Int. J . Peptide Protein Res. 8, 1976, 305-316 Published by Munhgaard. Copenhagen. Denmark N o part may be reproduced by any process without written permission from the author@)
THE PRIMARY STRUCTURE OF MUSKRAT PANCREATIC RIBONUCLEASE HENKVAN DIJK, BOELESLOOTS, ANNELIES VAN DEN BERG, WIMGAASTRA AND JAAP J. BEINTEMA Biochemisch Laboratorium, Zernikelaan, Rijksuniversiteit, Groningen, the Netherlands Received 3 July 1975 Pancreatic ribonucleasefrom muskrat (Ondatra zibethica) was isolated and its amino acid sequence was determined from tryptic digests of the performic acid-oxidized and the reduced and aminoethylated enzyme. The peptides have been positioned in the sequence by homology with other ribonucleases. This could be done unambiguouslyfor all peptides except Arg-Arg (tentative position 32-33) and Ser-Arg (tentative position 75-76). The amino acid sequences of the peptides were determined by the dansylEdman method, with the exception of residues 23-25 and 99-102, which were positioned by homology. The enzyme difers in 38 positions from the enzyme from rat and in 31-42 positions from other mammalianpancreatic ribonucleases, while rat ribonucleasedixers at 44-S2 positions from the other enzymes. These data point to a common ancestry of the enzymes from muskrat and rat and an increased evolution rate of rat ribonuclease afrer divergence of the ancestors of both species. Muskrat ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn- Val-Thr (6264). The primary structures of the pancreatic ribonucleases from a number of mammalian species, mainly artiodactyls and rodents are known (1-10). Ribonucleases from different mammalian orders differ in 21-28% of the positions. The pancreatic ribonuclease from the rat, a member of the myomorph suborder of the rodents, differs from all other ribonucleases, including the enzymes of species from the hystricomorph suborder of the rodents, in 3440% of the amino acid residues (10). This indicates that rat pancreatic ribonuclease has evolved at an increased rate after divergence of the hystricomorph and myomorph rodents. It is unknown whether this deviating position of the rat enzyme is a general characteristic of the myomorph rodents or a special feature of the rat. For this reason we started the investigation of the primary structure of the ribonuclease from another myomorph rodent, viz. the muskrat (Ondatra zibethica). F
Both the rat and the muskrat belong to the superfamily of the Muroidae, but to different families: the Muridae and the Cricetidae, respectively (1 l), which have diverged from each other approximately at the transition from the Eocene to the Oligocene, about 40 x lo6 years ago (12). MATERIALS AND METHODS
Muskrat pancreatic tissue was obtained from the Netherlands Committee for Muskrat Control*. Sephadex G-25 fine was a product of Pharmacia
* Muskrats were introduced to fur-farms in Europe from America in the first decades of this century. Escaped animals founded the European muskrat population. During the last few years these animals have been infiltrating the Netherlands across the Belgian and German borders. Since they make their 305
HENK VAN DlJK
(Uppsala), CM-cellulose CM-32 of Whatman (Maidstone), Aminex A-5 of Biorad Laboratories (Richmond) and Dowex 50-X2 of Fluka (Buchs). Trypsin (pig, 3 x cryst.) was obtained from Miles-Seravac (Maidenhead), a-chymotrypsin (bovine, 3 x cryst.) and carboxypeptidase A from Worthington (New Jersey), thermolysin from Calbiochem (San Diego), aminopeptidase M from Rohm & Haas (Darmstadt), “Glu” enzyme7 was a gift from Dr. G. Drapeau (Montreal). All other chemicals were analytical grade products. Isolation of ribonuclease The isolation of muskrat pancreatic ribonuclease was performed as described for the rat enzyme (9) except for the ammonium sulfate fractionating procedure, since the muskrat enzyme precipitates between 50% and 100% saturation. After desalting on Sephadex G-25, the enzyme was purified by chromatography on CM-cellulose. The following gradients were used: A) 0.01 M sodium phosphate pH 6.0-0.1 M sodium phosphate pH 7.5 (13); B ) 0.05 M sodium acetate pH 5.0-0.5 M sodium acetate pH 5.0 (7). Ribonuclease activity was detected according to Campagne & Gruber (14) using high molecular weight yeast RNA isolated as described by Crestfield et al. (15). Amino acid analysis and determination of carbohydrate were performed as described previously (8). Enzymatic cleavages Oxidative cleavage of disulfide bonds was performed as described by Hirs (16); reduction and aminoethylation, and cleavages with trypsin, chymotrypsin, and thermolysin as described by Scheffer et al. (7). Digestion with the “Glu” enzyme, aminopeptidase M and carboxypeptidase A were performed according to Welling et al. (8).
