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Peptic digests of the I-peptide fractions of five strains of tobacco mosaic virus
VIROLOGY
1%
589-595
Peptic
Digests
(1960)
of the I-Peptide Fractions of Tobacco Mosaic Virus’
of Five Strains
DARCY M. SILVA~ ANU C. A. KNIGHT Virus
Laboratory,
ZTdversitv Accepted
of Calijorniu,
Berkeley,
California
October 14, 1960
The protein components of five strains of tobacco mosaic virus (TMV) were separated from the virus particles by treatment with acetic acid. The isolated proteins were digested with trypsin, and the tryptic digests were adjusted to pH 4.5, causing, in each case, precipitation of an I-peptide fraction. The I-peptide fractions were digested with pepsin and the resulting peptides were separated by a combination of paper electrophoresis and paper chromatography. The peptide spots were located by spraying with various indicator reagents. The patterns, or maps, of such spots were very similar for four of the strains but grossly different from the map for the Holmes ribgrass (HR) strain. INTRODUCTTON
When t,he protein components of several strains of tobacco mosaic virus (TMV) were digested w&h trypsin, it was observed t,hat lowering the pH to about 4 or 4.5 resulted in each case in a fractionation of the digests into soluble and insoluble peptides (Woody and Knight, 1959). Two-thirds to three-fourths of the weight of peptidic material in each digest was soluble at pH 4.5 and above, and t,hese soluble fractions were subjected to a two-dimensional process of paper electrophoresis and paper chromatography followed by staining to give for each strain a highly characteristic pattern or “map” of the soluble tryptic peptides. The balance of t,he material of the tryptic digests consists mainly of a large peptide, which because of its relative insolubility has been designated I-peptide (Gish et al., 1958; Anderer et al., 1960a), and one or two other sparingly soluble peptides (D. T. Gish, and H. G. Wittmann, 1 Supported in part by a research grant, E-634, from the National Institute of Institutes of Health, United States Allergy and Infectious Diseases, National Public Health Service. * Work performed while on leave from Instituto Agronomico, Campinas, Brazil, and while holding a Rockefeller Foundation Fellowship. 589
590
SILVA
,4ND
KSIGHT
personal communications), which may precipitate together with I-peptide at pH 4.5. We shall call this mixture of the less soluble tryptic peptides the “I-peptide fraction.” In order to complete the comparison of the tryptic peptides obtained from five selected strains of TMV, we have attempted to digest the I-peptide fraction of each strain with a protease other than trypsin and to subject the products to t,he same mapping procedure that had yielded such characterist’ic patterns with t,he soluble tryptic peptides. Among the enzymes tested for this purpose under a variety of conditions were chymotrypsin, papain, streptomyces protease, subtilisin, collagenase, and pepsin. Xone proved highly satisfactory, but pepsin gave fewer product’s and the most readily reproducible results and hence was adopted for this study. The products revealed by mapping the peptic digests of I-peptide fractions of strains of TMV appeared very similar in some cases and quite distinctive in others, and presumably reflect similarities and differences in the amino acid sequences in those portions of the strain prot’eins from which the I-pept,ides were derived. MATERIAL
AND METHOUS
Virus Strains The common strain of tobacco mosaic virus (TMV) and the M, YA, HR, and J14Dl strains previously studied (Woody and Knight, 1959) were employed. Highly purified preparations were obtained by the customary procedure of differential centrifugation. Protein preparations were obtained from the virus strains by treatment with cold 67 % acetic acid (Fraenkel-Conrat, 1957). Preparation of I-Peptide Fractions The strain proteins were digested with trypsin as previously described (Woody and Knight, 1959). After digestion the pH was lowered to 4.5 and the digest stored for 1 hour at 4”, except for YA digests which were stored overnight. The precipitated I-peptide fraction was sedimented in an angle centrifuge and the soluble fraction removed. The precipitate, suspended in distilled water, was dialyzed against several changes of distilled water at 5” for 12-24 hours and then dried from the frozen state. Peptic Digestion The I-peptide fraction at 5 mg per milliliter was adjusted to pH 2 and one part by weight of crystalline pepsin (Worthington Biochemical
PEPTIC
DIGESTS
OF I-PEPTIDE
FRACTIONS
OF TOBACCO
MOSAIC
VIRUS
591
-‘-ELECTROPHORESIS++
0 3
04 8
t , * ;+
8
U
53 /;\ \.a*’
07
49 0
FIG. 1. Composite diagram of the peptide maps obtained after peptic digestion of the I-peptide fractions of five strains of tobacco mosaic virus. See Table 1 for assignment of spots to the various strains. Solid lines indicate spots that were quite definite with ninhydrin, whereas dotted lines indicate a weak reaction.
