Synthesis and Degradation of Methylated Proteins of Mouse 0 rgans: Correlation With Protein Synthesis and Degradation Masaharu L-( Methyl-‘4C)-methionine
was
Miyake admin-
to mice, and the incorporation of radioactive methionine into proteins and methyllyrine and methylarginine residues formed by the transfer of the methyl-14C group of methionine were measured. Tissue protein was actively methylated in organs having a high activity of protein synthesis, and the in vivo methylating activity in organs was not correlated with the protein methylating activity of the organs determined in vitro. Puromycin inhibited both protein synthesis and protein methylation in mouse organs to a simistered
i.p.
and Yasuo
Kakimoto
ilar degree. Neither the formation of S-adenosyl-( methyl-‘4C)-methionine nor protein methylare was inhibited by puromycin. The data suggests that proteins are methylated immediately after protein synthesis, that is, newly synthesized proteins are the substrates of protein methylation. Radioactive methionine and the [Cl41 methyl groups of methyllysine and methylarginine residues of tissue proteins are degraded in parallel over a period of 3 wk, suggesting that protein methylation is an irreversible type of protein modification.
M
ANY PROTEINS formed in organisms are subjected to various types of modification such as phosphorylation, methylation, hydroxylation, acetylation, adenylation, and glucosylation. The modification of proteins may be classified into two general types: (1) Modification which occurs shortly after protein synthesis. Proteins are then transported and incorporated into cellular structures as structurally completed proteins. This type of the modification is irreversible. Hydroxylation and glucosylation belong to this type of modification. (2) Modification in a dynamic state between original and modified forms; the protein participating in rapid adaptive function. Phosphorylation is a typical type of this modification. Previous studied on protein methylation have been conflicting about this type of modification.‘-” The present experiments were performed to clarify: (1) when proteins are methylated after protein synthesis and (2) whether or not proteins, once methylated, are demethylated prior to protein degradation. In order to study relationships between protein metabolism and methylation, it is desirable to use a single marker, (methyl-‘4C)-L-methionine. Methionine is activated to either methionine adenylate or S-adenosylmethionine and utilized for protein synthesis and protein methylation, respectively. C 14-methionine derived from protein degradation is also reutilized for both reactions. Although only a few methylated proteins have been found in nature there are much larger number of proteins, probably more than a half of cellular protein From the Department of Neuropsychiatry. Ehime University School of Medicine. Shigenobu-cho, Omen-gun, Ehime, Japan. Received for publication October 13,1975. Reprint requests should be addressed to Dr. Yasuo Kakimoto, Department of Neuropsychiatry. Ehime University School of Medicine.Shigenobu-cho, Onsen-gun, Ehime. Japan. 8 1976 by Grune & Stratton, Inc. Metabolism,
Vol. 25,
No.
8 (August),
1976
885
MIYAKE
886
AND
KAHIMOTO
molecules exist as methylated proteins as will be discussed later. It was therefore possible to draw general conclusions about the relationship between protein metabolism and methylation from the experiments using tissue proteins as a whole. MATERIALS
AND METHODS
Materials Male mice (dd-strain) weighing about 25 g were used. L-(methyl-‘4C)-methionine mmole) and S-adenosyl-L-(methyl-‘4C)-methionine (52 mCi/mmole) were purchased England Nuclear Co. Calf thymus histone, type II, and puromycin dihydrochloride chased from Sigma and Makor Chemicals Co., respectively. Myelin basic protein was the method of Oshiro et al.” N ’-mono-, di- and trimethyllysines and NG-mono-, and and NG, N’G-dimethylarginines were prepared as reported previously.‘3V’4
(54 mCi/ from New were purpurified by NG,NG-di-
Separation of the Methylamino Acid and Methionine Residues of Tissue Proteins and Measurement of Radioactivity (Methyl-‘4C)-methionine was injected intraperitoneally and the animal killed by decapitation. Organs were dissected and immediately frozen in a dry ice-acetone solution. Organs were homogenized in IO-20 volumes of cold 20% trichloroacetic acid and the homogenate was centrifuged. The supernatant solution was used for the determination of free amino acids. The precipitate was processed to obtain the protein fraction according to the method of Allfrey et a1.l5 Ten to 100 mg of the protein fraction was hydrolyzed in 6 N HCl at 105” for 20 hr, the hydrolysate was diluted with 10 volumes of water. The filtrate was passed through a 1 x 3 cm column of Amberlite IR 120, H+ form, 100-200 mesh. The column was washed with 30 ml of water. Amino acids were eluted with 20 ml of 3 M ammonia. The eluate was evaporated to dryness under reduced pressure. The dried residue was dissolved in a minimum volume of water and passed through a 1 x 3 cm column of Amberlite IR 120, NH: form, 100-200 mesh and the column was washed with 20 ml of water to remove acidic and neutral amino acids. The adsorbed basic amino acids containing histidine, arginine, lysine, and their methyl derivatives were eluted with 20 ml of 3 M ammonia. The eluate was dried under reduced pressure. Recovery of the amino acids was quantitative. A 1 or 2 ml aliquot of the neutral and acidic amino acid fraction was subjected to liquid scintillation counting in 5-10 ml of toluene-nonionic surfactant system in a Nuclear Chicago Mark 1. Counting efficiency was determined by external standard (Channels ratio) method. The main radioactive substance in this fraction was methionine. Methionine and methionine sulfoxide comprised SO%-90% of the total activity of the fraction. The basic amino acid fraction was chromatographed on a 1 x 20 cm Amberlite IR 120, NH; form, 200-400 mesh, and eluted with 50 ml each of 0.1, 0.5, and I.0 M ammonia successively. Three milliliter fractions of the eluate were collected and 2 ml aliquot was subjected to the measurement of radioactivity. With this system, the elution pattern was: 3_methylhistidine, an unknown compound, N’-dimethyllysine, N’-monomethyllysine, N’-trimethyllysine, two kinds of NG-di-methylarginines and NG-monomethylarginine. The identity and purity of each peak was confirmed with paper chromatography and paper electrophoresis. The exception was the peak of 3-methylhistidine which is contaminated by a compound migrating differently from 3-methylhistidine on a paper electropherogram. A typical chromatogram is shown in Fig. 1. This method is simple and many specimens could be analyzed simultaneously without using an amino acid analyzer which has been used for the separation of the methylamino acids. Total activity of the methyllysines and methylarginines were taken as an indicator of protein methylation. Protein was determined according to the method of Lowry et al.16
Fractionation of Subcellular Components Brain and liver were fractionated
to subcellular particles according to the methods of Whit-
takerI7and Schneider et al.,” respectively.