holes in dikes and riverbanks, the presence of animals in the vulnerable, below sea-level situated western parts of the Netherlands may have severe consequences. Therefore, the Dutch government has felt compelled to appoint a number of muskrat catchers to fight these animals continuously along the frontiers of the country. t Abbreviation used: “Glu” enzyme: Staphylococcus aureus V8 proteinase. 306
Isolation of peptides The tryptic digests were fractionated on a column of Sephadex G-25 fine. The absorbances at 280 and 220 nm were measured and peptides were detected in aliquots of the fractions by paper electrophoresis at pH 3.5. The pools from the digest of performic acidoxidized ribonuclease were separated into their constituent peptides by chromatography on Aminex A-5 (small peptides up to 10 residues) or Dowex 50-X2 (large peptides) (7). For elution of the strongly basic dipeptide Arg-Arg from the Aminex A-5 column after completion of the gradient, 0.5 M sodium hydroxide was used. Preparative high voltage paper electrophoresis at pH 3.5, detection, and elution of the peptides from the pools of the digest of reduced and aminoethylated protein were performed as described by Welling et al. (8). Edman degradation and deterntination of amide positions Dansyl-Edman degradation of small peptides up to 6 residues was performed as described by Gray & Smith (17) and of larger peptides as described by Hartley (18). The state of amidation of glutamic and aspartic acid residues was determined by the method of Offord (19) or derived from amino acid analysis after cleavage of pzptides with aminopeptidase or carboxypeptidase, or from the specificity of the “Glu” enzyme.
RESULTS
In two separate isolations we used 660 g and 800 g of pancreatic tissue, from 240 and 150 individuals, respectively. The first isolation, in which only gradient A was used for the chromatography on CM-cellulose, yielded 57 mg of pure pancreatic ribonuclease (Fig. 1). An additional chromatography on CM-cellulose (gradient B) was required in the second isolation, probably because the starting material could not be freed from contaminating material. In this case we obtained 25 mg of ribonuclease. The specific activity of the muskrat enzyme on RNA is comparable to that of the bovine enzyme. The purity of the enzyme preparations was
PRIMARY STRUCTURE OF MUSKRAT RIBONUCLEASE
FIGURE1 Chromatography of muskrat ribonuclease on a column (55x 1.2 crn) of CM-cellulose. Elution was carried out as described by Aqvist & Anfinsen (13) with a gradient from 0.01 M sodium phosphate (pH 6) to 0.1 M sodium phosphate (pH 7.9, fractions of 10 ml were collected. enzymatic AZa0, -o-o-; activity ; --r.--~, -; conductivity; _._._. pH. - - - - -
1 1
1.5
1.0
0.5
‘280
- 7.0 - 611 10
40
I
20
30
3
FRACTION NUMBER
,
I
30
PH
- 8.0
*220
15
10
5
1
50 60 FRACTION NUMBER
FIGURE 2 Gel filtration of the tryptic digest of performic acid-oxidized muskrat ribonuclease on a column (150 x 1.5 cm) of Sephadex (3-25 (fine grade) .Elution was carried out with 0. I M acetic acid at a flow rate of 12 ml/h, 2 ml fractions were collected. A,,,, - - - - -;
filtration on Sephadex G-25 are given for both digests (Figs. 2 and 3). Fig. 3 also gives the peptide pattern of the pooled fractions of the second digest. Peptides T17, 18 and T20 were further digested with a-chymotrypsin, the peptide mixture T8+T8a, peptide T9 and peptide T14 with thermolysin, and peptide T9 with “Glu” enzyme. The amino acid compositions of the isolated peptides are given in Table 1. The results of the sequence determinations of the peptides are summarized in Fig. 4. Table 2 gives the state of amidation of the glutamic and aspartic acid residues. The peptides were positioned by homology with other ribonucleases (1-10) (Fig. 4). Muskrat pancreatic ribonuclease does not contain carbohydrate, as was proved by the orcinol test and confirmed by a lack of amino sugars in amino acid analysis. DISCUSSION
