J. Moi Biol. (1976) 102, 349-365

Primary Structure of Bacteriophage T4 Internal Protein H and Characterization of the Cleavage upon Phage Maturation Tosnv_~K~ ISOBE ~, L[NDSAY W . BLACK 2 A N D A~rRA TSUGITA s

Biozcntrum der Universitdt Basel CH 4056 Basel, Switzerland (Received 15 September 1975) Internal proteins I*, II* amt [II*]', found within the bacteriophage T4 head complexed with the DNA, and bheir precursors I P I, I P I I and I P I I I , found in head-mutant defective infected cells, were purified and compared. The complete primary structures of I P I I and I P II* have been determined. Clustering of basic amino acid residues in I P I I somewhat resembles histone sequences and may be related to the ai~rity of I P I I for the assembly core and DNA. We show that I P II* (91 residues) is derived from I P I I (106 residues) by a specific morphogenetic cleavage between residues Glul5 and Alal6. Since in vivo or in vitro processing of the other head precursor proteins P23, P22, I P I and I P I I I , is associated with the formation of N-terminal alanine and C-terminal glutamic acid (Tsugita etal., 1975), it appears that T4 head protein cleavages are limited to Glu-Ala sequences and that one enzyme may be responsible for all these cleavages. The dipeptide sequence Glu-Ala is found in three positions in the I P I I structure, and in considering the three extended sequences we discuss structural features which may lead to recognition of the specific morphogenetic cleavage site in I P I I by the head assembly dependent protease.

l. Introduction I n an early stage of bacteriophage T4 head morphogenesis, an acidic protein directed by gone 22 (P22) and three internal proteins (IP I, I I and I I I t ) form an assembly core on the membrane of the host cell (Showe & Black, 1973). During head formation, P22 (molecular weight 30,000) undergoes fragmentation into small peptides (Laemmli, 1970) and one of the internal proteins, I P I I I (molecular weight 21,000), is also cleaved, yielding the smaller protein I P I I I * (molecular weight 18,000) found in the head. The other two internal proteins, I P I (molecular weight 10,000) and I P I I (molecular weight 10,000), also were found to be cleaved, resulting in a molecular weight reduction of about I 5 % in both cases (Tsugita etal., 1975). A major capsid protein (P23, molecular weight 58,000) (Hosoda & Cone, 1970; Laemmli, 1970; Kellenberger & Kellenberger-van der Kamp, 1970; Dickson etal., 1970) and a minor 1 Dept. of Chemistry, Faculty of Science, Tokyo Metropolitan University, Tokyo, Japan. 2 Present address : Dept. of Biological Chemistry, School of Medicine, University of Maryland, Baltimore, U.S.A. a On leave of absence from Laboratory of Molecular Genetics, Medical School, Osaka University, Osaka, Japan. t IP II*, P23" etc. refer to forms of proteins IP II, P23 etc. resulting from normal head assembly dependent protein cleavage. 349

350

T. I S O B E , L. W. B L A C K A N D A. T S U G I T A

protein (P24, molecular weight 48,000) of the m a t u r e d head are also known to undergo apparent molecular weight reductions; to 48,000 for P23" and to 47,000 for P24". Such cleavage reactions are not unique to T4 phage morphogenesis but are aIso observed in the assembly of other bacteriophages, such as A (Kaiser etal., 1974), as well as in m a n y animal viruses, such as polio (Maizel etal., 1967), Sindbis (Schlesinger & Schlesinger, 1973), vaccinia (Moss & Rosenblum, 1973) and adenovirus (Ishibashi & Maizel, 1974). The question arises whether protein cleavage is an essential regulatory mechanism in morphogenesis. While this is an open question, it is apparent t h a t the cleavages must provide for new protein configurations and protein-protein interactions through alterations in charge, hych.ophobicity and spatial configuration, resulting in great structural mobility in passing through intermediate complexes to a final complicated structure. The cleavage problem is of interest not only because of its apparently fundamental role in morphogenesis of complex structures, but also because it represents a complex series of protein interactions and structural constraints on proteins, the genetic basis of which is of interest and most readily approachable in the phage system. The uncleared and cleaved proteins I P I, I P I*, I P I I , I P I I * , I P I I I and I P I I I * were purified and compared on gels containing sodium dodecyl sulphate. Although I P I I is non-essential for phage head assembly (Black, 1974), we have chosen I P I I as a model protein to study the structural basis for cleavage for the following reasons : (1) I P I I is one of the smallest T4 proteins among those cleaved and (2) the cleavage of I P I I appears to share both the in rive requirements and the structural specificity of cleavage of the other T4 proteins which are cleaved. I n this paper we present details of the sequences around the cleavage site and other sequences containing the same dipeptide which are not cleaved in rive. Comparing the extended sequences around these three peptide bonds, we are able to discuss specific requirements for the cleavage reaction. 2. M a t e r i a l s a n d M e t h o d s

(a) Purification of internal proteins Cleaved internal proteins were isolated from T 4 D ~ particles purified by repeated differential centrifugation. The purification was aecordhlg to the procedure of Black & Ahmad-Zadeh (1971), except that internal proteins were released from the phage particles by freeze-thawing 4 times, and the proteins were isolated in purified form after the first sodium acetate gradient chromatography on carboxy-methylcellulose. Uncloaved internal proteins were purified from Escherichia coli B ~ infected T4.21 (amN90).23(amHll).e(amH26). With this triple mutant we have no cleavage (gone 21, amN90), no major head protein (gone 23, a m H l l ) and no lysozyme production (gone e, amH26), which prevents lysis (Showe & Black, 1973; Onorato & Showe, 1975). Uncleaved internal proteins could not be satisfactorily purified on carboxy-methylcellulose under conditions established for the cleaved internal proteins (Black & AhmadZadeh, 1971) oven in high salt, apparently duo to aggregation. Therefore, the following procedures were employed for the purffication of uncleaved internal proteins. Cells (300 g), collected late after infection and frozen, were suspended in 700 ml 0.05 M-Tris-HC1 (pH 7.0), 10 -a M-MgS04 containing 4-2 mg pancreatic DNAase, and homogenized in an Omnimixer. After addition of 1-5 ml chloroform, the cell suspension was frozen and thawed once, incubated for 20 min at 37~ and then centrifuged for 12 h at 36,000 revs/ rain at 4~ Solid (NH4)2SO4 was added to 50% saturation to the supernatant solution, and the precipitate was dialyzed against 0.05 M-Tris.HC1 (pH 7.0), 10- a M-MgSOa. The dialysate (240 ml) was ehromatographod on a 1-1 Sephadex G150 column equilibrated with the same buffer, from which 360 ml following the void volume wore pooled. The purification

