Complete amino acid sequence of rabbit muscle glycogen phosphorylase

Proc. Natl. Acad. Sci. USA Vol. 74, No. 11, pp. 4762-4766, November 1977

Biochemistry

Complete amino acid sequence of rabbit muscle glycogen phosphorylase (glycogen metabolism)

KOITI TITANI, ATSUSHI KOIDE*, JACQUES HERMANNt, LOWELL H. ERICSSON, SANTOSH KUMAR, ROGER D. WADE, KENNETH A. WALSH, HANS NEURATH, AND EDMOND H. FISCHER Department of Biochemistry, University of Washington, Seattle, Washington 98195

Contributed by Hans Neurath, July 11, 1977

The sequence of the 841 amino acid residues ABSTRACT in each subunit (molecular weight 97,412) of rabbit muscle glycogen phosphorylase b (1,4-a-D-glucan:orthophosphate a-glucosyltransferase; EC 2.4.1.1) has been determined. The general strategy was based on limited proteolysis of native phosphorylase b by subtilisin BPN', yielding two large segments (light and heavy)which were fragmented by cleavage at methionyl-, asparaginyl-glycine, and aspartyl-proline bonds. Analysis of two cyanogen bromide fragments (CB14 and CB17) isolated from the intact molecule yielded the overlap between the light and heavy fragments and the remainder ofthe sequence. The residues involved in the covalent aind allosteric control of the enzyme, and in the binding of the cofactor pyridoxal 5'-phosphate, were identified as serine-14, tyrosine-155, and lysine-679,

fonylbenzamido)benzylthio]adenine-labeled rabbit muscle phosphorylase b (9, 10) was generously provided by Donald J. Graves and coworkers (Department of Biochemistry and Biophysics, Iowa State University). Nagarse (subtilisin BPN') was a gift of Teikoku Chemical Industry, Japan.

Limited proteolysis with subtilisin BPN' was performed essentially as described by Raibaud and Goldberg (7). The material obtained by trichloroacetic acid precipitation was reduced with dithioerythritol, carboxymethylated (11), and separated into the light (Ls) and heavy (Hs) segments on a column of Sephadex G-150 in 9% formic acid containing 7 M urea. Cyanogen bromide digests of the whole carboxymethylated protein and of segments Ls and Hs were prepared and fractionated essentially as described by Saari and Fischer (5). Cyanogen bromide fragments were designated according to their original numbering system (CB1-18), whereas new fragments (CB20-24) were numbered in the order of their isolation. The letters N and C were placed after a CB fragment number to identify amino- and carboxyl-terminal subfragments, respectively. Asn-Gly, Asp-Pro, and tryptophanyl bonds were chemically cleaved by procedures adapted from Bornstein (12), Fraser et al. (13), and Omenn et al. (14), respectively. Other enzymatic subdigestions were performed as needed (e.g., ref. 15). Enzymatic cleavages of glutamyl bonds with staphylococcal protease followed the procedure of Houmard and Drapeau (16). Amino acid analyses were performed on Durrum (model D-500) amino acid analyzers. Automatic Edman degradations utilized the Beckman Sequencer (model 890B) according to the method of Edman and Begg (17) as modified by Hermodson et al. (18), or a Sequemat (model 12) using the attachment procedures summarized by Laursen et al. (19). Phenylthiohydantoin derivatives of amino acids were identified by gas chromatography (18, 20), spot tests (18), or high-pressure liquid

respectively.

Muscle glycogen phosphorylase (1,4-a-D-glucan:orthophosphate a-glucosyltransferase; EC 2.4.1.1) is one of the key enzymes directly involved in the metabolism of glycogen. Upon activation through a cascade of successive enzymatic reactions, the enzyme catalyzes the conversion of glycogen and phosphate to glucose-i-phosphate. Since its isolation and crystallization (1), phosphorylase has attracted the interest of numerous investigators because it is the first enzyme in which activity was shown to be controlled by both covalent and allosteric modifications (2-4). A full understanding of the regulation and mechanism of action of the enzyme requires a detailed analysis of the structure of the subunits of the protein and of their molecular assembly. As an initial step, Saari and Fischer (5) isolated 18 fragments produced by cleavage of phosphorylase with cyanogen bromide. Titani et al. (6) showed that one of these fragments (CB14) is acetylated at its amino-terminus and that serine-14 becomes phosphorylated in the conversion of the enzyme from the b to the a form. The sequence determination described herein took advantage of a finding by Raibaud and Goldberg (7) that subtilisin cleaves the native enzyme into two reasonably homogeneous and complementary segments of molecular weight about 30,000 and 70,000. Further chemical cleavages of each of these segments at asparaginyl-glycine, aspartyl-proline, and methionyl bonds facilitated the completion of the sequence analysis. The general strategy followed for establishing the primary structure is presented herein; the experimental details will be published elsewhere.

