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Fractionation of amino acid-specific s-RNA from silkgland by methylated albumin column chromatography
222
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 95326
FRACTIONATION OF AMINO ACID-SPECIFIC s-RNA FROM SILKGLAND BY M E T H Y L A T E D ALBUMIN COLUMN CHROMATOGRAPHY
KEIKO MATSUZAKI
Department o[ Chemistry, Sericultural Experiment Station, Suginami, Tokyo (Japan) (Received
April 2oth,
1965)
SUMMARY
Silkgland s-RNA markedly incorporated glycine, alanine, tyrosine and serine, but lysine, leucine, methionine and phenylalanine to a lesser extent. The elution patterns of aspartic acid- and serine-specific s-RNA's of silkgland on a methylated albumin column were different from those of Escherichia coli, while glycine-, alanine- and tyrosine-specific s-RNA's corresponded in elution characteristics with the corresponding fractions from E. coll.
INTRODUCTION
The amino acid composition of fibroin obtained from Bombyx mori is characteristic and contains 46 % (mole %) of glycine, 29 °/o of alanine, II % of serine, 5 % of tyrosine, 0.5 % of leucine, and several minor amino acids 1. If s-RNA in the silkgland acts as amino acid carrier in the biosynthesis of fibroin, it is assumed that s-RNA in the silkgland would be combined more with glycine and alanine, dominant components in fibroin, than with leucine and other minor components. It has previously been reported that E14C~glycine is incorporated into s-RNA from the silkgland markedly, while E14C;leucine scarcely 2. The nucleotide composition and arrangement of s-RNA from the silkgland of Bombyx mori and Philosamia cynthia ricini did not indicate any notable differences. The incorporation of E14Clamino acids in addition to E14C~glycine and E14C~leucine and fractionation of [14C]amino acid-specific s-RNA's on a column of methylated albumin are reported in this paper.
MATERIALS AND METHODS
Preparation o[ silkgland s-RNA S-RNA of the silkgland was prepared from Bombyx mori on the 6th day of the fifth instar by using sodium dodeeyl sulfate-phenol2, 3. A b b r e v i a t i o n : s - R N A , soluble RNA.
Biochim. Biophys..4cta, 114 (1966) 222-22(7
K. MATSUZAKI
223
Preparation o/ aminoacyl RNA synthetase The aminoacyl RNA synthetase of the silkgland was prepared according to TAKANAMI for rat liveIa. The posterior silkglands of the silkworm on the 6th day of the fifth instar were dissected and frozen. The cells of the silkglands (20 g) were broken by grinding with quartz sand and suspended in 0.05 M Tris buffer (pH 7.6) containing MgC12 (7-5" IO-a M). The homogenate was centrifuged once for 15 rain at io ooo x g at 4 ° to remove cell debris. The resulting supernatant was centrifuged at lO5 ooo x g for 2 h and adjusted to pH 5. The resulting precipitate was dissolved in 0.05 M Tris buffer (pH 7.6) and adsorbed on to a column of DEAE-cellulose. The enzymic fraction was eluted with gradient concentration of KC1. The fractions eluted from the column at a concentration range of 0.3-0. 4 M KC1 were used as the aminoacyl RNA synthetase fraction.
Preparation o/amino acid-speci/ic s-RNA Aminoacylation of s-RNA of the silkgland was performed according to TAKANAMI for rat liver4. Silkgland s-RNA was incubated with 0.2 #mole of a E14C]amino acid, 20/,moles of MgC12, IO/,moles of ATP, 12o/~moles of KC1, 2/,moles of CTP, the pH 5-enzyme preparation (0.25 mg protein), and 19 remaining non-radioactive amino acids at 37 °. Uniformly labelled [14C]amino acids were supplied by the Radiochemical Centre, Amersham, England, with the following specific activities (mC/mmole); L-aspartic acid, 3o.1; L-glutamic acid, 63.3; L-glycine, 8.0; L-leucine, lO.7; L-lysine, 43.8; L-methionine, 6.28; L-phenylalanine, 6.8; L-serine, 22.2; L-tyrosine, 16.6. DL-[I-a4C]alanine (specific activity 2.8 mC/mmole) was the product of Daiichi Chemical Co., Japan. The reaction was stopped by chilling the mixture to o °. [14C]amino acid-specific s-RNA's were extracted by the phenol treatment and precipitated with ethanol at --15 ° after the aqueous phase was dialyzed against distilled water. The radioactivity of [14C]amino acid in amino acidspecific s-RNA was counted by a windowless, gas-flow counter.
