carboxypeptidase B (0.28 mg) to the sample after a 60-min incubation with carboxypeptidase A resulted in the sequential release of lysine, alanine, and threonine in major amounts as well as lesser amounts of other amino acids. The blank cor- rected values for the major amino acids detected in aliquots at 30 min were (moles of amino acid/mol of protein): Lys, 1.2;

Ala, 0.09; Thr, 0.08; those at 1 h were: Lys, 0.93; Ala, 0.25;

Thr, 0.16.

These results c o n f m the assignment of the sequence Thr- Ala-Lys as the COOH terminus of intact modulator protein thus completing its sequence.

The complete amino acid sequence of bovine brain modu- lator protein as established by these studies (Fig. 1) yields a calculated molecular mass of 16,680 daltons, in relative agree- ment with the molecular weights of 15,256 and 17,800 previ- ously deduced from physical studies (20). The amino acid composition of bovine brain modulator protein determined from this sequence agrees with that previously reported (20) for the intact protein when those values are normalized to a molecular mass of 16,680 g/mol as shown in Table 111.

DISCUSSION

The studies of bovine brain modulator protein presented here provide the first fully documented complete amino acid sequence of this protein from any source. Numerous reports have now appeared showing the troponin C-like physicochem- ical and functional properties of modulator proteins which we first described for the bovine brain protein (20). Comparison of the structure of bovine brain modulator protein with those previously reported for bovine cardiac (23) and rabbit skeletal (22) muscle TnCs as shown in Fig. 7 clearly demonstrates the troponin C-like character of modulator protein. By aligning the sequence of bovine brain modulator protein so that residue 1 corresponds to residue 9 in the cardiac muscle TnC and to residue 8 in the rabbit skeletal muscle protein, maximum homology to both troponin Cs is maintained throughout their sequences by introducing only two small gaps in the modulator protein sequence. The first of these is a single residue gap which we place at a position corresponding to cysteine 35 in bovine cardiac muscle TnC. A gap is also required in this region of the sequences of all skeletal muscle TnCs (42) for proper alignment to the bovine cardiac muscle protein. Van Eerd and Takahashi (23) originally placed this gap at a position corresponding to valine 28 in bovine cardiac TnC.

TABLE 111

Amino acid composition of bovine brain modulator protein Residues/molecule by analvsis" _cule Residues,mole- by sequence

Lysine Trimethyllysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoieucine Leucine Tyrosine Phenylalanine Tryptophan

7.2 1.0 6.2 1.3 22.2 11.0 26.7 4.7 10.8 2.0 10.7 0.0 7.2 9.0 7.6 9.4 2.0 7.8 0.0

7 1 1 6 23 12 27 4 11 2 11 0 7 9 8 9 2 8 0

Total residues 148

a Calculated from time course analysis previously reported (20) using 16,680 g/mol.

However, the alignment shown in Fig. 7 appears to be more in accord with maintaining maximum homology in this region of the sequence of all three proteins. This plus the lack of any clear functional role for cysteinyl residues in the TnCs (43) suggests to us that cysteine 35 in the bovine cardiac protein arose by insertion. The only other gap introduced in the modulator protein sequence corresponds to residues 90 to 92 in the bovine cardiac muscle TnC sequence and is required for alignment to both TnCs. The significance of the location of this 3-residue gap immediately before the NH2 terminus of domain 3 of modulator protein is considered further below.

Of the 148 residues of modulator protein compared with those at corresponding positions in bovine cardiac TnC, iden- tical residues are found at 74 positions, while those in 44 positions are both functionally and genetically conserved.

Similar numbers of residues are either identical or conserved between the modulator protein and skeletal muscle TnC. The two muscle TnCs are identical at 103 positions and conserved at 23 others. Quite strikingly, 63 positions are identical in all three proteins with most of these identities occuring in the fist three domains; amino acids at 38 positions found predom- inantly in the fourth domain are conserved. This result is particularly interesting in view of the fact that many of the invariant residues among these three proteins lie outside of their putative calcium binding structures in potential hinge regions which may be sites for calcium dependent interaction with other proteins (see below).

