THE JOURNAL OF BIOLOGICAL CHEMWPEY Vol. 246, No. 23, Issue of December 10, pp. 7175-7190, 1971

Printed in U.S.A.

Amino Acid Sequence and Sequence Variability of the

Amino-terminal Regions of Lysine-rich Histones*

(Received for publication, February 24,197l)

S. C. RALL AND R. DAVID COLE

From the Department of Biochemistry, University of California, Berlceley, California 94720

SUMMARY

The amino acid sequence has been determined for the first 72 residues of a lysine-rich histone from rabbit thymus. These are the residues contained in a fragment released from the histone by treatment with N-bromosuccinimide. Pep- tides derived by tryptic, thermolysin, and chymotryptic di- gestion of this 72-residue fragment were used to reconstruct the total sequence.

Analysis of the sequence revealed some unusual aspects of the structure of the fragment, which comprises about one- third of the histone molecule. Thirty of the first 40 residues are accounted for by lysine, alanine, and proline; this portion of the fragment includes sequences of 4,2, and 3 consecutive basic residues. The last 32 residues contain all the hydro- phobic amino acids (valine, isoleucine, leucine, tyrosine) of the fragment but have few basic residues and no prolines. The NH2 terminus of the histone is acetylated.

Comparison of the sequence of the NHz-terminal half of this lysine-rich histone with the entire sequences of the slightly lysine-rich and arginine-rich histones shows that all three have similar characteristics, that is, an NHz-terminal region rich in basic amino acids and a COOH-terminal por- tion rich in hydrophobic residues.

Tryptic peptides from the NHz-terminal N-bromosuccini- mide fragment of two other thymus lysine-rich histones have been isolated. These peptides (with partial sequences) were aligned by direct identity or analogy with the complete amino acid sequence of the NH%-terminal N-bromosuccinimide fragment of the lysine-rich histone studied previously. It was found that there were from 7 to 14 amino acid differences between fractions. One of the amino acid interchanges eliminates a major phosphorylation site.

The class of histone referred to as lysine-rich is generally termed Fl or I, but in actuality consists of a number of molecular

* This work was supported by Grants AM-06482, AM-12618, AM-8845. and GM-0031 from the United States Public Health Service and by the Agricultural Experimental Station. A pre- liminary version of the sequence was presented at the Annual Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, April 1970; the seouence has been revised significantly.

species. This multiplicity of lysine-rich histones exists even in single tissues (l), and there are differences in the chromatographic patterns of lysine-rich histones when one tissue is compared with another (2). Although some multiplicity may be intro- duced into lysine-rich histones by phosphorylation (3, 4), the multiplicity observed in our earlier work (5) appeared to be an expression of differences in amino acid sequences. It has been suggested that both sorts of heterogeneity coexist (3, S), and it seemed desirable to determine experimentally the nature and extent of the diversity in primary structure. Moreover, studies on the approximate distribution of amino acids in these histones revealed an interesting segregation of certain kinds of amino acids into different regions of the molecule (7, S), further arousing our interest in the determination of the details of amino acid sequence in lysine-rich histones.

The fragmentation of a lysine-rich histone by N-bromosuc- cinimide (9) yielded a large peptide extremely enriched in lysine content and another, smaller peptide relatively poor in content of basic amino acids. The former proved to be from the COOH- terminal portion of the histone, while the latter was from the NHn-terminal region. We began the determination of amino acid sequence by studying the smaller fragment because it offered obvious technical advantages over the larger, very basic piece. Further, the NH&erminal portion of the histone was of special interest because of the possibility that variations in primary structure from one lysine-rich histone to another might be re- stricted to that region (5).

Therefore, sequence studies were undertaken on the NH2- terminal fragments derived from various lysine-rich histones by N-bromosuccinimide treatment. In this paper we report first the complete amino sequence of the NH&,erminal N-bromosuc- cinimide fragment (Nz) derived from chromatographic Fraction 3 of rabbit thymus lysine-rich histone (RTL-3). Then as a simple test of amino acid differences, the compositions and partial sequences of tryptic peptides of NHz-terminal N-bromosuccini- mide fragments of two other lysine-rich histones were compared with the completely determined structure of RTL-3-Nz.l Cne comparison was made between two chromatographic fractions

l The abbreviations used are: RTL-3, rabbit thymus lysine-rich histone Fraction 3; RTL-4, rabbit thymus lysine-rich histone Fraction 4; CTL-1, calf thymus lysine-rich histone Fraction 1; N1, the large carboxyl-terminal fragment released from the histone by N-bromosuccinimide treatment; NI, the smaller NHz-terminal N-bromosuccinimide fragment; dansyl, l-dimethylaminonaph- thalene-5-sulfonyl; Ac-Ser, N-acetyl serine.

7175

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7176 Sequence of Lysine-rich Histones Vol. 246, No. 23

2 8

g 0 300 450 600

EFFLUENT VOLUME (mls)

FIG. 1. Chromatography of the tryptic peptides of RTL-3-Nz on Senhadex G-25. The column (2 X 190 cm) was eluted at room temperature with 0.02 N HCI at‘s flow rate of 30 ml per hour. 0, absorbance at 230 rnp; 0, absorbance at 260 rnp.

(RTL-3 and RTL-4) from a single tissue (thymus) of the rabbit, and the second comparison was made with the same tissue in different species, rabbit (RTL-3) and calf (CTL-1). Previously, RTL-3, RTL-4, and CTL-1 were shown to be homogeneous 0, 2, 5, 10).

MATERIALS AND METHODS

Lysine-rich histone from either rabbit thymus or calf thymus glands was prepared as described by De Nooij and Westenbrink (11) as modified by Kinkade (12). Lyophilized histone prepara- tions were chromatographed on columns of Amberlite IRC-50 as described by Kinkade and Cole (1) and Bustin and Cole (2). The individual fractions were pooled from several preparations and stored at -10”.

Subtilisin (Sigma protease type VII, crystallized and lyophil- ized) was dissolved in water before use. Digestions were carried out in 0.05% (w/v) NHIHCOI, pH 8.0, in a 37” water bath. Digestions were terminated by freezing.

Digestions with carboxypeptidases and leucine aminopeptidase (bovine lens) were performed as described (13).

Other enzymic digestions were carried out at room temperature (except where otherwise noted) at pH 8.0 in a pH-stat, and were terminated by the addition of a small amount of 50% acetic acid to bring the pH to 3. Bovine trypsin (Seravac Labora- tories, Inc., Maidenhead, England, crystalline, salt-free, treated with L-(1-tosylamido-2-phenyl) ethyl chloromethyl ketone) was dissolved in 4 mM HCl, 20 mM CaC12. Thermolysin (a gift from Dr. H. Matsubara, University of California, Berkeley) was dis- solved in 0.01 M Tris, 0.01 M Ca*, pH 8.0. Worthington chymo- trypsin (CDC 52) was dissolved in 1 mM HCl before use.

Sephadex G-25 (fine beads), G-50, and G-100 were allowed to swell for 24 hours at room temperature in 0.02 N HCl, which contained 0.1 %, 1 , 1,l ,-trichloro-2-methyl-2-propanol as a bacteriostatic agent. Chromatography on Sephadex G-25, G-50, and G-100 columns (2 x 190 cm) was done at room tem- perature with 0.02 N HCl as eluent. Flow rates were 30 ml per

hour (G-25), 25 ml per hour (G-50); and 12 ml per hour (G-100). Fractions of 3 ml were collected with the aid of a drop counter. Chromatography was monitored by absorbance at 230 rnp.

Amino acid analyses were performed on 5 to 30 nmoles of peptide or protein which had been hydrolyzed for 20, 22, or 24 hours in 6 N HCl at 110” in sealed, evacuated tubes (14).

In those cases where peptide fractions from columns were impure, they were resolved by high voltage paper electrophoresis in one of the following systems; formic acid-acetic acid-water, 2:8:90, pH 1.9; pyridine-acetic acid-water, 1:10:289, pH 3.5; or pyridine-acetic acid-water, 25 : 1: 225, pH 6.4.

In some cases end groups were determined by the dansyl- chloride method of Gray (15). The thin layer method of Woods and Wang (16) was used to identify the dansylated amino acid.

Amide assignments were made by the technique of Offord

(17). The Edman procedure employed for sequential degradation of

peptides was that suggested by Gray (18) except that the amino acid removed was determined by difference.

RESULTS’

In this section the isolation and sequence analysis of individual peptides will be described, while the arrangement of the peptides will be considered in the first paragraph of the “Discussion.”

Tryptic Peptides of RTL-S-N2

RTL-3 was subjected to treatment with N-bromosuccinimide as described previously by Bustin and Cole (9), and the reaction mixture was applied to a Sephadex G-100 column to separate the peptide fragments, Ni and N2 (19). The molecular weights of Ni and Ns have been refined from their previously reported values (9). Since histones and histone peptides are known to behave anomalously on Sephadex (20), their elution positions are not a rigorous method for estimating molecular weights. In the light of analyses of peptides derived from subsequent enzymic digestions on Nt, it was necessary to revise the molecular weight of N2 upward (from 6200 to 7000) and Ni downward (from 15,000 to 14,000).

Tryptic digestion was performed on 7.5 pmoles of RTL-3-NZ (0.25% in water). Trypsin (1 :lOO by weight) was added at time zero and again at 2 and 7 hours. The digestion was terminated after 21 hours. After concentration the digest was applied to a Sephadex G-25 column and eluted with 0.02 N

HCl (Fig. 1). Some peptide fractions required further purification by high

voltage paper electrophoresis at pH 6.4. The tryptic peptides are referred to by the letter T and are numbered according to their order in the final amino acid sequence.

A summary of the tryptic peptides of RTL-3-Nz is presented in Table I.