chromatography (21). Large water-insoluble peptides were separated on columns of Sephadex and ion exchange Sephadex in the presence of urea or guanidine-HCI. Small peptides were separated by combinations of gel filtration, ion exchange chromatography, and

preparative paper electrophoresis. RESULTS The strategy of sequence determination is shown diagrammatically in Fig. 1. The top bar represents the 841 residues of rabbit muscle phosphorylase and identifies the major sites of

METHODS muscle phosphorylase b was prepared by the Crystalline rabbit method of Fischer and Krebs (8). [14C]8-[m-(m-Fluorosul-

Abbreviations: HA, hydroxylamine fragment; AC, acid-cleavage fragment; CB, cyanogen bromide fragment; L,, light segment; Hs, heavy segment. * Present address: Eisai Research Laboratories, Eisai Co., Ltd., Koishikawa 4, Bunkyo-ku, Tokyo, Japan. t On leave from INSERM, Paris, France.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

4762

Titani et al.

Biochemistry:

Proc. Natl. Acad. Sci. USA 74 (1977)

$Subtilisin 6 17 Ac

I Subtilisin 264 265 319

133

78 0 xx

x *

x

x

91. 119 147 176 99

x

224

452 483

0

x

-x

x

349

241

x *

632

0

. 0 603 :617

683

x xx

427: 440

xx

x x x

..

678 691 712 681 698

614

4763

763

x

765

841

799

SOURCES OF MAJOR FRAGMENTS

I Limited Proteolysis with Subtilisin

Ls

Hs

Trypsin

W/ W/

w

IR Cleovage at Met j Act CB 14

CB14C

iMI,

CB-12 CB 21 0

CB 11

X

-I El

CB 20

i CB 17

C89

C88

--Z I X

m

ml

CB5 CB16

--I 841

265

M

CB 17C

//*E

2

I

24

CB18

CB17N

m Cleavage of L, and H. at Asp- Pro

CB15

CB2

1

C813

AC-4 CB17C-CB18 (CB9,CB15, CB8, CB2)CB24N

j C824C (CB3, CB7, CB4,CBIO.C822,CBICB23)CBl3

Arg deavage

CB 24C (CB3,CB7,CB4,CaBO)CB22N

CC C CB22CCB -CB23N

HA-3

HA-l

HA-2

C823

AC-2

x x C IC

IV Cleavage of H. at Asn - Gly

CB1

CB3 CB4 CB22

AC-3

AC--i AC-5

C87 CB1O

I BEl////mI la0

I

IC,...

CB15C -CB8 (CB2, CB24,CB3)CB7N C87C-CB4-CBIO(CB22,CBICB23-CB13

CB17C (CB18,CB9)CB15N

FIG. 1. Summary of fragments generated for the sequence analysis of rabbit muscle phosphorylase. The top bar represents the whole molecule and the residues that are important for its fragmentation. Residue numbers above this bar identify the two sites of cleavage by subtilisin, the location of three Asp-Pro bonds (0), and the location of four Asn-Gly bonds (0). Residue numbers below this bar identify the 21 methionyl residues (x). The hatched section of each bar indicates the portion of the sequence determined by automated Edman degradation of the intact fragment. CB, fragments generated by cyanogen bromide cleavage; AC, fragments generated by acid cleavage; HA, fragments obtained by treatment with hydroxylamine.