METHYLATED
Fig. 1.
PROTEINS
Chromatographic
887
separa-
tion of (methyl-“C) methylamino acids. An example of the chromatogram on Amberlite IR 120 column of the basic amino acid fraction of proteins of spleens of four mice m sacrificed 40 min after the injection I is : methionine of (methyl-“C) demonstrated. The amount of the x sample corresponded to 46.6 mg of B protein. Treatment of the sample and ‘c) chromatographic conditions are described in text. MH, 3-methylhistidine; MML, N’-monomethyllysine; DML, N’-triN’-dimethyllysine; TML, N ‘-monoMMA, methyllysine; methylarginine; DMA, NO, No- and (overN’,N”-dimethylarginine lapped as described in text).
3
2
1
o
100
50
Elution
volume
( ml
)
Measurement of Protein Methylases Protein rnethylase was measured with a 100,000 g supernatant solution of tissue homogenate with a sucrose solution, 0.32 M for brain and 0.25 M for other tissues. Enzyme activity was measured with the method described before” using histone and myelin basic protein as substrates.
RESULTS
Methylamino Acid Residues of Tissue Proteins After Injection of (Methyl-“C) Methionine (Methyl-14C) methionine (10 &i/mouse) was injected intraperitoneally. Mice were killed by decapitation 20, 40, 60, 120, and 240 min after injection. The methyl-amino acid residues of proteins of liver, kidney, spleen, brain, and striate muscle were analyzed. The results are shown in Fig. 2. In every tissue, N’-mono-, di-, and trimethyllysine and NC-monoand NG-dimethylarginine residues were found to contain radioactivity. The radioactivities and their time courses are different for organs. An unknown compound disappeared rapidly in every tissue. This compound seems to be a degradation product of methionine during acid hydrolysis. Correlation Between Protein Synthesis and Methylation Activities Radioactivities of neutral amino acid residues, mainly of methionine, were measured at 20 and 40 min after the intraperitoneal injection of 10 &i of (methyl-14C) methionine. The radioactivity was incorporated in increasing order in muscle, brain, liver, and kidney (Fig. 3A). Total radioactivity of the methylamino acid residues are in the same order as that of methionine (Fig. 3B). The activity of protein methylase was highest in the brain and lowest in the liver (Fig. 3E), while in vivo methylation of tissue proteins was highest in kidney and liver and lowest in brain and muscle (Fig. 3B). Radioactivities of free methionine and S-adenosylmethionine in these periods were not proportional either to protein synthetic rates or to methylating activities (Fig. 3C and D). The rate of protein methylation was considered to be determined by the rate of protein synthesis rather than the activity of protein
MIYAKE AND
KAHIMOTO
Kidney
Liver 5-
TML TML
/
DMA
.0
DML
.5-
I I I I 0- 204060
I
120
Ill
I
I
120 204060 14 Time after injection of [methyl- c]methion ine (min.) 240
Brain 5-
I 240
Muscle 30-
TML
.o-
zo-
5-
lo-
1 0:
Time after injection
1 -0; 104060
120
of [methyl- 14C]methionine
240 (min.)
Fig. 2. Chonge in the rodieoctivities of the mathylamino acids in protein fractions of mouse orgons ofter injection of (methyl-“C) methionine. (Methyl-“C) methionine wos injected introperitonaally to mice, and the mice were sacrificed in different intervals. Pmtein fractions of organs were hydrolyzed, and the basic amino acid fraction was separated. The fmction wos chmmotogmphed OS in Fig. 1. Abbreviations of the omino acids ore the same OS in Fig. 1. Each dot represents the mean value of four mice.
