rat pancreatic ribonuclease ii. amino acid sequence

tliochimica et Biophysica .4cta, 3Io (t973) r61-I73 (C-) Elsevier Scientific Publishing Company, Amsterdam - Printcd in The Netherlands

~BA 36394

RAT PANCREATIC RIBONUCLEASE

II. AMINO ACID SEQUENCE

J. J. B F I N T E M A " AXD M. ( ;RUBI' ;R

Biochendsch Laboratorium, Rijksuniversiteit, (;roningen (The Netherlands)

(Received November 8th, 1972 )

SUMMARY

"file amino acid sequence of rat pancreatic ribonuclease was determined using I2o mg of the enzyme oxidised with performic acid.

Tryptic and chymotryptic peptides were obtained by gel filtration and chromatography on ion exchangers, and if necessary cleaved to smaller fragments. In tile discussion of the results, emphasis is laid on specific problems encountered during this study, e.g. conversion of asparagine residues and N-terminal glutamine residues to products which interfere with the Dansyl-Edman procedure. All amino acid residues could be located unambiguously. The position of one amide group could not be established with certainty. The total sequence consists of three inner sections in addition to the N-terminal and C-terminal sections. Since the order of the three inner sections could not be determined directly, due to the lack of overlapping peptides, these three inner sections were ordered by homology with other ribonucleases.

Compared with bovine ribonuclease, rat r ibonuclea~ contains 3 amino acid residues extra at tile N-terminus, and 41 substitutions in the remainder of tile chain. However, all amino acids which are part of the active site, or are implicated in activity or conformation, are identical in both enzymes.

INTRODUCTION

The isolation of rat pancreatic ribonuclease has been described in the accom- panying paper ~. The elucidation of the primary structure of the enzyme was under- taken because of the striking differences between the rat and the bovine enzymes in several physicochemical and enzymic properties, and in amino acid composition and peptide maps ~. Knowledge of the amino acid sequence might yield valuable infor- mation about structure-function relationship of mammalian pancreatic ribonucleases, especially since the conformation of bovine ribonuclease has been determined a&

• This work is par t of a thesis * submit ted by J. J. Beintema in fulfillment of the require- ments for the degree of Doctor of Science.

162 J . J . I,IFANTFMA, 31. GI/UBI:.I~,

Another point of interest is the ew)lution of pancreatic ribonucleases since this protein proves to be one of the most variable ma,nmalian proteins studied thus far 5.

A short comnmnication of the work described in this article has been published earlier G. A comparison of the sequence of rat ribonuclease with the coifformation of bovine ribonuclease S has t×~en made t)v \Vvckof[ v.

MATERIALS AND .METttODS

Rat ribonuclease was prepared as described in the preceding paper".

Proteo@tic e~t<V,tes Trypsin (z times crystallised, salt free, lots 839 and 0118), a-chymotrypsin

(3 times crystallised, lot 6o18-19), carboxypeptidase A, treated with diisopropy,1- fluorophosphate (COA-DFP, lot 6134) and carboxypeptidase B treated with diiso- prot~ylfluorophosphate (COB-DFP, lot 32) were obtained from Worthington Bio- chemical ('orlx)ration. Papain (2 times crystallised) was a product from Nutritional Biochemical Corporation.

Column materials Dowex I-X2 (2oo-4oo mesh), Dowex 5oW-X2 (2oo-4oo mesh) (Fluka) and

cellulose phosphate (7.4 mequiv/g, batch No. 22; \Vhatnlan) were regenerated before use .

Sephadex 6-25 and Sephadex G-Io (Pharmacia) were pretreated as indicated by" the manufacturer before use. The volatile buffer salts were prepared from A.R. grade reagents (Merck, lcluka). The heterocvclic bases were distilled before use. Hvdrindantin was prepared as described in ref. 8. Phenylisothioeyanate (Eastman Organic Chemicals) was purified by distillation in vacuo. The pyridine used in the Dansvl-Edman procedure was refluxed with l)hthalic anhydride for 3 h and distilled. Trifluoroacetic acid and ethyl acetate (Merck) were distilled before use. Ethylene chloride (Merck) was purified according to Sj6quist 9. The purified phenylisothiocyanate, pyridine and triftuoroacetic acid were stored under nitrogen at --18 :'('; the ethylene chloride at 2 °C. All other reagents were A.R. grade.

Digestions Rat ribonuclease (about 4o rag) was oxidised with performic acid according to

Hits ~°, dissolved in 13 ml water and digested with o.4 mg trypsin at 37 °C. The trypsin solution was pretreated with o.o6 M HC1 during I8 h at 37 ~(; as described in ref. ii. The pH was maintained at 8.o by addition of o.i M NaOH with a pH-stat (pH-stat TTT I, Radiometer, Copenhagen). Tryptic peptides have been isolated in two separate runs. In the first, the digestion was terminated after 6 h by increasing the pH to 9.() and immediately starting chromatography on l)owex I. In the second run, a second o.4-mg portion of trypsin was added 2 h after the first one, and the reaction was terminated 4 h later by the addition of I ml acetic acid. After lyophilisation the digest was fractionated on Sephadex G-25.

In the same way, 4 ° mg of oxidised rat ribonuclease were digested with o.25 nag chymotrypsin. At 2 h, a second portion of o.25 mg enzyme wa~s added and 4 h later the reaction was terminated by the addition of t ml acetic acid.

Peptide "1"-5,6 and "I'-2, 3 were digested with chymotrypsin as described in ref. tz .