Reliability of the sequence determination We did not succeed in determining the complete sequence of two peptides. Residues 23-25 in the Aim, -. Fractions pooled for further fractionation are indi- C-terminal part of peptide T3 (11-26) were cated by bar. positioned by homology with the ribonucleases from rat (9) and mouse (Lenstra, J. A,, Gaastra, tested by dansylation. The only detectable N- W. & Beintema, J. J., unpublished), which are terminal amino acid was lysine. identical in this part of the sequence. DansylFifty mg ribonuclease was oxidized with Edman degradation of peptide T18 (96-104) was performic acid and digested with trypsin (To.- not very successful. Therefore, the identities of peptides) and 25 mg was first reduced and the residues at positions 99, 101, and 102 were aminoethylated before cleavage with trypsin derived in another way. The leucine at position (T-peptides). The elution patterns of the gel 102 was derived from the chymotryptic
307
HENK VAN DlJK
based on homology with the ribonucleases from hystricomorph rodents (10). The positioning of tryptic peptides by homology with other ribonucleases is reliable for peptides consisting of 4-5 or more residues, but less so for smaller peptides in variant regions of the sequence. There is little reason to doubt that the positions of peptides T2 (Phe-Glu-Arg; 8-10) and T15 (Leu-Lys; 86-87) are correct, but the positions of peptides T5,6 (Arg-Arg; 32-33) and T13 (SerArg; 75-76) instead of the other way round, were chosen solely because of the invariability of the serine at position 75 and the frequent occurrence of arginine at position 32.
REF.
0 Lfi
8 ARG
: 0 4 @2e17
080
i;;,,;;”
08
633 09 437
(g
020
w
818 0
011
b7
016a e7a
z 0 GLU
0.2
2.0
‘280
0s
10
--
~
I
,
_--
Analysis by paper electrophoresis at pH 3.5 of the pooled fractions. Bottom of figure: Gel filtration of the tryptic digest of reduced and aminoethylated muskrat ribonuclease on a column (200x 1 cm) of Sephadex G-25 (fine grade). Elution was carried out with 0.1 M acetic acid at a flow rate of 10 ml/h, 2 ml fractions were collected. AZa0,- - - -; Aizo,
-.
Fractions pooled for further fractionation are indicated by bar. susceptibility of the peptide bond 102-103 and by analogy with the leucine at this position in dromedary and camel ribonucleases (8). The Thr-Ser-Gln (99-101) sequence is tentative and 308
The absence of carbohydrate Muskrat ribonuclease contains no carbohydrate, although the polypeptide chain contains a recognition site for carbohydrate ‘attachment in the sequence Asn-Val-Thr (62-64). Other ribonucleases with a recognition site at position 62 are horse ribonuclease with the sequence Asn-Ile-Thr (7) and guinea-pig ribonuclease A with the sequence Asn-Val-Ser (10). Horse ribonuclease is completely glycosidated at position 62 with a complex carbohydrate chain consisting of at least 18 monosaccharide residues (7), while guinea-pig ribonuclease A is carbohydrate-free like the enzyme from muskrat (10). Evolution of rodent pancreatic ribonucleases The sequences of the ribonucleases from rat and muskrat are shown in Fig. 5. The two enzymes differ at a strikingly large number of positions, many more than was anticipated at the beginning of these studies. The difference matrix of the enzymes from cow, pig, horse and five rodent species is given in Table 3. As mentioned in the introduction, ribonucleases from different mammalian orders generally differ in 21-28% of the positions. Rat ribonuclease, however, is an exception and differs in 3 4 4 0 % of the positions from the others. It is obvious from the data in Table 3 that muskrat ribonuclease conforms to the generally observed difference percentage of about 25 %. Nevertheless, the difference between rat and muskrat in 29% of the positions points to a common ancestry of these two myomorph rodent ribonucleases. This conclusion is corroborated by an analysis of the rat and muskrat
PRIMARY STRUCTURE OF MUSKRAT RIBONUCLEASE
-
-10: o!
0-0
309
1.9 (2)
1.0 (1)
Glu Pro G~Y
Ala Val
8-10
180
400 1-7
nrnoles
position in seq.