S E Q U E N C E OF T4 I P I I AND I T S C L E A V A G E

351

to this stage is similar to t h a t described for the isolation of the aggregate I complex between P22 a n d the internal proteins (Showe & Black, 1973), b u t the internal proteins are n o t highly purified at this stage. To the Sephadex G150 fraction diluted to 500 ml with the same buffer, 40 ml fresh 2"5~/o rivanol (6,9-diamino-2-ethoxy-acridine lactate; Sigma) were added, a n d after 45 rain at 4~ the precipitate was removed b y centrifugation (20 rain at 10,000 rcvs/min). For the removal of rivanol (necessary for chromatography} a n d acidic proteins, the s u p e r n a t a n t solution, which is now highly enriched for the three internal proteins, was adjusted to p H 4.3 with acetic acid a n d loaded on an IRC-50 column (equilibrated with 0.05 ~-KPO4, p H 6.4) from which the internal proteins were eluted in 0.1 M-KPO4 (pH 6.4), 1 ~-KC1. The internal proteins freed of rivanol were concentrated b y a n addition of solid (NH4)2S04 to 80~/o saturation a n d t h e n dialyzed against 0.05 ~-KPO4 (pH 6.5). The dialysate was loaded on a n IRC column (1 cm • 8 cm) equilibrated with the same buffer, a n d the internal proteins were eluted b y a linear, 1-1 gradient between 0-05 ~-KPO4 (pH 6.5) and 0-05 M-KPO4 (pH 6-5) -~ 0.6 ~-KC1. The internal proteins were separately eluted from the column in the order, I P I, I P II, I P I I I . I n t e r n a l proteins were assayed during purification b y double diffusion on Ouchterlony plates using antibodies prepared against the separated, cleaved proteins. The purifications yield internal proteins which appear nearly homogeneous on sodium dodecyl sulphate a n d acetic acid-containing polyacrylamide gels (Fig. 1), except for I P I, which contains minor contaminants. The yield of I P I is also somewhat low in the rivanol precipitation step. The internal protein preparations also appear nearly homogeneous on isoelectric focusing gels, except for I P I (unpublished observations). Only I P I* is changed significantly in charge b y cleavage, going from a p I value of approx. 7 to approx. 9. I P II* a n d I P I I I * show only slight differences from the uncleaved species on focusing gels to more basic forms, b u t the cleaved and u n c l e a r e d proteins all appear to have isoelectric points near 10. (b) Amino acid analysis The analysis of protein hydrolysates was carried out with a Beckman amino acid analyser model 4255 at flow rates of 30 ml/h, according to Spackmann et al. (1958). Proteins (200 ~g) were hydrolysed at 108~ q- 1 deg. C in 1 ml twice distilled 5.7 ~r-HC1 for 24 h or 144 h in evacuated sealed tubes. Peptides (1 to 3 nmol) were hydrolysed under the same conditions, or in 6 N-HC1 containing 50% acetic acid for acid-resistant peptides. The flow rate for peptide analysis was 100 ml/h. The analyser was modified to the expanded scale (10 nmol for full scale detection). (c) Maleylation The sample (0"7 ~mol) was dissolved in 2-0 ml 0-2 •-NaP207 buffer (pI-I 9"0), to which was added 0.1 ml maleie anhydride/benzene solution (0.4 ml of which contained 0.1 mCi [1-14C]maleic anhydride (spec. act. 18 Ci/mol; CEA-France Co.) a n d 10 mg cold maleic anhydride}, 4 times at 10-min intervals, while stirring vigorously at 2~ After the final addition, the mixture was left for a further 30 m i n and was t h e n dried on a rotary evaporator (Butler et al., 1969). The maleylated sample was isolated b y passing it through a column (1.8 cm • 37 cm) of Sephadex G25 equilibrated with 0-025 ~-ammonia. The spec. act. of the modified protein was 2.1 • 104 cts/min per nmol of amino residue. The extent of the reaction was determined b y measuring the level of free lysine residues remaining after digestions with trypsin followed b y carboxypeptidase B. (d) Tryptic digestion and fractionation of the digest Purified I P I I (8 mg) in 5 ml l~/o ~H4HCO3 (pH adjusted to 7.0 with formic acid) was incubated with 0.02 mg T,-1-tosylamino-l-phenyl-ethyl-chloromethylketene-treated trypsin (2)< crystallized, W o r t h i n g t o n Biochem. Co.) at 37~ for 15 h. After the digestion, the p H of the digest was lowered to 3.0 b y the addition of formic acid a n d the digest was lyophilized. The lyophilized residue was dissolved in 2 ml of the first buffer (see legend to Fig. 2) and the p H was adjusted to 2.5 with formic acid. I t was applied to a column of Dowex