Corporation, Icreehold, New Jersey) was added per 67 parts I-peptide. The digestiou was continued for 20-24 hours at 39”. These condit’ions were about maximal as judged by ninhydrin reackion. Addition of one or two fresh charges of enzyme part, way through the digestion seemed not t,o alter significantly the patterns of spots subseyuemly observed upon mapping the digests. Peptide Mapping
The two-dimensional procedure of paper electrophorexis and paper chromatography previously described (Woody and Knight,, 1959) was employed to separate the peptides obtained by peptic digestion of the buffer I-peptide fra&ons of the strains. The y-collidine/HOAc/HzO previously employed in the clectrophoresis step was tried at, pH values of 4, 7, 8, and 10, but the best separat,ions seemed to occur at pH 7 and
Strain TMV”
M --____
YA Cationic
1-15
1-15
HK peptides
1-15
at pH 7
lA, 3, -1, 5, lOA, 11, 12A, 13A, 15, 23. 24, 25, 26, 27, 28, 29, 30
Isoelectric
peptides
J14Dl
2215
at pH 7
31, 32, 33 Anionic 16-21
1621
peptides
16, 17, 18, WA, 20, 21, 22
at pH 7 19A, 21,22,3-L, 35
19, 20A, 21,35
a Numbers were arbitrarily assigned to the TMV peptides as shown in Fig. 1; for the other strains, the appearance on the peptide maps of spots in the same areas and giving the same color reactions was assumed to indicate identity, and hence the same numbers were assigned to such spots. New numbers were assigned to distinctive spots occurring in any of the strains. Letters added to numbers signify that the peptide occupies a position characteristic for one of the peptides of TMV or any other strain listed to the left of it in the table but differs from it in some respect, as indicated by distinctive color tests. Electroosmotic displacement was ignored in listing the peptides as anionic, etc.
PEPTIC
DIGESTS
OF I-PEPTIDE
FRACTIONS
OF TOBACCO
MOSAIC
VIRUS
5%
hence this condition was used throughout. Maps of the peptide spots were obtained by spraying the papers wit’h ninhydrin, or with suitable reagent’s reacting with arginine, tyrosine, tryptophan, and peptide bonds, respectively, as previously described (Woody and Knight, 1959). RESULTS
Fourteen major and seven minor spot’s were detected (by spraying with ninhydrin) on a typical map obt)ained from the peptic digest of the I-peptide fraction of TMV. This is close to t’he number to be expected from a peptic digest of I-peptides as shown by sequence studies (Anderer et al., 196Oa, b). The number and character of the spots was found to be reproducible with digest’s from different preparations of TMV I-peptide fraction, and hence the procedure seemed applicable to the comparison of strains. A diagram summarizing the distribution of all the spots observed on maps for the five strains is shown in Fig. 1. Numbers were arbitrarily assigned to the TMV peptide spots as shown in Table 1, and in the cases of t’he other strains, the appearance on the peptide maps of spots in the same areas and giving the same color reactions was assumed to indicate identity, and hence the same numbers were assigned to such spots. In this system, a number represents an area on the map; when a demonstrably different peptide is found t.o occupy this area, a distinction is made by adding a letter to t,he number, and when a new area is involved, a new number is assigned. The peptic peptides thus ident,ified in the digests of the st’rain 1 peptide fract,ions are summarized in Tables 1 and 2. The dat,a in Tables 1 and 2 show a decided similarity between TMV TABLE PEPTIDES
Spots with:
SHOWING
COLOR
TMV”
2
REACTIONS SPECIFIC AND TYROSINE M
YA
Arginine
1, 3, 4, 5
1, 3, 4, 5
1, 3, 4, 5
Tryptophan Tyrosine
10, 17, 20
10, 17, 20
21
21
10, 17 19A, 20 21
FOR AROININE,
TRYPTOPHAN,
HR
1-k 3, 4, 5, 25, 26, 28 lOA, 12A, 19A, 31 lOA, 12A, 13A, 22, 26
J14Dl
3, 4, 5 10 21
n The peptides are those ohtained hy peptic digestion of the I-peptide fractions of strains of tobacco mosaic virus. They are numbered arbitrarily as indicated in Tahle 1 and as shown, in part, in Fig. 1.