Fractionation o/amino acid-speci/ic s-RNA o[ silkgland The methylated albumin column was prepared according to SUEOKA5. A linear saline gradient in phosphate buffer was established with 200 ml of starting buffer (0.2 M NaC1, 0.05 M Na-phosphate buffer, pH 6.7) and 200 ml of final buffer (i.o M NaC1, 0.05 M Na-phosphate buffer, pH 6.7). Silkgland s-RNA (2 mg)was subjected to the methylated albumin column (1.5 x I o cm). The amount of s-RNA in the effluent was estimated using a Beckman model DU spectrophotometer and taking the value of the specific absorbance to be E 1%=24° at 260 m#. Fractions (3 ml each) were collected and to each 0.3 mg of RNA (high molecular weight RNA of the silkgland or yeast) was added as a carrier and the whole was precipitated with 3 volumes of cold (95 ~o, v/v) ethanol and kept at --15 ° overnight. The precipitate was centrifuged and dissolved in I ml of water. The radioactivity of the amino acidspecific s-RNA was counted in a windowless, gas-flow counter. RESULTS AND DISCUSSION
As shown in Table I, ElaC]glycine was incorporated into s-RNA obtained from Biochim. Biophys. Acta, 114 (1966) 222-226
224
FRACTIONATION
TABLE
I
AMINO
ACID
INCORPORATION
INTO
SILKGLAND
OF
SILKGLAND
s-RNA
s-RNA
Amino acid
Co~nts/mi~7 per mg s - R N A
Amino acid per mg s - R N A (re#moles)
Glycine Alanine Serine Tyrosine Aspartic acid Glutamic acid Leucine Lysine Methionine Phenylalanine
i I 425 3395 355 ° 2315 3665 668 175 200 i o5 163
1.14 o.95 o.2o o. I o.i2 0.05 o.oi o.o o.o i o.o i
the posterior silkgland. On the other hand, however, [14Clglycine was not incorporated into s-RNA obtained from the ribosomal RNA z. The incorporation of [14Clamino acid into s-RNA in the posterior silkgland was characteristic of the organism. Silkgland s-RNA took up more glycine, alanine, and tyrosine, and less leucine, lysine, methionine, and phenylalanine than other organisms 6-s. It seems that there is a correlation between the incorporation of [14C]amino acid into the s-RNA of silkgland and the amino acid composition of fibroin, but it is not quantitative. In the amino acid incorporation into s-RNA from the silkgland, the addition of CTP and a treatment with a Tris-buffer (pH 8) prior to alcoholic precipitation of the silkgland s-RNA did not affect the formation of amino acid-specific s-RNA at all. The elution pattern on a methylated albumin column of bulk RNA of the silkgland is shown in Fig. I. Fractionation on the methylated albumin column of
3.0
Ribsomal RNA
12 1.0
_t 2.0
08 g O6
s-RNA
'~
°o Fig. i.
lO
/
20
A260 rnp
30 40 Tube number
50
0.4
CoO
Elution pattern of silkgland RNA on a methylated albumin column.
ten kinds of amino acid- specific s-RNA's obtained from the silkgland is shown in Fig. 2. The elution patterns were different from those of amino acid-specific s-RNA's from E. coli reported by SUEOKA 10, but peaks of glycine-, alanine-, and tyrosinespecific s-RNA's seemed to correspond with those in E. coli s-RNA reported by SUEOKA. Several investigators have reported the existence of multiple components Biochim. Biophys..4cta,
1i 4
(1966)222
226
04A,on,n
K. MATSUZAKI
225
Q4
50
0.2
Lysine
/
Q2
10 010
20
30
40
0
0
20
10
30
0
40
04
Aspartic acid
]
Methionine
04
200
0.2
r
100
0
0
0
10
20
30
t:
40
600
300
/
Phenelalanine
Q6
20
[ v
Q4 02
o j~
o.l! lO 04
J. . ZU
.