352

T. I S O B E , L. W. B L A C K A N D A. T S U G I T A

50X2 and an elution gradient of increasing p H and ionic strength was made with a 9chamber Varigrad apparatus. The compositions and the mode of elutions of which are given in the legend to Fig. 2. F o r the chromatographic profile, 0.1 ml (1/20) of eluate from each of 10 tubes was subjected to complete acid hydrolysis, and the hydrolysates were analysed with a 5-cm column of the amino acid analyser operated on the expanded scale. This procedure is much more sensitive t h a n the conventional manual ninhydrin m e t h o d and is also convenient for further sequential work, since it provides more information for the compositions (a total a m o u n t of neutral and acidic amino acids, a sum of pheiaylalanine and tyrosine residues, lysine, histidine and arginine) and for the homogeneities of the peptides. (e) Chymotryptic digestion and fractionation of the digest I P I I (2.5 mg) in 5 ml of l~/o p y r i d i n e / 1 % colhdine acetate buffer (pH 8.0} was digested with 50 ~g a-chymotrypsin (Worthington, Biochem.) at 37~ for 6 h. The digest, after the p H was lowered to 3 by the addition of formic acid, was lyophilized. The lyophilized residue was dissolved in 1 ml of t h e first buffer for Dowex 50 chromatography (see legend to Fig. 2), and the p H of the solution was adjusted to 2.5 with formic acid. I t was applied to a small column (0"9 cm • 7 cm) of SP-Sephadex C25 (pyridine form, Pharmacia) and eluted by a gradient of pyridine acetate buffer at a flow rate of 7 ml/h. The compositions of the buffers and the mode of elution were the same as those for the Dowex 50 chromatography described above, except 50 ml of buffers were used in each chamber instead of 200 ml. The elution profile was obtained by acid hydrolysis of 0" 1 ml (1/15) of eluate from each of 5 tubes, followed by analysis with a 5-cm column of the amino acid analyser. (f) Cyanogen bromide cleavage and fractionation of the fragments I P I I (8 rag) in 5 ml 70~/o formic acid was incubated with 1 ml 0.2% CNBr in 70~/o formic acid at room temperature for 24 h. The reaction m i x t u r e was then reduced in volume to 1 ml on a rotary evaporator and p ut on a column (1-8 e m • 90 cm) of Bio-Gel P10 (400 minus mesh), which was equilibrated with 20~o formic acid. El u t i o n was carried out with 20% formic acid at a flow rate of 3 ml/h, and each 1.25 ml of the eluate was collected. The analysis was made on a 5-cm column of the amino acid analyser as described above. (g) Sequence analysis of peptides F o r determination of N-terminal residues and sequences, dinitro-phenylation (Sanger & Tuppy, 1951) and E d m a n degradation-subtraction methods (Konigsberg & Hill, 1962) were employed. To proteins or peptides (1 nmol) dissolved in 0" 1 ml 4% NaHCO3, was added 0-2 ml ethanol containing 250 nmol [14C]dinitrofluorobenzene (17 Ci/mol; Amersham). After reaction for 3 h at 37~ the reaction mixture was extracted with ether. Then the residual water layer was acidified with HC1, extracted again with ether repeatedly, and dried under N2. T h e dried residue was hydrolysed with 0-5 ml 6 ~-HCt at 106~ for 16 h. The hydrolysate was extracted with ether and both water and ether layers were plated on silica gel thin-layer plates (Kieselgel F 254, Merck), together with adequate standard dinitrophenylated amino acids. F o r the ether layer, 2-dimensional chromatography was carried out. The first solvent was toluene/pyridine/2-ehlorethane/0.2 N-ammonia ( 100:30:60:60, by vol.); the second solvent was chloroform/benzyl alcohol/glacial acetic acid (70:70:3, by vol.). F o r the water layer, a solvent composed of N-propanol/34% ammonia (7:3, v/v) was used (Brenner et a/., 1961). The plates were exposed to K o d a k R P R 5 4 X - r a y film and the radioactive spots were identified by comparison with the standard, scraped off and assayed with a scintillation counter. F o r the E d m a n degradation, peptides (1 to 2 nmol/eycle) in 500 ~l 50% pyridine solution were reacted with 20 ~l phenylisothiocyanate at 50~ for 30 rain. Cyclization was carried out in 0-2 ml trifiuoroacetic acid for 1 h at room temperature. After r em o v al of phenylthioearbamyl derivatives, a portion of the residue was hydrolysed and analysed directly for amino acid composition. The derivatives were, if necessary, converted to

S E Q U E N C E OF T4 I P I I AND I T S CLEAVAGE

353

phenylthiohydantoin-treated amino acids and identified by thin-layer chromatography on doubly coated polyamide plates (Cheng Cin Trading Co., Taipei, Taiwan} according to the procedure of Summers e$ aL (1973}. Carboxypeptidase digestion and hydrazinolysis (Tsugita eta/., 1960} were carried out for C-terminal sequences. Peptides or proteins (1 nmol} in 0"5 ml 0.2 ~-sodium bicarbonate buffer (pH 8.0), containing 0-1 M-NaC1 were incubated with carboxypeptidases A and/or B (diisopropylfluophosphate-trcated, Worthington Biochem. Co.} in an enzyme to substrate molar ratio of 1/20 to 1/80 at 37~ overnight, unless otherwise noted. Peptides containing acidic amino acid residues near the C-terminus were digested in 0.2 ~-sodium acetate buffer (pH 6.0) containing 0.1 ~-NaC1. Digestion with carboxypeptidase Y (donated by Dr Hayashi, Kyoto University, Japan) in 0.1 M-pyridinium acetate (pH 5.5} was used for peptides containing glutamyl and/or prolyl residues near the C-termlnus. Digestion with acid carboxypeptidase from Penlcillium janthinellum (abbreviated as Cpase P, donated by Dr Ichishima, Tokyo Noko University, Tokyo I in 0.1 M-pyridine formate (pH 2.5) was used for peptides containing aspartyl and/or prolyl residues. In all cases, digestion was stopped by the addition of 1 drop of glacial acetic acid or boiling, and the analysis was made directly with the amino acid analyser. For hydrazinolysis, peptide (2 nmol) was dried completely under a high vacuum over concentrated H2S04 and P205, and then 0.5 ml anhydrous hydrazine (Pierce Chem. Co.I was added. The reaction was at 106~ • 1 deg. C for 8 to 10 h in a sealed test tube. Excess reagent was again removed over H2S04 and P205. The completely dried residue was applied to the amino acid analyser immediately after dissolving in the p H 2.2 sodium citrate sample buffer. (h) Further degradation o/laeptidc8 Peptides were subjected to further degradation when necessary. ~-Chymotrypsin (3 • crystallized, Worthington Biochem. Co.}, thcrmolysin (donated by Daiwa Kasei Inc. Japan) or Staphylococcus aureus protease (donated by Dr Drapeau, Montreal University, Canada) were used in an enzyme to substrate molar ratio of 1/50. The reaction was carried out at p H 7.5 to 8.0, in 0.1 M-pyridine acetate buffer containing 10 rn~-CaC12 or in 0.1 ~-N-H4HCOs. Dilute acid hydrolysis was used to cleave peptide bonds specifically at aspartyl residues (Schultz e$ al., 1962). Peptides in 0.01 N-HC1 (adjusted to p H 2.0 exactly) were sealed in evacuated tubes and kept at 106~ • 1 dog. C for 24 h. The partially degraded products from the above treatment were evaporated and subjected to further sequential processes, such as Edman degradation or carboxypeptidase digestion, or were fractionated by gel chromatography. Chromatography was on Bio-Gel P2, P4 or P6 (each 400 minus mesh) or on Sephadex GS0, according to the expected molecular size of the degraded peptide fragments. The eluant generally used was 1%