Collins and co-workers (44) first noted that the amino acid sequence of rabbit skeletal muscle TnC was composed of four homologous domains. The amino acid sequence of bovine brain modulator protein also possesses internal homology as shown in Fig. 8. Although all four domains are related in sequence, the level of homology is greatest when the first domain is aligned with the third domain and the second domain is aligned with the fourth. Of the 33 residues compared in each pair of domains, 18 residues are identical and 6 are conservative replacements between Domains 1 and 3. There are 13 identical residues and 13 conservative replacements between Domains 2 and 4. This level of internal homology appears to be greater than that observed within the muscle TnCs (42). As noted above, the 3-residue gap introduced in

Complete Sequence of Calmodulin

10 20

969

30 40 50

A: Leu-GlytAlatGlu-Asp-Gly-Cys B: Lys-Asp+GlytAsn-Gly-Thr"---

,"-,

C: Ala-Asp+Glykly-Gly-Asp---- I _ - _ I

60 70 80

90 100 110

A : Ser-Lys-Gly-Lys 8: Asp--- C: Ala-Lys-Gly-Lys

120 130 140

A: Ile/M;t-Le;tGln-Ala-Thr G l u - A s p - A s p - I l e - G l u ~ G l u + ~ e u - M e t - L ~ s - A s p - G l y - A ~ p ~ L y s ~ ~ ~ ~ A s ~ ~ ~ ~ A r g ~ ~ ~ ~

"""_""_

B: HistVal-MettThr-Asn-Leu Asp-Glu-Glu-Val-AsptGlu~Met-Ile-Arg-Glu-Ala-Asn~Ile~Asp~Gly Asp-Gly GlutVal- C: GlutIle-PhetArg-Ala-Ser Asp-Glu-Glu-Ile-Glu~Ser+Leu-Met-Lys-Asp-Gly-Asp~Lys~AsntAsn Asp-Gly Argtlle-

I - - - J _""""__"I I ^""""""" "_I

150

- - - - - -

-

FIG. 7. Comparison of the amino acid sequence of modulator protein with the amino acid sequence of muscle troponin C. A , the amino acid sequence of bovine cardiac muscle troponin C reported by Van Eerd and Takahashi (23); B, the amino acid sequence of bovine brain modulator protein as determined in this report (see Fig.

1); C, the amino acid sequence of rabbit skeletal muscle troponin C as reported by Collins and co-workers (22). The numerals refer to

M * *

positions in the bovine cardiac troponin C sequence. Solid line boxes indicate positions a t which all three proteins are identical. Dashed line boxes indicate positions at which all three proteins are assumed to be related by functionally conserved replacements. ---, gaps introduced into the sequences for the purpose of alignment; they are discussed in the text.

* * * * 4 0

0 u m . i i n 1: - l ; l z ~ - l l ~ - A l ~

$ 1 i l o r n ~ i n 3 : -Ser-GlL-Glu

"""

4 4 76

." - - - - - - - -

FIG. 8. Internal sequence homology in modulator protein.

The amino acid sequence of bovine brain modulator protein (Fig. 1) can be divided into four homologous domains as shown. Whereas all four domains are related in sequence, the level of homology is greatest when Domain 1 is aligned with Domain 3 and Domain 2 is aligned with Domain 4. The numerals refer to positions in the modulator the modulator protein sequence solely for the purpose of alignment occurs before the NH2 terminus of domain 3. These data and the shorter NH2-terminal sequence of modulator protein suggest that it may have arisen by duplication of a slightly smaller two domain precursor than that from which the striated muscle TnCs arose. Detailed computer analyses support these conclusions and suggest that modulator protein is closely related to the original four-domain ancestral protein of this family of calcium-modulated proteins (26).

