- Email: [email protected]
The amino acid composition of turnip yellow mosaic virus and the associated abnormal protein
VIROLOGY
4, 126-129
The Amino Virus
(1957)
Acid Composition and the Associated
of Turnip Abnormal
Yellow Mosaic Protein’
Amino acid analyses of two preparations each of the “top” and “bottom” components of turnip yellow mosaic virus preparations show them to be parallel in composition. This is consistent with the hypothesis that the “top” component is actually virus protein and the “bot,tom” component complet,e virus consisting of this same protein combined wit,h nucleic acid. INTRODUCTION
In 1949 Markham and Smith reported finding, in plants infect’ed wit’h turnip yellow mosaic virus, a protein similar in many properties to t,hc infectious virus particles, but it was noninfectious and contained no nucleic acid. Although other plant virus infections have led to concomitant production of specific proteins, the turnip yellow mosaic virus system is unusual in that, unlike the “X-protein” from plants infect’ed with tobacco mosaic virus (Takahashi and Ishii, 1952; Newmark and Fraser, 195(i), t’he turnip yellow mosaic protein is of the same general size and appearance as t’he virus itself. The question, then, is whether these protein particles are simply incomplete viruses in which the ribonucleic acid (R?JA) is somehow missing. In recently reported work, Cosentino et al. (1956) have discussed the isolation and purification of both the protein and infectious components and have compared a number of their properties. The present, work consist’s of a comparison of the amino acid composit’ion of the int,act \-irus and that of t,he noninfectious prot#ein. 1 Aided hg grant,s from the National Foundation for Infantile Rockefeller Foundation. 2 Present address: Bact~eriologp Department,, Indiana University, t,on, Indiana. 3 Present address : Istituto di I’atologia Veget,ale, Universita I’crugia, Italy.
Paralysis
and
the
126
Bloomingdi
Perugia,
AMINO
ACIDS
OY
TUILA-11’
YELLOW
MATERIALS
AND
VIRUS
AND
127
1’ltOTElN
METHODS
The purified protein and virus were prepared by the methods previously described (Cosentino et al., 1956). The purification entailed density gradient separation and final purification by electrophoresis, selecting material which appeared homogeneous in the Perkin-Elmer apparatus. Data on the purity of the materials are included in the previous paper. The amino acid analyses were run essentially by the ion exchange chromatographic technique of Moore and Stein (1948, 1951) as modified by Him et al. (1954) and by the hydrolysis procedure described by Fraser (1957). RESULTS
Because of the very great difficulty experienced in obtaining highly purified preparations of the two components, particularly the “top” component or protein (Cosentino et al., 1956), it was possible to perform only a very few analyses. The results shown in Table 1 are derived from TABLE Ac~u
AMIXO TrfE
COMPOSITION “TOP COMPUNENT”
1
OF TURNIP ISOI,ATED Grams
Amino
acid
“Top”
Alanine Arginine Aspartic acid Cystine Glutamic acid Glycine Hist,idine Leucine Isoleucine Lysine Methionine Phenyla!anine Proline Swine Threonine Tyrosine Valine
5.1 2.4 5.9 0 7.7 2.5 2.2 8.5 7.4 5.0 2.1 3.5 9.7 6.3 11.3 2.0 6.2 Totalp
q Total
range
of the
analyses
YELLOW FROM of amino
(protein)
f f f
oa 0.4 0
f 0.3 f 0 f 0.1 f 0.1 * 0 It 0.3 f
0.1
* I!C & f f f
0 0.6 0.1 0.2 0.3 0.2
87.8 to nearest
1CZosa1c INFECTED acid
VIRUS PLANTS
per 100 I: of Drotein “Bottom”
5.4 2.2 6.3 0 8.0 3.8 1.6 8.6 7.4 5.0 2.1 3.6 11.8 6.7 12.2 2.2 6.2 93.1
0.1%.
ANI)
(virus)
f f f
O.lG 0.2 0
f f f & f f f f f f f f f
0 0 0 0.3 0.3 0.1 0.2 0.4 0.4 0.2 0.1 0.4 0.1
128
FRASER
SND
COSENTINO
ten samples in all, varying from 0.806 to 0.817 mg of protein each. These were used in two analyses of the protein and three of the virus for the neutral and acidic amino acids and also for two and three analyses of the protein and virus, respectively, for the basic amino acids obtained from a separate column. Only a very few individual determinations (Table 1) differed sufficiently to deserve comment; i.e., only a few were different beyond the error we have experienced in replicate determinations of identical samples of other proteins. The amount of glycine obtained from the virus is appreciably greater than that obtained from the protein. This was to be expected as a result of decomposition of the nucleic acid of the virus, originally described by Smith and Markham (1950). The differences in proline values were not surprising when the quite low color yield of the ninhydrin method for determination of this part’icular amino acid (Moore and Stein, 1948) was considered; our replicate determinations for this amino acid showed considerable scatter. The divergent values for histidine, also, were within the variance of these particular determinations. It should be noted that we have not, in these analyses, been able to compensate for the decomposition of serine, t’hreonine, and tyrosine (cf. Fraser, 1957) because of scarcity of material. These values, then, although comparable with each other because of identical hydrolytic conditions, were certainly somewhat low, probably accounting for the slightly low total recoveries. The necessarily separate determinations of tryptophan and cysteine have not been possible with the amounts of material available. DISCUSSION
The question under discussion is whether this protein is to be considered an extraneous by-product of virus replication or whether the protein is in actuality virus protein devoid of ribonucleic acid and hence noninfectious. By the present’ analyses one could not, of course, prove the identity of the two prot,eins, but the methods used should have demonstrated nonidentity if the amino acid compositions of the two components had differed appreciably. Actually, the analysis of the virus agrees with that of the protein to a remarkable degree; in fact, the agreement is to someextent fortuitous in view of the errors we have observed and in view of the fact that the analyses represent. a very few determinations of two different preparations each of virus and protein.