RAT I 'AN( 'REATIC I,UBONUCI.I.IAS]-. II 1 6 3

Peptides T-a, T-I6 and T-I4, I5b were digested with 0. 5 mg chymotrypsin per #mole peptide during 4 h at 37 °C in a w~lume of 1.5 ml. The reaction mixture was maintained at pH 8.o by the addition of o.oi M NaOH with a pH-stat. Tryptic digestions of the peptides C-I and C-2 were performed in the same way. The re.action was started with 15/~g enzyme per IOO nmoles peptide and an equal portion of trypsin was added I h later. Peptide ]'-4 was digested with papain as described by Hirs ~a %r the digestion of the homologous peptide (O-Tryp 4)Chy I of bovine ribonuclease. Peptides T-4-P-c and -d and T-lob (the latter after having been subjected to two Edman cycles) were hydrolysed with o.o3 M HC1 at Io5 °(" in vacuo during 5 h~a.

Column cl, r , malography Column chromatographic separations of peptide mixtures on Dowex I, Dowex

5o and cellulose phosphate were performed as described by Hilse and Braunitzer 15. Some minor modifications, for instance slight changes in buffer compositions, may be found in ref. I (pp. 99-m2). Gel filtrations on Sephadex G-a 5 and Sephadex G-io were perfi)rmed according to Eaker ct alJ 6. In a few instances, we used paper chromatography for the purification of a peptide followed by elution from the paper with 5% pyridine.

The column eluates were analysed by performing alkaline hydrolysis ~7 followed by the ninhydrin reaction ~ on o.oi o.4-ml samples from the collected fractions. The peak fractions were collected, evaporated to dryness, dissolved in a small amount of water and evaporated again in vacuo over P,,Os and solid KOtt to remove completely all buffer salts (which interfere in the dansyl reaction). Collidine and lutidine acetate are difficult to remove by' evaporation from peptides eluting between pH 6 and 8 from Dowex I; these buffer salts were removed by gel filtration in o.2 M acetic acid on Sephadex G-Io.

The purity of the separated peptides was checked by paper chromatography x~ of the peptides, and both paper chromatography ~s and high-voltage electrophoresis (at 5ooo \.q0 with an electrophorator from Gilson Medical Electronics) of the peptide hydrolysates. Paper electropherograms of the hydrolysates of pure peptides were scanned with a Spinco Analytrol serve-type paperstrip scanner. From these, the amino acid compositions of several short i~ptides was calculated (see "fables I -V') . The amino acid compositions of the other peptides were determined as described in the preceding paper 2. Samples hydrolysed for 2o h were analysed, with the exception of peptide "1"-16, which had to be hydrolysed fl)r 72 h since it contains three isoleucine residues in succession.

l ) a n s v l - E d m a n degradation

The amino acid sequences of tile peptides were elucidated by the Dansvl- Edman procedure"°,2h Identification of the Dansyl amino acids was performed by paper electrophoresis as described in ref. 2I with a few minor modifications a. The dansyl amino acids were liberated by hydrolysis during 5 h, with exception of the derivatives of isoleucines-io6 and -lO 7 from the sequence Ile -Ile-Ile (io6-1o8), which were hydrolysed during 48 h.

We also identified many amino acid residues as their phenylthiohydantoin derivatives. Therefl)re, the cleavage reaction in the Dansyl-Edman procedure was

" See f l ) o t n o t c o n flfllowin~4 pa~e .

164 J.J . BEINTEMA, M. GRVBER

performed in the following way: the dry phenylthioearbamyl peptide (obtained after the coupling reaction and extraction with benzene) was incubated during 3o rain at 50 °C with 25/~1 trifluoroacetic acid. After evaporation of the trifluoroacetic acid in vacuo, the residue was dissolved in o.I5 ml water, extracted twice with ethylene chloride (2 ml and I ml) and dried in vacuo over solid KOtt and P2Os. The dry residue was used for the next Dansyl reaction and Edman degradation step. The combined ethylene chloride layers were used for the preparation "2 and identification "a of phenylthiohydantoin derivatives of amino acids.

The (?-terminal re.sidues of the peptides T-4-P-ca and C-8,9 were determined with carboxypeptidase A, and those of peptide 1"- 5 with carboxypet)tidases A and B.

Nomenclature of the peptides and purification scheme Arabic numerals with prefix T refer to tryptic peptides, numbered from the N-

terminus of the molecules (an exception is peptide T-a which is a mixture of T-8, 9 and T-8). Arabic numerals with prefix C refer to chymotryptic peptides in the same way. Peptides obtained by further cleavage are denoted by a subscript C (ehymo- trypsin), T (trypsin) or P (papain) followed by a letter in small tyIx ' according to their elution position at, e.g. ion-exchange chrolnatography.

(;enerally, all primary digestion products were purified by fractionation on Dowex I and Dowex 50 successively (tryptic peptides; Run I) or on Sephadex G-25 followed by the two ion exchangers (tryptic peptides; Run 2 and chymotryt~tic peptides). The purification procedure of the secondary digestion products has been less uniform. Usually, fewer steps were used. (Details may be found in ref. I (pp. I2O, 121, 124 and 126); for systematical reasons, however, we changed the nomenclature of the peptides.)

RESULTS AND DISCUSSION

Tile amino acid compositions of the peptides and the results of the l)ansyl- Edman degradation and carboxypeptidase digestions are given in Supplementary data, Tables I -V ' and Fig. 12. Figs I - I I illustrate the purification procedures used for the isolation of the peptides ; a choice has been made of the most important eolunm chromatographic steps. In the following, only a few aspects of the experimental work will be discussed.