NH4HCOs. 3. R e s u l t s (a) Characterization of cleaved and uncleared internal proteins Comparison of T4 internal proteins purified from mature virus particles with those from m u t a n t infected cells in which head assembly a n d associated protein cleavages are blocked (see Materials and Methods, section (a)) shows t h a t all three internal proteins are altered upon incorporation into the head. On urea/polyacrylamide gels containing sodium dodeeyl sulphate or acetic acid all three packaged internal proteins have higher mobilities t h a n internal proteins purified from head m u t a n t defective infected cells, and therefore appear to be cleaved during head assembly (Fig. 1). The precursor-product relationship of I P I and I T I*, I P I I and I P I I * , and I T I I I and I P I I I * is established b y reaction of the uncleaved forms with antibodies prepared against I P I*, I P H * and I P I I I * , as well as b y amino acid and end-group analysis of both from Tsugita et al. (1975). Cleavage of all three internal proteins in rive appears ~

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FIG. 1. Comparison of cleaved and uneleaved internal proteins on polyacrylamide gels. Purified cleaved internal proteins from mature T4 phage particles, and uncleaved internal proteins from T4 head mutant defective infected bacteria (Materials and Methods) were compared on polyacrylamide slab gels. Sodium dodecyl sulphate/urea gradient discontinuous polyacrylamide gels were made according to the procedure of ]Dr C. Castillo (manuscript in preparation), with

SEQUENCE OF T4 I P I I AND ITS CLEAVAGE

365

to depend upon normal head assembly in the same way as does cleavage of P23, P22 and P24 (unpublished observations). I P I I and I P II* appeared adequately pure, by gel electrophoresis, to permit structural studies. The molecular weight of I P II* was previously estimated to be about 9000 from gel electrophoresis in the presence of sodium dodecyl sulphate and sedimentation equilibrium studies (Black & Ahmad-Zadeh, 1971), as can also be estimated from the urea]sodium dodecyl sulphate gradient gel (Fig. 1). I P I I is only slightly retarded relative to I P II* on the urea/sodium dodecyl sulphate gel, as compared to the cleavage-associated changes in I P I and I P I I I mobility (Fig. 1); the mobility difference between I P I I and I P II* is more marked on acetic acid/urea gels (Fig. 1, insert A). Although the sodium dodecy] sulphate gel analysis suggested t h a t only a short peptide is removed from I P I I by cleavage, subsequent work on the primary structures suggests that the mobility change on this is anomalous. This might be explained b y the removal of two lysine residues from I P I I upon cleavage, with reduced sodium dodecyl sulphate binding density offsetting the molecular weight decrease (IP I cleavage removes a similar fraction of the molecule and appreciably changes the charge, but no lysines are removed and the mobility change reflects the molecular weight change). (b) Amino acid compositions of I P I I and I P II* The amino acid compositions of these two proteins are listed in Table 1. For the cleaved protein I P II*, the number of alanine residues was set at 15.0 on the basis of the molecular weight of I P II* and the molar concentration of alanine. Values for other amino acids were calculated as molar ratios relative to alanine. Taking into account extrapolation to zero time of hydrolysis for acid-labile amino acid residues, such as serine, and values from 144 hours hydrolysates for acid-resistant residues (valine and isoleueine) and average values for the rest of the residues, the predicted number of residues for each amino acid is obtained and agrees with the values given by Black & Ahmad-Zadeh (1971). The molecular weight was recalculated from this composition to be from 9950 to 10,210. The composition of uncleared I P I I was determined, assuming t h a t no amino acid residues in I P I I could be less than those in I P II*. For this to be the case, the alanine residues must be increased to 17.0. The rest of the amino acids were calculated on the basis of this number of alanine residues. This led to predicted residue numbers for all amino acids as listed in Table 1, and a minimum possible molecular weight of I P I I of 12,010 to 12,500. The eighth column in Table 1 indicates the differences in amino acid residues between the uncleared (IP II) and cleaved (IP II*) proteins, which was useful as a guide to sequencing of the cleaved region. Tryptophan and half cystine residues were absent as determined by spectrophotometric measurement (Goodwin & Morton, 1946) or b y the analysis after performic acid oxidation (Hirs, 1956), respectively.

gel buffers and sample preparation according to Laemmli (1970), and 25% acetic acid, 5 M-urea gels (insert A) were made according to the procedure of Takayama e~ al. (1966). The gels were stained for protein in 0.15% Coomassie blue in methanol]acetic acid]water (6:1:6, by vol.) at 37~ for 90 min, and were diffusion destained in 5% acetic acid, 20% methanol. Samples: a,!P I; b, IP I*; c, IP I -t- IP I*; d, IP If; e, IP II*; f, IP II -t- UP II*; g, IP III; h, IP III*;i, IP III ~- IPIII*; j, IP I -~ I P I I ~ IP III; k, IP I* -t- IPII* ~ IP III*; 1, T4_D phage particles./nsert A: m, IP II*; n, IP II; o, IP II ~ IP II*. The arrow indicates the origin of electrophoresis.

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(c) Terminal analysis of I P I I and I P II* The N-terminals of IP II and IP II* were analysed by Sanger's dinitrophenylation method. From IP II, no dinitrophenylated amino acid was detected, while alanine was identified as the N-terminal residue for IP II* in a reasonable recovery (80~/0). Edman degradation did not result in any N-terminal amino acid for IP II, while alanine was confirmed by this degradation as the N-terminal residue of 1~ II*. The C-terminal residues of these proteins were determined by treatment with carboxypeptidase B and carboxypeptidase A plus B. Nearly one mol of lysine per mol of protein was recovered from either IP II or IP II*. Hydrazinolysis failed to yield any C-terminal residue. This is consistent with C-terminal lysine, since the hydrazide derivatives of non C-terminal amino acids interfere with the analysis of basic amino acids. The end-group determinations, together with the differences in amino acid composition of IP II and IP II*, indicate that the peptide bond cleavage is located in the N-terminal portion of the uncleared protein, IP II. (d) Sequence of the uncleared protein, I P I I The tryptic digest of 1-P II was first fractionated on a Dowex 50 column (Fig. 2). The homogeneity of each peptide peak was estimated by determining amino acid compositions of samples from tubes on both sidewof individual peaks. Inhomogeneous peptides were subjected to further purification step(s) by gel chromatography (BioGel P2, P4, P6 or P10). As a result, 22 pure peptida fragments, including eight overlapping fragments, were isolated. Sequential analysis was carried out for each peptide. The results are shown in Figure 4(a) and (b). To order these peptide fragments, IP II was fragmented by two other methods, cyanogen bromide cleavage and chymotryptic digestion. By the cyanogen bromide cleavage, two peptide fragments Lys7 to l~Iet87, (B t 7-87) and Asn90 to Lysl06 (B 90-106) were obtained by gel chromatography on Bio-Gel P10, while an expected hexapeptide from the N-terminal portion B 1-6 and a dipeptide B 88-89 were not recovered. After the amino acid composition and terminal amino acid analysis of B 7-87 were determined, it was subjected to [14C]maleylation. The maleylated peptide was digested with trypsin, since only three arginine peptide bonds (17, 42 and 55) were known to be significantly split by trypsin digestion of the whole protein (Fig. 3). The digest was fractionated on Bio-Gel P6 and rechromatographed on Bio-Gel P10 or Sephadex G75, according to the sizes of expected peptides. As expected, four peptides were isolated, i.e. Lys7 to Argl7 (BT 7-17), Vall8 to Arg42 (BT 18-42), Lys43 to Arg55 (BT 43-55) and Ile56 to Met87 (BT 56-87). The N to C-terminal order of BT 18-42 and BT 43-55 was determined from the tryptie peptide T 31-43 as well as from the chymotryptic peptide C 35-46. The details of the sequence of the N-terminal region, Glnl to Argl7 are presented in Figure 4(a). This region was believed to cover the cleavage site from the differential composition of the two proteins (Table 1). The peptide T 8-12 was purified on Bio-Gel P2, while T 1-5, T 1-7 and T 13-17 were purified on Bio-Gel 1)4. From the compositions and partial sequences, T 1-5 and T 1-7 seemed to overlap. This overlapping was confirmed by a chymotryptic peptide C 5-9. T 8-12 was possibly derived by chymotryptic activity B peptide, peptide resulting from CNBr cleavage of protein; T and C peptides, peptides resulting from digestion with trypsin and chymotrypsin, respectively; BT peptide, peptide resulting from digestionwith trypsin of maleylated CNBr peptide.