Peptide Tl (Residues 1 to 15), Ac-Ser-Glu-Ala-Pro-Ala-Glu- Thr-Ala-Ala,-Pro-Ala-Pro-Ala-Glu-Lys-This peptide gave an extremely weak ninhydrin response on paper and had no free amino group as judged by failure of both the Edman and dansyla-

2 All the data on Edman degradations referred to in this paper, if not included in the text are available on microfilm. Request NAPS document No. 01638 from the ASIS National Auxiliary Publications Service, CCM Information Corporation, 866 Third Avenue, New York, New York 10022, remitting in advance (pay- able to CCMINAPS) $2.00 for each microfiche or $5.00 for each photocopy.

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TABLE I

Amino acid composition of tryptic peptides of RTL-3-Nz

7177

Amino ads

Lysine ..............

Arginine. ........... Aspartic acid. ...... Threonine .......... Serine .............

Glutamic acid ....... Proline ............. Glycine ...........

Alanine. ......... Valine .............. Isoleucine. .........

Leucine. .......... Tyrosine ............ Total residues.

Apparent yield ($&) Position on Sephadex

G-25. .............

Mobility, pH 6.40. .. Charge, pB 6.4. ....

.I

(

(

1

1

(

!

(

Tl T2 T3 L, 7b, 12

- T4

11. 1.04 (1: .06 (1: .o (2) .o 0)

TS

.09 (1)

T6

.lO (2, .07 (1: .93 (1:

1.90 (1: I.97 (1: 2.96 (3: 2.98 (3;

1.26 5.95 (6:

.29

.75 (1: .22

I.90 (1:

.16 (1: 1.18

.83 (2)

_-

)I 0

1

)

.09 (1,

.89 (2

.81 (3,

I.19 .27

15 4 2 1 3 8 2

90 100 59 148 76 71 43

Peak II

--0.66 -3.0

Peak Peak

VIb VP +0.45 S1.R +1.0 +1.9

Peak Peak Peak Peak

VIb VP IV VI’ f1.01 +0.55 +0.61 +1.15 f1.0 +0.9 $2.1 +2.0

i

0.82 (1: 1.03 (1:

0.91 (1: 1.89 (2: 1.08 (1; 2.13 (2;

0.92 (1: 0.78 (1; 1.01 (1:

1 12

25 59

Peak Peak

VII” III +0.88 <0.05 +1.0 0

I1

I

IO 1

) 1

12 )O

) )

T9 TlO T13’=

.oo (1) .35 .95 (1)

.26

.81 (1)

Tll

-.

.06 0)

.83 (1)

.95 (1) .68 .05 (1)

:.86 (3: I.90 (1;

.06 (1) .04 (2)

.82 (2)

.28 (3)

.50

.Ol (3)

6 2 9

75 43 36

.50 (1)

(1) 7

54

Peak Peak Peak Peak

Vb VII6 Vb VIb

+0.39 0 +0.30 0 +1.0 0 +1.1 0

Q It was not necessary to use this contaminated fraction to determine the structure of RTL-3-N%. The major peptide present proba- bly comprised the COOH-terminal 7 residues of RTL-3-N*. See text.

b Further purified by paper electrophoresis at. pH 6.4. c Aspartic acid = -1.00.

tion procedures. It is known that lysine-rich histones have a hypochlorite reagents and so it was isolated by the same tech- blocked NHz-terminus; the blocking group has been assumed to niques as used for TlSl. To deduce the sequence of Peptide be acetyl since Phillips (21) found 1.2 moles of acetyl (per 21,000 TlS2, hydrazinolysis was performed on 0.36 pmole according to molecular weight) associated with the lysine-rich histones of the method of Fraenkel-Conrat and Tsung (23). After hydrazine calf thymus. was removed, 0.2 ml of Hz0 was added and the sample was

Digestion with carboxypeptidase A plus B for 2 hours at 37” divided into two parts. One part was examined for free amino released lysine (82yo) but no acidic or neutral amino acids. The acids by electrophoresis (pH 1.9) and by the amino acid analyzer. electrophoretic mobility of Tl at pH 6.4 showed that all three Both analyses showed glutamic acid (with a trace of serine). glutamic acid residues must be in the carboxyl, rather than the The other part was examined for hydrazides by chromatography amide, form. in collidine-Hz0 (5: 1, v/v) as described by Narita (24). Hy-

Since the peptide could not be degraded from the NHa- drazides were detected by the spray of Andreae (25). Two in- terminus, it was fragmented into a smaller set of peptides by tense spots were detected, one of which chromatographed exactly digestion with subtilisin for 4 hours at 37” at an enzyme to with authentic acetyl hydrazide (RF 0.35). The other spot substrate ratio of 1: 50 (w/w). One part of the digest was sub- (Rp 0.10) was likely serine hydraaide or that of some other jetted to paper electrophoresis at pH 6.4 and then pH 3.5 (where amino acid with a charged amino group since its RF was too appropriate) to isolate the ninhydrin-positive peptides. The low for that of formyl or proprionyl hydrazide. These results other part of the digest was subjected to paper electrophoresis at were consistent only with the structure Ac-Ser-Glu for Peptide pH 1.9 to isolate the ninhydrin-negative peptides. Although TlS2. Because of the acetylated NHz-terminus, Tl must be these peptides were not detected analytically, they were pre- the NH%-terminal peptide of RTL-3-Nz (and of the intact dieted to migrate in the neutral position at pH 1.9. The paper histone).

which would contain this neutral band was then subjected to Peptide T2 (Residues 16 to 19), Ser-Pro-Ala-Lys-Two steps electrophoresis at pH 6.4 to achieve further purification. A of the Edman degradation showed the sequence Ser-Pro-. summary of the peptides isolated from subtilisin digestion of Treatment with carboxypeptidase B for 2 hours at 25” released peptide Tl is presented in Table II. lysine (109%).

Peptide TlSl was negative to both the ninhydrin and hypo- Peptide TS (Residues 20 to 21), Lys-Lys-This peptide con- chlorite (22) reagents, so it was isolated by electrophoresis at tained only lysine by amino acid analysis. That it was Lys-Lys pH 1.9 and pH 6.4 after prediction of its mobilities by the and not free lysine or Lys-Lys-Lys was deduced by the theoreti- method of Offord (17). This could be done since it was known cal mobility at pH 6.4 relative to free lysine (17): free lysine, that all glutamic acid residues of Tl were in the carboxyl form. 1.00; Lys-Lys, 1.22; Lys-Lys-Lys, 1.43; Peptide T3, 1.15. No further attempt was made to deduce the sequence of this Peptides T4, T7b, and Ti2 (Residues 22, 35, and 65), Lys- peptide. Analysis of equal aliquots of this fraction with and without

Peptide TlS2 was also negative to both the ninhydrin and hydrolysis showed lysine in equivalent amounts.

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TABLE II Subtilisin peptides derived from Tl

Summary : Ac-Ser-Glu-Ala-Pro-Ala-Glu-Thr-Ala-Ala-Pro-Ala-Pro-Ala- I I I I

TlSl TlS4

I I I I I II I TlS2

Glu-Lys l-l

TlS8

Subtilisin peptides

T

TlSl (52%)

TlS2 (44%)

TlS3 (36%)

TlS4 (47%)

TlS5 (24%)

TlS6 (29%)

TlS7 (16%) TlS8 (91%)

TlS3 TlS5 TlS6

I--l TlS7

Mobility - Composition and structural features

rn8.P

-0.8

-1.1

-- Thr 0.14, Ser 0.85 (l), Glu 0.97

(1)) Pro 0.85 (1)) Ala 2.00 (2) Ser 0.82 (I), Glu 1.00 (I), Ala

0.23

0

-0.3!

IO.26

5

-0.6

Hydrazinolysis: glutamic acid, acetyl hydrazide

Pro 1.05 (I), Ala 1.95 (2) Edman degradation : alanine Thr 0.84 (l), Glu 1.01 (l), Pro

1.98 (a), Ala 3.80 (4) Edman degradation: Glu-Thr-

Ala-Ala-Pro-Ala-Pro-Ala Thr 1 .OO (I), Glu 0.6gd (I), Ala

1.00 (1) to.23

j-o.20 to.62

Pro 2.10 (2), Ala 2.88 (3) Edman degradation : alanine Ala 1.0 (1) Lys 1.04 (I), Glu 0.96 (1)

-

a At pH 6.4, mobility relative to ) aspartic acid = -1.0. 6 At pH 3.5, mobility relative to lysine = +l.O. c At pH 1.9, mobility relative to serine = +l.O. d Composition determined after partial reaction with ninhy-

drin; low value for glutamic acid indicates glutamic acid is the NHt-terminal residue.

Peptide T5 (Residues .% to .%?5), Ala-Ala-Lys-One step of the Edman degradation showed alanine to be NHt-terminal. Car- boxypeptidase B treatment for 2 hours at 25” released lysine

(74%).

Peptide TlO (Residues 54 to 55), Glu-Arg-One Edman degradation was performed on TlO and glutamic acid was shown to be NHB-terminal. The electrophoretic mobility of TlO in- dicated that the glutamic acid residue was in the carboxyl form.

Peptide Tii (Residues 56 to 64), Asn-(Gly, Leu, Ser)-(Leu, Ala, Ala, Leu)-Lys-The electrophoretic mobility of Tll in- dicated that the aspartic acid residue was amidated. One step of the Edman degradation showed asparagine NH%-terminal.

Treatment with carboxypeptidase A plus B at 37” released lysine (101 %), alanine (128 %), and leucine (138%) in 30 min and lysine (95y0), alanine (162%), and leucine (163%) in 2 hours.

Peptide TlS (Residues 66 to 72) (Ala, Leu, Ala, Ala, Gly, Gly, Tyr)-Although the recovery of this peptide from Sephadex appeared to be 54% based on leucine content, its recovery from paper was extremely low and it was grossly contaminated. However, the deduction of the sequence of the COOH-terminal seven residues did not require the use of this peptide.