Two separate digests of tryptic peptides (Run I and Run 2) and one chvm-- tryptic digest have beelT used for the isolation of peptides; the second tryptic digestion differed from the first in the addition of a second portion of trypsin after 2 h. This caused some minor differences in the relative recoveries of several tryptic peptides and, also, the presence in Run 2 of one tryptie peptide (T-14) which had been absent in Run I. The preliminary fractionation of the tryptic peptides on Sephadex G-25 in Run 2 proved to be a real improvement of the purification proce.dure: During acidification of the tryptic digest, before the gel filtration a precipitate insoluble in 0.2 M acetic acid formed, which proved to be the C-terminal peptide. "I'-~6 in a

• Supplementary data to this article giving details of the amino acid compositions of the peptides isolated are deposited with, and can be obtained from: Flsevier Scientific l'ublishing Co., BBA Data l)el)osition, P.(). Box 33 ° , Amsterdam, The Netherlands. Refert'ncc should 1)c made to BBA/l)D/oo4[36304/31o (1973) t(~o.

RAT PANCRliATIC RIBONUCI.EASE. II 16 5

H _ ~ I GRADIENT pH 7.4

A570 T-3=T-6 Q.6- T-11

DOWEX 1

0/.- T- S , 6

T- 7a T-9

I I L T-13

ivy' Y~v~,, " vw_.,.~ b I , c 100

I . , pH 6.6 pH G.0 ~ pH 5.5 ~ pH &.5 - ~ l e ~"

I ~. ~ pH

o . . . .

T-1 ~ °" ~o~ ~ T-16

T-2 T-14,15~" " 5 T-5 T-12,13 , - ,b I / , , ~

T-lOa [~ . . ,,ls.T-lobl ~ / I

l ' ~ _ L - h,__ _ I _ 26o 3bo

FRACnON NU.BER

Fig. r. Fract ionat ion of tryptic peptides (Run z) on I)owex i (44 c m x z.o cm) at 37 °C. Equili- brat ion of the column anti elutinn of the first zo fractions at pH 9.0. Gradient elution with a cons tant -volume mixing chamber (r5o ml). p t [ values of the buffers in the open chamber art; given in the top of the figure. Flow rate, 6. 5 ml/h; fraction size, 1.6 ml; ninhydrin reaction on o. t -ml samples from each fraction. The fractions indicated by a horizontal bar were combined.

recovery of 50% and contaminated with very little other material• In addition a first separation of the peptides according to size proved to be very useful. The elution of the soluble peptides from Sephadex G-25 is shown in Fig. 2. In Peak a, a mixture of peptidcs T-8 and T-8, 9 (about one-quarter of the molecule) was obtained free from all other pept ides Its high recovery (9o%) made possible the elucidation of the

A570

1.0"

0.5-

AS70

1.0-

SEPHABEX G-25 0.5-

' ~ a , b [ c | d , t ~

5'0 100 FRACTION NUMBER

A~ 7_1 IGRAOIENT u[ J..- 0.5 M ~ 1.0 M~2.0M.

024 T-:,b • ] T-Ta

CELL-® ~, ~ \

. , I P m ,

20 ~,0 FRACTION NUMBER

Fig. 2. Fract ionat ion of the t rypt ic peptides (Run ~) soluble in 0.2 M acetic acid on Sephadex (;-25 (x4o cm × x cm). Elution with o.2 M acetic acid, 2. 5 ml/h; fraction size, z.o ml; ninhydrin reaction on o.o5-ml samples frmn each fraction.

Fig. 3. Fract ionat ion of peak b (Dowex i ; Run i ; Fig. I) on cellulose phosphate (26 cm × 0.3 cm) at room temperature . Equil ibrat ion of the column and elution of the first 5 fractions with o.o 5 M pyridine formate buffer, pH 2.5. Gradient elution with cons tant -volume mixing chamber (to ml). Concentrat ions of the buffers in the open chamber are given in tile top of the figure (the pH values of these pyridine formate buffers are given in Fig. 4). Flow rate, 2. 5 ml/h; fraction size, 0. 7 ml; ninhydrin reaction on o.I -ml samples from each fraction.

I ( )0 J . J . BI,:INTI.:MA, M. ( ;RUBI .R

A57D I J GRADIENT

0.41 ~,.- 0.27 M ~ - 0.5 M ~ 1.0 M- -~ 2.0 M ~ - - pH-3.2 pN-3.4 pH- 3.4 pH- 3.7

OOWE 0.2 , , .

T - 7 . T-9 I I I~

io ,:'o 6'0 FRACTION N~.k~4BE R

0,~

A570

0.2,

] GRAOiENT I.,,--0.27 M~0.5 M ~ I . 0 M

DOWEX 50 T-~ D ~

ti T4~,13 :l ! !i i

' t

2'0 4'0 FRACTION NUMBER

Fig. 4. F r a c t i o n a t i o n of l ' e a k c (l)<)wex i ; R u n i ; Fig. r) on l)<)wex 5o (~7 cm ". o.9 cm) a t 50 ( ' . V o l u m e of m i x i n g c h a m b e r , z5 m l ; e q u i l i b r a t i o n b u t t e r a n d buffers used in t he g r a d i e n t e lut i<m as in Fig. 3. F l o w ra te , 0 ml,"h ; f r a c t i o n size, 1-3 ml ; n i n h v d r i n r e a c t i o n ml o. I -ml s a m p l e s f rom each f r ac t i on .

Fig. 5. F r a c t i o n a t i o n of t he t r y p t i c p e p t i d e s o b t a i n e d a f t e r c h r o m a t o g r a p h y on S e p h a d e x G-2 5 ( P e a k s c a n d d; R u n z; Fig. 2) a n d I ) o w e x i ( peaks e l u t i n g a t p t l 7.5; see Fig. l ; p e a k e), Stl~,'ces- s ive ly , <m l ) o w e x 5 ° ( 8 c m :< 0. 3 c m ) . V<)lume of m i x i n g c h a m b e r , 2 o m l ; o t h e r con<l i t ions as in F ig . . I .