METHYLATED
PROTEINS
889
Methionine
Free
Residue
M&hionine
S-Adenosylmethionine
Methylamino Residue
Acid
Methylase
Activity
I
x
Kidney
Liver
Muscle
Brain
afi
0
Kidney
Liver
Muscle
Brain
Fig. 3. Radioactivities of methionine and methylamino acid residues and of free methionine and S-adenosylmethionine in mouse organs and activities of protein methylares. (Methyl-“C) methionine was iniected intraperitoneally to two groups of four mice, and the mice were sacrificed 20 and 40 min later. Organs were homogenized with trichloroacetic acid. Protein fraction WCIS processed as described in text. Radioactivities of free methionine and S-adenosylmethionine were measured by passing the trichloroacetic acid-soluble fmction of tissue through 1 x 3 cm column of Amberlite IR 120, H+ form. The column was washed with water. Methionine was eluted with 15 ml of 1 M pyridine, and S-adenosylmethionine was then eluted with 20 ml of 3 M ammonia. Paper electrophonsis revealed that the radioactivity in this fraction represents that of S-adenosylmethionine and its degradation product formed in alkaline condition. Aliquots were applied to the measurement of radioactivity. Activity of protein methylases was measured as previously reported.20 iI, 20 min. 40 min, 0, histone methylating activity; m myelin basic protein methyloting activity;l, standard error.
methylase. This may be explained proteins are methylated in situ.
by postulating
the only newly
synthesized
Correlation Between the Radioactivities of Methionine and of Methyl Group of Methylamino Acid Residues of Proteins of Subcellular Fractions If proteins are methylated shortly after protein synthesis and then transported to intracellular structures, the methionine and methylamino acid residues are expected to be labeled proportionally in different subcellular fractions.
MIYAKE
AND
KAHIMOTO
4 hr
‘“Jr
1C Liver
Brain 6 _Methionine OMethylamino
Acids
4
lic.Mit.Nuc.
Fig. 4. Distribution of radioactivities of methionine and the methylamino acids in subcellular fractions of mouse liver and brain. Four hours after the injection of ( methyl-14C) methionine to mice, liver and brain were homogenized in sucrose and separated into subcellular fractions. The radioactivities of the methionine and the methylamino acid residues of the protein were measured. Distribution of the radioactivity of each fraction was expressed as percentage of those of whole organs. Sup., supernatant fraction; Mic., microsome; Mt., mitochondria; NW., nuclei; Mye., myelin; Syn., synaptosome.
Ill
L ll-ln-
up.Mlc.Mit.Nuc.Mye.
syn.
If all tissue proteins are methylated nonselectively and continuously after protein synthesis, the previously stated proportionality would not be observed. Four hours after intraperitoneal injection of (methyLL4C) methionine (20 &i/ mouse) to four mice, liver and brain were dissected and subjected to subcellular fractionation. Both the radioactivities of methionine and the methylamino acid residues were roughly proportionally distributed in subcellular fractions of the liver and brain, except nuclear fractions, as shown in Fig. 4. Although the correlation is only approximate as stated, distribution of radioactivities of the methylamino acids is quite different from distribution of protein methylase which is almost exclusively localized in the cytoplasmic fraction.20 This finding is also supportive of the hypothesis that newly synthesized, and not preexisting, proteins are mainly methylated. Cell nuclei contained, however, a relatively high proportion of labeled methylamino acid residues. This may be explained by abundance of methylated proteins.8 Another explanation that methylation of the nuclear proteins is continuing for some time after protein synthesis can not be excluded. Simultaneous Inhibition of Protein Synthesis and Protein Methyiation by Puromyrin If the rate of protein synthesis determines the rate of protein methylation, inhibition of the protein synthesis should decrease protein methylation simultaneously. Puromycin dihydrochloride (0.3 mg/g body weight) was injected to two groups of four mice intraperitoneally 1 and 2 hr prior to (methyl-‘4C) methionine (10 jKi/mouse) injection, and mice were killed 20 min after the injection of the methionine. The radioactivity of the methylamino acid and methionine residues of proteins of kidney, spleen, liver, testis, and brain were measured to obtain the results shown in Table 1. Saline was injected to the control group instead of puromycin. Puromycin inhibited both the rates of protein synthesis and methylation of lysine and arginine residues in each organ.
METHYLATED
PROTEINS
891
Table 1.Inhibition of Protein Synthesis and Protein Methylation by Puromycin* Radioactivity Monomethyl-
Liver
4
None
Puromycin Brain
Trimethyl-
Monomethyl-
Dimethyl-
lyrine
lysine
arginine
Spleen
argininet
207
5.1
12.8
7.5
5.5
Kidney
210+
61
1.3
4.1
1.6
2.8
3.3
2 hr
4
476 i-
91
2.2
4.9
2.3
3.7
4.4 2.9
5
7BOzt
131
1.5
4.7
1.8
1.6
1 hr
4
562i
118
1.1
2.5
1.5
1.2
I .3
2 hr
4
495 +
157
1.1
t
1.1
1 .o
1.2
5
9604
i
1894
199.7
154.6
78.8
30.9
i hr
4
1171 i
366
28.6
22.0
5.6
4.6
12.9
2 hr
4
2521
i
338
6.4
21.1
6.9
5.0
21.0 15.6
Testis
5
4710
*
1100
7.5
32.8
18.3
6.7
4
2352
*
842
3.2
21.4
5.6
3.7
5.6
2 hr
4
1515 zt
348
2.4
13.6
3.9
2.8
5.6
4
1687 f
326
9.3
13.3
8.5
5.2
15.0
40
5.4
4.9
2.2
4.0
7.0
151
4.6
5.6
4.3
4.3
6.5
NCVle
Puromycin
1 hr
4
245 zt
2 hr
4
579i
*(Methyl-‘4C)-L-methionine they
war
injected to two
groups
of four or five mice 1 and 2 hr after
were killed 20 min later. Tissue proteins were hydrolyzed
methionine.