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Fzo. 2. Elution profile of the tryptic peptides of I P I I on Dowex 50X2. The tryptic digest from 8 m g of I P I I was applied to a Dowex 50X2 column (0.9 cm • 47 cm). Elution was performed with a gradient of pyridlne acetate buffers established with a 9-chamber Varigrad apparatus. The buffers were m a d e as follows (Canfield, 1963). 1st buffer, 0.1% (pH 3.8); 2nd buffer, 0.25% (pH 3.97); 3rd buffer, 0.75% (pH 4.25); 4th buffer, 1.5% (pH 4.47); 5th buffer 2.5% (pH 4.65); 6th buffer 5.0% (pH 4.87); 7th buffer, 7.5% (pH 4.96); 8th buffer, 2 0 % (pH 5.18); 9th buffer, 30% (pH 6.0). The first 2 chambers each contained 200 ml of t h e 1st buffer a n d t h e remaining chambers contained 200 ml of t h e 2nd to 8th buffers. The elution was first carried out a t 37~ with a flow rate of 20 ml/h a n d 1.5-ml fractions were collected. The final elution was a gradient between 150 ml each of the 8th a n d 9th buffers a t 50~ Other details of the c h r o m a t o g r a p h y are given in the text. The eluted position of each peptide is indicated b y a n arrow. - - ~ - - @ ~ , l~eutral a n d acidic amino acids; - - • 2 1 5 lysine, a n d - - O - - O - - , arginine residues. The llne (. . . . . ) indicates the expected height of t h e peak from a peptide t h a t contains one lysine or one arginine residue, showing t h e approximate yield of a tryptie peptide which possesses 1 tool of lysine or arginine.

in the trypsin preparation used. These peptides were aligned by using the tryptic peptide BT 7-17, derived from the cyanogen bromide peptide B 7-87. Edman degradation was carried out on the peptide BT 7-17 after preincubation for three days in 0.2 M-pyridinium acetate buffer (pit 3"5) at 50~ to remove the maleic acid residues. Without such a preincubation, the Edman reagent did not react with it, indicating that the ~-amino group of the terminal lysine had been blocked. From the above results, the sequence of peptide fragment 7-17 was established as Lys-Thr-Tyr-GlnGlu-Phe-Ile-Ala-Glu-Ala-Arg. This sequence, together with the sequence of T 1-7, the specificity of cyanogen bromide cleavage and the chymotryptic peptide C 5-9, provided the sequence of the amino terminal region of IP II 1-17. The amino terminus of the uncleared protein IP II seemed to be blocked, because dinitrophenylation and Edman degradation did not work. The N-terminals of both peptides T 1-5 and T 1-7 were also found to be blocked by the above tests. These observations led to the

r

~o

9"4

"~

~o~, ,

~.~

N

~.~

|

T. I S O B E , L. W. B L A C K A N D A. T S U G I T A

360

/

Jo)

I0 15 I 25 ZO 29 G i n - P r o - A I o - Leu - L y s - M e t - Lys - T h r - T y r - G I n - G l u - P h e - I l e - A l o - G l u - A I o - A r g o V o I - G l y - A l o - G l y - L y s - / e u - G l u - A l o - A l o - V o I - A s n - L y s I

5

T I-5

T 8-]2

T 13-17

r 18-29

T I-7

T 18-28 i

i

C 5"9

i

, J

C 10"12

C f3"17

C I R 12 8

C 24 "~8" B7-87

9 T 7-17

OT 19-42 mTI6-17

85 (b)

90

- Phe - Gly-Met-Ser-

Mel-Asn

95 -Asp-

Leu-GIn-GIn-Ile-

BTS 18-24

I00 Glu-Alo-Ala-Alo-Alo-Lys-Alo-Ile-

T 95-101

106 Lys-OH

T 102-106 ,

T Th 85 - 93

105 Lys-Asp-

TTh 9 5 - 9 7

TTh98 - I01

i

T 102 - 104 v--

C 86-106 B 7-87

9 90 - 106

BC 8 6 - 8 7

9Th 103 -106

Fro. 4. Am;no acid sequences around the dipeptides Glu-Ala, between (a) residues 1 and 29 and (b} residues 85 and 106. An arrow ( ~ ) indicates the cleavage site. Determined sequences are shown with solid rectangles, inferred sequences are shown with hatched rectangles. The parts of sequences shown with open rectangles have not yet been established. The peptide numbers are written below the rectangles. The abbreviations used are as follows: T, tryptic peptide; TTh thermolytic peptide of T; C, ehymotryptic peptide; B, peptido isolated with cyanogen bromide cleavage; BTh, thermolytic peptide of B; BC, chymotryptie peptide of B; BT, tryptic peptide of the maleylated derivative of cyanogen bromide peptide; BTS, peptide isolated with S. aureus protease digestion of BT; *mT, tryptic peptide of the maleylated derivative of IP II*. The numbers after these abbreviations indicate from which residue they start and where they end. ( . ) The sequence proved by Edman degradation; ( ~ ) and (,) indicate the sequence proved by carboxypeptidase Y and A + B digestion, respectively. (. .) The sequence proved by dilute acid hydrolysis. (-) C-terminal amino acid r~idue proved by hydrazinolysis. Details of the methods are given in the text. idea t h a t the N - t e r m i n u s of I P I I was blocked by pyrolidonation of the g l u t a m y l residue or w i t h an additional u n k n o w n acyl group. This problem is still under investigation.