Peptide T6 (Residues 26 to SS), Lys-Pro-Gly-Ala-Gly-Ala- Ala-Lys-Seven steps of the Edman degradation established the sequence of T6. Carboxypeptidase A plus B treatment for 2 hours at 37” released lysine (640/,), alanine (25%), and a trace of glycine.

Peptide T7 (Residues 34 to S5), Arg-Lys-This peptide could be separated from free lysine and Lys-Lys by prolonged elec- trophoresis at pH 6.4. Dansylation showed only a spot where both e-dansyl-lysine and dansyl-arginine run in the thin layer system used (16). After one step of the Edman degradation, the aqueous phase remaining after extraction was applied to the analyzer without hydrolysis and the only free amino acid found was lysine.

Thermolysin Peptides of RTL-S-N2

Thermolysin digestion was performed on 2.3 pmoles of RTL- 3-N* (0.2% in water). The digestion was carried out for 2 hours at 40” at an enzyme-substrate ratio of 1:200 (w/w). After the digestion, the mixture was concentrated and applied to a Sepha- dex G-25 column and eluted with 0.02 N HCl (Fig. 2).

Impure fractions were further purified by paper electrophoresis at pH 6.4. The thermolysin peptides isolated are referred to by the letters Th and are numbered according to their order in the sequence.

A summary of the thermolysin peptides of RTL-3-Nz is pre- sented in Table III.

Peptide T7a (Residue 34), Arg-Analysis with and without Peptide Thi (Residues 1 to dl), (AC-Ser, Glu, Ala, Pro, Ala, hydrolysis showed that all of T7a was free arginine. Glu, Thr, Ala, Ala, Pro, Ala, Pro, Ala, Glu, Lys, Ser, Pro, Ala)-

Peptide T8 (Residues 36 to 47), Ala-Ala-Gly-Pro-Pro-Val- Xer-Glu-Leu-Ile-Thr-Lys-The electrophoretic mobility of T8 showed that the glutamic acid residue was in the carboxyl form. Eight Edman degradations were performed on T8 and the results indicated the sequence: Ala-Ala-Gly-Pro-Pro-Val-Ser-Glu-.

Treatment of the peptide with leucine aminopeptidase released 1.45 molar equivalents of alanine in 3 hours at 40”. The speci- ficity of leucine aminopeptidase is such that it stops at the residue preceding proline in the peptide chain, so this result is consistent with the sequence Ala-Ala-Gly-Pro.

Treatment of T8 with carboxypeptidase A plus B at 37” released lysine (119%), threonine (12yo), and isoleucine (8%) in 30 min and lysine (103%), threonine (26%), and isoleucine (26%) in 2 hours, which suggests a COOH-terminal sequence of -Ile-Thr-Lys. With this result and the Edman results, the leucine residue can be placed by difference before isoleucine and after glutamic acid.

In one series of experiments an 8-hour tryptic digestion (en- zyme-substrate 1: 100 for 5 hours, and an additional 1 :lOO for the next 3 hours) was fractionated by Dowex 50 chromatography to yield an extra peptide which was like T8 except that it had an extra lysine residue which will be shown later to be at the NHz-terminus. This suggests a sequence Arg-Lys- or Lys-Lys- immediately preceding T8.

Peptide TQ (Residues 48 to 53), Ala-Val-Ala-Ala-Xer-Lys- Four steps of the Edman degradation established the sequence Ala-Val-Ala-Ala-. Carboxypeptidase A plus B treatment for 30 min at 37” released lysine (99%), serine (74%), and alanine 01%).

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Thl which was not detected by analytical paper electrophoresis. To test this hypothesis, 0.25 pmole of Thl was subjected to tryptic digestion for 8 hours at 37” in 0.05 M NH~HCOZ, pH 7.5, at an enzyme-substrate ratio of 1:25 by weight. The digest was subjected to paper electrophoresis at pH 6.4. The peptides isolated are presented in Table IV.

The peptides ThlTl, ThlT2, and ThlT3, corresponding to the tryptic Peptides Tl, T2, and T3, were found, but there was also a small amount of a peptide (ThlT5) not isolated from tryptic digests of the entire Nz fragment. The presence of this peptide suggests that Thl is really two peptides, the major one terminating in -Lys-Lys-Lys, but another t.ermmating in

TABLE IV

Tryptic peptides derived from Thl Summary: Ac-Ser-Glu-Ala-Pro-Ala-Glu-Tbr-Ala-Ala-Pr~Ala-Pr~~a-Glu-Lys- I

ThlTl

F-Pro-Ala-Lyg-f+-Lys-(Lys-Ala\

ThlT2 TF”ThlT5

Thk’ k/IT4

Lys-Lys-Lys-From its composition, this peptide appeared to contain the NH&erminal tryptic peptide. The electrophoretic mobility of Thl confirmed the blocked NHz-terminus and the assignment of all three glutamic acid residues as carboxyl forms. The peptide was slightly ninhydrin-positive due to the e-amino groups of lysines. Treatment with carboxypeptidase B alone for 2 hours at 37” released lysine (197%). Treatment with carboxypeptidase A plus B at 37” released lysine (1680/,) and alanine (15%) in 30 min and lysine (272%) and alanine (32%) in 2 hours.

The carboxypeptidase results and the analysis for alanine (Table III) suggested heterogeneity at the COOH-terminus of

0.4

t

I

Mobility’” Tryptic peptides

ThlTl (69%)

ThlTP (42%)

ThlT3 (22%)

ThlT4 (18%)

ThlT5 (17%)

Lys 0.90 (l), Thr 0.90 (l), Ser 1.05 (l), Glu 2.83 (3), Pro 3.00 (3), Ala 6.1 (6)

Lys 1.13 (l),,Ser 0.91 (l), Pro 0.95 (l), Ala 1.00 (1)

Lys 2.0 (2)

Lys 1.0 (1)

Lys 1.02 (l), Ala 0.98 (1)

-0.53

+0.45

+1.16

+1.00

+0.63

0’ I I I

300 450

EFFLUENT VOLUME (mls)

FIG. 2. Chromatography of the thermolysin peptides of RTL- 3-Nz on Sephadex G-25. The column and elution conditions were the same as those for Fig. 1. a At pH 6.4, mobility relative to aspartic acid = -1.0.

TABLE III Amino acid compositions of thermolysin peptides of RTL-S-N2 -

.-

-

-

-

-

Thl Thla Th2 Th3 Th4 Th5 Th6 Th7 Th& Amino acids

Lysine ................... Arginine ................. Aspartic acid. ........... Threonine ............... Serine ................... Glutamic acid ............ Proline .................. Glycine .................. Alanine .................. Valine ................... Isoleucine ................ Leucine .................. Tyrosine ................. Total residues ............ Apparent yield (%) ...... Position on Sephadex G-2: Mobility, pH 6.4 ......... Charge, pH 6.4. .........

5 See text.

3.82 (4) 4.02 (4) 3.82 (4) 0.93 (1)

1.08 (1)

1.05 fl) 1.74 (2) 2.88 (3)

4.29 (4)

0.92 (1) 0.78 (1) 1.04 ‘1) 3.10 (3) 0.3 5.10 (5)

0.18

7.50 (7)*

1.23 (1) 1.97 (2) 4.50”

0.97 (1) 1.01 (1)

2.05 (2) 1.03 (1) 1.38”

1.00 (1)

0.10 0.99 (1)

1.01 (1) 0.87 (1)

21-23a 14 12-130 8-9” 4

54 40 49 100 88 Peak I Peak 11~ Peak II. Peak VC Peak VIc

0 +o.eco +0.99 -0.31 1-0.46

0 f2.9 +4.4 -1.0 +1.0

1.07 (1) 0.95 (1) 0.84 (1)

1.08 (1) 1.04 (1)

1.03 (1) 1.95 (2) 1.01 (1)

0.27 2.13 (2)

0.91 (1)

9 3

44 93 Peak IVc Peak VII

+0.30 0 +1.1 0

2.29 (2)

0.26

0.18

1.00 (1) 0.18

0.86 (1)

4

72 Peak VC

+0.90

+2.0

1.85 (2)

2.29 (2)

0.85 (1) Ob (1)

6

10 Peak VIc

0 0

5

b Tyrosine does not appear in these analyses because of its previous reaction with N-bromosuccinimide. c Further purified by paper electrophoresis at pH 6.4.

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7180 Sequence of Lysine-rich H&ones Vol. 246, No. 23

-Lys-Lys-Lys-Lys-Ala. This minor form was apparently missed during analytical paper electrophoresis; this is not sur- prising since it was at the most only 30% of the total and the ninhydrin response of Thl was very weak. The presence of the tryptic Peptides Tl, T2, and T3 in Thl and the carboxypeptidase results established their positions in the sequence.

Pep&e Thla (Residues 8 to 21), Ala-(Ala, Pro, Ala, Pro, Ala, Glu, Lys, Ser, Pro, Ala, Lys, Lys, Lys)-The mobility of Thla was consistent with the assignment of the glutamic acid residue as the carboxyl form. One Edman degradation established alanine as the NHz-terminal amino acid.

Peptick Th2 (Residues 24 to 35 and 24 to 36), Ala-(Lys, Lys, Pro, Gly, Ala, Gly, Ala, Ala)-(Lys, Arg, Lys) and Ala-(Lys, Lys, Pro, Gly, Ala, Gly, Ala, Ala)-(Lys, Arg, Lys)-Ala-It was obvious from the composition that Th2 contained the sequence of T6 in its sequence. One step of the Edman degradation established alanine as the only NHz-terminal residue. Two subsequent Edman degradations gave no reduction in any of the acidic or neutral amino acids; only the lysine value varied. This indicated the peptide began Ala-Lys-Lys-.

Carboxypeptidase A plus B treatment at 37” released lysine (116%) and arginine (58%) in 10 min. After 1 hour carboxy- peptidase A plus B released lysine (124%) and arginine (60%) which was essentially no increase over that of digestion for 10 min. This result indicated that the peptide terminated in LyszArgl, but the order could not be established even with very short digestion times.