and
neutral
Basic amino acid fractions of each group were combined
each methylamino
101.7
1 hr
None
Puromycin
9.1
4
None
Puromycin
f
I hr
NOW
Puromycin
3007
protein)
Dimethyl-
Methionine
animals
Treatment
SE/mg
lysine
Number of Orqanr
(dpm f
amino
and
injection of puromycin,
acid fraction
chromotographed
to
was
separated
measure
to
and count
radioactivity
of
acid.
tSum of NG,NG-and
NG,N’
G
-dtmethylarginine.
$An accurate value was not obtained
because of poor resolution from an unknown peak.
Before concluding that the decrease in protein methylation results from inhibition of protein synthesis, other possibilities must be excluded. (1) Protein methylases might be inhibited directly by puromycin. As shown in Table 2, protein methylase activities of the cytosole fractions of liver and brain were not inhibited by 127 pg/ml of puromycin. (2) If the turnover rates of protein methylases are rapid, amounts of tissue protein methylases are reduced by puromycin. Protein methylase activities, in the liver, brain, spleen, and kidney of mice injected with puromycin or saline were not different as shown in Table 3. (3) Formation of S-adenosyl(methyl-r4C) methionine may be reduced by the injection of puromycin. Radioactivity of S-adenosyl-methionine was not decreased in the brain, liver, spleen, kidney, and testis 20 min after the injection of (methyl-%) methionine as shown in Table 4. These observations are consonant with the hypothesis that puromycin decreased the rate of protein methylation by inhibiting protein synthesis, thereby reducing the amount of newly synthesized proteins. Newly synthesized proteins are probably the substrates of protein methylases. Table 2. Effect of Puromycin on the Activity of Protein Methylares Histone Methylore
Number of Puromycin
Experiments
+
3
0.87 f 0.24 0.14
_
3
0.83&0.10
The with as
cytoplasmic calf
reported
tration
was
thymus
Brain
fractions histones
previously’9 127 pg/ml.
of
brains, bovine
ztO.05
presence
The activity
livers, myelin ond
is expressed
Brain
Spleen 1.65
0.12ztO.02
and
m
Myelin Basic Protein Methylase
Liver
ho.28
1.74
2.23zkO.40 and
spleens
basic absence as (dpm
of
+
three as
mice substrates.
puromycin SE)
ztO.08
0.06
1.87&0.10
protein of
Liver
x
ho.04
0.10~1~0.03 were
used
The
dihydrochloride
10m3/hr/mg
Spleen
protein.
as
activity of
4.65
f
0.64
3.60+0.39 enzyme was which
sources measured concen-
892
MIYAKE AND
Table 3. Activities of Protein Methylares Number Treatment
Saline
Histone
of
Animals
Brain
0.87
3
ho.24 Puromycin
4
After the Injection of Puromycin
Methylose
Liver
My&n
Spleen
Brain
Kidney
0.14
1.65
0.39
hO.05
l o.za
l O.lO
0.86
0.14
+O.lO
zto.04
KAHIMOTO
1.61 ho.56
Cytoplasmic fractions of mouse orgons were obtained
1.74 ko.08
0.40
2.90
ho.08
zto.45
Basic Protein Liver
Methylare
Spleen
Kidney
0.06
4.65
0.64
*to.04
zt0.64
hO.21
0.04
4.82
*0.02
*0.22
from mice killed 1 hr after
0.65 ho.24
the injection of
puromycin (0.3 mg/g body weight) or saline, and the activity of protein methyloses were measured.”
The
activity is expressed os (dpm + SE) x 10m3/hr/mg protein.
Degradation of the Methylated Proteins If protein methylation is reversible and if methylation-demethylation is a cyclic process, methyl-‘4C group on lysine and arginine residues of a protein should be diluted more rapidly than the 14C-methionine residue of the protein. If irreversible, both radioactivities should decrease in parallel. (Methyl-14C) methionine was administered to mice (7.2 &i/mouse) intraperitoneally, and the change in the radioactivities of the tissue proteins was observed for 3 wk. The radioactivity of methionine and methylamino acid residues of tissue protein of brain, liver, and striate muscle decreased in parallel in every organ as shown in Fig. 5. This finding suggests that protein methylation is an irreversible process. In another experiment, proteins from homogenates of rat liver and brain were methylated in vitro with S-adenosyl-(methyl-%) methionine in a manner of 1 g of tissue in 5 ml of 0.1 M reported previously. 2’ Thus the homogenate phosphate buffer, pH 7.4, was incubated with 10 &i of (methyl-‘4C) S-adenosylmethionine for 3 hr at 37”, and 1 ml aliquot was used for the determination of the radioactivity of the methylamino acid residues. The remaining homogenate was dialyzed overnight against 500 ml of the phosphate buffer and was incubated at 37” after the addition of 5 ml of the freshly prepared homogenate. Two milliliter aliquots were taken at 1 hr intervals up to 3 hr, and subjected to analysis of radioactivities of the methylamino acid residues. No decrease in the radioactivities was observed after the addition of the fresh homogenate. Any Table 4.