1.2 ( I )
1.8 (2)
0.9 ( I )
400 11-26
1.1 ( I ) 1 .o (1)
1 .o (1)
0.1 (-) 1 .o (1)
200 27-31
1.1 ( I )
1.5 (2)
85
150 32 = 33 =
32-33
50
34-40
175
0.7 (1)
0.3 (-) 1.2 ( I ) 0.2 (-)
1.3 ( I )
1.2 ( I )
0.9 (I)
T7
0.2 (-)
2.0 (2)
T5,6
1.5 (2)
1 .O (1)
T5 = T6 = T14b
0.9 (1)
1.4 ( I ) b
1.2 (1) 2.0 (2) 5.4 (6) 1.2 (I) 1.0 (1) 1.1 (1)
0.2 (-)
0.9 ( I )
T4
T3
T2
LYS AetCys His Arg
Leu Tyr Phe
Ile
Met
1.1 (1) 1.1 (1)
Thr Ser
ASP
TI
25 34-39
0.7 ( I )
0.9 (1)
1.3 (1)
}
lo00 41-58 40-58
+ (1-2)
+ (1) + (1)
0.1 (-1 0.s ( I )
0.1 (-) 1 .o (1)
0.4 (-) 2.3 (2) 2.3 (2) 0.1 (-) 1.9 (2) 2.8 (4)
0.3 (-) 1.6 (2)
1.1 (1)
2.7 (2)
T8a T8
0.7 (1)
T7'
59-65
735
0.1 (-1 0.7 (1)
0.1 (-) 0.3 (-)
0.9 (1)
2.2 (2) 0.2 (-) 0.2 (-)
1.1 (1) 1 .o ( I ) 1.2 (1)
T9
66
200
1.0 (1)
TI0
T11
67-72
160
0.3 (-) 0.9 (1)
1.1 (1)
1.1 (1)
2.9 (3)
TABLE 1B Amino acid compositions of tryptic peprides from reduced and aminoethylated muskrat ribonuclease
73-74
200
1.0 (1)
1 .o ( I )
0.1 (-)
TI2
75-76
1.3 (1) 500
0.8 (1)
TI3
ASP
0.4(-) 0.7 (1) 0.8 (1) 1 .o (1) 270 77-85
1.0 (1)
200 88-91
225 86-87
0.9(I)
1 .o (1)
1.1 (1)
T16
1.1 (1)
0.8(1) 1.0 (1) .0.9(1)
0.3 (-) 1.1 (1)
1.3 (1) 1.2 (1) 1.3 (1) 0.3 (-)
T15
20 88-89
0.9 ( I )
1.1 (1)
T16a
60 90-91
0.8(1)
1.1 (1)
T16b
T18
60 92-95
0.6 (1)
1.0 (I) 0.3(-)
T17,18'
250 220 96-104 92-104
1.1 (1)
0.9 (1) 0.8 (1)
0.2(-)
1.3 (1) 1.2 (1) 0.9(1) 1.4(1) 0.4 (-) 2.7 (3) 1.2(1) 0.2(-) 0.4 (-)
T17
260 105-110
0.4 (-) 0.9(1)
0.6(l)c 0.1 (-) 0.4(-)
0.1(-1 1.1 ( I ) 1 .O (2)c
0.8(-) 0.3 (-) 0.8 (-) 2.0 (1)
T19'
530 111-124
0.1 (-) 0.8 (1)
0.2(-) 1.8 (2)
1.2 (1) 0.3 (-) 2.9 (2)d 1.1 (1) 1.9 (2) 1.1 (1) 1.0 (1) 2.4 (3) 0.1 (-)
T20
Peptides T17,18,and T19 were only partly resolved by paper electrophoresis at pH 3.5 (see Figure 2). The analysis of peptide T17,18 only gave qualitative evidence about the presence. of this peptide. The impurities in peptide T19 can be accounted for by the presence of about 0.3 equivalent of peptide T17,18. me results of the Dansyl-Edman degradation were in agreement with this contamination. Including methionine sulphoxide. c The Val-Ile-Val sequence. (106-108)is only partly hydrolyzed after 20 h. High serine value from minor contamination by peptide T3.
Ser Glu PI0 G~Y Ala Val Met Ile Leu TYr Phe LYS AetCys His Arg nmoles positioninseq.