The analysis for alanine in Th2 (Table III) and the failure of carboxypeptidase to release more arginine and lysine than it did, suggested heterogeneity at the COOH-terminus of Th2. (At- tempts to separate the two components failed.) It was sur- prising that carboxypeptidase A did not release any alanine, but apparently its failure to do so restricted the ability of carboxy- peptidase B to release all the lysine and arginine, since carboxy- peptidase B would not act on the form of the peptide terminating in alanine until the alanine is released by carboxypeptidase A. Edman results argued against heterogeneity at the NHz-terminus of Th2.

The carboxypeptidase results indicated that Peptide T7 is at the COOH-terminus of Th2 and follows T6 in the sequence. The NHz-terminal alanine of Th2 suggested that T5 precedes T6 in the sequence, but this was somewhat uncertain until further confirmation was obtained. Residues 22 to 23 were not recovered from the thermolysin digest, except as a minor part of the COOH-terminus of Thl, a complication which made assign- ment of the position of T5 less certain at this point than would otherwise have been possible.

Peptide Th3 (Residues 36 to 44 and 37 to 44), Ala-Ala-Gly- Pro-Pro-Val-Ser-Glu-Leu and Ala-Gly-Pro-Pro-Val-Ser-Glu- Leu-Amino acid analysis suggested heterogeneity of the peptide with regard to the alanine content (Table III). Since the two forms could not be separated they were studied together. Treatment of Th3 with leucine aminopeptidase for 3 hours at 40” released 1.39 molar equivalents of alanine and no other amino acids. This result was in agreement with amino acid analysis regarding the relative amounts of the two forms of the peptide and confirmed that the heterogeneity of Th3 was at the NHt-terminus. Treatment with carboxypeptidase A for 3 hours at 37” released leucine (87%) and glutamic acid (14%), establishing the COOH-terminal sequence of Th3 as -Glu-Leu.

Seven steps of the Edman degradation were performed on

Th3. The data showed a partial reduction of each residue re- moved at two consecutive steps, confirming the heterogeneity at the NHz-terminus as well as establishing the sequences of the two forms of Th3. The sequence of Th3 is contained in the NHz-terminal portion of Peptide T8.

Peptide Th4 (Residues ~$5 to .@), Ile-Thr-&s-Ala-This peptide includes the COOH-terminus of Peptide T8. Two steps of the Edman degradation established the sequence Ile- Thr-. From the carboxypeptidase results on peptide T8, the order of the lysine and alanine in Th4 can only be as written.

Peptide Th5 (Residues 49 to 57), Val-Ala-Ala-Ser-Lys-Glu- (Arg, Asn, Gly)-The mobility of Th5 suggested one amide and one carboxyl side chain for the asparaginyl and glutamyl residues, consistent with the previous assignments from TlO and Tll. Th5 contains most of T9, all of TlO, and the NHt-terminal portion of Tll in its sequence.

Six steps of the Edman degradation confirmed the presence of the last five residues of T9 in the peptide and further placed TlO after T9 in the total sequence. By difference Peptide Tll must then follow TlO because of the existence of the single asparaginyl residue of Nz in Tll and Th5.

Peptide Th6 (Residues 60 to 6d), Leu-Ala-Ala-One step of the Edman degradation was sufficient to establish the sequence of Th6, since leucine was found to be NHz-terminal. Th6 is con- tained in Tll.

Peptide Th7 (Residues 63 to 66), Leu-Lys-Lys-Ala-Three steps of the Edman degradation established the sequence of Th7. Th7 contains the COOH-terminal region of Tll and together with Th6 establishes the COOH-terminal sequence of Tll as -Leu-Ala-Ala-Leu-Lys.

Peptide Th3 (Residues 67 to X9), (Leu, Ala, Ala, Gly, Gly, Tyr)-This peptide was recovered in very low yield. Because the tyrosine is converted to the spirolactone derivative during the N-bromosuccinimide reaction, analysis of RTL-3-NZ and peptides derived from it will not show the presence of tyrosine even when it occurs in a peptide. This fact makes the place- ment of Th8 at the COOH-terminus of RTL-3-Nz somewhat uncertain, but it appears to be the only position in the sequence the peptide can fit. Moreover its assignment here reconciles the present data with the earlier studies of Bustin and Cole (8).

Chymotryptic Peptides of RTL-S-N2

Chymotryptic digestion was performed on 6.6 pmoles of RTL- 3-Nz (0.2% in water) as described in “Methods.” The digestion was carried out for 2 hours at an enzyme-substrate ratio of 1: 150 by weight. After the digestion was terminated, the sample was applied to a Sephadex G-50 column and eluted with 0.02 N HCl (Fig. 3).

Impure fractions were further purified by paper electrophoresis. Chymotryptic peptides are referred to by the letter C and are numbered according to their order in the sequence.

A summary of the chymotryptic peptides of RTL-3-Nn is presented in Table V.

Peptide Cl (Residues 1 to 44), (AC-Ser, Glu, Ala, Pro, Ala, Glu, Thr, Ala, Ala, Pro, Ala, Pro, Ala, Glu, Lys, Ser, Pro, Ala, Lys, Lys, Lys, Lys, Ala, Ala, Lys, Lys, Pro, Gly, Ala, Gly, Ala, Ala, Lys, Arg, Lys, Ala, Ala, Gly, Pro, Pro, Val, Ser, Glu, Leu)- Tryptic digestion was performed on 1.8 pmoles of Cl for 16 hours at 37” in 0.05 M NHdHCOa, pH 7.5, at an enzyme-substrate ratio of 1:25 An aliquot of the digest was subjected to paper electrophoresis at pH 6.4; nine bands were detected, one of which

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contained two peptides which could be separated by pH 3.5 electrophoresis. A summary is presented in Table VI.

Peptides corresponding to Tl, T2, T3, T4, T5, T6, T7, and T7a were isolated and are numbered ClTl, ClT2, ClT3, ClT4, ClT5, ClT6, ClT7, and ClT7a, respectively. This confirms the presence of these tryptic peptides in the NHz-terminal portion of RTL-3-Nz. In addition, two other peptides were isolated. ClT8 corresponds to the NHn-terminal portion of T8 and is identical with one of the Th3 peptides. Since ClT8 is

the only tryptic peptide of Cl which lacks a basic residue, it must be the COOH-terminal peptide of Cl. Peptide ClT4a arose from an incomplete tryptic split next to ClT4. Because of the known specificity of trypsin ClT4a must have either the sequence Lys-Ala-Ala-Lys or Ala-Ala-Lys-Lys. That the composition of ClT4a was LyszAlaz and not LysrAlal was de- termined readily by its mobility at pH 6.4 (17).

Peptide Cia (Residues 1 to bl), (AC-Ser, Glu, Ala, Pro, Ala, Glu, Thr, Ala, Ala, Pro, Ala, Pro, Ala, Glu, Lys, Ser, Pro, Ala,

’ II I Lys, Lys, Lys)-This peptide is the same as the major form of

1 Thl. Cla (2.5 pmoles) was digested with subtilisin for 4 hours

0.8 -

0.6 -

E‘

a

z c 0.4 -

x

5 2

at 37” at an enzyme-substrate ratio of 1: 100. The digest was then subjected to paper electrophoresis at pH 6.4. All of the bands detected corresponded to subtilisin peptides of Tl with one exception. This unique peptide (ClaS4) was isolated (20% yield) and its composition determined: Lys 2.88 (3), Ser 1.00 (l), Glu 1.09 (I), Pro 1.08 (l), Ala 0.91 (1). The mobility of +0.58 (relative to Asp = -1.0) indicates a charge of +1.9 at neutral pH for ClaS4. One step of the Edman degradation on ClaS4 showed glutamic acid was NH&erminal. The structure of ClaS4 is likely Glu-Lys-Ser-Pro-Ala-Lys-Lys, and confirms the overlap of the tryptic Peptides Tl and T2.

Pep&k Cl b (Residues .%Y to 44), Lys-(AEa, Ala, Lys, Lys, Pro, Gly, Ala, Gly, Ala, Ala, Lys, Arg, Lys, Ala, Ala, Gly, Pro, Pro, Val, Ser, Glu, Leu)-Dansylation showed that lysine was the NHz-terminal residue of Clb. Tryptic digestion was performed on 0.35 Mmole of Clb for 8 hours at 37” in 0.05 M NHdHCOa,

HHH - k----,H-c--l k--4- pH 7.5, at an enzyme-substrate ratio of 1:25 by weight. The 0

300 450 600 digest was subjected to paper electrophoresis at pH 6.4. A ml Effluent summary of the peptides isolated is presented in Table VI.

FICA 3. Chromatography of the chymotvptic peptides of RTL- 3-Nz on Sephadex G-50. The column (2 X 190 cm) was eluted at

peptide ClbTl corresponds to C1~8 and Th3; ClbT2 to T5 and

room temperature with 0.02 N HCl at a flow rate of 25 ml per hour. ClT5; ClbT3 to T6 and ClT6; ClbT4a to ClT4a; ClbT4b to

Solid bars indicate the fractions pooled. T7a and ClT7a; and ClbT5 to T4. A peptide corresponding

TABLE V

Amino acid compositions of chymotryptic peptides of RTL-S-N2

Amino acids Cl Ch Clb c2 c3

.- -

Lysine.................. Arginine . Aspartic acid. Threonine . . . . Serine . Glutamic acid.. . Proline. . . . Glycine Alanine................. Valine Isoleucine.. . Leucine. . Tyrosine.

Total residues.. . . Apparent yield (%) Position on Sephadex G-E Mobility, pH 6.4..

Charge, pH 6.4. . .