cleavage. The lower bars represent the major fragments isolated for sequence determination. Two large segments, L, and H,, were isolated from a 17-hr digest of native phosphorylase b with soluble subtilisin BPN' under mild conditions (2.7 mg of subtilisin per g of substrate at 50, pH 8.5, in the presence of 10 mM 2-mercaptoethanol and 2 mM AMP). Their molecular weights (about 30,000 and 70,000) and amino acid compositions (not shown) were essentially identical to those of fragments II and I of Raibaud and Goldberg (7). Although sequenator analysis of the subtilisin digest indicated one major and several minor cleavage sites, the preponderant products were segments L,, representing approximately the amino-terminal third of the molecule, and segment H,, representing the carboxyl-terminal two-thirds. Segment L, started mainly with glycine-17 (within CB14) and H, mainly with amino-terminal alanine-265 (within CB17). Cleavage of segment L, with cyanogen bromide yielded four major fragments, CB5, CB11, CB12, and CB16, previously isolated by Saari and Fischer (5), and four new fragments, i.e., CB20, CB21, CB14C, and CB17N. The sequences of these peptides and of several smaller fragments produced by their further chemical and enzymatic cleavage were determined largely by automatic sequence analyses. These fragments were aligned (Fig. 1) with the aid of (a) two minor cyanogen bromide fragments which overlapped either CB21 and CB20 or CB1 1 and CB17N; (b) one large fragment (AC-1, residues 79-264), obtained by cleavage at an Asp-Pro bond; and (c) five methi-

onine-containing tryptic peptides (residues 94-138, 139-160,

170-184, 215-234, and 235-242). An analogous experiment was performed

on

[14C]8-

(m-fluorosulfonylbenzamido)benzylthio]adenine-labeled phosphorylase b in order to locate the tyrosine residue previously reported to be involved in the allosteric control of the enzyme (10). The radioactive enzyme (22.9 mg, 29.9 X 10P cpm/,gmol) was digested with subtilisin; the light segment was isolated and cleaved with cyanogen bromide. Most of the radioactivity was recovered in the light segment and specifically in CB21 (residues 148-176). During sequenator analysis of CB21 (using ethyl acetate as extractant), 62% of the radioactivity was recovered from cycle 8, 13% from cycle 9, and 3% from cycle 10, indicating that tyrosine-155 (the eighth residue in CB21) had been modified by affinity labeling of phosphorylase b. Segment Hs yielded eleven cyanogen bromide fragments previously isolated by Saari and Fischer (5) (CB1, CB2, CB3, CB4, CB7, CB8, CB9, CB1O, CB13, CB15, and CB18) and four new fragments, CB22, CB23, CB24, and CB17C. While the sequences of these fragments were readily determined, their alignment proved to be more difficult than in the case of segment Ls, and some of the overlaps need further corrobora[m-

tion.

Treatment of Hs with hydroxylamine yielded three large fragments, HA-I (residues 484-683), HA-2 (residues 265-452), and HA-3 (residues 684-841). Subsequent cleavage with cy-

Proc. Natl. Acad. Sci. USA 74 (1977)

Biochemistry: Titani et al.

4764

D

1

40

20

50

AC-S-R-P-L-S-D-Q-E-K-R-K-Q-I-S-V-R-G-L-A-G-V-E-N-V-T-E-L-K-K-D-F-D-R-H-L-H-F-T-L-V-K-N-R-N-V-A-T-P-R-D90

70

100

Y-Y-F-A-H-A-L-T-V-R-D-H-L-V-G-R-W-I-R-T-Q-Q-H-Y-Y-E-K-D-P-K-R-I-Y-Y-L-S-L-Q-F-Y-M-G-R-T-L-Q-N-T-M-V140

120

150

N-L-A-L-E-N-A-C-D-E-A-D-Y-Q-L-G-L-D-M-E-E-L-E-E-I-E-E-D-A-G-L-G-N-G-G-L-G-R-L-A-A-C-F-L-D-S-M-A-T-L(i)