. JU
.
4U
0
Glutamic acid
0
20
10
30
40
0
Serine
04
20 0.2
010 0.4
20 20
30
40
0
02
0 10 (24
Leucine Q2
20
02
10
010
20
30
40
0
10 20
~
0 10
20
30
40
0
I00 50
30
40
0
Tube n u m b e r
Fig. 2. Elution patterns of IO amino acid-specific s-RNA's from the silkgland on the methylated albumin column. with reference to leucine-, methionine-, serine-, and threonine-specific s-RNA'sg, 1°. The elution profiles of serine-, and aspartic acid-specific s-RNA's were different from those of E. coli. Glycine-specific s-RNA from the silkgland on a methylated albumin column was eluted prior to other amino acid-specific s-RNA's as is found in the case of E. coli and yeast s-RNA. In the case of E. coli s-RNA, the peak of glycinespecific s-RNA overlapped with those of leucine-, lysine-, and aspartic acid-specific s-RNA. However, the initial fractions of the peak corresponding to the glycinespecific s-RNA from the silkgland were just about resolved from the peaks of leucineand aspartic acid-specific s-RNA. It is assumed, from the results presented here, that the silkgland s-RNA contains a large amount of glycine-specific s-RNA. Unless certain aminoacyl RNA synthetases from the silkgland are lost during isolation 12, the specificity of the amino acid incorporation into the silkgland s-RNA appears to Biochim. Biophys. Acta, 114 (1966) 222-226
226
FRACTIONATION OF SILKGLAND
s-RNA
depend on the amount of amino acid-specific s-RNA in the silkgland s-RNA. Further, it is to be expected that if the amino acid incorporation into silkgland s-RNA is proportional to the amount of amino acid-specific s-RNA, then the fraction of glycine-specific s-RNA in silkgland s-RNA when eluted from a methylated albumin column should scarcely contain other amino acid-specific s-RNA's.
ACKNOWLEDGEMENT
The author is grateful to Dr. K. MIURA for much helpful advice, and wishes to thank Dr. M. SHIMIZU for his encouragement. REFERENCES I J. KIRIMURAAND T. SUZUKI,J. Agr. Chem. Soc. Japan., 36 (i962) 336. 2 3 4 5 6
7 8 9 IO 11 12
K. MATSUZAKI, J. Bioehem. (Tokyo), 53 (1963) 326. K. MIURA AND K. MATSUZAKI, Biochim. Biophys. Acta, 91 (1964) 427 • M. TAKANAMI, Bioehim. Biophys. Aeta, 51 (1961) 85. IN~.SUEOKA AND T. Y. CHENG, J. Mol. Biol., 4 (1962) 141. M. B. HOAGLAND, M. L. STEPHENSON, J. J. SCOTT, L. I. I~IECHT AND P. C. ZAMECNIK, J. Biol. Chem., 231 (1961) 241. F. LIPMANN, W. C. HUELSMANN, G. HARTMANN, H. G. BOMAN AND (;. ACS, J. Cellular Comp. Physiol., 54, Suppl. I (1959) 75, P. BERG F. H. BERGMANN, E. J. OFENGAND AND M. DIECKMANN, ,]. Biol. Chem., 236 (1961) 1726. T. YAMANE AND iX~. SUEOKA, Proe. Natl. Acad. Sei., U.S., 5 ° (1963) lO93. N. SUEOKA AND T. YAMANE, Proe. Natl. Acad. Sci., U.S., 48 (1962) 1454. N. SUEOKA ANn T. YAMANE, In/ormational Macromolecules, Academic Press, N e w Y o r k , 1963, p. 2o5. G. L. CANTONI, H. V. GELBOIN, S. W. LUBORSKY, H. H. [~ICHARDS AND ~'VI.F. SINGER, Bioehim Biophys. Acta, 61 (1962) 354.