8.60 (9) 1.03 0)

3.60 (4) 4.86 (5) 0.94 (1)

1.36 (1) 2.78 (3) 3.70 (4) 7.25 (7)

3.00 (3) 14.5 (14)

1.5 (1)

1.07 (1) 1.66 (2)

2.63 (3) 3.87 (4)

1.98 (2)

1.00 (1) 0.73 (1) 0.81 (1) 1.51a (1)

0.89 (1)

1.00 (1) 0.83 (1) 0.19

7.46 (7) 0.12

1.31 (1)

1.02 (1) 2.76 (3) 2.82 (3)

6.90 (7) 0.95 (1)

0.93 (1) 0.97 (1)

0.96 (1)

2.80 (3) 1.02 (1) 0.64 (1)

0.88 (1)

2.06 (2) 0.24 2.14 (2)

1.93 (2) 1.00 (1) 1.00 (1)

i0

-

44 21 23 14 5 2 3

27 44 28 60 30 8 18

Peak II Peak 111~ Peak 111~ Peak IV” Peak VIC Peak VIId Peak VIc

+0.45 0 +0.68 +0.39 0 0 0

+4.8 0 +4.5 +2.0 0 0 0 - - - -

* See text. * Tyrosine does not appear in analyses because of its previous reaction with N-bromosuccinimide.

c Further purified by paper electrophoresis at pH 6.4. d Further purified by paper electrophoresis at pH 3.5.

- - - C3b

-

-

-

c4

2.00 (2)

1.01 (1)

1.00 (1)

4 10

Peak VC

+0.90 +2.0

CS

1.93 (2) 2.07 (2)

Ob (1) 5

13 Peak VIId

0 0

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7182 Sequence of Lysine-rich H&tones

TABLE VI Peptides derived from Cl, Cla, and Clb

Vol. 246, No. 23

I i Clss4

I Clb

I

H-l II I, t I iXb!$ ClbT2 . .

I

!Ry-ptic Peptides: Mobilitya

ClTl (4%) -0.53

ClT2 (53%) +0.47

ClT3 (43%) +1.18

cm4 (78%) +1*cQ

cl*a (10s) +0.79

ClT5 (61s) +0.58

~1~6 $3) +0.63

cm7 (20%) +1.09

ClT7a (41s) +0.79

ClT8 (558) -0.26

Cm!1 (3Y%) -0.27

ClbT2 (28$) +0.95

ClbTs (40%) +0.61

Clbha (Us) +-a79

Clb!&b (50%) a.79

ClbT5 (4%) +1.00

ClbT3 Clblhb

ClbT5

ClbTl

Composition

4s Ala

4s

4s

4s

4s

4s

4s

4’s

1.00(l), Thr O.Y2(1), Ser 0.92(l), Glu 2.92(3), Pro Z!b(3), 6.50(6) l&O(l), Ser 0.72(l), Pro 0.92(l), G13r 0.20, Ala l.lJ.(l)

2.0( 2)

LO(l)

2.10(2), Ala 1.92(2)

l&(l), Gly 0.22, Ala 1.88(2)

2.04(2), pro 0.96(l), GW 1.95(2), - 3.10(3)*

1.08(l), Arg 0.931).

Jm3

FS

1.0(l)

0.26, Ser 1.22(l), Glu l.lO(l Ala 1.92(2), Val 1.01(l), Leu 0. 98

, Pro 2.00(2), Gly 1.13(l), (l),

Ser 1.12(l), Glu 1.00(l), Pro l&(2), Gly 1.08(l), Ala 1.92(2), val LOO(l), Leu 0.95(l).

4s 1,07(l), Ala 1.93(2)

4s l.%(2), Pro l&(l), Gly 2.00(2), Ala 3.03(3).

4s 2.02(2), Ala 2.00(2)

Arg LO(l)

4s LO(l)

At pH 0.4, mobility relative to aspartic acid = -1.0.

to ClT7 and T7 was not isolated, presumably because it ran too lysine. However, because of the overlap provided by Th2, far and was lost during electrophoresis. ClbT4a must be the NH&erminal tryptic peptide of Clb.

The COOH-terminal tryptic peptide of Clb must be ClbTl Cla and Clb combine to equal the composition of Cl. A because it is the only tryptic peptide lacking a basic COOH- summary of the studies on the tryptic peptides of Cl and Clb terminal residue. The NH%-terminal tryptic peptide of Clb is presented in Table VI. could be either ClbT3 or ClbT4a, since both have NHrterminal The unusual peptide bond hydrolysis by chymotrypsin at

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TABLE VII

Tryptic peptides derived from C.2 Summary :

Ile-Thr-Lys-Ala-Val-Ala-Ala-Ser-Lys-Glu-Arg-Asn-Gly-Leu I I I I I-1 I I

C2Tl

Tryptic peptides

C2Tl (42%) C2T2 (53%)

C2T3 (53%) C2T4 (50%)

__

-

C2T2 C2T3 C2T4

Mobility

ma2 ?m.bb __-

Composition

+0.75 Lys 1.12 (l), Thr 0.91 (l), Ile 1.00 (1) i-0.40 +0.59 Lys 1.09 (1)) Ser 1.05 (I), Ala 2.84 (3),

Val 0.85 (1) 0 +0.59 Arg 1.01 (l), Glu 0.99 (1)

+0.12 Asp 0.98 (I), Ser 0.12, Gly 1.09 (l), Val 0.10, Leu 1.00 (1)

Edman degradation: Asn-Gly-Leu

D At pH 6.4, mobility relative to aspartic acid = -1.0. b At pH 3.5, mobility relative to lysine = +l.O.

residues 21 to 22 was not totally unexpected. It is known that commercial preparations of chymotrypsin can be contaminated with small amounts of trypsin (26). Furthermore, even in preparations free of trypsin, hydrolysis by chymotrypsin at bonds it normally does not attack often occurs when there are basic residues preceding or following these bonds (27). Yet another explanation might be advanced for this unusual cleavage. It is conceivable in a sequence of consecutive lysine residues, that one or more pK values of the e-amino groups of lysine could be low enough because of their environment so that at the pH of chymotryptic digestion the usual polar nature of the lysine chains could be overpowered by the hydrophobic character of adjacent lysine side chains, overpowered in the sense that chymo- trypsin could tolerate a small charge in binding to a significantly hydrophobic site. The situation could be analogous to the cleavage of a leucyl bond where leucine is preceded by lysine. The peptide bond 21 to 22 occurs in a sequence of four consecu- tive lysine residues and is sensitive to hydrolysis by chymotryp- sin and thermolysin, both of which preferentially hydrolyze bonds involving hydrophobic amino acids. Inherent non- specificity of the enzymes cannot be ruled out, but there may be something special about the 21 to 22 linkage.

Peptide C.3 (Residum ~$5 to 58), Ik-Thr-(Lys, Ala, Val, Ala, Ala,Ser,Lys,Glu,Arg)-Asn-Gly-Leu-Two steps of the Edman degradation established the sequence Ile-Thr- for Peptide C2. Tryptic digestion was performed on 1.0 pmole of C2 for 3.5 hours at 37” in 0.05 M NH4HC03, pH 7.5, at an enzyme-substrate ratio of 1:25. The digest was subjected to paper electrophoresis at pH 3.5. A summary of the peptides isolated is presented in Table VII.

C2Tl corresponds to the COOH-terminal region of T8 and is the NHz-terminal tryptic peptide of C2. C2T2 corresponds to T9 and C2T3 to TlO. C2T4 corresponds to the NHt-terminal portion of Tll. Edman degradation on C2T4 established its sequence: Asn-Gly-Leu.

Peptide C2 allows the assignment of overlaps for the tryptic peptides T8, T9, TlO, and Tll. The order is TS-T9-TlO-Tll. The serine value in the analysis of C2 was 1.5, but only one residue of serine was found in the tryptic peptides of C2. The high serine value is probably attributable .to the contaminant which was removed from C2T4 by a second electrophoresis.

I H i---i~Hkk-+t---+~ ,

I 300 450 600

ml Effluent FIG. 4. Sephadex G-25 chromatography of the tryptic digest

of RTL-4-N*. The column @‘X 190 cm) was eluted with 0.02 N

HCl at room temperature at a flow rate of 30 ml per hour. Solid bars indicate the fractions-pooled.

I I I I J 300 450 600

mls Effluent

FIG. 5. Sephadex G-25 chromatography of the tryptic digest of CTL-I-N2. Conditions were identical with those used for the RTL-4-Nz tryptic digest. Solid bars indicate the fractions pooled.

Peptide Cs (Residues 59 to 63), Ser-Leu-Ala-Ala-Leu-Three Edman degradations established the sequence Ser-Leu-Ala-. I f leucine is COOH-terminal (according to chymotrypsin speci- ficity), the sequence of C3 is as written. C3 is contained in Tll and now allows the complete sequence for Tll to be written Asn-Gly-Leu-Ser-Leu-Ala-Ala-Leu-Lys.

Peptide CSa (Residues 69 to SO), Xer-LeuyPeptide C3a is contained in the NHz-terminal portion of C3 and,must have the sequence Ser-Leu.

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7184 Sequence of Lysine-rich H&ones

TABLE VIII Amino acid compositiona of tryptic peptides of RTL-Q-N,

Vol. 246, No. 23

Amino acids Tl T2 T2a __-__

Lysine.. . . . . 1.00 2.14 1.10 (1) (2) (1)

Arginine

Aspartic acid.. .I / 1

Threonine.

Serine. .........

Glutamic acid.

Proline. ........

Glyeine ........

Alanine. .......

Valine. .........

Isoleucine ......

1.00

0)

0.86 0.79 0.90

(1) (1) (1) 0.97 1.78 1.73

0) (2) (2) 1.91 1.00 1.10

(2) 0) (1) 2.90 1.82 2.00

(3) (2) (2) 1.01 0.97

0) (1) .6.13 1.20 1.20

(6) If) (1) 0.94 1.00

(1) (1) 0.75 0.80

(1) (1) 0.77 0.85

0) (1)

1.83 (2)

1.05 0)

2.12 (2)

0.80

(1)

Leucine . 1.72 (2)

Tyrosine . Total residues. . 14 Apparent yield (%) 69

13 12 32 25

9 1 57

Position on Sepha- / dex G-25. . . . . Peak Peak Peak Peak dex G-25. . . . .IPeak /Peak /Peak Peak

II II IV v Mobility, pH 6.4L.. -0.44 $0.24 0 $0.2 Charge, pH 6.4 . . . . . -2.0 +l.l 0 +i.a

0 See Footnote b, Table III. 0 See Footnote b, Table III. b Relative to aspartic acid = -1.0. b Relative to aspartic acid = -1.0.