G-L-A-A-

170

190

200

G-Y-G-I-R-Y-E-F-G-I-F-N-Q-K-I-C-G-G-W-Q-M-E-E-A-D-D-W-L-R-Y-G-N-P-W-E-K-A-R-P-E-F-T-L-P-V220

240

250

270

290

300

H-F-Y-G-R-V-E-H-T-S-Q-G-A-K-W-V-D-T-Q-V-V-L-A-M-P-Y-D-T-P-V-P-G-Y-R-N-N-V-V-N-T-M-R-L-W-S-A-K-A-P-N-

D-F-N-L-K-D-F-N-V-G-G-Y-I-Q-A-V-L-D-R-N-L-A-E-N-I-S-R-V-L-Y-P-N-D-N-F-F-E-G-K-E-L-R-L-K-Q-E-Y-F-V-V320

340

350

A-A-T-L-Q-D-I-R-R-F-K-S-S-K-F-G-C-R-D-P-V-R-T-N-F-D-A-F-P-D-K-V-A-I-Q-L-N-D-T-H-P-S-L-A-I-P-E-L-M-R370

390

400

V-L-V-D-L-E-R-L-D-W-D-K-A-W-E-V-T-V-K-T-C-A-Y-T-N-H-T-V-L-P-E-A-L-E-R-W-P-V-H-L-L-E-T-L-L-P-R-H-L-Q420

440

450

I-I-Y-E-I-N-Q-R-F-L-N-R-V-A-A-A-F-P-G-D-V-D-R-L-R-R-M-S-L-V-E-E-G-A-V-K-R-I-N-M-A-H-L-C-I-A-G-S-H-A470

490

500

V-N-G-V-A-R-I-H-S-E-I-L-K-K-T-I-F-K-D-F-Y-E-L-E-P-H-K-F-Q-N-K-T-N-G-I-T-P-R-R-W-L-V-L-C-N-P-G-L-A-E520

540

550

I-I -A-E-R- I-G-E-E-Y-I -S -D-L-D-Q-L-R-K-L-L-S-Y-V-D-D-E-A-F-I-R-D-V-A-K-V-K-Q-E-N-K-L-K-F-A-A-Y-L-E-R570 590 600

E-Y-K-V-H-I-N-P-N-S-L-F-D-V-Q-V-K-R-I-H-E-Y-K-R-Q-L-L-N-C-L-H-V-I-T-L-Y-N-R-I-K-K-E-P-N-K-F-V-V-P-R620

640

650

670

690 700 F-M-L-N-G-A-L-T-I-G-T-M-D-G-A-N-V-E-M-A-E-

T-V-M-I-G-G-K-A-A-P-G-Y-H-M-A-K-M-I-I-K-L-I-T-A-I-G-D-V-V-N-H-D-P-V-V-G-D-R-L-R-V-I-F-L-E-N-Y-R-V-SL-A-E-K-V-I-P-A-A-D-L-S-E-Q-I-S-T-A-G-T-E-A-S-G-T-G-N-M 720

740

750

770

790

800

820

840

E-A-G-E-E-N-F-F-I-F-G-M-R-V-E-D-V-D-R-L-D-Q-R-G-Y-N-A-Q-E-Y-Y-D-R-I-P-E-L-R-Q-I-I-e-Q-L-S-S-G-F-F-S-

P-K-Q-P-D-L-F-K-D-I-V-N-M-L-M-H-H-D-R-F-K-V-F-A-D-Y-E-E-Y-V-K-C-Q-E-R-V-S-A-L-Y-K-N-P-R-E-W-T-R-M-V-

I-R-N-I-A-T-S-G-K-F-S-S-D-R-T-I-A-Q-Y-A-R-E-I-W-G-V-E-P-S-R-Q-R-L-P-A-P-D-E-K-I-P FIG. 2. The amino acid sequence of rabbit muscle phosphorylase. One-letter amino acid abbreviations are used; A (alanine), C (cysteine), D (aspartic acid), E (glutamic acid), F (phenylalanine), G (glycine), H (histidine), I (isoleucine), K (lysine), L (leucine), M (methionine), N (asparagine), P (proline), Q (glutamine), R (arginine), S (serine), T (threonine), V (valine), W (tryptophan), andY (tyrosine). Overlaps of the cyanogen bromide fragments at residues 427441 and 614-618 are tentative (see the text). Circled symbols locate the site of phosphorylation by ATP (P), the site labeled by an analog of AMP (see text), and the site of binding of pyridoxal 5'-phosphate (PLP).