Biochim. Biophys. Acta, 114 (1966) 222-226
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 95326
FRACTIONATION OF AMINO ACID-SPECIFIC s-RNA FROM SILKGLAND BY M E T H Y L A T E D ALBUMIN COLUMN CHROMATOGRAPHY
KEIKO MATSUZAKI
Department o[ Chemistry, Sericultural Experiment Station, Suginami, Tokyo (Japan) (Received
April 2oth,
1965)
SUMMARY
Silkgland s-RNA markedly incorporated glycine, alanine, tyrosine and serine, but lysine, leucine, methionine and phenylalanine to a lesser extent. The elution patterns of aspartic acid- and serine-specific s-RNA's of silkgland on a methylated albumin column were different from those of Escherichia coli, while glycine-, alanine- and tyrosine-specific s-RNA's corresponded in elution characteristics with the corresponding fractions from E. coll.
INTRODUCTION
The amino acid composition of fibroin obtained from Bombyx mori is characteristic and contains 46 % (mole %) of glycine, 29 °/o of alanine, II % of serine, 5 % of tyrosine, 0.5 % of leucine, and several minor amino acids 1. If s-RNA in the silkgland acts as amino acid carrier in the biosynthesis of fibroin, it is assumed that s-RNA in the silkgland would be combined more with glycine and alanine, dominant components in fibroin, than with leucine and other minor components. It has previously been reported that E14C~glycine is incorporated into s-RNA from the silkgland markedly, while E14C;leucine scarcely 2. The nucleotide composition and arrangement of s-RNA from the silkgland of Bombyx mori and Philosamia cynthia ricini did not indicate any notable differences. The incorporation of E14Clamino acids in addition to E14C~glycine and E14C~leucine and fractionation of [14C]amino acid-specific s-RNA's on a column of methylated albumin are reported in this paper.
MATERIALS AND METHODS
Preparation o[ silkgland s-RNA S-RNA of the silkgland was prepared from Bombyx mori on the 6th day of the fifth instar by using sodium dodeeyl sulfate-phenol2, 3. A b b r e v i a t i o n : s - R N A , soluble RNA.
Biochim. Biophys..4cta, 114 (1966) 222-22(7
K. MATSUZAKI
223
Preparation o/ aminoacyl RNA synthetase The aminoacyl RNA synthetase of the silkgland was prepared according to TAKANAMI for rat liveIa. The posterior silkglands of the silkworm on the 6th day of the fifth instar were dissected and frozen. The cells of the silkglands (20 g) were broken by grinding with quartz sand and suspended in 0.05 M Tris buffer (pH 7.6) containing MgC12 (7-5" IO-a M). The homogenate was centrifuged once for 15 rain at io ooo x g at 4 ° to remove cell debris. The resulting supernatant was centrifuged at lO5 ooo x g for 2 h and adjusted to pH 5. The resulting precipitate was dissolved in 0.05 M Tris buffer (pH 7.6) and adsorbed on to a column of DEAE-cellulose. The enzymic fraction was eluted with gradient concentration of KC1. The fractions eluted from the column at a concentration range of 0.3-0. 4 M KC1 were used as the aminoacyl RNA synthetase fraction.
Preparation o/amino acid-speci/ic s-RNA Aminoacylation of s-RNA of the silkgland was performed according to TAKANAMI for rat liver4. Silkgland s-RNA was incubated with 0.2 #mole of a E14C]amino acid, 20/,moles of MgC12, IO/,moles of ATP, 12o/~moles of KC1, 2/,moles of CTP, the pH 5-enzyme preparation (0.25 mg protein), and 19 remaining non-radioactive amino acids at 37 °. Uniformly labelled [14C]amino acids were supplied by the Radiochemical Centre, Amersham, England, with the following specific activities (mC/mmole); L-aspartic acid, 3o.1; L-glutamic acid, 63.3; L-glycine, 8.0; L-leucine, lO.7; L-lysine, 43.8; L-methionine, 6.28; L-phenylalanine, 6.8; L-serine, 22.2; L-tyrosine, 16.6. DL-[I-a4C]alanine (specific activity 2.8 mC/mmole) was the product of Daiichi Chemical Co., Japan. The reaction was stopped by chilling the mixture to o °. [14C]amino acid-specific s-RNA's were extracted by the phenol treatment and precipitated with ethanol at --15 ° after the aqueous phase was dialyzed against distilled water. The radioactivity of [14C]amino acid in amino acidspecific s-RNA was counted by a windowless, gas-flow counter.