Effect of Puromycin Injection on the Radioactivity S-Adenorylmethionine
Number Treatment
of
in Mouse Orgons
of Liver
animals
Brain
Spl&?ll
Kidney
Testis
dpm & SE/mg protein Without puromycin
5
29+
5
161 +21
a52 f
137
4
55 f
11
157 f 36
1033 f 279
4
66*
7
145 f 28
710 + 148
658 f
151
444 f
185
699+
80
388+2lo
632 &
97
463 +219
1 hr after puromycin 2 hr after puromycin
One and 2 hr after the injection of puromycin (0.3 mg/g body weight) (methyl-?)
methionine (10 rCi/
mouse) was injected. A fraction containing S-odenosylmethionine was seporoted from trichloroacetic soluble fraction of organs
qs
acid
described under Fig. 3. The radioactivity of S-odenosylmethionine increased
linearly to the highest value at about 20 min after the injection of methionine in every orgon and was taken as on indicator of the rate of formation of S-adenosylmethionine.
METHYLATED
893
PROTEINS
Methionlne
Mechglamino
acids
l/11111111111 0
4
8
12
16
0
20
days
after
injection
of
4
8
12
16
20
Imethyl-14Clmethlonlne
Degradation of proteins and decrease in the radioactivity of methylomino acid residues. Fig. 5. (Methyl-“C) methionine was injected intraperitoneally to mice, and radioactivities of methionine and the methylamino acid residues were measured in the protein fractions of the liver, brain, and muscle from the 1st to the 21st day. Each point represents the value obtained from one mouse.
detectable amounts of radioactive methylamino trichloroacetic acid soluble fraction.
acids were not
found
in the
DISCUSSION
Presence of mono-, di-, and trimethyllysine residues22-26 and methylarginine residues20.27 in histones, NG,NrG-dimethyl-, and NG-monomethylarginine28 in myelin basic proteins, methyllysine in flagellin,29 monomethyllysine in S 50 residues in actin, 3’ 3_methylhistidine, microsomal protein, 3o 3-methylhistidine mono- and trimethyllysine and NG,NG-dimethylarginine in myosine,32 and trimethyllysine in bacterial and vegetable cytochrome C33 has been reported. We have found the occurrence of the methylamino acid residues in proteins of bacteria, insects, plants, and animals. Amounts of total methyllysine and methylarginine residues of rat tissue proteins ranged from 3 to 10 pmoles/g protein. The concentrations of methylamino acids in histone or myelin basic protein are 5-15 pmoles/g protein and that of myosin is 2 pmoles/g protein. These concentrations are not very different from the average concentrations of methylamino acid residues of tissue proteins. This means that many other proteins exist in tissue as methylated proteins. Methylamino acid residues of proteins distribute widely in every subcellular component. When (methyl-‘4C) methionine was injected intraperitoneally to rats or mice, radioactivity of methylamino acid residues were one tenth to fiftieth the radioactivity of methionine residues. Postulating the average number of methionine residues in protein being 2-3 of 100 amino acid residues, one of about 500 amino acid residues is calculated to be methylated on the assumption that the
894
MIYAKE
AND
KAHIMOTO
specific radioactivity of free methionine is roughly the same as that of free Sadenosylmethionine. The concentration of S-adenosylmethionine is about 40% of that of methionine in rat brain and liver on molar basis.34 This ratio is in accordance with that shown in Fig. 3C and D. From the above calculation 10 /Imoles of amino acid residues per gram of tissue proteins are the amino acid residues which are acceptable sites for methylation. This also indicates that many proteins are capable of being methylated. In an additional experiment, rats were injected with (methyl-14C) methionine, and microsomal and nuclear fractions of the testis were subjected to SDS-acrylamide gel electrophoresis. Radioactivity of methionine and of the methylamino acid residues of proteins were distributed in parallel on the electropherogram. The previously stated experimental findings indicate that protein methylation is universal for a wide variety of tissue proteins and that our present method of treating tissue proteins as a whole is justified in order to study the relationship between protein metabolism and methylation. Our first conclusion that protein methylation takes place not long after protein synthesis is drawn from the fact that methylation is active in organs having high protein synthetic activity and vice versa and that inhibition of protein synthesis by puromycin inhibited protein methylation. The site of protein methylation is unknown. Cytoplasm contains the highest activity of methylating proto assume that proteins synthesized on teins,‘9,35 so it would be reasonable ribosomes are methylated there or during protein transport. The present findings have been preceeded by other workers using different sources and methods. Reporter’ has demonstrated that the selective protein methylation of nascent myosin begins at ribosome using cultured muscle cells. Duerre et al.’ have injected H3-lysine and (methyl-14C) methionine to rats, and purified four kinds of histones. Specific activities of 3H-lysine and “C-methylamino acid residues of the four histones were the same. This finding indicates histone synthesis and methylation takes place within a short interval. In these two studies the relation of protein methylation to protein synthesis was examined for the specific proteins, myosin and histone, respectively. The hypothesis that nascent proteins are selectively methylated was shown to be applied generally to a wide variety of tissue proteins in our present study. Other indirect evidences are from the studies of protein methylase III which increases in a variety of proliferating cell systems such as hepatoma,3 fetal brain,4 immature rat organs5 regenerating rat liver,6 and HeLa S-3 ce11.36,37Matsuoka’ has shown that an increase in proteins is accompanied by an increased amount of methylated proteins keeping the ratio of the amounts of methylamino acid residues of proteins constant in regenerating rat liver and during growth of bacterial cells. Kakimoto et a1.8 have shown that NC-mono, and NG,N’G-dimethylarginine residues in acid soluble protein fraction of rat brain increase concomitantly with myelination which also provides indirect evidence for the hypothesis that protein methylation takes place mainly on nascent proteins. Allfrey et a1.9 incubated (methyl-14C) methionine with isolated nuclei of calf thymus. Addition of puromycin to the reaction mixture did not inhibit histone methylation. The finding however may not necessarily reflect series of reactions occurring in situ. It is not known whether histones are synthesized and methylated in cell nuclei or not. Recent evidence indicates that synthesis of histone
METHYLATED
PROTEINS
895
occurs predominantly in perikaryon. 38-4oThe products of histone methylation in vitro are mainly methylarginine residues4’ but Allfrey et al. determined the radioactivity of methyllysine residues. The significance of their experiment is not clear. Boyoet” has observed the time course of the radioactivities of methylamino acids and methionine of arginine rich histone from rat spleen Novikoff hepatoma, and hamster’s ovary and concluded that protein methylation is irreversible. Thomas et al.” has reached a similar conclusion by the observation of turnover of the methyl group of histone of Ehrlich ascites tumor cells. These findings are restricted to histone, and our conclusion drawn from the observation of whole tissue proteins of mouse organs is in accordance with theirs. Paik and Kim42 found demethylase of N-alkyllysines and methylated histones, but this enzyme reaction seems to be minor one for the metabolism of the methylated proteins and protein hydrolysis is considered to be a major pathway of breakdown of the methylated proteins. REFERENCES I. Reporter M: Protein synthesis in cultured muscle cells: methylation of nascent proteins. Arch Biochem Biophys 158:577. 1973 2. Duerre JA, Lee CT: In vivo methylation and turnover of rat brain histones. J Neurothem 23:541, 1974 3. Paik WK, Lee HW, Morris HP: Protein methylases in hepatome. Cancer Res 32:37, 1972 4. Pdik WK, Kim S, Lee HW: Protein methylation during the development of rat brain. Biochem Biophys Res Commun 46:933, 1972 5. Paik WK. Lee HW, Lawson D: Age dependency of various protein methylases. Exp Geront 6:271, 1971 6. Lee HW, Paik WK: Histone methylation during hepatic regeneration in rat. Biochim Biophys Acta 277: 107, 1972 7. Matsuoka Y: N’-Methylated lysines and NC;-methylated arginines III. Existence and distribution in nature and mammals. Seikagaku 44~364, 1972 8. Kakimoto Y, Matsuoka Y, Miyake M, Konishi H: Methylated amino acid residues of proteins of brain and other organs. J Neurothem 24:893. 1975 9. Allfrey VG, Faulker R, Mirsky AE: Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis. Proc Nati Acad Sci USA 51:786, 1964 IO. Boyoet P: In vivo turnover and distribution of radio-N-methyl in arginine rich histones from rat tissue. Arch Biochem Biophys 152: 887, 1972 11. Thomas G, Lange HW, Hempel K: Relative Stabilitlt Lysine-gebundener Methylatgruppen bei den argininreichen Histonen und
ihren Unterfraktionen von Ehrlich AscitesTumorzellen in vitro. Hoppe-Seyler’s Z Physiol Chem 353: 1423, 1972 12. Oshiro Y, Eylar EH: Allergic Encephalomyelitis: Preparation of the encephalitogenic basic protein from bovine brain. Arch Biochem Biophys 138:392. 1970 13. Kakimoto Y. Akazawa S: Isolation and identification of NG,NG-and NG,N’G-dimethylarginine, N’-mono, -di, and tri-methyllysine, and glucosylgalactosyland galacotosyl-6hydroxyllysine from human urine. J Biol Chem 245:575 I, 1970 14. Nakajima T, Matsuoka Y, Kakimoto Y: Isolation and identification of NG-monomethyl, NG,NG-dimethyl and NG,NtG-dimethylarginine from the hydrolysate of proteins of bovine brain. Biochim Biophys Acta 230:212, 1971 15. Allfrey tein synthesis Physiol40:45
VG, Mirsky in isolated I, 1957
AE, Osawa cell nuclei.