594
SILVA
AND
KNIGHT
and strains M, YA, and J14D1, but a pronounced difference between all of the strains and HR. DISCUSSION
Mapping of the tryptic peptides soluble at pH 4.5 (Woody and Knight, 1959) from the same strains involved in the present study, showed many similarities, and some striking differences, among the strain proteins. Those findings are extended by the present result’s, which essentially complete the survey of the tryptic peptides obtained from these strain proteins. As previously noted, there are limitations to this approach which cannot be removed until complete sequences are determined for the amino acid residues in each tryptic peptide and each peptide is then located in it,s proper sequence in the protein subunit. However, this task is enormous and cannot be readily accomplished. In the meantime, the results of the mapping procedure permit tentative conclusions to be drawn with import,ant implications regarding the primary structure of the strain proteins. In the present study, the TMV and M strain peptides were found to be indistinguishable; this was also the result of the earlier studies on the soluble tryptic pept,ides. Combining results, it appears likely t’hat TMV and M proteins are identical, although complete sequential analyses might reveal small differences. A similar conclusion was reached by Wittmann (1959) after comparing the compositions of the peptides from TMV and a so-called nitrous acid mutant. The general nature of the present maps of YA and J14Dl also resemble the previous results obtained with the soluble tryptic peptides, namely, the maps for these strains show a great many similarities with the peptide pattern of TMV, but also demonstrate a few distinct differences. These resulm support the former suggestion t,hat the few differences in amino acid content found between TMV, YA, and J14Dl strains (Knight, 1947) are localized and that the rest of the protein chain is probably identical for the three strains. The present peptide map for the HR strain, like the previous ones for the soluble tryptic peptides, is grossly different from t’he maps for TMV and the other strains. This is also consistent with the results of the amino acid analyses which show HR protein to be very different from TMV and the other strains (Knight,, 1947). REFERENCES ANDERER, F. A., WEBER, E., and UHLIG, H. (1960a). Aminosiirwen Tabak-mosaikvirus. I. Die N-terminale aminoshrefolge von 2. Naturforsch lSb, 79-85.
in Protein des Posit,ion l-41.
PEPTIC
DIGESTS
OF I-PEPTIDE
FRACTIONS
OF TOBACCO
MOSAIC
VIRUS
595
F. A., UHLIG, H., W~BER, E., and SCHRAMM, G. (1960b). Primary structure of the protein of tobacco mosaic virus. Nature 186, 922-925. FRAENKEL-CONRAT, H. (1957). Degradation of tobacco mosaic virus with acetic acid. Virology 4, l-4. GISH, D. T., RAMACHANDRAN, L. K., and STANLEY, W. M. (1958). Studies on the amino acid sequence of tobacco mosaic virus (TMV) protein. I. Fractionation of products of tryptic hydrolysis by countercurrent distribution. Arch. &o&em. Biophys. 78, 433-450. KNIGHT, C. A. (1947). The nature of some of the chemical differences among strains of tobacco mosaic virus. J. Biol. Chem. 171, 297-308. WITTMANN, H. G. (1959). Vergleich der Proteine des Normalstamms und einer Nitritmutante des Tabakmosaikvirus. 2. Vererbungslehre 90, 463-475. WOODY, B. R., and KNIGHT, C. A. (1959). Peptide maps obtained with tryptic digests of the proteins of some strains of tobacco mosaic virus. Virology 9, 359374.