(e) Amino terminal sequence of the cleaved protein, I P I1" F r o m the difference in amino acid c o m p o s i t i o n b e t w e e n I P I I and I P I I * and the terminal analysis, it appeared t h a t the in vivo cleavage t o o k place in the a m i n o terminal region of the u n c l e a r e d protein, near to the 18th amino acid from the Nterminus. K n o w i n g the sequence o f ] P I I and the N - t e r m i n a l residue of I P I I * to be alanine, one m a y predict t h a t the cleavage site is A l a l 4 , A l a l 6 or (unlikely) Ala20 in I P I I . Therefore, the following e x p e r i m e n t was undertaken. The protein I P I I * w a s m a l e y l a t e d and digested w i t h trypsin. One m a y e x p e c t to isolate either Ala(14)Glu-Ala-Arg(17) or Ala(16)-Arg(17), besides the peptides 18-42, 43-55, 56-106, and possibly 20-42. I P I I * (1.5 mg) was m a l e y l a t e d and digested w i t h trypsin as was I P II. The digest

SEQUENCE OF T4 I P I I AND ITS CLEAVAGE

361

was chromatographed on Bio-Gel P6, and a peptide having equimolar amounts of both alanine and arginine was found in the elution position expected for a dipeptide, besides three larger peptides. The sequence of the new dipeptide was determined by E d m a n degradation (Fig. 4(a)). The results given above now show that the sequence Ala-Arg is at the N-terminal end of the cleaved protein I P II* and that the cleavage takes place in vivo at the peptide bond between Glul5 and Alal6 in the uncleared protein, I P II. (f) Other Glu-Ala sequences in I P I I Since the in vivo cleavage was found to occur at the bond Glul5, we looked for an identical dipeptide sequence in other parts of the protein molecule. Two other parts were found to be identical: one is located near the cleavage site between residues 24 and 25 and the other is near the C-terminus between residues 96 and 97. The details of the sequencing work around these residues are summarized in Figure 4(a) and (b), respectively. The tryptic peptides T 18-29 and T 18-28 were fractionated by Dowex 50 and purified by Bio-Gel P6 and P4 gel chromatography, respectively. The analysis of T 18-29 showed the sequence to be Val(18)-Gly-Ala-(Gly,Lys)Leu-Glu,(Ala,Ala,Val)Asn-Lys(29). The peptide T 18-28 was considered to be derived from T 18-29 by splitting the bond Asn28-Lys29 with chymotryptic activity in the trypsin preparation used. The sequence 24-28 was determined by the chymotryptic peptide C 24-28, which was purified by SP-Sephadex followed by Bio-Gel P6 chromatography, where the N-terminal glutamic acid was confirmed by leucine amino peptidase digestion as well as identification of the corresponding phenylthiohydantoin amino acid. The sequence Val-Gly-Ala-Gly, which was found by Edman degradation of BT 18-42, showed t h a t this peptide contains T 18-29, T 18-28 and C 24-28 in its N-terminus. An N-terminal peptide BT-S 18-24 was isolated from the digest of BT 18-42 with Staphylococcus aureus protease (which is known to be specific for glutamyl bonds; Howard & Drapeau, 1972) with Bio-Gel P4 chromatography (Fig. 4(a)). From the analysis of this peptide, the sequence 18-29 was concluded to be Val(18)-Gly-AlaGly-Lys-Leu-Glu-Ala-Ala-Val-Asn-Lys(29). The peptide T 85-101 was obtained by Dowex 50 chromatography of tryptic digests of I P I I and further purified on Bio-Gel P6. The peptide is characteristic for the high content of glutamyl and alanine residues. After the preliminary sequential work, the peptide was subjected to digestion with thermolysin for further sequencing. Three peptide fragments, Th 85-93, Th 95-97 and Th 98-101, were obtained by Bio-Gel P4 chromatography. The sequential works on these fragments clear up the sequence of original peptide T 85-101. The peptide T 102-106 was isolated by Dowex 50 chromatography followed by Bio-Gel P6. The overlapping peptide B 90-106 for T 85-101 and T 102-106 was obtained by cyanogen bromide cleavage of I P II. The carboxyl terminus of B 90-106 was found not to be homocystein nor its lactone but a lysine (and only lysine) residue b y carboxypeptidase A and B digestion. Since liberation of one tool of lysine residue (but no other amino acid) was observed in the same enzyme digestion of the protein I P II, the peptide B 90-106 was concluded to be derived from the carboxyl end of the protein. Thus from the above consideration, together with the sequential works shown in the Figure, we may conclude that the carboxyl terminal sequence of I P I I 24

362

T. I S O B E ,

L . W . B L A C K A N D A. T S U G I T A

is Phe(85)-Gly-lY[et-Ser-Met-Asn-Asp-Leu-Gln-Gln-Ile-Glu-Ala- Ala- Ala-Ala-Lys-AlaIle-Lys-Asp-Lys. The aspartie acid residue second from the carboxyl terminus was not susceptible for carboxypeptidase digestion in the cases of peptides and proteins tested here, probably because of fl-carboxyl group inhibits the action. The above sequences involve the Glu-Ala bond clearly, as in the case of the cleavage site. The entire sequence of internal protein II including the cleavage site has been determined. Of 12 aspartyl residues, five residues are found to be in the amide form. Five glutamine and six glutamic acid residues are identified, while an additional glutamyl residue is located at the N-terminus in a blocked form. The amino acid composition calculated from the sequential study is listed in Table 1 (ninth column). The number of amino acid residues in IP II is 106, and 15 of the residues are removed by cleavage. The molecular weights of IP II and IP II* are 11,730 and 9970, respectively. Details of the remainder of the sequence will be described elsewhere (Isobe & Tsugita, manuscript in preparation).