AMINO
ACIDS
OF
TURNIP
YELLOW
VIRUS
AiXD
129
PROTIEN
ACKNOWLEDGMENTS The sistance
authors wish to express of Barbara Corts Rawls
their and
appreciation Mary Brown
of the Lawson.
skillful
technical
as-
REFERENCES COSENTINO, V., PAIGEN, K., and STEERE, R. L. (1956). Electron microscopy of turnip yellow mosaic virus and the associated abnormal protein. Virology 2, 139-148. FRASER, D. (1957). Comparison of the amino acid composition of T2 and T3 bacteriophages. J. Biol. Chem. (in press). HIRS, C. H. W., STEIN, W. H., and MOORE, S. (1954). The amino acid composition of ribonuclease. J. Biol. Chem. 211, 941-950. MOORE, S., and STEIN, W. H. (1948). Photometric ninhydrin method for use in the chromatography of amino acids. J. Bio2. Chem. 170, 367-388. MOORE, S., and STEIN, W. H. (1951). Ch romatography of amino acids on sulfonated polystyrene resins. J. Biol. Chem. 192, 663681. MARKHAM, R., and SMITH, K. M. (1949). Studies on the virus of turnip yellow mosaic. Parasitology 39, 330-342. NEWMARK, P., and FRASER, D. (1956). Composition of an abnormal protein present in tobacco plants infected with tobacco mosaic virus. J. ilm. Ckem. Sot. 78, 1588-1590. SMITH, J. D., and MARKHAM, R. (1950). Chromatographic studies of nucleic acids. 2. The quantitative analysis of ribonucleic acids. Biochem. J. 48, 509513. TAKAHASHI, W. N., and ISHII, M. (1952). An abnormal protein associated with tobacco mosaic virus infection. Nature 189. 419-420.
4, 126-129
The Amino Virus
(1957)
Acid Composition and the Associated
of Turnip Abnormal
Yellow Mosaic Protein’
Amino acid analyses of two preparations each of the “top” and “bottom” components of turnip yellow mosaic virus preparations show them to be parallel in composition. This is consistent with the hypothesis that the “top” component is actually virus protein and the “bot,tom” component complet,e virus consisting of this same protein combined wit,h nucleic acid. INTRODUCTION
In 1949 Markham and Smith reported finding, in plants infect’ed wit’h turnip yellow mosaic virus, a protein similar in many properties to t,hc infectious virus particles, but it was noninfectious and contained no nucleic acid. Although other plant virus infections have led to concomitant production of specific proteins, the turnip yellow mosaic virus system is unusual in that, unlike the “X-protein” from plants infect’ed with tobacco mosaic virus (Takahashi and Ishii, 1952; Newmark and Fraser, 195(i), t’he turnip yellow mosaic protein is of the same general size and appearance as t’he virus itself. The question, then, is whether these protein particles are simply incomplete viruses in which the ribonucleic acid (R?JA) is somehow missing. In recently reported work, Cosentino et al. (1956) have discussed the isolation and purification of both the protein and infectious components and have compared a number of their properties. The present, work consist’s of a comparison of the amino acid composit’ion of the int,act \-irus and that of t,he noninfectious prot#ein. 1 Aided hg grant,s from the National Foundation for Infantile Rockefeller Foundation. 2 Present address: Bact~eriologp Department,, Indiana University, t,on, Indiana. 3 Present address : Istituto di I’atologia Veget,ale, Universita I’crugia, Italy.