A570[ T 'a 'C

sT°l I

_ , : ; ' ' 20 20 20 40

FRACTION NUMBER FRACTION NUMtBER FRACTION NUM6ER

Figs ~> a n d 7. F r a c t i o n a t i o n of t he t r y p t i c p e p t i d e s o b t a i n e d a f t e r c h r o m a t o g r a p h y on S e p h a d e x (;-,~5 ( P e a k d ; R u n -'; Fig. 2) a n d D o w e x I, s u c c e s s i v e l y , on l ) o w e x 5 ° (4 cm . o. 3 cm) . ( ) t h e r c o n d i t i o n s as in Fig. 5- Fig. ¢>: t r y p t i c p e p t i d e m i x t u r e e l u t i n g a t p t l ().4 f rom l ) o w e x l (see Fig. x ; P e a k g). Fig. 7: t r y p t i c p e p t i d e m i x t u r e e l u t i n g a t p l l 6.0 f rom l ) o w e x i (see FiR. 1 ; P e a k h).

Fig. 8. V r a c t i o n a t i o n of c h . w n o t r y p t i c p e p t i d e s f rom the t r y p t i c p e p t i d e T-a (T-a-C p e p t i d e s ) on S e p h a d e x ( ; - ' 5 ( t . to cm ;.. o. 3 cm) . l~,lution w i t h <~., .~1 ace t i c ac id , I m l / h ; f r a c t i o n size, o.z m l ; n i n h v d r i n r e a c t i o n on O.Ol-ml s a m p l e s f rom each f rac t ion .

A$70 A570 1.0 .1.0

A570 ]GRADIENT J SEPHADEX G- 25 I~--~'--DH 6 7 ~ D H 60~pH5.0"I pH 0.5, -0.5

/ T a ' C d - ' ° - d I

., , .L, ! 20 40 5'o 100

FRACTION NUMBER FRACTION NUMBER

Fig. q. F r a c t i o n a t i o n of p e a k d (Fig. 8) on l ) o w e x i (4 cm > 0. 3 cm) a t 37 ~('. V o l u m e of m i x i n g c h a m b e r , 3 ° m l . C o l u m n e q u i l i b r a t e d a t pH 7.4. F low ra te , 3.5 m l / h ; f r a c t i o n size, T.t m l ; nin- h y d r i n r e a c t i o n on o . z - m l s a m p l e s f rom each f r ac t ion .

Fig. lo. F r a c t i o n a t i o n of t he c h y m o t r y p t i c p e p t i d e s on S e p h a d e x (;-- '5. S iune c o n d i t i o n s as in Fig. -.

R A T P A N C R E A T I C R I B O N U C I . E A % I , ~ . I I 16 7

sequence of this part of the molecule. Peak b contained peptide T-I4,I 5 and a small amount of T- 4. Most other peptides were present in Peaks c and d. In Run z, it had been rather difficult to separate the short acidic peptides T-I2 and T-Io from the larger peptides, but in Run e, these two short peptides could tx; recovered rather easily from Peak d of the gel filtration. Peak e contained two short peptides (T-z and T-9), and free arginine from two positions in the molecule.

Several lysyl and arginy'l bonds were cleaved incompletely or not at all in rat ribonuclea.~e. As expected, there was no cleavage of the I .vs-Pro (4I-4 2) bond. But

I - H ~ GRADIENT J pH P = ~ p H T . / ~ - - pH6.6 pH 6.0 , , pH5.5 • • pH/,.5~ 9

A570 - --o . . . . . . . . . o--,, \~Ep H 0.6j -a ~o~

-°t DOWEX 1 5

50 100 150 FRAC'riON NUMBER

Fig. r r. Fractionation of Peak c of Fig. zo on Dowex x (20 cm x 0. 5 cm) at 37 °C. Volume of mixing chamber, 5 ° ml; ttow rate, 3.5 ml/h; fraction size, z.2 ml; ninhydrin reaction on o.o5ml- samples from each fraction; other conditions as in Fig. i.

we also observed no cleavage of the l ,ys-Phe (7--8) bond and only very limited cleavage of the L y s - T y r (91-92) bond. The Lys-Asn (66-67), Arg-Leu (85-86), Lys - Arg (9-1o) and Lys Arg (31-32) bonds were only partially cleaved. These incomplete cleavages resulted in a much larger number of tryptic peptides than expected from the amino acid composition. We did not observe non-specific cleawige by trypsin.

Overlappi,zg sequences Generally, we experienced no special problems in obtaining pure tryptic pep-

tides from the whole molecule in sufficient amounts. The fractionation of chvmo- t rypt ic peptides, however, was more difficult, probably because of many non-specific cleavages. Therefore, we were unable to isolate all chymotrypt ic peptides, necessary fnr establishing all overlapping sequences between t rypt ic peptides. For this reason, the evidence for the sequence of rat ribonuclease consists of five independent parts (Fig. 12). Only one of these sequences contains N-terminal glycine, which was demonstrated to be the N-terminus of the intact protein. The absence of a basic C-terminal residue in the last sequence shown in Fig. 12 locates it at the C-terminus. The other three sequences were put in the order shown bv homology with beef and pig ribonuclease.