ANDERER,
1%
589-595
Peptic
Digests
(1960)
of the I-Peptide Fractions of Tobacco Mosaic Virus’
of Five Strains
DARCY M. SILVA~ ANU C. A. KNIGHT Virus
Laboratory,
ZTdversitv Accepted
of Calijorniu,
Berkeley,
California
October 14, 1960
The protein components of five strains of tobacco mosaic virus (TMV) were separated from the virus particles by treatment with acetic acid. The isolated proteins were digested with trypsin, and the tryptic digests were adjusted to pH 4.5, causing, in each case, precipitation of an I-peptide fraction. The I-peptide fractions were digested with pepsin and the resulting peptides were separated by a combination of paper electrophoresis and paper chromatography. The peptide spots were located by spraying with various indicator reagents. The patterns, or maps, of such spots were very similar for four of the strains but grossly different from the map for the Holmes ribgrass (HR) strain. INTRODUCTTON
When t,he protein components of several strains of tobacco mosaic virus (TMV) were digested w&h trypsin, it was observed t,hat lowering the pH to about 4 or 4.5 resulted in each case in a fractionation of the digests into soluble and insoluble peptides (Woody and Knight, 1959). Two-thirds to three-fourths of the weight of peptidic material in each digest was soluble at pH 4.5 and above, and t,hese soluble fractions were subjected to a two-dimensional process of paper electrophoresis and paper chromatography followed by staining to give for each strain a highly characteristic pattern or “map” of the soluble tryptic peptides. The balance of t,he material of the tryptic digests consists mainly of a large peptide, which because of its relative insolubility has been designated I-peptide (Gish et al., 1958; Anderer et al., 1960a), and one or two other sparingly soluble peptides (D. T. Gish, and H. G. Wittmann, 1 Supported in part by a research grant, E-634, from the National Institute of Institutes of Health, United States Allergy and Infectious Diseases, National Public Health Service. * Work performed while on leave from Instituto Agronomico, Campinas, Brazil, and while holding a Rockefeller Foundation Fellowship. 589
590
SILVA
,4ND
KSIGHT
personal communications), which may precipitate together with I-peptide at pH 4.5. We shall call this mixture of the less soluble tryptic peptides the “I-peptide fraction.” In order to complete the comparison of the tryptic peptides obtained from five selected strains of TMV, we have attempted to digest the I-peptide fraction of each strain with a protease other than trypsin and to subject the products to t,he same mapping procedure that had yielded such characterist’ic patterns with t,he soluble tryptic peptides. Among the enzymes tested for this purpose under a variety of conditions were chymotrypsin, papain, streptomyces protease, subtilisin, collagenase, and pepsin. Xone proved highly satisfactory, but pepsin gave fewer product’s and the most readily reproducible results and hence was adopted for this study. The products revealed by mapping the peptic digests of I-peptide fractions of strains of TMV appeared very similar in some cases and quite distinctive in others, and presumably reflect similarities and differences in the amino acid sequences in those portions of the strain prot’eins from which the I-pept,ides were derived. MATERIAL
AND METHOUS
Virus Strains The common strain of tobacco mosaic virus (TMV) and the M, YA, HR, and J14Dl strains previously studied (Woody and Knight, 1959) were employed. Highly purified preparations were obtained by the customary procedure of differential centrifugation. Protein preparations were obtained from the virus strains by treatment with cold 67 % acetic acid (Fraenkel-Conrat, 1957). Preparation of I-Peptide Fractions The strain proteins were digested with trypsin as previously described (Woody and Knight, 1959). After digestion the pH was lowered to 4.5 and the digest stored for 1 hour at 4”, except for YA digests which were stored overnight. The precipitated I-peptide fraction was sedimented in an angle centrifuge and the soluble fraction removed. The precipitate, suspended in distilled water, was dialyzed against several changes of distilled water at 5” for 12-24 hours and then dried from the frozen state. Peptic Digestion The I-peptide fraction at 5 mg per milliliter was adjusted to pH 2 and one part by weight of crystalline pepsin (Worthington Biochemical
PEPTIC
DIGESTS
OF I-PEPTIDE
FRACTIONS
OF TOBACCO
MOSAIC
VIRUS
591
-‘-ELECTROPHORESIS++
0 3
04 8
t , * ;+
8
U
53 /;\ \.a*’
07
49 0
FIG. 1. Composite diagram of the peptide maps obtained after peptic digestion of the I-peptide fractions of five strains of tobacco mosaic virus. See Table 1 for assignment of spots to the various strains. Solid lines indicate spots that were quite definite with ninhydrin, whereas dotted lines indicate a weak reaction.