T3 T4 TS

2. .oo

(2) 00

(1)

.94 .96

(1) (1)

.29

.17 (1)

.07 (3)

.76 (3)

.66 (1)

7 6 43 95

‘eak V

to.7 t2.1

‘eak V

to.4 t-1.(

T6

.21

Tl T8 T9 TlO

1.04 00 98 (1) (1) (1)

J.94 (1)

.34

1.00 .96 (1) (1)

.20 0)

.83 (4)

0.16

0.95 (1)

.oo

(1)

1 (1) 7

38 4

30

Peak Peak VI VI 0 +0.4 0 +1.1

Peptide C3b (Residues 61 to 63), Ala-Ala-Leu-Edman degra- dations established the sequence of C3b as Ala-Ala-Leu. C3a and C3b combine to equal C3.

Peptide C4 (Residues 64 to 67), Lys-Lys-Ala-Leu-Dansyla- tion showed lysine to be the NHz-terminal residue. C4 has the same composition as Th7, but not the same end group. There- fore, C4 probably terminates in leucine (chymotrypsin speci- ficity) and thus has the sequence as written.

Peptide C5 (Residues 68 to 7.@, Ala-Ala-Gly-Gly-Tyr-This peptide was isolated in low yield and as expected amino acid analysis failed to reveal the presence of tyrosine. In order to determine the structure Edman degradations were performed on Peptide nl rather than on C5; Peptide nl was previously (8) isolated from a chymotryptic digestion of intact RTL-3 (not peptide Nz), and contained the tyrosine residue unmodified. Peptide nl has the composition GlyzAlazTyrl (8). The Edman degradations on nl established the sequence Ala-Ala-Gly- Gly-Tyr.

Tryptic Peptides of RTL-4-N2

RTL-4 was isolated and subjected to N-bromosuccinimide cleavage as previously described for RTL-3 (9). The pattern obtained from Sephadex G-100 chromatography was similar to

.oo (1)

.14 (1)

4 30

‘eak VI

l-0.: j-l.1

.02 (1)

2 17

‘eak VI

kO.6 t-o.9

1.06 (1)

0.92 (1)

2 ?ot deter-

mined

Peak VI

$1.06 +1.9

2.

II1

0 (2)

T12 T13 II4 T1.5

.O (1)

03 0)

03 (1)

0.

1.

09

0 0)

.98 (1)

.98 (1)

2 1 2 2 1 41 82 82 22 28

‘eak VI

Cl.1 Cl.9

leak VI

to.9 j-1.0

‘eak VI 0 0

‘eak VI.

-0.6 -l.a

I I 17 - I - -

‘eak VII

to.88 t1.0

the one derived from RTL-3 (9, 19). The yields of the two fragments were: N1, 93%; Nz, 86%.

RTL-4-Nz (2.1 pmoles, 0.1% in water) was digested with trypsin as described under “Methods.” Trypsin (1 :lOO by weight) was added at zero time and again at 2 hours. The di- gestion was terminated after 5 hours. The digest was chroma- tographed on Sephadex G-25 in a manner identical with that used for the RTL-3-Nz tryptic digest. The pattern obtained is pre- sented in Fig. 4.

All peptide fractions were further purified by paper electro- phoresis at pH 6.4. The peptides isolated are preceded by the letter T (the numbering is arbitrary). A summary of the tryptic peptides of RTL-4-Nz is presented in Table VIII and Table IX.

Tryptic Peptides of CTL-I -Nz

CTL-1 was isolated and subjected to N-bromosuccinimide cleavage in the same manner as described for the other histone fractions. The pattern obtained from Sephadex G-100 chro- matography of the N-bromosuccinimide reaction mixture was similar to that from RTL-3 and RTL-4. The yields of Ni and Nz were calculated to be 70% and 92%, respectively.

CTL-l-N2 (1.4 pmoles, 0.04% in water) was digested with trypsin as described under “Methods.” Trypsin (1 :lOO by

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TABLE IX Partial sequences of trgptic peptides from RTL-4-N%

and CTL-l-N2

Peptide

ItTL-4-N*

T2a

T3

T4

T6

T7

CTL-l-N2

T4

-

sequence

Ala-Ser-(Gly, Pro, Pro, Val, Ser, Glu, Leu, Ile, Thr, Lys) Leucine aminopeptidase, 3 hrs, 40” Ala 1.0, Ser 0.3 residues

Ser-Gly-Val-(Ser, Leu, Ala, Ala, Leu, Lys) Edman degradation

Lys-Ser-Ala-Gly(Ala, Ala, Lgs) Edman degradation

Ala-Leu-Ala-Ala-Ala-(Gly, Tyr) Edman degradation

Thr-Pro-(Val, Lys) Edman degradation

Lys-Pro-Ala-Gly-(Ala)-Arg Edman degradation Carboxypeptidase B, 2 hrs, 37”: Arg 0.97 residues

Amino acids

Lysine. ..............

Arginine .............

Aspartic acid. ........

Threonine ............

Serine. ...............

Glutamic acid. .......

Proline. ..............

Glycine. .............

Alanine. .............

Valine ................

Isoleucine ............

Leucine .............

Tyrosine ............. Total residues ........ Apparent yield (%). .. Position on Sephadex

G-25. .............

Mobility, pH 6.4. ... Charge, pH 6.4 ......

Tl

.I

- -

.95

0)

__-

1.83 1.01 (2) (1)

.94

(1) .93

(1) .96

(2) .87

(4)

.35 03)

15 22

1.04 0.99 (1) 0)

1.61 1.69 (2) (2)

1.01 1.00 (1) (1)

1.85 1.90 (2) (2)

1.07 1.06 (1) (1)

1.29 1.25 (1) (1)

0.95 0.93 (1) (1)

0.82 0.86 (1) (1)

0.91 0.99 (1) (1)

13 12 17 45

‘eak Peak Peak II II III

-0.3 +0.22 0

-1.9 +1.0 0

weight) was added at zero time and again at 2 hours. The di- gestion was terminated after 20 hours and the digest was chro- matographed on Sephadex G-25 in the same manner as RTL- 3-Nz and RTL-4-Nz tryptic digests. The pattern obtained is presented in Fig. 5.

All peptide fractions were further purified by electrophoresis at pH 6.4. The peptides isolated are preceded by the letter T (the numbering is arbitrary). A summary of the tryptic pep- tides of CTL-l-N2 is presented in Table IX and Table X.

DISCUSSION

Construction of Total Sequence of RTL-S-Nz---The arguments for establishing the total sequence of RTL-3-Nz can be followed by reference to Fig. 6. Peptide Tl is placed at the NHz-terminus because of its N-acetylation. Studies on Thl placed T2 and T3 after Tl. Studies on Peptide Cl allowed the grouping of tryptic Peptides Tl through T8 to be made. The order of T4 through T8 was established in the following manner: Th2 overlaps T5, T6, and T7, and along with studies on Clb allows their order to be established. Studies on Clb allow T4 to be placed before T5. T8 must therefore follow T7 by difference. The order of the tryptic peptides T8 through Tll was established in the following manner: Peptide C2 overlapped T8, T9, TlO, and Tll and established that a portion of T8 was at the NHi-terminus of C2 and a portion of Tll was at the COOH-terminus of C2. Th4 overlaps T8 with T9, and Th5 overlaps T9 with TlO and Tll, thus allowing the order of T8 through Tll to be established.

TABLE X

Amino acid compositions of tryptic peptides of CTL-I-N2

T2 T2a T3 T4

04

(1)

.09

(1) .97

(1)

.77 (2)

.02 0)

.Ol (2)

.99

(1)

.03 (1)

.oo

(1) .92

(2)

.95

(2)

9 6 44 31

‘eak IV

-0.3 -1.0

‘eak V

kO.7 k1.9

TS T6 __-

.oo

(1)

1.64

.76 3 ii’

(3) '(4) .84 0.44

(1)

1.04 (1)

0 (1) 6 7

62 18

‘eak Peak V VI

-0.42 0

-1.1 0

T7

1.12 (1)

3.82 (1)

3.15

0.92

(1) 0.22

0.31

0.82 (1)

0.30

4 42

Peak VI

+0.4: +1.0

T8 T9 T10

.oo .16 0) (1)

.ll (1)

.89 (1)

-

2.

Tll T12 T13

- T14 TlS

.o (2) .o (1)

.05 0)

.21 (1)

1 .o (1)

.73 (1)

.95

(1) .oo

(1)

.12 .84 (1) (1)

-

_- 1

1 .79

(21

.24

4 2 2 2 1 2 3 1 39 28 16 40 84 48 47 32

‘eak VI

-0.4! -1.0

‘eak VI

t-0.6 t-1.0

‘eak VI

k1.l b2.0

‘eak ‘eak ‘eak VI VI VII

-1.22 t1.00 0 -2.0 l-1.0 0

F

-I -I

-

‘eak I VI

-0.6( I- -1.0

-

‘eak VII

t-o.73 l-O.85

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7186 Sequence of Lysine-rich Histones Vol. 246, No. 23

AC - Ser - Glu - Ata - Pro - Ala - Gju -Thr - Ala -Ata - F?o - Ala -fro - Ato - Gtu - Lys - Ser - Pro - Ato - TI T2

* Thl cii--------------ii ---_-----__----__- ---

Thla -------------------------

*---- ----------------------------------- Cl0 *------------- ---__---- --_--------------d-e

LyS-Lg-LyS-Lys Ato-Ata-Lys-Lys-Pro-Gly-Ata-$y-Ata-Ato- Lys-Arg-Lys-Ata- T3 T4 1.5 T6

-v-p. z T7 zc- 7h2 c w --,---,,--,------------* Th2 ---- *------A---------- .-

Cl -------------------- -------_-----_--I Clb

--- -------------.------------------

Alo-Gly-Pro-~~-Vol-Ser-Gtu-Leu-Ile-Thr-Lys-Ala-Vol-A~~-Ala-Ser-Lys-Gtu- T8 T9 _ .TIO

Th3 Th4 Th5 4 p*..---*

Th3 .‘,

t c2

,----------------CT---- ___--------------

---------- --m-w*

iWrg L ‘Am- Glr<-‘-Leu - S6r - L”,“, -Ala - Alo i Leu!i Lys - Lys - Alo - Leu - Alo - Ala - $y - Gly - Tyr.