Based on the crystal structure of parvalbumin and its amino acid sequence homology to troponin C, Kretsinger and Barry (45) have predicted a three-dimensional structure of rabbit skeletal muscle TnC in which they identify putative calcium- binding residues in each domain. The asterisks in Fig. 8 denote the corresponding positions in modulator protein. All four domains possess a potential calcium liganding residue at each of these positions. In addition, the regions of sequence

protein sequence (Fig. 1). Solid line boxes indicate positions at which the two domains compared are identical. Dashed line boxes indicate positions a t which the two domains compared are related by func- tionally conservative replacements. Asterisks indicate putative cal- cium binding residues predicted by the Kretsinger model (45).

immediately adjacent to these putative calcium-binding resi- dues contain the appropriate hydrophobic residues necessary for the helices predicted by Kretsinger and Barry (45) as essential constituents in the formation of functional calcium- binding structures. The presence of four potential calcium- binding domains is in agreement with the previous observa- tions that bovine brain (20) and other (46-48) modulator proteins bind 4 atoms of calcium/molecule. However, it should be noted, that, while the TnCs possess both calcium-specific and mixed Ca2+- or Mg"+-binding sites, the four sites in mod- ulator protein appear to bind only calcium (49).

As noted earlier, preliminary communications have de- scribed sequence analyses of two other modulator proteins.

The amino acid sequence of bovine uterine smooth muscle modulator protein, reported by Grand and Perry (24), is identical to that reported here except for assignments of the amidation states of 2 aspartyl residues. We have concluded

that residues 24 and 97 in bovine brain modulator protein are asparagines, based on the results of aminopeptidase digestion and automated sequence analyses as described in the mini- print supplement. Grand and Perry (24) assigned these resi- dues as aspartic acid by virtue of the electrophoretic proper- ties of the pure peptides (50). The reasons for these minor differences in the proposed sequences of bovine modulator proteins are unclear.

The sequences of the bovine modulator proteins differ at a number of positions from the partial sequence published for the rat testis protein (25). These are: 1) difference in amidation states at residues 3,42,53, 111, 137, and 142 and 2) placement of an asparagine residue at position 57 in the bovine protein instead of alanine and, conversely, alanine replacing aspara- gine at position 60. It seems likely that these latter differences result from mistaken assignments. Position 60 represents the Z octahedral binding coordinate in the second domain cal- cium-binding site (Fig. 8) predicted as noted above. An amino acid residue with an oxygen containing side chain, or a glycyl residue when a water molecule is a potential liganding species, is found at this position in all other calcium-modulated pro- teins whose primary structures have been reported (42). In addition, the amino acid sequence of this region of modulator protein from the sea invertebrate Renilla reniformis is iden- tical to that of the bovine brain modulator protein reported here (51).

The level of homology between modulator protein and the TnCs indicates that their overall three-dimensional structures must be quite similar, despite the shorter sequence of modu- lator protein at the NH2 termini of its first and third domains.

In fact, substantial variation has previously been reported (52) in the NHp termini of the otherwise highly conserved se- quences of skeletal muscle TnCs from different species. The results of detailed physicochemical analyses (46-48,53-57) as well as computer-based modeling analyses (56) further suggest the overall similarities in structure of both the calcium-lig- anded and -unliganded forms of TnCs and modulator protein.

It is, therefore, not surprising that modulator protein possesses TnC-like functional properties.

Clearly, modulator protein possesses numerous unique reg- ulatory activities not shared by muscle TnCs to any significant extent. The most obvious structural difference between mod- ulator protein and TnC whlch might correlate with modulator protein-specific functions is the region containing the single trimethyllysyl residue found uniquely in modulator protein.

To date, no exact role has been established for the methylated amino acids found in modulator protein and other proteins.

However, detailed studies of bovine brain modulator protein and rabbit skeletal muscle TnC as inhibitors of kinase-me- diated phosphorylation of troponin I suggest that a region of amino acid sequence encompassing Domain 3 in modulator protein (residues 81 to 116) represents a site for specific interaction with troponin I (58). Examination of the align- ments of this region of the sequence of TnCs and bovine brain modulator protein (Fig. 7) shows that most identities in all three proteins lie adjacent to either side of the predicted helix- loop-helix-forming residues in this third domain. The se- quence -Ser-Glu-Glu-Glu- (positions 93 to 96 in the cardiac protein sequence shown in Fig. 7) is particularly noteworthy as this sequence is identical in all TnCs and modulator pro- teins thus far studied (42). It seems likely that this sequence may form a portion of the troponin I recognition site in this domain and may represent a common site for calcium-de- pendent interaction with other proteins. Studies are currently in progress to test this possibility.