4. D i s c u s s i o n

Internal protein II is the second phage T4 protein following lysozyme to be sequenced (Tsugita & Inouye, 1968). Internal protein II is a non-essential protein with no known enzymatic activity; it binds to the acidic P22 protein in assembly core formation (Showe & Black, 1973), and also binds to DNA, apparently nonspecifically and weakly (fully dissociated above 0.15 M-NaC1) (Black, unpublished results). Notable features of the IP H sequence include clusters of basic amino acids (e.g. Lys(40)-Asp-Arg-Lys-Lys-Lys(45)) which are not common but resemble those found in histones (De Lange et al., 1969), although IP I I has a lower basic amino acid content and weaker DNA binding than the histones. Also found in the sequence are clusters of polar amino acids (e.g. Asp(52)-Arg-Glu-Arg(55)). The basic amino acid contents of IP I I and T4 lysozyme are similar, although only the former has affinity for DNA. This difference may be associated with the presence of clustered basic amino acids in IP I I but not in lysozyme. From the determined primary sequence, five a-helical (2-18, 23-37, 54-59, 69-78 and 92-106) and one fl-sheet (46-51) conformation were predicted in the IP II molecule based on the method of Chou & Fasman (1974). Strong a-helix breakers such as proline and glycine residues would then be localized in the central hydrophilic and non-helical portion, but the tertiary structure of the protein remains to be studied. General structural features of IP II and IP II* do not clearly differ. Neither the basicity (one positive charge created when the blocked N-terminal amino acid is removed and a free a-NI-I2 group is produced by cleavage), nor the hydrophobicity (42.5% --> 41.8%) of the molecule are appreciably altered by cleavage. Antigenicity of IP II and IP II* are also very similar, which suggests that no major structural transformation occurs upon cleavage. However, the predictive analysis of Chou & Fasman (1974) suggested that the N-terminal region of the uncleared protein IP II would be in ~-helica] conformation (2-18), while the cleaved protein IP II* would lose this a-helix, resulting in a random coil conformation in its N-terminus (16-18). We have clearly demonstrated that the primary structure of IP II is altered during the process of normal head morphogenesis. Cleavage occurs at one peptide bond

SEQUENCE

O F T 4 I P II AND I T S

CLEAVAGE

363

located near the N-terminus, Glul5-Alal0. The extended sequence on the N-terminal side of the cleavage site Phe(12)-Ile-Ala(14) is very rich in hydrophobic amino acids. Head maturation protease binding at this site may be correlated with the inhibitory effect of relatively non-polar organic solvents such as chloroform or n-butanol on the in vitro cleavage reaction (Tsugita et al., 1975). The products of genes 23, 22, 24, I P I, I P I1, I P I I I , and B1 undergo cleavage during head maturation (Laemmli, 1970; Hosoda & Cone, 1970; Kellenberger & Kellenberger-van dcr Kamp, 1970; Dickson et al., 1970; Coppo et al., 1973; Tsugita et al., 1975). Terminal analyses and amino acid compositions of the precursor and matured forms of P23, IP III, IP II IP I indicate the loss of N-terminal portions and generation of N-terminal alanine in all these proteins (Tsugita et al., 1975). The in vitro digestion of the purified product of gene 22 with the 23ts aberrant head protease suggested appearance of N-terminal alanine and C-terminal glutamic acid accompanied proteolysis (Tsugita et al., 1975). These results suggest that cleavage of all the head precursor proteins occurs at Glu-Ala peptidc bonds, and that only one enzyme is responsible for all of the head assembly dependent cleavages. A candidate for this enzyme is the product of gene 21 (Onorato & Showe, 1975). A protease with specificity for Glu Ala peptide bonds has not been reported until now, although enzymes were reported with cleavage specificity for Glu X or Asp---X, where X can be almost any amino acid (Garg & Virupaksha, 1970; Howard & Drapeau, 1972). The cleavage fragment from the N-terminus of IP II has not been found, although it should be trichloroacetic acidsoluble and present in relatively large amounts (Eddleman & Champe, 1966). Although the in vitro cleavage experiments on P22 suggest no exopeptidase activity, it would appear that the cleaved peptide from IP II may undergo further degradation in the cell. Our finding that assembly dependent head precursor protein cleavages are apparently limited to Glu-Ala sequences suggests an unusual degree of primary sequence specificity, but the extent of this specificity is not yet determined. The dipeptide Glu-Ala is commonly found in proteins. In fact, it occurs three times in IP II in the following extended sequences: 9

(1) Tyr-Gin-Glu-Phe-Ile-Ala

15

16

I

22

-Glu-Ala -Arg-Val-Gly-Ala-Gly-Lys I

18

2~:

25

]

31

(2) Val-Gly-Ala-Gly-Lys-Leu -Glu-Alal-Ala-Val-Asn-Lys-Lys-Ala I 90

96

97

I

103

(3) Asn-Asp-Leu-Gln-Gln-Ile -Glu-Ala -Ala-Ala-Ala-Lys-Ala-Ile I

What structural features distinguish the first Glul5-Alal6 sequence which is cleaved from the two that arc not? Although the first is most N-terminal and the cleavage sites in IP I, IP II, IP I I I and P23 are all located towards the N-terminus, it appears extremely unlikely that the free a-amino group participates in selection of the specific site for the morphogenetic protease, because (1) in IP II the a-NH2 group is blocked; (2) the uncleared Glu24-Ala25 would be proximal to a free a-NH2 group following cleavage of the first site; and (3) in P23 the cleavage site is at lea~t 100 residues from the N-terminus.