Fractionation o/amino acid-speci/ic s-RNA o[ silkgland The methylated albumin column was prepared according to SUEOKA5. A linear saline gradient in phosphate buffer was established with 200 ml of starting buffer (0.2 M NaC1, 0.05 M Na-phosphate buffer, pH 6.7) and 200 ml of final buffer (i.o M NaC1, 0.05 M Na-phosphate buffer, pH 6.7). Silkgland s-RNA (2 mg)was subjected to the methylated albumin column (1.5 x I o cm). The amount of s-RNA in the effluent was estimated using a Beckman model DU spectrophotometer and taking the value of the specific absorbance to be E 1%=24° at 260 m#. Fractions (3 ml each) were collected and to each 0.3 mg of RNA (high molecular weight RNA of the silkgland or yeast) was added as a carrier and the whole was precipitated with 3 volumes of cold (95 ~o, v/v) ethanol and kept at --15 ° overnight. The precipitate was centrifuged and dissolved in I ml of water. The radioactivity of the amino acidspecific s-RNA was counted in a windowless, gas-flow counter. RESULTS AND DISCUSSION
As shown in Table I, ElaC]glycine was incorporated into s-RNA obtained from Biochim. Biophys. Acta, 114 (1966) 222-226
224
FRACTIONATION
TABLE
I
AMINO
ACID
INCORPORATION
INTO
SILKGLAND
OF
SILKGLAND
s-RNA
s-RNA
Amino acid
Co~nts/mi~7 per mg s - R N A
Amino acid per mg s - R N A (re#moles)
Glycine Alanine Serine Tyrosine Aspartic acid Glutamic acid Leucine Lysine Methionine Phenylalanine
i I 425 3395 355 ° 2315 3665 668 175 200 i o5 163
1.14 o.95 o.2o o. I o.i2 0.05 o.oi o.o o.o i o.o i
the posterior silkgland. On the other hand, however, [14Clglycine was not incorporated into s-RNA obtained from the ribosomal RNA z. The incorporation of [14Clamino acid into s-RNA in the posterior silkgland was characteristic of the organism. Silkgland s-RNA took up more glycine, alanine, and tyrosine, and less leucine, lysine, methionine, and phenylalanine than other organisms 6-s. It seems that there is a correlation between the incorporation of [14C]amino acid into the s-RNA of silkgland and the amino acid composition of fibroin, but it is not quantitative. In the amino acid incorporation into s-RNA from the silkgland, the addition of CTP and a treatment with a Tris-buffer (pH 8) prior to alcoholic precipitation of the silkgland s-RNA did not affect the formation of amino acid-specific s-RNA at all. The elution pattern on a methylated albumin column of bulk RNA of the silkgland is shown in Fig. I. Fractionation on the methylated albumin column of
3.0
Ribsomal RNA
12 1.0
_t 2.0
08 g O6
s-RNA
'~
°o Fig. i.
lO
/
20
A260 rnp
30 40 Tube number
50
0.4
CoO
Elution pattern of silkgland RNA on a methylated albumin column.
ten kinds of amino acid- specific s-RNA's obtained from the silkgland is shown in Fig. 2. The elution patterns were different from those of amino acid-specific s-RNA's from E. coli reported by SUEOKA 10, but peaks of glycine-, alanine-, and tyrosinespecific s-RNA's seemed to correspond with those in E. coli s-RNA reported by SUEOKA. Several investigators have reported the existence of multiple components Biochim. Biophys..4cta,
1i 4
(1966)222
226
04A,on,n
K. MATSUZAKI
225
Q4
50
0.2
Lysine
/
Q2
10 010
20
30
40
0
0
20
10
30
0
40
04
Aspartic acid
]
Methionine
04
200
0.2
r
100
0
0
0
10
20
30
t:
40
600
300
/
Phenelalanine
Q6
20
[ v
Q4 02
o j~
o.l! lO 04
J. . ZU
.