S: ProJ Gen
16. Lowry OH, Rosebrough NJ, Farr AL. Randall RJ: Protein measurement with the folin phenol reagent. J Biol Chem 193:256, 195 I 17. Whittaker VP: The isolation and acterization of acethylcholine-containing ticles from brain. Biochem J 72:694, 1959
charpar-
18. Schneider WC, Hogeboon GH: Intracellular distribution of Enzymes V. Futher studies on the distribution of cytochrome C in rat liver homogenates. J Biol Chem 183:123. 1950 19. Miyake M, Kakimoto Y: Protein lation by cerebral tissue. J Neurochem 1973 20. Miyake
M: Methylases
of myelin
methy20:859, basic
a96
protein and histone in rat brain. J Neurochem 24909, 1975 21. Kakimoto Y: Methylation of arginine and lysine residues of cerebral proteins. Biochim Biophys Acta 243:31, 1971 22. Dalange RJ, Fambrough DM, Smith EL, Bonner J: Complete amino acid sequence of pea seedling histone IV: Comparison with the homologous calf thymus histone. J Biol Chem 2445669, 1969 23. Ogawa Y, Quagliarotti G, Jordan J, Taylor CW, Starbuck WC, Busch M: Structural analysis of the glycine-rich, arginine-rich Histone III Sequence of the amino-terminal half of the molecule containing the modified lysine residues and the total sequence. J Biol Chem 244:4387, 1969 24. Delenge RJ, Hooper JA, Smith EL: Complete amino-acid sequence of calf-thymus histone III. Proc Nat1 Acad Sci USA 69:887, 1972 25. Brandt WF, Holt CV: The complete amino acid sequence of histone F, from chicken erythrocytes. FEBS Letter 23:357, 1972 26. Patthy L, Smith EL: Histone III V. the amino acid sequence of pea embryo histone III. J Biol Chem 248:6834, 1973 27. Paik WK, Kim S: e-N-methylarginine in histones Biochem Biophys Res Commun 40: 224,197O 28. Deibler GE, Martenson RE: Determination of methylated basic amino acids with the amino acid analyzer. J Biol Chem 248:2387, 1973 29. Glazer AN, Delange RJ, Martine RJ: Identification of c-N-methyllysine in spirillum serpens flagella and of t-N-dimethyllysine in salmonella typhimurium flagella. Biochim Biophys Acta 188:164, 1969 30. Alix JH, Hayes D: Properties of ribosomes and RNA synthesized by Escherichia coli grown in the presence of ethionine III, methylated proteins in 50s ribosomes of E cob EA2. J Mol Biol86: 139, 1974
MIYAKE
AND
KAHIMOTO
31. Asatoor AM, Armstrong MD: 3-methylhistidine, a component of actin. Biochem Biophys Res Commun 26: 168,1967 32. Reporter M. Corbin JL: NGNG-dimethylarginine in myosin during muscle development. Biochem Biophys Res Commun 43:644, 1971 33. Delange RJ, Glazer AN, Smith EL: Presence and location of an unusual amino acid, r-N-trimethyllysine, in cytochrome c of wheat germ and neurospora. J Bioi Chem 244: 1385,1969 34. Gaitonde MK: Sulfer amino acids, in Laitha A (ed): Handbook of Neurochem, vol 3. New York. Plenum Press. 1970, p 225 35. Paik WK. Kim S: Protein methylation. Science 174:114, 1971 36. Borun TW, Pearson D, Paik K: Studies of hitsone methylation during HeLa S-3 cell cycle. J Biol Chem 247:4288, 1972 37. Lee HW, Paik WK, Borun T: The periodic synthesis of S-adenosylmethionine: protein methyltransferases during the HeLa S-3 cell cycle. J Biol Chem 248:4194, 1973 38. Gallwitz D, Mueller GC: Histone synthesis in vitro on HeLa cell microsomes. J Biol Chem 24415947, 1969 39. Gallwitz D, Mueller CC: Histone synthesis in vitro by cytoplasmic microsomes from HeLa cells. Science 163:1351, 1969 40. Oliver D, Granner D, Chalkley R: Identification of a distinction between cytoplasmic histone synthesis and subsequent histone deposition. Biochemistry 13:746, 1974 41. Paik WK, Kim S: Enzymatic methylation of protein fractions from calf thymus nuclei. Biochem Biophys Res Commun 29:14, 1967 42. Paik WK, Kim S: c-Alkyllysinase, new assay method, purification, and biological significance. Arch Biochem Biophys 165:369, 1974
was
Miyake admin-
to mice, and the incorporation of radioactive methionine into proteins and methyllyrine and methylarginine residues formed by the transfer of the methyl-14C group of methionine were measured. Tissue protein was actively methylated in organs having a high activity of protein synthesis, and the in vivo methylating activity in organs was not correlated with the protein methylating activity of the organs determined in vitro. Puromycin inhibited both protein synthesis and protein methylation in mouse organs to a simistered
i.p.
and Yasuo
Kakimoto
ilar degree. Neither the formation of S-adenosyl-( methyl-‘4C)-methionine nor protein methylare was inhibited by puromycin. The data suggests that proteins are methylated immediately after protein synthesis, that is, newly synthesized proteins are the substrates of protein methylation. Radioactive methionine and the [Cl41 methyl groups of methyllysine and methylarginine residues of tissue proteins are degraded in parallel over a period of 3 wk, suggesting that protein methylation is an irreversible type of protein modification.
M
ANY PROTEINS formed in organisms are subjected to various types of modification such as phosphorylation, methylation, hydroxylation, acetylation, adenylation, and glucosylation. The modification of proteins may be classified into two general types: (1) Modification which occurs shortly after protein synthesis. Proteins are then transported and incorporated into cellular structures as structurally completed proteins. This type of the modification is irreversible. Hydroxylation and glucosylation belong to this type of modification. (2) Modification in a dynamic state between original and modified forms; the protein participating in rapid adaptive function. Phosphorylation is a typical type of this modification. Previous studied on protein methylation have been conflicting about this type of modification.‘-” The present experiments were performed to clarify: (1) when proteins are methylated after protein synthesis and (2) whether or not proteins, once methylated, are demethylated prior to protein degradation. In order to study relationships between protein metabolism and methylation, it is desirable to use a single marker, (methyl-‘4C)-L-methionine. Methionine is activated to either methionine adenylate or S-adenosylmethionine and utilized for protein synthesis and protein methylation, respectively. C 14-methionine derived from protein degradation is also reutilized for both reactions. Although only a few methylated proteins have been found in nature there are much larger number of proteins, probably more than a half of cellular protein From the Department of Neuropsychiatry. Ehime University School of Medicine. Shigenobu-cho, Omen-gun, Ehime, Japan. Received for publication October 13,1975. Reprint requests should be addressed to Dr. Yasuo Kakimoto, Department of Neuropsychiatry. Ehime University School of Medicine.Shigenobu-cho, Onsen-gun, Ehime. Japan. 8 1976 by Grune & Stratton, Inc. Metabolism,
Vol. 25,
No.