Dansyl-Edman degradation We combined tile Dansyl -Ednlan procedure with identification of tile amino

108 j . j . BI.HNTETqA, M. (;RUBI'R

-3 1 5 ~O 15 ~0 25 30

ZK ,r~, ~. > ( ¢~-v-~-----.-) ( ¢_% ~ > - ' - - ' - " T ' . . . . . . ,1,

. . %~ ^ =ix't = r¢ { -"~ "-7 ---7 ~'---0- - 5, ~ -~ ~-'~( KT-2~yc.~ ~ . tO.C3 I; ~Cl "7 --7

( c-, ) ( c-, ~ > ( c-~t )

35 40 4~ -G 1 n -G 1~ -~e t -~hr-L~ i~-~ ly -~l r-Cy e-~y e-Pro-Va 1 -Aen-Thr-Phe-

( T-7a ) l ~ T-~ " T-8,9 + T~

( C-T= )l ~" clkyt~o t r.,/~ e ~. n ' ] "-'=~ - " l -'-~ --"7 -4, ( e--~-c--.~ )

:::X - " --" - - ' - " - - --¢ --"

< c-a > 50 D. ~ 60 65 ~0

-Va I -H z e-G I u-Pr o-L~u-G I u-A ~-Va : -G I r.-A : &-I I *-¢y e--~% r-G : n-C Iy-G in-Va I -Th~-Cy 8-Ly s-A sr.-G ~ rg-Ae ~en~y~ ~ e-~ s-

* ) }~--.---¢-,0, > T ~ - ~ t u t ~ 0£ T-8,9 + T-~ ) ~-"t-'-/

-T- I Ob-----'-~

(

- z - - , - - - - - z - z - - - , - - - - - , - , 7 , ~,.~_~, --, > - , - ~ "-Z -z - ' , --, --, --" - ' , - ' ,

(-.--¢.-,..c-r, > ( - - : - . - e - .~ -~ " - " -"Z - 7 " - "

75 80 8~ 90 95 ~00 -Se r . ~ , r . ~ e r-r~nr-L*u-lrg-I 1 *-Thr-As p-~ye-Arg-Leu-l~ve~3 ~ * ~ e ~ o ~ r @ ~ e n ~ y s ~ r ~ s ~ r ~ I n ~ e ~ I u - ~ e -

( ~_, >( ~_~.~,. ---)~ ~-~4-----4 --7---,---7--7... a ---: --~ ----~ ----y ---'r--'p ~ ¢-~4.~, )

( ¢-~2 ) <__~_,~ ---~ - - - t .--p - - 7 - - - 7 - ' - ,

- ' ~ -- '2 " - ' / "= - / - - -7 "--1 ( t1'-14' 15b ) ( ¢ - ~ )-'-* "--7 ( c-5~ ) --"7 "-'7. - '7 - '7

.T-14,15b-C-b • 10 120 124 lo5 115 ( = ~ u t * ) -H t e-I i, -I le-I ~ e-~ : ~-Cy *-Aep-~ ly-Aen-Fro Jb~r -v& I -Prc-Va l-Ri s'~e-Ae p'~: &-~e ~Va I

( . . . . . ~'-~ > < e - ~ - - -"~ -"/ "-7 chyl~*, ry'plz n t ~[~ ( C"~, 7--

-c-~( .<-~ ) ( c-*,9 ) __: _.-( --~ --Z ---, ---~ ---, ---, ---, v--

c-~.., ) ( c-~ > < c-9 )

I;ig. ~ 2. Amino acid sequence of rat pancreatic ribonuclease. ~ . . . . , peptides isolated; , . secondary cleavage; - ,, identified ~ dansyl derivative; ..... , identified as phenyl th iohydan- toin derivative ; _.'.. identified both as dansyl and as phenylthioh.vdantoin derivative ..--, cleaved ILv carboxypeptidasc. Cvs and Met were ahvavs identitied as cysteic acid and methionine suI- time, respectively, or their derivatives.

a c i d s sp l i t off as t h e i r p h e n y l t h i o h y d a n t o i n d e r i v a t i v e s . T h e i d e n t i f i c a t i o n o f t h e

p h e n y l t h i o h y d a n t i o n d e r i v a t i v e s w a s o n l y o c c a s i o n a l l y p o s s i b l e b e c a u s e o f t h e s m a l l

a m o u n t s o f t h e p e l ) t i d e s a v a i l a b l e .

Sequence 88-ro 4 T h e low r e c o v e r i e s o f p e p t i d e s T - i 4 , t 5, C-6 a n d C-6, 7 m a d e t h e s e q u e n c e d e t e r -

r u i n a t i o n o f t h i s p a r t o f t he m o l e c u l e less u n a n a b i g u o u s t h a n t h a t o f tile ()t i ler p a r t s .

T h e a m o u n t s o f p e p t i d e a v a i l a b l e w e r e t o o s m a l l to l )er f l ) rm all t h e r e q u i r e d a n a l y t i c

p r o c e d u r e s ( a m i n o ac id a n a l y s i s , D a n s y l - E d m a n d e g r a d a t i o n ) .

W e d id n o t s u c c e e d in o b t a i n i n g p e p t i d e C-6, 7 in a p u r e s t a t e . A f t e r c h r o m a t o -

RAT PANCREATIC RIBONUCLEASE. II 16 9

graphy on Sephadex G-25 and Dowex I, the amino acid analysis ('Fable V) suggested the presence of two peptides (98-I~5 and i o I - I I 5 ) still contaminated with other material. After another purification step on Dowex 5o, an N-terminal Asx-Ser sequence was demonstrated in the remainder of the material.

Peptide T-I4, I5b was digested with chymotrypsin, and the resulting peptides eluting from Dowex I at neutral pH were subjected to Dansyl--Edman degradation. We found Asx as N-terminal amino acid, both Thr and Glx at the third position, but no Dansyl amino acids after the next step. These results, and the presence of peptide (;-6, point to an unexpectedly high susceptibility of the Thr-Asx (~oo- ioi) bond to chymotryptic digestion. From this chymotryptic susceptibility and from homology with other ribonucleases (sheep and goat (Welling, (;. W., Schefler, A. J. and Beintema, J. J., unpublished); horse24), we concluded that the amide group in the neutral sequence ~oI-IO4 is located on Asx-IoI and not on Glx-Io3.

Both tyrosines were placed in peptide "1"-14,15 at positions 92 and 97 by homology with the other ribonueleases. By difference, the two threonines which are left must be placed at positions 96 and 99.