Paralysis
and
the
126
Bloomingdi
Perugia,
AMINO
ACIDS
OY
TUILA-11’
YELLOW
MATERIALS
AND
VIRUS
AND
127
1’ltOTElN
METHODS
The purified protein and virus were prepared by the methods previously described (Cosentino et al., 1956). The purification entailed density gradient separation and final purification by electrophoresis, selecting material which appeared homogeneous in the Perkin-Elmer apparatus. Data on the purity of the materials are included in the previous paper. The amino acid analyses were run essentially by the ion exchange chromatographic technique of Moore and Stein (1948, 1951) as modified by Him et al. (1954) and by the hydrolysis procedure described by Fraser (1957). RESULTS
Because of the very great difficulty experienced in obtaining highly purified preparations of the two components, particularly the “top” component or protein (Cosentino et al., 1956), it was possible to perform only a very few analyses. The results shown in Table 1 are derived from TABLE Ac~u
AMIXO TrfE
COMPOSITION “TOP COMPUNENT”
1
OF TURNIP ISOI,ATED Grams
Amino
acid
“Top”
Alanine Arginine Aspartic acid Cystine Glutamic acid Glycine Hist,idine Leucine Isoleucine Lysine Methionine Phenyla!anine Proline Swine Threonine Tyrosine Valine
5.1 2.4 5.9 0 7.7 2.5 2.2 8.5 7.4 5.0 2.1 3.5 9.7 6.3 11.3 2.0 6.2 Totalp
q Total
range
of the
analyses
YELLOW FROM of amino
(protein)
f f f
oa 0.4 0
f 0.3 f 0 f 0.1 f 0.1 * 0 It 0.3 f
0.1
* I!C & f f f
0 0.6 0.1 0.2 0.3 0.2
87.8 to nearest
1CZosa1c INFECTED acid
VIRUS PLANTS
per 100 I: of Drotein “Bottom”
5.4 2.2 6.3 0 8.0 3.8 1.6 8.6 7.4 5.0 2.1 3.6 11.8 6.7 12.2 2.2 6.2 93.1
0.1%.
ANI)
(virus)
f f f
O.lG 0.2 0
f f f & f f f f f f f f f
0 0 0 0.3 0.3 0.1 0.2 0.4 0.4 0.2 0.1 0.4 0.1
128
FRASER
SND
COSENTINO
ten samples in all, varying from 0.806 to 0.817 mg of protein each. These were used in two analyses of the protein and three of the virus for the neutral and acidic amino acids and also for two and three analyses of the protein and virus, respectively, for the basic amino acids obtained from a separate column. Only a very few individual determinations (Table 1) differed sufficiently to deserve comment; i.e., only a few were different beyond the error we have experienced in replicate determinations of identical samples of other proteins. The amount of glycine obtained from the virus is appreciably greater than that obtained from the protein. This was to be expected as a result of decomposition of the nucleic acid of the virus, originally described by Smith and Markham (1950). The differences in proline values were not surprising when the quite low color yield of the ninhydrin method for determination of this part’icular amino acid (Moore and Stein, 1948) was considered; our replicate determinations for this amino acid showed considerable scatter. The divergent values for histidine, also, were within the variance of these particular determinations. It should be noted that we have not, in these analyses, been able to compensate for the decomposition of serine, t’hreonine, and tyrosine (cf. Fraser, 1957) because of scarcity of material. These values, then, although comparable with each other because of identical hydrolytic conditions, were certainly somewhat low, probably accounting for the slightly low total recoveries. The necessarily separate determinations of tryptophan and cysteine have not been possible with the amounts of material available. DISCUSSION
The question under discussion is whether this protein is to be considered an extraneous by-product of virus replication or whether the protein is in actuality virus protein devoid of ribonucleic acid and hence noninfectious. By the present’ analyses one could not, of course, prove the identity of the two prot,eins, but the methods used should have demonstrated nonidentity if the amino acid compositions of the two components had differed appreciably. Actually, the analysis of the virus agrees with that of the protein to a remarkable degree; in fact, the agreement is to someextent fortuitous in view of the errors we have observed and in view of the fact that the analyses represent. a very few determinations of two different preparations each of virus and protein.
AMINO
ACIDS
OF
TURNIP
YELLOW
VIRUS
AiXD
129
PROTIEN
ACKNOWLEDGMENTS The sistance
authors wish to express of Barbara Corts Rawls
their and
appreciation Mary Brown
of the Lawson.
skillful
technical
as-
REFERENCES COSENTINO, V., PAIGEN, K., and STEERE, R. L. (1956). Electron microscopy of turnip yellow mosaic virus and the associated abnormal protein. Virology 2, 139-148. FRASER, D. (1957). Comparison of the amino acid composition of T2 and T3 bacteriophages. J. Biol. Chem. (in press). HIRS, C. H. W., STEIN, W. H., and MOORE, S. (1954). The amino acid composition of ribonuclease. J. Biol. Chem. 211, 941-950. MOORE, S., and STEIN, W. H. (1948). Photometric ninhydrin method for use in the chromatography of amino acids. J. Bio2. Chem. 170, 367-388. MOORE, S., and STEIN, W. H. (1951). Ch romatography of amino acids on sulfonated polystyrene resins. J. Biol. Chem. 192, 663681. MARKHAM, R., and SMITH, K. M. (1949). Studies on the virus of turnip yellow mosaic. Parasitology 39, 330-342. NEWMARK, P., and FRASER, D. (1956). Composition of an abnormal protein present in tobacco plants infected with tobacco mosaic virus. J. ilm. Ckem. Sot. 78, 1588-1590. SMITH, J. D., and MARKHAM, R. (1950). Chromatographic studies of nucleic acids. 2. The quantitative analysis of ribonucleic acids. Biochem. J. 48, 509513. TAKAHASHI, W. N., and ISHII, M. (1952). An abnormal protein associated with tobacco mosaic virus infection. Nature 189. 419-420.