anogen bromide yielded CB17C, CB18, CB9, and CB15N from fragment HA-2; CB15C, CB8, CB2, CB24, CBS, and CB7N from fragment HA-1; and CB7C, CB4, CB10, CB22, CB1, CB23, and CB13 from fragment HA-S. Sequenator analysis indicated that fragment HA-3 started within CB7 at glycine684 and overlapped CB4 and CB10. Fragment CB13 was placed at the carboxyl-terminus, as already discussed by Saari and Fischer (5). In addition to the above cyanogen bromide fragments, another, which was isolated from HA-3 in low yield, overlapped CB23 and CB13. Analysis of HA-1 and HA-2 identified the cyanogen bromide fragments at their amino- and carboxyl-termini. In addition, a large peptide (residues 601-638) was isolated from a tryptic digest of succinylated HA-1 and sequenator analysis aligned CB15-CB8-CB2-CB24, but these results need confirmation. Cleavage of segment Hs at two Asp-Pro bonds yielded three major fragments, namely AC-2 (residues 633-841), AC-4 (residues 320-632), and AC-S (residues 265-319), and a minor fragment, AC-3 (residues 265-632) which overlapped AC-5 and AC-4 (see Fig. 1). Cyanogen bromide fragments were isolated from peptides AC-4 and AC-2, and identified as summarized in Fig. 1. Sequenator analysis of AC-4 suggested that CB17 was

followed by CB18. Two methionine-containing peptides (residues 649-713 and 739-769, as judged by compositions) were isolated from a tryptic digest of succinylated AC-2. One appeared to overlap CB24C, CB3, CB7, CB4, CB10, and CB22; the other placed CB1 between CB22 and CB23. These overlaps, however, are based on composition. The results obtained by the two chemical cleavages described above, and an overlap between CB24 and CBS already reported by Forrey et al. (22), yields the alignment illustrated in Fig. 1. However, the overlaps for CB9 and CB2 require confirmation. The complete amino acid sequence of the 841 residues is shown in Fig. 2. The amino acid composition calculated from the sequence is Asp5l, Asn45, Thrs5, Ser29, Glu64, Gln31, ProM, Gly48, Ala63, Cys9, Val62, Met2l, Ile4g, Leu79, Tyr36, Phe38, Trp12, Lys48, HiS22, and Arg63. Except for proline and tryptophan, this composition is in good agreement with that recalculated from the data of Sevilla and Fischer (23) on the basis of the molecular weight of 97,412 obtained here (including a phosphoryl group, an acetyl group, and pyridoxal 5'-phosphate). The molecular weight calculated from the present data is also in good agreement with the values of 92,500 for the

Biochemistry:

Titani et al.

Proc. Natl. Acad. Sc. USA 74 (1977)

4765

Table 1. Locations of functional sites in rabbit muscle phosphorylase

Residue Serine-14 Cysteine-108 Cysteine-142 Tyrosine-155 Lysine-679

Surrounding sequence -Gln-Ile-Ser-Val-Arg-Asn-Ala-Cys-Asp-Glu-Ala-Ala-Cys-Phe-Leu-Ala-Ala-Tyr-Gly-Tyr-Asn-Met-Lys-Phe-Met-

subunit obtained by ultrgcentrifugal analyses by Seery et al. (24, 25) and of 100,000 using sodium dodecyl sulfate gel electrophoresis (26).