Conversion of asparagine and N-terminal glutamine residues In three tryptic peptides ('1"-9, T-Io and T-I4,I5) deamidation of asparagine

residues occurred. This deamidation occurs at low pH values, (e.g. during chromatography on Dowex 50) ; it is not a simple hydrolysis, but proceeds via the cyclic imide to formation of a fl-peptide bond, with liberation of the tz-carboxyl group 2s.

Evidence for the formation of fl-peptide bonds comes from the results of the 1)ansyl-Edman procedure with these peptides. An N-terminal/3-aspartyl residue is l)ansyl positive, but fails to undergo phenylisothiocyanate degradation, causing a negative Dansyl reaction for the next residue. It may be expected that a peptide with an N-terminal fl-aspartyl residue is only weakly bound to Dowex 50 at low pH values because of the low pK value of a free ~-carboxylic group at the N-terminus. Therefore, these peptides will resemble peptides lacking a free cz-amino group in their elution behaviour on Dowex 50.

The elution of peptide T- 9 (Asn-Gly-Arg; 67-69) in two peaks on I)owex 5o is in agreement with the observation that also in bovine~S, 27 and porcine ribonuclease 2~, asparagine-67 readily undergoes conversion. Also, like Jackson and Hirs 28 we found that both the more alkaline and the more acidic forms of this peptide fail to undergo phenylisothiocyanate degradation. These authors suggested that the more alkaline form is the imide and the more acidic one, the fl-aspartyl t))rm 28. Like Jackson and Hirs "~, we had no difficulty demonstrating glycine at position 69 when this -\sn-- Gly sequence occurred in an internal position in a peptide (peptide T-a-C-if).

Peptide "f-lo (Asp-Asn-Cys-His-Lys; 7o-74 ) eluted at two positions from Dowex I (Fig. I). It was difficult to deduce from the results of the dansyl- Edman procedure which aspartic acid residue in this peptide was originally the amidated one, for two residues from the N-terminus could be identified in T-Ioa, but only one in T-rob (Fig. I2).

Proof of the proposed sequence was obtained by subjecting peptide T-Iob after two Edman cycles (with no cleavage during the second cycle in any case) to hydrolysis with o.o 3 M HCI. The hydrolysate contained a large amount of a peptide with an N-terminal sequence CySOaH-His trot essentially no free aspartic acid. This result

I7o j . j . BEINTEMA, M. GRUBER

must be expected after hydrolysis with dilute acid of a peptide with N-terminal ~- phenylthiocarbamyl--(~-Asp) (;ySO3H (formed from Asp-Asn-CySOaH-His-Lys) but not of one with N-ternlinal a-phenylthiocarbamyl-({/-Asp) Asp-( 'ySOatt (formed from Asn-Asp-( 'ySOzH-His-1.ys).

The third position at which we observed partial deamidation of asparagme was Ash-94 in peptide T-~4,I5: this wpt ide also eluted at two positions from Dowex I (Fig. I). Similar results were obtained by Slnyth et al. '26 with a bovine ribonuclease peptide c~mtaining Asn-94.

N-terminal glutamine is easily converted to N-terminal pyrrolidone carboxylic acid. Peptides with this residue are Dansyl negative and show a higher mobility at paper chromatography and a weaker bonding to cation exchangers than the original peptides with N-terminal glutamine. Complete conversion of N-terminal glutamine occurred at chromatography on I)owex 5o (peptides T-4 and T-7a in Run I) and also occurred in the dilute acetic acid used for the chromatographies on Sephadex (peptides T- 4 and T- 7 in run 2, peptide C-4). During Run I, we observed two peaks of peptide T- 4 on Dowex I (Fig. I) and two peaks of peptide 3"- 7 on cellulose phosphate (Fig. 3), indicating partial conversion of the N-terminal glutainines under these conditions. Peptide T-Tb, which still contained an N-terminal glutamine residue, could be used for the Dansyl -Edman procedure.

Our results with peptide T- 4 are identical with those obtained by others with tile homologous peptides from bovine 26,':~, and porcine a° ribonuclease containing N-terminal (iln-i I.

A ~nide groups

The elution of a peptide from Dowex I is mainly governed by its charge al,aa. Therefore, it should be possible to deduce the number of amide groups in a peptide from its elution pH from Dowex I. "rhe validity of this procedure was checked by tYerforming a chromatographic separation of the tryptic peptides of performic acid- oxidised bovine ribonuclease under identical conditions. The results were as expected, with the exception of peptide 92 98, which was retarded bv its two tyrosine residues. There is an additional indication that the procedure is warrauted: peptides containing no aspartate, glutamate or their amides, overlapping peptides (resulting from incomplete tryptic or chymotryptic cleavages), and peptides which occur in two forms because of conversion of asparagine or N-terminal glutamine residues, all eluted at the expected pH values. In most cases this determination of amide groups was unambiguous.

The determinat i ,n of amide groups in the sequence 47-(~(), howew~r, was more difflt ult. From the results of the digestion with chymotrypsin it could be concluded that positions 55 and 62 are occupied by glutamine. Peptide T-a-C-db is a very weakly acidic peptide (Fig. 9) which contains cysteic acid and no basic amino acid residues, so both of its glutamic acids, including the one at position 0o, nmst be amidated. Peptide T-a-C-dd eluted from Dowex I at pH O.I, a very low pH value for a peptide with two positive charges (from His-4S and the N-terminus). For comparison, peptide T-4-P-d, with only one positiw: charge (from His-I2) and a blocked N-terminus, elutes at about the same pH value (6.o) and contains three free carboxylic groups (Asp-I 4, Glu-I6 and the C-terminus). Therefl~re, peptide T-a-C-dd must contain fl)ur flee carboxylic groups, viz. the (;-terminus and the amino acid

RAT PANCREATIC RIBONUCLEASE. II I 7 I

residues at positions 49, 52 and 53. The failure of chymotrypsin to cleave rat ribonuclease next to Leu-51 can be explained by the hindering effect of the negative charge on Glu-52. (The homologous bond Leu-Ala is cleaved, although slowly, in both bovine x3 and porcine "s ribonuclease.)