TII T12 T13 ------------------ --c---------------W, Th6 ---a--c + _ ‘r’iti *+-- -A!3 ---w--c

c3 c4 CS --+ * c C3a _ _ Cyb-

-cty-----.

FIG.; 6. Construction of thkJtot* +quence of RTL-3-N*. -, residues determined by sequen&. - - -, residues determined by

comdiiion only. See the &xt for hoinenclature of the individual peptides.

Studies on Th7 and C4 allow the sequence following Tll to be written as -Lys-Ala-&u. COOH-terminal Peptides Th8 and C5 allow the completion of the sequence following Tll to be written: -Lys-Ala-Leu-Ala-AlaX.+ly-Gly-Tyr. Thus: the en- tire sequence of RTL3-NI is established.

drophobic in nature and contains no prolines and is therefore presumably capable bf assuming some compact conformation.

Some unusual features of the structure of RTL-3-N* are ap- parent from Fig. 6. All 7 proline residues occur in the first 40 residues. From previous studies (7, 8) it was known that the first proline in the COOH-terminal portion of RTL3 does not occur before residue 105. Therefore, there is a sequence in RTL3 of at least 65 consecutive residues which does not contain any proline.

These studies further extend previous work (7-9) regarding the distributidn of the viriou‘s types of amino acids in RTL-3 and confirm the earl&;qonclusions about the remarkable segre- gation of amino:acidg into various portions of the molecule.

The fjrst’ 40 residues of RTL3-Nz are also unusual in that lysine, akanine, and proline account for 75% of the amino ahd residues. This over-all composition is’ similar to that of the last 110 residues of the whole molecule (7, 8). The 21 residues from residue number 15 to 35 contain 10 basic amino acids, including sequences of 4, 2, and 3 consecutive bases.

The,region from residue 41 to the tyrosine, on the other hand; has only 5 basic residues and no proline whatsoever. Further- more, all 9 hydrophobic amino acids (valine, isoleucine, leucine, and tyrosine) of RTL3-Nz occur in thii region.

It is possible to consider RTL3-NZ as containing two major

Comparison of Sequences of Lytine-rich Histone with Slightly Lysine-rich and Arginine-rich H&ones-Analysis of the sequences of the arginine-rich histone (histone IV) (28) and the slightly lysine-rich histone (histone IIb) (29) revealed some unusual amino acid distributions. It was found that when these se- quences were compared with the NHz-terminal region of the lysine-rich &stone (histone I), many similarities became ap- parent. Comparisons of the distribution of the different types of amino acids are shown in Fig. 7. Included for the lysine-rich histone are another 34 residues beyond NZ encompassing the region between the single tyrosine and single phenylalanine. .The partial sequence for this region is known* (8).

In all three histones there is a high proportion of basic residues in the NHz-terminal 40 residues, while in the remainder of the peptide chain this proportion is much lower. (Of course it must be remembered that only half of the lysine-rich histone molecule is shown in Fig. 7 and that the region not shown is extremely high

regions. The NH&erminal region is highly basic in nature and! a in basic amino acid residues.)

contains several prolines, which.restrict the conformations this The distribution of the hydrophobic amino acids in the three

r&on can assume. The COOH-terminal region is rather hy- a G. M. T. Jones and R. D. Cole, unpublished experiments.

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Issue of December 10, 1971 8. C. Rail and R. D. Cole 7187

IV *** * *cnw* *w.wr 4*\ * .*ilu(1 de+

Ilb l w*mB#4Bwa* *e4 a 4 *I)w l e 44 4eH4

I *www wo 4 44 ,w +*** e * -S-W-

BASIC AMINO ACIDS

IV A

Ilb A A ik A A

I A && A A AA -------)

PROLINES

I I I I I I 20 40 60 80 100 120

RESIDUE NUMBER Fro 7. Comparison of amino acid distributions in arginine- not included in the comparison. 0, positions of the basic residues

rich (IV), slightly lysine-rich (ZZb), and lysine-rich (Z) histones. The hatched bars indicate the peptide chains. The dashed arrow

lysine, histidine, and arginine; I, positions of the hydrophobic residues, valine, methionine, isoleucine, leucine, tyrosine, and’

for histone I indicates that the peptide chain continues but was phenylalanine; A, positions of the proline residues.

histones is also quite similar. In all cases, the hydrophobic DNA molecule while the other basic region is combined with a residues are concentrated in the latter half or two-thirds of the second DNA molecule, a process which could lead to the forma- peptide chain shown. There are no hydrophobic residues in the tion of nucleohistone complexes of any size. The physiological first 40 amino acids of the lysine-rich histone and only 1 in the process would be the condensation of chromatin. first 35 amino acids of the slightly lysine-rich histone. The In any case, the suggestion is that the lysine-rich histone has highest concentration of hydrophobic residues occurs in the been redesigned from the fundamental structure of the other region from residue 40 to residue 100 in all three histones. histones in order to carry out a different function. In light of the

As pointed out previously, there are 7 proline residues in the fact that arginine-rich histones are almost invariant in structure first 40 residues of the lysine-rich histone and then none for at from tissue to tissue (31), while lysine-rich histones exhibit a least another 65 residues. The proline distribution of the slightly much greater degree of variability, it seems reasonable to assume lysine-rich histone is quite similar to that of the lysine-rich his- the two classes have diverged in both structure and function. tone in this respect. There are 4 prolines in the first 10 residues It has been suggested previously that the two classes of histones and there is a space of over 50 residues coinciding with that of the may function differently in repression and derepression (2). lysine-rich histone which has no proline at all. The arginine- Comparison of Amino-terminal Regions of Lysine-rich Histones rich histone has only 1 proline, so discussion of its proline distri- -For a majority of the tryptic peptides of RTL-4-N2 and CTL- bution is of little consequence. 1-Nz, it was possible to make alignments with the RTL-3-Nz

These comparisons all point to a functional similarity for the sequence by visual inspection using the criteria of (a) direct various regions of each of the histone molecules. Furthermore, identity or (b) analogy of the composition and partial sequences. they suggest the possibility that all classes of histones may have In a few cases this could not be done because the peptides from been derived from a single prototype histone. one fraction had no obvious counterpart in the other fractions.

Whereas in the arginine-rich and slightly lysine-rich histones Since in this study overlapping fragments to order the tryptic the NHt-terminal portion of the molecule is the most likely peptides of RTL-4-Nz and CTL-l-N2 were not obtained, align- binding site for DNA, in the lysine-rich histone the extremely ments that were not immediately obvious were made in two basic COOH-terminal region appears to be the most likely site different ways. In one case, alignments were made on a con- for DNA binding. However, both basic-rich regions of the secutive residue basis (as far as was possible), so that the least lysine-rich histone (the first 40 residues and the last 100) may number of amino acid differences was obtained. In the other be sites for DNA binding. It has been suggested from physical case, alignments were made so that the basic residues (lysine studies of nucleohistones (30) that lysine-rich histones cross- and arginine) and the proline residues occurred in the same link chromatin. One basic region could be combined with one positions in the three sequences. To do this, it was necessary to

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718X Xeqaettce oj Lysine-rich Histones Vol. 246, h’o. 23