Thr
T14
TABLE1B (con?.)
m
III
F
0
5
5
w
g
5x
0 %
32
c"
7
THE PRIMARY STRUCTURE OF MUSKRAT PANCREATIC RIBONUCLEASE HENKVAN DIJK, BOELESLOOTS, ANNELIES VAN DEN BERG, WIMGAASTRA AND JAAP J. BEINTEMA Biochemisch Laboratorium, Zernikelaan, Rijksuniversiteit, Groningen, the Netherlands Received 3 July 1975 Pancreatic ribonucleasefrom muskrat (Ondatra zibethica) was isolated and its amino acid sequence was determined from tryptic digests of the performic acid-oxidized and the reduced and aminoethylated enzyme. The peptides have been positioned in the sequence by homology with other ribonucleases. This could be done unambiguouslyfor all peptides except Arg-Arg (tentative position 32-33) and Ser-Arg (tentative position 75-76). The amino acid sequences of the peptides were determined by the dansylEdman method, with the exception of residues 23-25 and 99-102, which were positioned by homology. The enzyme difers in 38 positions from the enzyme from rat and in 31-42 positions from other mammalianpancreatic ribonucleases, while rat ribonucleasedixers at 44-S2 positions from the other enzymes. These data point to a common ancestry of the enzymes from muskrat and rat and an increased evolution rate of rat ribonuclease afrer divergence of the ancestors of both species. Muskrat ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn- Val-Thr (6264). The primary structures of the pancreatic ribonucleases from a number of mammalian species, mainly artiodactyls and rodents are known (1-10). Ribonucleases from different mammalian orders differ in 21-28% of the positions. The pancreatic ribonuclease from the rat, a member of the myomorph suborder of the rodents, differs from all other ribonucleases, including the enzymes of species from the hystricomorph suborder of the rodents, in 3440% of the amino acid residues (10). This indicates that rat pancreatic ribonuclease has evolved at an increased rate after divergence of the hystricomorph and myomorph rodents. It is unknown whether this deviating position of the rat enzyme is a general characteristic of the myomorph rodents or a special feature of the rat. For this reason we started the investigation of the primary structure of the ribonuclease from another myomorph rodent, viz. the muskrat (Ondatra zibethica). F
Both the rat and the muskrat belong to the superfamily of the Muroidae, but to different families: the Muridae and the Cricetidae, respectively (1 l), which have diverged from each other approximately at the transition from the Eocene to the Oligocene, about 40 x lo6 years ago (12). MATERIALS AND METHODS
Muskrat pancreatic tissue was obtained from the Netherlands Committee for Muskrat Control*. Sephadex G-25 fine was a product of Pharmacia
* Muskrats were introduced to fur-farms in Europe from America in the first decades of this century. Escaped animals founded the European muskrat population. During the last few years these animals have been infiltrating the Netherlands across the Belgian and German borders. Since they make their 305
HENK VAN DlJK
(Uppsala), CM-cellulose CM-32 of Whatman (Maidstone), Aminex A-5 of Biorad Laboratories (Richmond) and Dowex 50-X2 of Fluka (Buchs). Trypsin (pig, 3 x cryst.) was obtained from Miles-Seravac (Maidenhead), a-chymotrypsin (bovine, 3 x cryst.) and carboxypeptidase A from Worthington (New Jersey), thermolysin from Calbiochem (San Diego), aminopeptidase M from Rohm & Haas (Darmstadt), “Glu” enzyme7 was a gift from Dr. G. Drapeau (Montreal). All other chemicals were analytical grade products. Isolation of ribonuclease The isolation of muskrat pancreatic ribonuclease was performed as described for the rat enzyme (9) except for the ammonium sulfate fractionating procedure, since the muskrat enzyme precipitates between 50% and 100% saturation. After desalting on Sephadex G-25, the enzyme was purified by chromatography on CM-cellulose. The following gradients were used: A) 0.01 M sodium phosphate pH 6.0-0.1 M sodium phosphate pH 7.5 (13); B ) 0.05 M sodium acetate pH 5.0-0.5 M sodium acetate pH 5.0 (7). Ribonuclease activity was detected according to Campagne & Gruber (14) using high molecular weight yeast RNA isolated as described by Crestfield et al. (15). Amino acid analysis and determination of carbohydrate were performed as described previously (8). Enzymatic cleavages Oxidative cleavage of disulfide bonds was performed as described by Hirs (16); reduction and aminoethylation, and cleavages with trypsin, chymotrypsin, and thermolysin as described by Scheffer et al. (7). Digestion with the “Glu” enzyme, aminopeptidase M and carboxypeptidase A were performed according to Welling et al. (8).