Several features of the trypsin cleavage pattern of modula- tor protein were noteworthy. The Asp-Lys-Asp sequences at

residues 20 to 22 and 93 to 95 were relatively resistant to trypsin cleavage. Trypsin cleavage did not occur at the single trimethyllysyl residue as this residue was isolated exclusively in a larger peptide containing a COOH-terminal arginine.

Also, no evidence of monomethyllysine or dimethyllysine was found. More surprisingly, unusual trypsin cleavages were ob- tained between methionyl residues 71 and 72 and following the methionyl-methionine sequence at residues 144 and 145.

Similar trypsin cleavages have also been observed with muscle troponin C (22). This observation would seem particularly important in view of the fact that modification of a limited number of methionyl residues in modulator protein leads to complete loss of activity (59). It seems likely that either unusual structural constraints or local electrostatic effects make these Met-Met sequences susceptible to trypsin cata- lyzed cleavage.

Another interesting observation made during the course of these analyses was the elution properties of peptides CNBr- 1A and CNBr-1B. It is evident that pool 1 from the initial fractionation of the cyanogen bromide digest contained pep- tides that eluted earlier than expected. Peptide CNBr-1A contained 33 residues and peptide CNBr-1B contained only 15 residues (Table I). However, they both eluted as CNBr-1 from Sephadex G-75 (Fig. 3) before peptide CNBr-2, which contained 36 residues. It is possible that peptides CNBr-1A and -lB eluted as an aggregate from the column. However, it seems more likely these two peptides which are contiguous in the sequence of modulator protein, eluted as a single large peptide, due to inefficient cleavage at the methionyl-threonine bond between residues 109 and 110, as has previously been reported for the bovine uterine smooth muscle (24) and rat testes (25) proteins. This methionyl-threonine bond then would have had to cleave upon subsequent treatment and chromatography of CNBr-1. In this regard, it has been re- ported (60) that methionyl-threonine bonds are inefficiently cleaved by cyanogen bromide.

In conclusion, the evidence summarized here suggests that the TnCs are highly specialized derivatives of the more gen- eralized calcium receptor, modulator protein. Not only does it appear that modulator protein arose early in eukaryotic evo- lution as a mediator of calcium second messenger function, but the interaction sites on both modulator protein and the enzymes it regulates have been highly conserved. It is further suggested that modulator protein-regulated enzymes may share at least limited regions of amino acid sequence with muscle troponins I and T. Clearly, this latter proposal awaits detailed structural analyses of modulator protein regulated enzymes for confiiation. However, it appears likely that modulator protein and the enzyme systems it regulates have evolved in parallel, endowing eucaryotic cells with the ability to respond to external stimuli using calcium flux as a second messenger signal (27).

Acknowledgments-We thank Ms. Delores Johnson for expert technical assistance and Ms. Ann Allen for great patience and skill in typing this manuscript.

REFERENCES

1. Berridge, M. J. (1975) Adu. Cyclic Nucleotide Res. 6, 1-98 2. Greengard, P. (1978) Science 199, 146-152

3. Cheung, W. Y. (1970) Biochem. Biophys. Res. Commun. 38,533- 4. Kakiuchi, 538 S., Yamazaki, R., and Nakajima, H. (1970) Proc. Jpn.

Acad. 46, 589-592

5. Brostrom, C. O., Huang, Y.-C., Breckenridge, B. McL., and Wolff, D. J. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 64-68

6. Cheung, W. Y., Bradham, L. S., ^Lynch, T. J., Lin, Y. M., and Tallant, E. A. (1975) Bcochem. Biophys. Res. Commun. 66, 1055-1062