Corporation, Icreehold, New Jersey) was added per 67 parts I-peptide. The digestiou was continued for 20-24 hours at 39”. These condit’ions were about maximal as judged by ninhydrin reackion. Addition of one or two fresh charges of enzyme part, way through the digestion seemed not t,o alter significantly the patterns of spots subseyuemly observed upon mapping the digests. Peptide Mapping
The two-dimensional procedure of paper electrophorexis and paper chromatography previously described (Woody and Knight,, 1959) was employed to separate the peptides obtained by peptic digestion of the buffer I-peptide fra&ons of the strains. The y-collidine/HOAc/HzO previously employed in the clectrophoresis step was tried at, pH values of 4, 7, 8, and 10, but the best separat,ions seemed to occur at pH 7 and
Strain TMV”
M --____
YA Cationic
1-15
1-15
HK peptides
1-15
at pH 7
lA, 3, -1, 5, lOA, 11, 12A, 13A, 15, 23. 24, 25, 26, 27, 28, 29, 30
Isoelectric
peptides
J14Dl
2215
at pH 7
31, 32, 33 Anionic 16-21
1621
peptides
16, 17, 18, WA, 20, 21, 22
at pH 7 19A, 21,22,3-L, 35
19, 20A, 21,35
a Numbers were arbitrarily assigned to the TMV peptides as shown in Fig. 1; for the other strains, the appearance on the peptide maps of spots in the same areas and giving the same color reactions was assumed to indicate identity, and hence the same numbers were assigned to such spots. New numbers were assigned to distinctive spots occurring in any of the strains. Letters added to numbers signify that the peptide occupies a position characteristic for one of the peptides of TMV or any other strain listed to the left of it in the table but differs from it in some respect, as indicated by distinctive color tests. Electroosmotic displacement was ignored in listing the peptides as anionic, etc.
PEPTIC
DIGESTS
OF I-PEPTIDE
FRACTIONS
OF TOBACCO
MOSAIC
VIRUS
5%
hence this condition was used throughout. Maps of the peptide spots were obtained by spraying the papers wit’h ninhydrin, or with suitable reagent’s reacting with arginine, tyrosine, tryptophan, and peptide bonds, respectively, as previously described (Woody and Knight, 1959). RESULTS
Fourteen major and seven minor spot’s were detected (by spraying with ninhydrin) on a typical map obt)ained from the peptic digest of the I-peptide fraction of TMV. This is close to t’he number to be expected from a peptic digest of I-peptides as shown by sequence studies (Anderer et al., 196Oa, b). The number and character of the spots was found to be reproducible with digest’s from different preparations of TMV I-peptide fraction, and hence the procedure seemed applicable to the comparison of strains. A diagram summarizing the distribution of all the spots observed on maps for the five strains is shown in Fig. 1. Numbers were arbitrarily assigned to the TMV peptide spots as shown in Table 1, and in the cases of t’he other strains, the appearance on the peptide maps of spots in the same areas and giving the same color reactions was assumed to indicate identity, and hence the same numbers were assigned to such spots. In this system, a number represents an area on the map; when a demonstrably different peptide is found t.o occupy this area, a distinction is made by adding a letter to t,he number, and when a new area is involved, a new number is assigned. The peptic peptides thus ident,ified in the digests of the st’rain 1 peptide fract,ions are summarized in Tables 1 and 2. The dat,a in Tables 1 and 2 show a decided similarity between TMV TABLE PEPTIDES
Spots with:
SHOWING
COLOR
TMV”
2
REACTIONS SPECIFIC AND TYROSINE M
YA
Arginine
1, 3, 4, 5
1, 3, 4, 5
1, 3, 4, 5
Tryptophan Tyrosine
10, 17, 20
10, 17, 20
21
21
10, 17 19A, 20 21
FOR AROININE,
TRYPTOPHAN,
HR
1-k 3, 4, 5, 25, 26, 28 lOA, 12A, 19A, 31 lOA, 12A, 13A, 22, 26
J14Dl
3, 4, 5 10 21
n The peptides are those ohtained hy peptic digestion of the I-peptide fractions of strains of tobacco mosaic virus. They are numbered arbitrarily as indicated in Tahle 1 and as shown, in part, in Fig. 1.