holes in dikes and riverbanks, the presence of animals in the vulnerable, below sea-level situated western parts of the Netherlands may have severe consequences. Therefore, the Dutch government has felt compelled to appoint a number of muskrat catchers to fight these animals continuously along the frontiers of the country. t Abbreviation used: “Glu” enzyme: Staphylococcus aureus V8 proteinase. 306
Isolation of peptides The tryptic digests were fractionated on a column of Sephadex G-25 fine. The absorbances at 280 and 220 nm were measured and peptides were detected in aliquots of the fractions by paper electrophoresis at pH 3.5. The pools from the digest of performic acidoxidized ribonuclease were separated into their constituent peptides by chromatography on Aminex A-5 (small peptides up to 10 residues) or Dowex 50-X2 (large peptides) (7). For elution of the strongly basic dipeptide Arg-Arg from the Aminex A-5 column after completion of the gradient, 0.5 M sodium hydroxide was used. Preparative high voltage paper electrophoresis at pH 3.5, detection, and elution of the peptides from the pools of the digest of reduced and aminoethylated protein were performed as described by Welling et al. (8). Edman degradation and deterntination of amide positions Dansyl-Edman degradation of small peptides up to 6 residues was performed as described by Gray & Smith (17) and of larger peptides as described by Hartley (18). The state of amidation of glutamic and aspartic acid residues was determined by the method of Offord (19) or derived from amino acid analysis after cleavage of pzptides with aminopeptidase or carboxypeptidase, or from the specificity of the “Glu” enzyme.
RESULTS
In two separate isolations we used 660 g and 800 g of pancreatic tissue, from 240 and 150 individuals, respectively. The first isolation, in which only gradient A was used for the chromatography on CM-cellulose, yielded 57 mg of pure pancreatic ribonuclease (Fig. 1). An additional chromatography on CM-cellulose (gradient B) was required in the second isolation, probably because the starting material could not be freed from contaminating material. In this case we obtained 25 mg of ribonuclease. The specific activity of the muskrat enzyme on RNA is comparable to that of the bovine enzyme. The purity of the enzyme preparations was
PRIMARY STRUCTURE OF MUSKRAT RIBONUCLEASE
FIGURE1 Chromatography of muskrat ribonuclease on a column (55x 1.2 crn) of CM-cellulose. Elution was carried out as described by Aqvist & Anfinsen (13) with a gradient from 0.01 M sodium phosphate (pH 6) to 0.1 M sodium phosphate (pH 7.9, fractions of 10 ml were collected. enzymatic AZa0, -o-o-; activity ; --r.--~, -; conductivity; _._._. pH. - - - - -
1 1
1.5
1.0
0.5
‘280
- 7.0 - 611 10
40
I
20
30
3
FRACTION NUMBER
,
I
30
PH
- 8.0
*220
15
10
5
1
50 60 FRACTION NUMBER
FIGURE 2 Gel filtration of the tryptic digest of performic acid-oxidized muskrat ribonuclease on a column (150 x 1.5 cm) of Sephadex (3-25 (fine grade) .Elution was carried out with 0. I M acetic acid at a flow rate of 12 ml/h, 2 ml fractions were collected. A,,,, - - - - -;
filtration on Sephadex G-25 are given for both digests (Figs. 2 and 3). Fig. 3 also gives the peptide pattern of the pooled fractions of the second digest. Peptides T17, 18 and T20 were further digested with a-chymotrypsin, the peptide mixture T8+T8a, peptide T9 and peptide T14 with thermolysin, and peptide T9 with “Glu” enzyme. The amino acid compositions of the isolated peptides are given in Table 1. The results of the sequence determinations of the peptides are summarized in Fig. 4. Table 2 gives the state of amidation of the glutamic and aspartic acid residues. The peptides were positioned by homology with other ribonucleases (1-10) (Fig. 4). Muskrat pancreatic ribonuclease does not contain carbohydrate, as was proved by the orcinol test and confirmed by a lack of amino sugars in amino acid analysis. DISCUSSION