. JU
.
4U
0
Glutamic acid
0
20
10
30
40
0
Serine
04
20 0.2
010 0.4
20 20
30
40
0
02
0 10 (24
Leucine Q2
20
02
10
010
20
30
40
0
10 20
~
0 10
20
30
40
0
I00 50
30
40
0
Tube n u m b e r
Fig. 2. Elution patterns of IO amino acid-specific s-RNA's from the silkgland on the methylated albumin column. with reference to leucine-, methionine-, serine-, and threonine-specific s-RNA'sg, 1°. The elution profiles of serine-, and aspartic acid-specific s-RNA's were different from those of E. coli. Glycine-specific s-RNA from the silkgland on a methylated albumin column was eluted prior to other amino acid-specific s-RNA's as is found in the case of E. coli and yeast s-RNA. In the case of E. coli s-RNA, the peak of glycinespecific s-RNA overlapped with those of leucine-, lysine-, and aspartic acid-specific s-RNA. However, the initial fractions of the peak corresponding to the glycinespecific s-RNA from the silkgland were just about resolved from the peaks of leucineand aspartic acid-specific s-RNA. It is assumed, from the results presented here, that the silkgland s-RNA contains a large amount of glycine-specific s-RNA. Unless certain aminoacyl RNA synthetases from the silkgland are lost during isolation 12, the specificity of the amino acid incorporation into the silkgland s-RNA appears to Biochim. Biophys. Acta, 114 (1966) 222-226
226
FRACTIONATION OF SILKGLAND
s-RNA
depend on the amount of amino acid-specific s-RNA in the silkgland s-RNA. Further, it is to be expected that if the amino acid incorporation into silkgland s-RNA is proportional to the amount of amino acid-specific s-RNA, then the fraction of glycine-specific s-RNA in silkgland s-RNA when eluted from a methylated albumin column should scarcely contain other amino acid-specific s-RNA's.
ACKNOWLEDGEMENT
The author is grateful to Dr. K. MIURA for much helpful advice, and wishes to thank Dr. M. SHIMIZU for his encouragement. REFERENCES I J. KIRIMURAAND T. SUZUKI,J. Agr. Chem. Soc. Japan., 36 (i962) 336. 2 3 4 5 6
7 8 9 IO 11 12
K. MATSUZAKI, J. Bioehem. (Tokyo), 53 (1963) 326. K. MIURA AND K. MATSUZAKI, Biochim. Biophys. Acta, 91 (1964) 427 • M. TAKANAMI, Bioehim. Biophys. Aeta, 51 (1961) 85. IN~.SUEOKA AND T. Y. CHENG, J. Mol. Biol., 4 (1962) 141. M. B. HOAGLAND, M. L. STEPHENSON, J. J. SCOTT, L. I. I~IECHT AND P. C. ZAMECNIK, J. Biol. Chem., 231 (1961) 241. F. LIPMANN, W. C. HUELSMANN, G. HARTMANN, H. G. BOMAN AND (;. ACS, J. Cellular Comp. Physiol., 54, Suppl. I (1959) 75, P. BERG F. H. BERGMANN, E. J. OFENGAND AND M. DIECKMANN, ,]. Biol. Chem., 236 (1961) 1726. T. YAMANE AND iX~. SUEOKA, Proe. Natl. Acad. Sei., U.S., 5 ° (1963) lO93. N. SUEOKA AND T. YAMANE, Proe. Natl. Acad. Sci., U.S., 48 (1962) 1454. N. SUEOKA ANn T. YAMANE, In/ormational Macromolecules, Academic Press, N e w Y o r k , 1963, p. 2o5. G. L. CANTONI, H. V. GELBOIN, S. W. LUBORSKY, H. H. [~ICHARDS AND ~'VI.F. SINGER, Bioehim Biophys. Acta, 61 (1962) 354.
Biochim. Biophys. Acta, 114 (1966) 222-226