DISCUSSION Despite recent progress in the determination of the amino acid sequence of proteins, the elucidation of the sequence of a protein as large as phosphorylase (subunit molecular weight of 97,000) is still extremely complex. The main difficulty proved to be the isolation of large homogeneous fragments of the molecule for subsequent degradation. As an initial step, Saari and Fischer (5) used cleavage with cyanogen bromide, one of the most reliable procedures for obtaining large fragments in high yield. However, they encountered difficulty in separating all of the 22 fragments expected from the composition, especially those of an intermediate size (30-60 residues). Following an observation by Nolan et al. (27) that phosphorylase appears to have very few regions susceptible to limited proteolysis, Raibaud and Goldberg (7) succeeded in cleaving the protein with subtilisin into two reasonably homogeneous and complementary segments. Additional cyanogen bromide fragments were isolated from these separated segments. Other large peptides were generated by tryptic digestion of succinylated fragments or by chemical cleavage of Asn-Gly or Asp-Pro bonds (12, 13). Once homogeneous fragments were available in appropriate quantity, the structural analysis proceeded rapidly. The amino acid sequence of the rabbit phosphorylase subunit thus obtained (Fig. 2) is complete except that the evidence for placing fragments CB9 and CB2 is based only on compositions. It appears very unlikely, however, that CB9 is located elsewhere, since this fragment was isolated from both AC-4 and HA-2, as shown in Fig. 1, and the linkage of CB17 to CB18 was established. Likewise, from other alignments it is most likely that CB2 is located between CB8 and CB24. The polypeptide chain of 841 residues begins with Nacetylserine and terminates with proline. It has already been reported (6) that the amino-terminus is blocked, as is the case in many other muscle proteins. Since the carboxyl-terminal proline could not be identified either by hydrolysis with carboxypeptidases A and B or by hyrazinolysis, it was once believed that the carboxyl-terminus was blocked as well (2). Lysyl-isoleucine was reported to be at the carboxyl-terminus of rat phosphorylase (24). The rabbit enzyme terminates with -LysIle-Pro. The locations of functional residues closely related to the enzymatic activity or regulation are listed in Table 1. Serine-14, the site phosphorylated in the conversion of phosphorylase b to a, immediately precedes one of the sites cleaved by subtilisin. Since subtilisin cleaves the b but not the a form of the enzyme, it seems likely that the amino-terminal region of the molecule is exposed in phosphorylase b but undergoes drastic conformational changes upon phosphorylation so as to become shielded against proteolysis. In fact, the recent x-ray crystallographic analyses of phosphorylases a and b (28, 29) showed a clear difference between the two forms of the enzyme in their

Reported function

Ref.

Phosphate binding (covalent control) Association and activity Association and activity AMP binding (allosteric control) Binding of pyridoxal 5'-phosphate

6, 27 30, 31 30, 31 9, 10 22

amino-terminal regions. This portion of the polypeptide chain is extended outwards in phosphorylase b but is folded in phosphorylase a so that serine-14 moves closer to the active site region. The second site of interest is tyrosine-155. The radioactivity of the AMP analog was incorporated into phosphorylase b (9, 10) and quantitatively recovered on tyrosine-155. This finding attests to the specificity of the affinity label towards the allosteric site of the enzyme. It is noteworthy too that both tyrosine-155 and the phosphorylated site are located near cysteine-142, which upon blocking with iodoacetate renders the enzyme inactive (30,31). Current x-ray analyses of the structure of the enzyme (28, 29) have identified these three functional sites within a fairly small area of the molecule, but studies now in progress promise to identify the substrate binding site and other relevant portions of the molecule. Among the functional residues listed in Table 1, only lysine-679, to which pyridoxal 5'-phosphate is bound, is remote from the others in the linear amino acid sequence. This cofactor, present in all eukaryotic and prokaryotic glycogen phosphorylases so far isolated, is indispensable for enzymatic activity (2) even though some of these enzymes lack covalent or allsteric control. The recent crystallographic analysis of Sygusch et al. (32) has placed pyridoxal 5'-phosphate in a location consistent with its direct participation in catalysis by the enzyme. Thus, detailed analysis of the primary and tertiary structure of this enzyme is providing the chemical framework for an understanding of its catalytic and regulatory functions. We thank Dr. D. J. Graves and coworkers for donation of [14C]8[m-(m-fluorosulfonylbenzamido)benzylthioladenine-labeled rabbit