Reliability of the sequence determination We did not succeed in determining the complete sequence of two peptides. Residues 23-25 in the Aim, -. Fractions pooled for further fractionation are indi- C-terminal part of peptide T3 (11-26) were cated by bar. positioned by homology with the ribonucleases from rat (9) and mouse (Lenstra, J. A,, Gaastra, tested by dansylation. The only detectable N- W. & Beintema, J. J., unpublished), which are terminal amino acid was lysine. identical in this part of the sequence. DansylFifty mg ribonuclease was oxidized with Edman degradation of peptide T18 (96-104) was performic acid and digested with trypsin (To.- not very successful. Therefore, the identities of peptides) and 25 mg was first reduced and the residues at positions 99, 101, and 102 were aminoethylated before cleavage with trypsin derived in another way. The leucine at position (T-peptides). The elution patterns of the gel 102 was derived from the chymotryptic
307
HENK VAN DlJK
based on homology with the ribonucleases from hystricomorph rodents (10). The positioning of tryptic peptides by homology with other ribonucleases is reliable for peptides consisting of 4-5 or more residues, but less so for smaller peptides in variant regions of the sequence. There is little reason to doubt that the positions of peptides T2 (Phe-Glu-Arg; 8-10) and T15 (Leu-Lys; 86-87) are correct, but the positions of peptides T5,6 (Arg-Arg; 32-33) and T13 (SerArg; 75-76) instead of the other way round, were chosen solely because of the invariability of the serine at position 75 and the frequent occurrence of arginine at position 32.
REF.
0 Lfi
8 ARG
: 0 4 @2e17
080
i;;,,;;”
08
633 09 437
(g
020
w
818 0
011
b7
016a e7a
z 0 GLU
0.2
2.0
‘280
0s
10
--
~
I
,
_--
Analysis by paper electrophoresis at pH 3.5 of the pooled fractions. Bottom of figure: Gel filtration of the tryptic digest of reduced and aminoethylated muskrat ribonuclease on a column (200x 1 cm) of Sephadex G-25 (fine grade). Elution was carried out with 0.1 M acetic acid at a flow rate of 10 ml/h, 2 ml fractions were collected. AZa0,- - - -; Aizo,
-.
Fractions pooled for further fractionation are indicated by bar. susceptibility of the peptide bond 102-103 and by analogy with the leucine at this position in dromedary and camel ribonucleases (8). The Thr-Ser-Gln (99-101) sequence is tentative and 308
The absence of carbohydrate Muskrat ribonuclease contains no carbohydrate, although the polypeptide chain contains a recognition site for carbohydrate ‘attachment in the sequence Asn-Val-Thr (62-64). Other ribonucleases with a recognition site at position 62 are horse ribonuclease with the sequence Asn-Ile-Thr (7) and guinea-pig ribonuclease A with the sequence Asn-Val-Ser (10). Horse ribonuclease is completely glycosidated at position 62 with a complex carbohydrate chain consisting of at least 18 monosaccharide residues (7), while guinea-pig ribonuclease A is carbohydrate-free like the enzyme from muskrat (10). Evolution of rodent pancreatic ribonucleases The sequences of the ribonucleases from rat and muskrat are shown in Fig. 5. The two enzymes differ at a strikingly large number of positions, many more than was anticipated at the beginning of these studies. The difference matrix of the enzymes from cow, pig, horse and five rodent species is given in Table 3. As mentioned in the introduction, ribonucleases from different mammalian orders generally differ in 21-28% of the positions. Rat ribonuclease, however, is an exception and differs in 3 4 4 0 % of the positions from the others. It is obvious from the data in Table 3 that muskrat ribonuclease conforms to the generally observed difference percentage of about 25 %. Nevertheless, the difference between rat and muskrat in 29% of the positions points to a common ancestry of these two myomorph rodent ribonucleases. This conclusion is corroborated by an analysis of the rat and muskrat
PRIMARY STRUCTURE OF MUSKRAT RIBONUCLEASE
-
-10: o!
0-0
309
1.9 (2)
1.0 (1)
Glu Pro G~Y
Ala Val
8-10
180
400 1-7
nrnoles
position in seq.