594
SILVA
AND
KNIGHT
and strains M, YA, and J14D1, but a pronounced difference between all of the strains and HR. DISCUSSION
Mapping of the tryptic peptides soluble at pH 4.5 (Woody and Knight, 1959) from the same strains involved in the present study, showed many similarities, and some striking differences, among the strain proteins. Those findings are extended by the present result’s, which essentially complete the survey of the tryptic peptides obtained from these strain proteins. As previously noted, there are limitations to this approach which cannot be removed until complete sequences are determined for the amino acid residues in each tryptic peptide and each peptide is then located in it,s proper sequence in the protein subunit. However, this task is enormous and cannot be readily accomplished. In the meantime, the results of the mapping procedure permit tentative conclusions to be drawn with import,ant implications regarding the primary structure of the strain proteins. In the present study, the TMV and M strain peptides were found to be indistinguishable; this was also the result of the earlier studies on the soluble tryptic pept,ides. Combining results, it appears likely t’hat TMV and M proteins are identical, although complete sequential analyses might reveal small differences. A similar conclusion was reached by Wittmann (1959) after comparing the compositions of the peptides from TMV and a so-called nitrous acid mutant. The general nature of the present maps of YA and J14Dl also resemble the previous results obtained with the soluble tryptic peptides, namely, the maps for these strains show a great many similarities with the peptide pattern of TMV, but also demonstrate a few distinct differences. These resulm support the former suggestion t,hat the few differences in amino acid content found between TMV, YA, and J14Dl strains (Knight, 1947) are localized and that the rest of the protein chain is probably identical for the three strains. The present peptide map for the HR strain, like the previous ones for the soluble tryptic peptides, is grossly different from t’he maps for TMV and the other strains. This is also consistent with the results of the amino acid analyses which show HR protein to be very different from TMV and the other strains (Knight,, 1947). REFERENCES ANDERER, F. A., WEBER, E., and UHLIG, H. (1960a). Aminosiirwen Tabak-mosaikvirus. I. Die N-terminale aminoshrefolge von 2. Naturforsch lSb, 79-85.
in Protein des Posit,ion l-41.
PEPTIC
DIGESTS
OF I-PEPTIDE
FRACTIONS
OF TOBACCO
MOSAIC
VIRUS
595
F. A., UHLIG, H., W~BER, E., and SCHRAMM, G. (1960b). Primary structure of the protein of tobacco mosaic virus. Nature 186, 922-925. FRAENKEL-CONRAT, H. (1957). Degradation of tobacco mosaic virus with acetic acid. Virology 4, l-4. GISH, D. T., RAMACHANDRAN, L. K., and STANLEY, W. M. (1958). Studies on the amino acid sequence of tobacco mosaic virus (TMV) protein. I. Fractionation of products of tryptic hydrolysis by countercurrent distribution. Arch. &o&em. Biophys. 78, 433-450. KNIGHT, C. A. (1947). The nature of some of the chemical differences among strains of tobacco mosaic virus. J. Biol. Chem. 171, 297-308. WITTMANN, H. G. (1959). Vergleich der Proteine des Normalstamms und einer Nitritmutante des Tabakmosaikvirus. 2. Vererbungslehre 90, 463-475. WOODY, B. R., and KNIGHT, C. A. (1959). Peptide maps obtained with tryptic digests of the proteins of some strains of tobacco mosaic virus. Virology 9, 359374.
ANDERER,