muscle phosphorylase b, and Drs. J. C. Saari, M. A. Hermodson, and D. L. Enfield and Mrs. S. Balian for contributions to the initial phase of this work. We thank Drs. N. B. Madsen, R. J. Fletterick, and their colleagues for providing us with the results of their crystallographic analyses prior to publication. Thanks are also due to Mrs. R. M. McDonald, Mr. R. Olsgaard, and Mr. R. G. Granberg for excellent technical assistance throughout this work. This work has been supported by Grants GM 15731 and AM 7902 from the National Institutes of Health. J. H. is a recipient of a NATO Scholarship. 1. Green, A. A. & Cori, G. T. (1943) J. Biol. Chem. 143,21-29. 2. Fischer, E. H., Pocker, A. & Saari, J. C. (1970) Essays Biochem. 6,23-68. 3. Fischer, E. H., Heilmeyer, L. M. G., Jr. & Haschke, R. H. (1971) Curr. Top. Cell. Regul. 4,211-251. 4. Graves, D. J. & Wang, J. H. (1972) Enzymes 3rd Ed. 7, 435482. 5. Saari, J. C. & Fischer, E. H. (1973) Biochemistry 12, 52255232. 6. Titani, K., Cohen, P., Walsh, K. A. & Neurath, H. (1975) FEBS Lett. 55, 120-123. 7. Raibaud, O. & Goldberg, M. E. (1973) Biochemistry 12, 5154-5161. 8. Fischer, E. H. & Krebs, E. G. (1958) J. Biol. Chem. 231, 6571. 9. Anderson, R. A. & Graves, D. J. (1973) Biochemistry 12,

4766

Biochemistry: Titani et al.

1895-1900. 10. Anderson, R. A., Parrish, R. F. & Graves, D. J. (1973) Biochemistry 12, 1901-1906. 11. Crestfield, A. M., Moore, S. & Stein, W. H. (1963) J. Biol. Chem. 238,622-627. 12. Bornstein, P. (1970) Biochemistry 9,2408-2421. 13. Fraser, K. J., Poulsen, K. & Haber, E. (1972) Biochemistry 11, 4974-4977. 14. Omenn, G. S., Fontana, A. & Anfinsen, C. B. (1970) J. Biol. Chem. 245, 1895-1902. 15. Yaoi, Y., Titani, K. & Narita, K. (1964) J. Biochem. Jpn. 56, 222-229. 16. Houmard, J. & Drapeau, G. R. (1972) Proc. Nati. Acad. Sci. USA 69,3506-3509. 17. Edman, P. & Begg, G. (1967) Eur. J. Biochem. 1, 80-91. 18. Hermodson, M. A., Ericsson, L. H., Titani, K., Neurath, H. & Walsh, K. A. (1972) Biochemistry 11, 4493-4502. 19. Laursen, R. A., Bonner, A. G. & Horn, M. J. (1975) in Instrumentation in Amino Acid Sequence Analysis, ed. Perham, R. N. (Academic Press, New York), pp. 73-110. 20. Pisano, J. J. & Bronzert, T. J. (1969) J. Biol. Chem. 244, 55975607. 21. Bridgen, P. J., Cross, G. A. M. & Bridgen, J. (1976) Nature 263,

Proc. Nati. Acad. Sci. USA 74 (1977) 613-614. 22. Forrey, A. W., Sevilla, C. L., Saari, J. C. & Fischer, E. H. (1971) Biochemistry 10, 3132-3140. 23. Sevilla, C. L. & Fischer, E. H. (1969) Biochemistry 8, 21612171. 24. Seery, V. L., Fischer, E. H. & Teller, D. C. (1967) Biochemistry 6,3315-3327. 25. Seery, V. L., Fischer, E. H. & Teller, D. C. (1970) Biochemistry 9,3591-598. 26. Cohen, P., Duewer, T. & Fischer, E. H. (1971) Biochemistry 10, 2683-2694. 27. Nolan, C., Novoa, W. B., Krebs, E. G. & Fischer, E. H. (1964)

Biochemistry 3,542-551. 28. Fletterick, R. J., Sygusch, J. Semple, H. & Madsen, N. B. (1976) J. Biol. Chem. 251, 6142-6146. 29. Johnson, L. N., Madsen, N. B., Mosley, J. & Wilson, K. S. (1974) J. Mol. Biol. 90,703-717. 30. Batell, M. L., Zarkadas, C. G., Smillie, L. B. & Madsen, N. B. (1968) J. Biol. Chem. 243,6202-6209. 31. Avramovic-Zikic, O., Smillie, L. B. & Madsen, N. B. (1970) J. Biol. Chem. 245, 1558-1565. 32. Sygusch, J., Madsen, N. B., Kasvinsky, P. J. & Fletterick, R. J. (1977) Proc. Nati. Acad. Scd. USA, in press.