Peptide T-I4,I5a (with Asn-94 not yet in the fl-peptide form) must be neutral. Nevertheless, this peptide elutes from Dowex i together with the weakly acidic peptides like the neutral peptide 92-98 from oxidi.~d bovine ribonuclease.

During the Dansyl-Edman degradation of peptide C- 7 .~everal residual peptides were submitted to paper electrophoresis at pH 6.5. Peptide l I I 115 was negatively charged at this pH value and gave a blue colour with ninhydrin, while the peptides 112-115, 113-115 and r 14-115 were neutral and yielded a yellow col(mr. This evidence points to aspartate at position I I r and asparagine at position 113.

Disulfide bonds zX.ll half-cystine residues in rat ribonuclease occur at exactly the same positions

as in bovine and l~)rcine ribonuclease. Phelan and Hirs 3a have shown that the disulfide bonds in porcine ribonuclease occur between the same half-cystine residues as in bovine ribonuclease. There is every reason to believe that the half-cystine residues in rat ribonuclease are paired in exactly the same way. A different way of pairing would mean that rat ribonuclease has a conformation completely unlike the beef enzyme, and this is most improbable because of the evident homology between both enzymes. Other families of homologous proteins also show identical pairing of half- cystine residuesa4, aS.

Structure of rat ribo~,uclease For the purpose of this discussion it is assumed that the structural configuration

of the peptide backbone in rat ribonuclease is identical with that found by X-ray crystallography in bovine ribonuclease A and ribonuclease S. A comparison of the sequence of rat ribonuclease with the conformation of bovine ribonuclease has already been made by WyckoffL He found that all differences between both enzymes can be easily accommodated in the model of ribonuclease S, with little or no change in the backbone confi~rxnation, and that none of the differences involves residues in the active site or in contact with the competitive inhibitor 3'-I.JMP. Yet there must be slight differences in the geometry of the active site caused by amino acid substitutions elsewhere in the molecule, since there exist striking differences between the rat and bovine enzymes in their specific activities towards cyclic 2',3'-CMP and cyclic 2',3'- UMP as substrates 2. In addition, the p/( values of the histidine residues in both enzymes as determined by NNR spectroscopy are different 3~.

There are 3 extra amino acid residues in rat ribonuclease at the N-terminus and 41 substitutions in the remainder of the chain, including 19 changes of charge. Wyckoff 7 obserw~d that 14 of the 19 charge differences can be grouped into seven mutually compensating pairs, for the side chains of each pair approach each other closely in the conformation of bovine ribonuclease. For instance, the side chain of residue 8o (See' in beef; Arg" in rat) approaches the side chain of residue IO3 (Asn ° in beef; Glu in rat).

The other 22 substitutions involve neutral residues, mostly at the surface of the molecule. An interesting substitution is the Ser-Pro change at residue 5o, which

I 7 2 J . J . B E I N T E M A , M. G R U B E R

is at the amino te rmina l end of a helical region: here a sharp bend occurs in ribonuclease S, into which a proline residue fits perfect lyL

There are 3 subs t i tu t ions in the hydrophobic core of the molecule. Two internal valine residues (at posi t ions 57 and lO8) are replaced by isoleucines, requir ing ex t ra room for two methylene groups, l.ee and Richards '~7, however, observed tha t bovine r ibonuclease S conta ins three in ternal cavi t ies and tha t the ex t ra room required bv these subs t i tu t ions is provided by two of these cavit ies.

I f the amino acid sequences of bovine "~, porcine ~8 and ra t r ibonuclease are compared, three highly variable regions are ev iden t : the N- te rmina l region-3 to (J and the sequences 15- 24 and 96 lO3. These are ex te rna l par ts of the molecule, far from the act ive site. Other modera t e ly var iable regions are 61-8o and 31-39, also at the surface of the molecule. Al though there are many subs t i tu t ions in the sequence 31-39, including several charged residues, the net charge of this sequence is not changed and is t 3 in all three enzymes. Ev iden t ly , the presence of a number of posi t ive charges is impor t an t in this par t of the sequence, but not the exac t posi t ions of these charges.

The exac t agreement between the d a t a on synthe t ic S-pept ides ob ta ined by the groups of Ihf fmann 3s and Scoffone "~9, and the amino acid subs t i tu t ions in the N- te rmina l par t of several pancreat ic r ibonucleases, is str iking. Exac t ly those amino acid residues tha t according to the syn the t ic S-pept ide s tudies are impor t an t for the ac t iv i ty of the enzyme or for the binding of the S-pept ide to the S-pr~tein, are identical in bovine and ra t r ibonuclease, while all o ther residues are highly variable, especial ly the 6 residues at the C-terminus of the S-pept ide .

The number of differences between ra t and bovine r ibonuclease (41 ; which is 33'.',0 of 124) is very large; these two m a m m a l i a n proteins differ more than cvto-

20-~¢~ o/ chromes c of different t axa like the ve r t eb ra tes and the insects ( o~/o difference, (2Z /o, ref. 5). Thus, pancrea t ic ref. 5) or the t'1 and y chains of human haemoglobin o,

r ibonuclease proves to be one of the more var iable m a m m a l i a n proteins, and the most var iable mammal i an enzvme s tudied to date.