1.2 ( I )
1.8 (2)
0.9 ( I )
400 11-26
1.1 ( I ) 1 .o (1)
1 .o (1)
0.1 (-) 1 .o (1)
200 27-31
1.1 ( I )
1.5 (2)
85
150 32 = 33 =
32-33
50
34-40
175
0.7 (1)
0.3 (-) 1.2 ( I ) 0.2 (-)
1.3 ( I )
1.2 ( I )
0.9 (I)
T7
0.2 (-)
2.0 (2)
T5,6
1.5 (2)
1 .O (1)
T5 = T6 = T14b
0.9 (1)
1.4 ( I ) b
1.2 (1) 2.0 (2) 5.4 (6) 1.2 (I) 1.0 (1) 1.1 (1)
0.2 (-)
0.9 ( I )
T4
T3
T2
LYS AetCys His Arg
Leu Tyr Phe
Ile
Met
1.1 (1) 1.1 (1)
Thr Ser
ASP
TI
25 34-39
0.7 ( I )
0.9 (1)
1.3 (1)
}
lo00 41-58 40-58
+ (1-2)
+ (1) + (1)
0.1 (-1 0.s ( I )
0.1 (-) 1 .o (1)
0.4 (-) 2.3 (2) 2.3 (2) 0.1 (-) 1.9 (2) 2.8 (4)
0.3 (-) 1.6 (2)
1.1 (1)
2.7 (2)
T8a T8
0.7 (1)
T7'
59-65
735
0.1 (-1 0.7 (1)
0.1 (-) 0.3 (-)
0.9 (1)
2.2 (2) 0.2 (-) 0.2 (-)
1.1 (1) 1 .o ( I ) 1.2 (1)
T9
66
200
1.0 (1)
TI0
T11
67-72
160
0.3 (-) 0.9 (1)
1.1 (1)
1.1 (1)
2.9 (3)
TABLE 1B Amino acid compositions of tryptic peprides from reduced and aminoethylated muskrat ribonuclease
73-74
200
1.0 (1)
1 .o ( I )
0.1 (-)
TI2
75-76
1.3 (1) 500
0.8 (1)
TI3
ASP
0.4(-) 0.7 (1) 0.8 (1) 1 .o (1) 270 77-85
1.0 (1)
200 88-91
225 86-87
0.9(I)
1 .o (1)
1.1 (1)
T16
1.1 (1)
0.8(1) 1.0 (1) .0.9(1)
0.3 (-) 1.1 (1)
1.3 (1) 1.2 (1) 1.3 (1) 0.3 (-)
T15
20 88-89
0.9 ( I )
1.1 (1)
T16a
60 90-91
0.8(1)
1.1 (1)
T16b
T18
60 92-95
0.6 (1)
1.0 (I) 0.3(-)
T17,18'
250 220 96-104 92-104
1.1 (1)
0.9 (1) 0.8 (1)
0.2(-)
1.3 (1) 1.2 (1) 0.9(1) 1.4(1) 0.4 (-) 2.7 (3) 1.2(1) 0.2(-) 0.4 (-)
T17
260 105-110
0.4 (-) 0.9(1)
0.6(l)c 0.1 (-) 0.4(-)
0.1(-1 1.1 ( I ) 1 .O (2)c
0.8(-) 0.3 (-) 0.8 (-) 2.0 (1)
T19'
530 111-124
0.1 (-) 0.8 (1)
0.2(-) 1.8 (2)
1.2 (1) 0.3 (-) 2.9 (2)d 1.1 (1) 1.9 (2) 1.1 (1) 1.0 (1) 2.4 (3) 0.1 (-)
T20
Peptides T17,18,and T19 were only partly resolved by paper electrophoresis at pH 3.5 (see Figure 2). The analysis of peptide T17,18 only gave qualitative evidence about the presence. of this peptide. The impurities in peptide T19 can be accounted for by the presence of about 0.3 equivalent of peptide T17,18. me results of the Dansyl-Edman degradation were in agreement with this contamination. Including methionine sulphoxide. c The Val-Ile-Val sequence. (106-108)is only partly hydrolyzed after 20 h. High serine value from minor contamination by peptide T3.
Ser Glu PI0 G~Y Ala Val Met Ile Leu TYr Phe LYS AetCys His Arg nmoles positioninseq.
Thr
T14
TABLE1B (con?.)
m
III
F
0
5
5
w
g
5x
0 %
32
c"
7