RTL-3-N2: AC - Ser - Glu - Ala - Pro -Ala 5

A) RTL-4-N2: AC -Ser - Glu -Ala - Pro - Ala -

CTL- I - Nz: AC -Ser -Glu -Ala-Pro-Ala-

Glu - Thr - Alo - Ala - Pro - Ala - Pro - Ala - Glu - Lys L5

Glu -Thr-Ala -Ala -Pro-Alo -Pro-Ala-o -Lys-

Glu - Thr - Ala - Ala - Pro - Ala - Pro- Ala - Pro - Lys -

RTL- 3-N2: AC - Ser - Glu - Ala - Pro - Ala 2

6) RTL-4-N2: AC - Ser - Glu - Ala - Pro -Ala -

CTL-I -N2: AC - Ser-Glu- Ala- Pro-Ala-

Glu - Thr - Ala - Ala - Pro- Ala - Pro -Ala - Glu - Lys15

Glu-Thr- Ala-Ala-Pro-Ala- Pro-Ala-- -Lys-

Glu - Thr - Ala - Ala - Pro- Ala - Pro - Ala - Pro - Lys-

Ser - Pro - Ala - Lys - Lys - Lys - o - Lys -Ala - Ala E5

Ser - Pro - Ala - Lys - Thr - Pro - Val - Lys - Ala - Arg -

Ser - Pro - Ala - Lys - Thr - Pro - Val - Lys - Ala - Alo -

25 Ser - Pro-Ala - Lys- Lys- Lys- o - o - o - Lys-

Ser - Pro- Ala - Lys- Lys- Lys-Thr - Pro- Val- Lys-

Ser - Pro - Ala - Lys - Lys- Lys - Thr - Pro - Val - Lys-

35 35 Lys - Lys - Pro - Gly - Ala - Gly - Ala - Ala - Lys - Arg -

Lys - Lys- Lys- Ser - Ala - Gly - Alo - Alo - Lys- Arg -

Lys - Lys- Lys- Lys - Pro - Alo - Gly - Ala - Arg - Arg -

45

Alo - Ala - Lys- Lys- Pro- Gly - Ala- Gly- Ala - Ala-

- - Alo - Arg - Lys- 0 - Ser - Ala - Gly - Ala - Alo -

Ala- Ala - Lys- Lys- Pro --o - Ala - Gly - Ala - n -

45 Lys - Ala - Ala - Gly - Pro - Pro - Val - Ser - Glu - Leu -

Lys - Ala - Ser - Gly - Pro - Pro - Val - Ser - Glu - Leu -

Lys - Ala - Ser - Gly - Pro- Pro- Val - Ser - Glu - Leu-

I le - Thr- Lys- Ala -Val - Ala - Ala - Ser - Lys- Glu !5

Ile -Thr- Lys-Alo-VaI-Alo-Ala-Ser-Lys-Glu-

I le - Thr- Lys- Alo- Val- Ala - Ala - Ser- Lys-Glu -

65

Lys-Arg-Lys-Ala- Ala-Gly- Pro- Pro- Val-Ser-

Lys-Arq-Lys-Ala-Ser-Gly- Pro- Pro-Val- Ser-

Arg-Arg- Lys-Ala-Ser-Gly- Pro- Pro-Val- Ser-

Glu - Leu- I le -Thr - Lys- Ala- Val - Ala - Ala - Ser F5

Glu - Leu- I le - Thr- Lys - Ala - Val -Ala - Ala - Ser -

Glu - Leu- I le - Thr - Lys - Ala - Val - Ala - Alo - Ser-

65 Arg - Asn- Gly - Leu- Ser - Leu- Ala - Ala - Leu - Lys-

Arg - Ser- Gly - Vol - Ser - Leu- Alo- Ala - Leu- Lys-

Arg - Ser - Gly - Val - Ser - Leu - Ala - Ala - Leu - Lys -

Lys- Glu - Arg - Asn- Gly - Leu- Ser - Leu- Ala - Ala -

Lys-Glu - Arg- Ser- Gly- Val- Ser- Leu-Ala -Ala -

Lys- Glu - Arg - Ser - Gly - Vol - Ser - Leu- Ala - Ala -

73 75 Lys - Ala - Leu - Ala - Ala - Gly - Gly - Tyr.

Lys- Ala- Leu- Ala- Ala -Ala - Gly -Tyr.

Lys - Ala - Leu- Ala - Ala - Ala - Gly - Tyr.

Leu - Lys - Lys- Ala - Leu- Ala - Ala - Gly - Gly - Tyr.

Leu- Lys- Lys- Ala- Leu- Ala - Ala - Ala - Gly - Tyr.

Leu- Lys- Lys- Ala - Leu- Ala- Ala- Ala- Gly - Tyr.

FIG. 8. Alignment of RTL-4-N% and CTL-l.N2 tryptic peptides with the total sequence of RTL-3-Nz. A, alignment made on a con- secutive residue basis which yields the minimum number of amino acid differences; B, alignment, made to yield maximum identity of the basic residues (lysine and arginine) and proline residues. See the text for details.

TABLI~ XI

Amino acid compositions of NP peptides from various lysine-rich histones

Amino acids

Lysi ne. .............

Arginine ............ Aspartic acid. ....... Threonine. .......... Serinec. .............

Glutamic acid ....... Proline. .............

Glycine. ............ Alanine. ............ Valine ............... Isoleucine ........... Leucine ............. Tyrosine ............

Total. ..............

RTL-3&z RTL-4-N% CTL-l-N?

P

13-14 2

1 2 5

4-5 7 6

22

2-3 1

4-5

1

70-74

116

13 13 12 14 2 3 3 3 1 0 0 0 2 3 3 3

5 6 8 6-7 5 4 4 4 7 8 7 7-8 6 5 4 4

22 21 21 20 2 4 4 4 1 1 1 1 5 4 4 3-4

1 1 1 1

72 73 72 70-73 I-

I II I II

12 3

0 3 7 4

9 4

21

4 1 4

1

73

* I, residues expected from amino acid analysis for molecular weight 7000.

b II, residues found from peptide and sequence analysis. c Serine not corrected for hydrolytic losses.

make gaps in each of the three sequences in several places.

These two possible alignments (there are, of course, numerous alternatives) are depicted in Fig. 8.

After tryptic digestion of a calf thymus lysine-rich histone similar to CTL-I, Murray (32) isolated peptides equivalent to our Tl, T5, T7, T13, and T14. Butler, Johns and Phillips (33) report the isolation of peptides like our T6, T8, and T13 from tryptic digests of the whole mixture of calf thymus lysine-rich histones. Indeed the composition assumed for our obviously contaminated T6 is that reported by the latter workers.

The peptides T9 (Ala-Lys) from RTL-4-Nz and CTL-l-N2 are not included in the comparisons. The choice to omit these peptides was made because of the unpublished experiments of Jones and Cole.3 They found that several equivalents of Ala- Lys are released from peptide N, (the carboxyl portion of lysine- rich histone) by tryptic digestion. If Fraction NB were even slightly contaminated by Ni, subsequent tryptic digests of Nz could give the peptide Ala-Lys in high enough yield to allow its isolation while t.he other peptides of Ni (at much lower levels) are below the level of detection.

The comparison made here points out that there are distinct differences in the primary structure of the three histone frac- tions studied. From amino acid analyses on Ns from the three histones, some primary structural differences were expected. The compositions expected from amino acid analyses were found

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Issue of December 10, 1971 X. C. Rail and R. D. Cole 7189

to be in close agreement with those found from peptide analyses (Table XI). The number of differences that are shown in Fig. 8 are as follows: from Fig. 8A ; 11 differences when RTL-3.Nz is compared with RTL-4-N2; 14 comparing RTL-3-Nz with CTL- l-N*; 7 comparng RTL-4-N* with CTL-l-N*; and, from Fig. 8B; 12 comparing RTL-3-Nz with RTL-4-Nz; 11 comparing RTL-3-Nt with CTL-I-Ns; 7 comparing RTL-4-N, with CTL- 1-Nz.

It should be remembered that these are minimal differences, since alignments have been made to masimize identities among t.he three histories. The number of d,fferences could conceivably be greater than those enumerated here. Furthermore, it should be remembered that the portion of the histones studied accounts for only one-third of the total histone molecule. As yet little is known about differences in the COOH-terminal regions of lysine- rich histories.

While the studies presented here clearly demonstrate that the lysine-rich histones resolved chromatographically do differ significantly in primary structure, it is not possible to state that thme structural differences alone are responsible for chromato- graphic resolution. It can be stated, however, that side-chain modifications alone do not account for the resolution, since the histones resolved are not otherwise identical. Indeed, neither the in v&o phosphorylation of lysine-rich histone (34) at residue 40 (Fig. 8B) nor the treatment of the histone with alkaline phosphatase (6) alters the chromatographic pattern appreciably.

It was surprising that of the three histones studied, it was RTL-4-Nz and CTL-l-N2 which were seen to resemble one another rnost closely in primary st.ructure. This is particularly intriguing because RTL-4 is the last of the rabbit thymus lysine- rich histones to emerge from Amberlite IRC-50 columns while CTL-I is the first of the calf thymus lysine-rich histones to be eluted. Furthermore, Bustin and Cole (2) showed that RTL-4 and CTL-1 are almost indistinguishable from each other in poly- acrylamide gel electrophoresis but have higher mobilities than RTL-3. Since mobilities in gels depend on both charge and effective size, there must be multiple factors for determining the relative positions of the various histories in gels and on columns. It may be concluded, however, that primary structural differences are a major contributing factor.

Certain other aspects of the structural differences can be dis- cussed here. It can be seen in Fig. 8 that there are certain re- gions which appear to be much more highly conserved than others. The region of 27 residues from position 14 to 40 in the three histones accounts for almost all of the amino acid dlffer- ences, including all of those which are not of a strictly conserva- tive nature. On the other hand, the region from residue 41 to the tyrosine seems highly conserved. RTL-4-Nz and CTL-l-N, have identical sequences in this region and differ from RTL-3-N, in only three amino acids. These interchanges are conservative replacements.

A possible reason for the high conservation of the sequence of this region is that it may be the recognition site for phosphorylat- ing enzymes. A major site for phosphorylation of thymus lysine-rich histone has been shown to be at the same serine resi- due (residue 40, Fig. 8B) in calf thymus by Langan (35) and in rabbit thymus by Langan, Rall, and Cole (34). The kinase enzymes have a high degree of specificity for this particular serine residue, so there must be some factor in the histone which imparts this specificity, either a particular primary sequence or a particular conformation generated by the sequence. It should

be noted that one of the amino acid replacements (alanine for serine at residue 40, Fig. 8B) eliminates this major site of phos- phorylation in RTL-3 (34).

The variability from pos;tion 14 to 40 might. be corisidered as due to the specific involvement of this region in several func- tionally distinct molecular interactions. In contrast, it could be argued that there was a lack of selective pressure on tliis region during evolution because litt)lr specZicity is involved in its molecular interactions, and that multiple, slightly varied geircs (for lysine-rich histones) are carried in the animal. The evidence favors the not’ion of functional diversity among lysinc-ricli histories: (a) the quantitative balance of the subfract.ions of this histone class vary from tissue to t,issue (36) ; (b) the subfractioiis have different turnover rates (37); (c) the effect of hormones on rates of amino acid incorporation varies from one subfraction to another (38). The multiplicity of lysine-rich histones thus stems to have physiological significance and it is not unreasonable to postulate that the differences observed in primary structure are the basis for the diversity in function.

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S. C. Rall and R. David ColeLysine-rich Histones

Amino Acid Sequence and Sequence Variability of the Amino-terminal Regions of

1971, 246:7175-7190.J. Biol. Chem. 

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