364

T. ISOBE, L. W. B L A C K

AND

A. T S U G I T A

In our sequencing work we noticed that in the chymotryptic digestion of IP II, the peptides Ilel3 to Argl7 and Vail8 to Asn28 were isolated, while the expected peptides IleI3 to Leu23 or Ilel3 to Ash28 were not (see Fig. 4). This m a y indicate that Arg17-Val18, which is near the morphogenetic cleavage site,is highly susceptible to protease activity because it is exposed on the surface of the protein molecule. In contrast, Lys22 and Leu23 which are near one uncleared Glu24oAla25 sequence, were hardly accessible to trypsin and chymotrypsin, respectively, and in both cases peptides Vail8 to Ash28 were isolated. Leu92 also seemed to be resistant to chymotrypsin and thermolysin digestion, since chymotryptie digestion of IP II yielded the p~ptide Gly86 to Lysl06 instead of Gly86 to Leu92 and Gin93 to Lysl06 and, since thermolytie digestion of Phe85 to Lysl01 yielded Phe85 to Gin93 instead of the expected peptide Phe85 to Asp91. However, the argument is not unequivocal, because thermolytie peptide Ile95 to Ala97 was isolated. Nevertheless, taken together, these results suggest the presence of some structural rigidity around Glu24-Ala25 and Glu96-Ala97, which remain uncleared in vive. Further studies by in vitro digestion of denatured ver.susnative IP II with the protease which is responsible for specific cleavage would give a more precise answer to this question. These works are in progress in our laboratory. Alternatively, considering the extended sequences around the three Glu-Ala dipeptides in IP II, the amino acids around the cleavage site appear more hydrophobic, particularly those (Phel2-11el3-Alal4) on the N-terminal side of the Glul5-Alal6 bond which is cleaved. Recognition of such an extended primary sequence could be the basis for limiting cleavage to only certain specific Glu-Ala sequences. Whether or not such extended primary sequence recognition, or a combination of primary (Glu-Ala sequence) and tertiary structure is the basis of the specificity of the head assembly related morphogenetie cleavages awaits the sequencing of cleavage sites in other head proteins. It appears more likely that specificity of the latter type determines head precursor protein morphogenetic cleavage. This is believed to be the basis for m a n y biologically important cleavages, such as apoenzyme activation (Wright r al., 1968), cleavage of peptide hormone precursors (Tager et al., 1973), blood clotting (Magnusson, 1971) and collagen maturation (Martin et al., 1975). Viral morphogenetic cleavages which accompany protein eonformational changes during structure formation would appear to be closely related to these other biological phenomena. However, morphogenetic protein cleavages which accompany bacteriophage and animal virus maturation are only now being investigated, and it is of interest to determine the generality of the proteolytie mechanisms which produce them and their interrelationship with the eonformational changes that occur during assembly. W e are very grateful for donations of enzymes: earboxypeptidase from Penicillium by Dr E. Ichishima (Tokyo Noko University), carboxypeptidase from yeast by Dr l~. Hayashi (Kyoto University) and staphylococcal protease by Dr R. Drapeau (University of Montreal). Also, we would like to express our appreciation to Drs E. Kellenberger and M. K. Showe for discussions and for arranging for the visit of one of us (L.W.B.) to Basel. W e thank M. Cakil for his technical assistance. Part of this work was supported by the Swiss National Foundation for Scientific Research (grant 3.189.73). One of us (L.W.B.) was supported by the Roche Research Foundation for ScientificExchange and Biomedical Collaboration with Switzerland and by a grant from the National Institutes of Health, U.S.A., Division of Allergy and Infectious Diseases.

S E Q U E N C E O F T4 I P I I A N D I T S C L E A V A G E

365

I~EFERENCES Black, L. W. {1974}. Virology, 60, 166-179. Black, L. W. & A h m ~ l - Z a d e h , C. {1971}. J. Mol. Biol. 5"/, 71-92. Brenner, M., Niederwieser, A. & Pataki, G. (1961). Experientia, 17, 145-153. Butler, P. J. G., Harris, J. I., H a r t l e y , B. S. & Leberman, R. (1969). Biochem. J. 112, 679-689. Canfield, 1~. E. {1963). J. Biol. Chem. 238, 2691-2697. Chou, P. Y. & Fasman, G. D. (1974}. Biochemistry, 13, 222-245. Coppo, A., Manzi, A., Pulitzer, J. F. & Takahashi, H. {1973}. J. Mol. Biol. 76, 61-87. DeLange, R. J., F a m b r o u g h , D. H., Smith, E. L. & Bonner, J. (1969}. J. Biol. Chem. 244, 319-334. Dickson, R. C., Barnes, S. L. & Eiserling, F. A. (1970}. J. Mol. Biol. 53, 461-473. Eddleman, H. L. & Champe, S. P. (1966}. Virology, 30, 471-481. Garg, G. K. & Virupaksha, T. K. {1970}. Eur. J. Biochem. 17, 13-18. Goodwin, T. W. & Morton, R. A. (1946}. Biochem. J. 40, 628-632. Hirs, C. H. W. {1956). J. Biol. Chem. 219, 611-621. Hosoda, J. & Cone, R. (1970). Prec. Nat. Acad. Sci., U.S.A. 66, 1275-1281. Howard, J. & Drapeau, G. R. (1972). Prec. Nat. Acad. Sci., U.S.A. 69, 3506-3509. Ishibashi, M. & Maizel, J. V. J r (1974). Virology, 57, 409-424. Kaiser, A. D., Syvanen, M. & Masuda, T. (1974). J. Supramol. Struct. 2, 318-328. Kellenberger, E. & Kellenberger-van der K a m p , C. (1970). _~EBS Letters, 8, 140-144. Konigsberg, W. & Hill, R. J. (1962). J. Biol Chem. 237, 2547-2561. Laemmli, U. K. (1970). Nature (London), 227, 680-685. Magnusson, S. (1971). I n The Enzyme (Boyer, P. D., ed.), 3rd edit., pp. 277-321, Academic Press, New York. Maizel, J. V. Jr, Phillips, B. A. & Summers, D. F. (1967). Virology, 32, 692-699. Martin, G. R., Byers, P. H. & Piez, K. A. (1975). Advan. Enzymol. 42, 167-191. Moss, B. & Rosenblum, E. N. (1973). J. Mol. Biol. 81, 267-269. Onorato, L. & Showe, M. K. (1975). J. Mol. Biol. 92, 395-412. Sanger, F. & Tuppy, H. (1951). Biochem. J. 49, 463-481. Schlesinger, M. J. & Schlesinger, S. (1973). J. Virol. 11, 1013-1016. Schultz, J., Allison, H. & Grice, M. (1962). Biochemistry, 1, 694-698. Showe, M. K. & Black, L. W. (1973). Nature New Biol. 242, 70-75. Spackmam~, D. H., Stein, W. H. & Moore, S. (1958). Anal. Chem. 3D, 1190-1206. Summers, M. R., Smythers, G. W. & Oroszlan, S. (1973). Anal. Biochem. 53, 624-628. Tager, H. S., Emdin, S. O., Clark, J. L. & Steiner, D. F. (1973). J. Biol. Chem. 248, 34763482. T a k a y a m a , K., MacLennan, D. H., Tzagoloff, A. & Stoner, C. D. (1966). Arch. Biochem. Biophys. 114, 223-230. Tsugita, A. & Inouye, M. (1968). J. Mol. Biol. 37, 201-212. Tsugita, A., Gish, D. T., Yong, J., Fraenkel-Conrat, H., Knight, C. A. & Stanley, W. M. (1960). Prec. Nat. Acad. Sei., U.S.A. 46, 1462-1469. Tsugita, A., Black, L. W. & Showe, M. K. (1975). J. Mol. Biol. 98, 271-275. Wright, H. T., K r a u t , J. & Wilcox, P. E. (1968). J. Mol. Biol. 37, 363-366.