AC lZt N( )WLE I)G M I.'NT.'q

We thank Miss G. M. van der Goot for her skilful technical assistance and Dr C. H. Monfoort (Labora tor ium w)or Fysiologische Chemie, Utrecht , the Nether- lands) for performing the amino acid analyses. Many thanks are due to Dr G. Brauni- tzer and D r J . Sj6quist for giving hosp i ta l i ty to one of us (J. J. B.) a t their labora tor ies in Miinchen (Germany) and Lund (Sweden) and in t roducing him to many techniques used in these invest igat ions. These two visi ts were made possible by gran ts from tlw Nether lands Organiza t ion for the Advancemen t of Pure Research (Z.W.O.).

I(EFEI~ENCES

I Bcintema, J. J. (I966) Ph. 1). Thesis, Groningen 2 Beintema., J. J.. ('ampagnt', R. N. and (;ruber, .M. (1973) lliochim. Hiophys. ,.lch~ 31o,

148 . , 6 o 3 I(artha, t;., Bello, J. and Harker, D. (1967) Nature 2r 3, 862-865 4 \Vyckott, H. ~V., Hardman, I(. I)., Allewell, N. M., Inagami, T., Johns(m, 1.. N. and Richards,

F..M. (1967) .[. lliol. Chem. 242, 3984-3988

RAT PANCREATIC RIBONUCLEASE. II 173

5 Dayhoff , M. O. (I969) Atlas o/Protein Sequence and Structure, Nat ional Biomedical Research Founda t ion , Silver Spring, Md.

(~ Be in tema , J. J. and Gruber , M. (1967) Biochim. Biophys. Acta 147, 612-614 7 Wyckoff , H. W. (1968) Brookhaven Symp. Biol. 21, 252-257 8 Moore, S. and Stein, W. H. (1954) J. Biol. Chem. 211, 9o7--913 9 Sj6quist , J. (1957) Arkiv Kemi 11, 15I-I6O

io | t i t s , C. H. XV. (1956) J. Biol. Chem. 219, 611-621 ix I.iebold, B. arid Brauni tzer , (;. (1959) Z. Physiol. Chem. 315, 271-277 12 Cantield, R. l~. (1963) ./. Biol. Chem. 238, 2698-27o7 13 Hits, C. t l . \V. (i96o) J. Biol. Chem. 235, 625-632 14 Tsung , C. M. and Fraenke l -Conra t , H. (I965) Biochemist~:v 4, 793 - 8 ° I 15 lli lse, K. and Brauni tzer , G. (I968) Z. Physiol. Chem. 349, 433-45 ° lb Eaker , D. I.., King, T. 1 ~. and Craig, I.. (~. (I965) Biochemistry 4, 1479-1486 17 Hirs, ('. H. XV., Moore, S. and Stein, W. H. (1956 ) J. Biol. Chem. 219, 623-642 18 Hilse, K. and Brauni tzer , G. (1962) Z. Physiol. Chem. 329, 213-129 19 l.)reyer, \V. J. and B ynum, E. (~907) in .VIethods in Enz~,mology (Hirs, C. II. V~r., ed.), Vol. XI ,

pp. 32-39, Academic Press, New York and London 2o Gray, VV. R. and Har t ley , t3. S. (1963 ) Biochem. J. 89, 379-380 21 Gray, XV. l~. (19(>7) in Methods in I-nzymology (l-lirs, C. 11. \\"., ed.), Vol. X[ , pp. I39 -15I ,

Academic Press, New York and I .ondon 22 Ilse, ]). and E d m a n , 1 >. (1963) Auslr. J. Chem. 16, 411-416 23 Stahl, E. (I962) Diinnschicht-chromatographie, pp. 44o-445, Springer-Verlag, Berlin 24 Schelter, A. J. and I~eintema, J. j . (1972) .4bstr. 8th Meet. FEBS, Amsterdam, p. 358, Nor th-

Holland, A m s t e r d a m 25 N a u g h t o n , M. A., Sanger, F., Har t ley , B. S. and Shaw, D. C. (t96o) Biochem. J. 77, 149-163 26 S m y t h , 1). G., Stein, W. H. and Moore, S. (1963) ./. Biol. Chem. 238 , 227-234 "7 Borns te in , P. and ]~alian, G. (197 o) J. Biol. Chem. 24.5, 4854-4850 28 Jackson , ll. L. and Hirs, C. H. W. ( i97 ° ) J. Biol. Chem. 245, 637-653 29 S m y t h , D. G., Stein, \V. H. and Moore, S. (1962) J. Biol. Chem. 237, I845-I85O 30 Jackson , H. I.. and Hits , C. H. W. (I97 o) ./. Biol. Chem. 245, 624-636 3I Rudloit , V. and Brauni tzer , G. (1961) Z. l'hysiol. Chem. 323, I29-144 32 Sehroeder, \V. A. (1967) in Method.~ in Enzymology (Hits, C. H. \ \ ' . , ed.), Vol. XI, pp. 361-369,

Academic I~ress, New York and l .ondon 33 Phelan, J. J. and Hirs, ('. H. \\:. (197 o) J. Biol. Chem. 245, 654-661 i~4 Shot ton , D. M. and '~Vat.son, H. C. (197 o) ,¥atz~re 22.5, 811-81o 35 V a n a m a n , T. ('., Brew, K. and Hill, [~. L. (197o) J. Biol. Chem. 245, 4583-459o 36 Migchelsen, ('. and Be in tema , J. J. (1972) .4bstr. 8th Meet. FEBS, Amsterdam, p. 416, Nor th°

Holland, A m s t e r d a m 37 Lee, B. and I~ichards, F. M. (1971) J. Mol. Biol. 55, 379-4 °0 38 Hofmann , K., Visser, j . I'. anti Finn, F. M. (197 o) J. Am. Chem. Soc. 92, 290o-29o9 39 Moroder, L., Marchiori, l:., Hocchi, l(., t ;nn tana , A. and Scoitone, E. (1969) J. Am. Chem.

Soc. 91 , 3921-3926

rat pancreatic ribonuclease ii. amino acid sequence

Documents