8 (August),
1976
885
MIYAKE
886
AND
KAHIMOTO
molecules exist as methylated proteins as will be discussed later. It was therefore possible to draw general conclusions about the relationship between protein metabolism and methylation from the experiments using tissue proteins as a whole. MATERIALS
AND METHODS
Materials Male mice (dd-strain) weighing about 25 g were used. L-(methyl-‘4C)-methionine mmole) and S-adenosyl-L-(methyl-‘4C)-methionine (52 mCi/mmole) were purchased England Nuclear Co. Calf thymus histone, type II, and puromycin dihydrochloride chased from Sigma and Makor Chemicals Co., respectively. Myelin basic protein was the method of Oshiro et al.” N ’-mono-, di- and trimethyllysines and NG-mono-, and and NG, N’G-dimethylarginines were prepared as reported previously.‘3V’4
(54 mCi/ from New were purpurified by NG,NG-di-
Separation of the Methylamino Acid and Methionine Residues of Tissue Proteins and Measurement of Radioactivity (Methyl-‘4C)-methionine was injected intraperitoneally and the animal killed by decapitation. Organs were dissected and immediately frozen in a dry ice-acetone solution. Organs were homogenized in IO-20 volumes of cold 20% trichloroacetic acid and the homogenate was centrifuged. The supernatant solution was used for the determination of free amino acids. The precipitate was processed to obtain the protein fraction according to the method of Allfrey et a1.l5 Ten to 100 mg of the protein fraction was hydrolyzed in 6 N HCl at 105” for 20 hr, the hydrolysate was diluted with 10 volumes of water. The filtrate was passed through a 1 x 3 cm column of Amberlite IR 120, H+ form, 100-200 mesh. The column was washed with 30 ml of water. Amino acids were eluted with 20 ml of 3 M ammonia. The eluate was evaporated to dryness under reduced pressure. The dried residue was dissolved in a minimum volume of water and passed through a 1 x 3 cm column of Amberlite IR 120, NH: form, 100-200 mesh and the column was washed with 20 ml of water to remove acidic and neutral amino acids. The adsorbed basic amino acids containing histidine, arginine, lysine, and their methyl derivatives were eluted with 20 ml of 3 M ammonia. The eluate was dried under reduced pressure. Recovery of the amino acids was quantitative. A 1 or 2 ml aliquot of the neutral and acidic amino acid fraction was subjected to liquid scintillation counting in 5-10 ml of toluene-nonionic surfactant system in a Nuclear Chicago Mark 1. Counting efficiency was determined by external standard (Channels ratio) method. The main radioactive substance in this fraction was methionine. Methionine and methionine sulfoxide comprised SO%-90% of the total activity of the fraction. The basic amino acid fraction was chromatographed on a 1 x 20 cm Amberlite IR 120, NH; form, 200-400 mesh, and eluted with 50 ml each of 0.1, 0.5, and I.0 M ammonia successively. Three milliliter fractions of the eluate were collected and 2 ml aliquot was subjected to the measurement of radioactivity. With this system, the elution pattern was: 3_methylhistidine, an unknown compound, N’-dimethyllysine, N’-monomethyllysine, N’-trimethyllysine, two kinds of NG-di-methylarginines and NG-monomethylarginine. The identity and purity of each peak was confirmed with paper chromatography and paper electrophoresis. The exception was the peak of 3-methylhistidine which is contaminated by a compound migrating differently from 3-methylhistidine on a paper electropherogram. A typical chromatogram is shown in Fig. 1. This method is simple and many specimens could be analyzed simultaneously without using an amino acid analyzer which has been used for the separation of the methylamino acids. Total activity of the methyllysines and methylarginines were taken as an indicator of protein methylation. Protein was determined according to the method of Lowry et al.16
Fractionation of Subcellular Components Brain and liver were fractionated
to subcellular particles according to the methods of Whit-
takerI7and Schneider et al.,” respectively.
METHYLATED
Fig. 1.
PROTEINS
Chromatographic
887
separa-
tion of (methyl-“C) methylamino acids. An example of the chromatogram on Amberlite IR 120 column of the basic amino acid fraction of proteins of spleens of four mice m sacrificed 40 min after the injection I is : methionine of (methyl-“C) demonstrated. The amount of the x sample corresponded to 46.6 mg of B protein. Treatment of the sample and ‘c) chromatographic conditions are described in text. MH, 3-methylhistidine; MML, N’-monomethyllysine; DML, N’-triN’-dimethyllysine; TML, N ‘-monoMMA, methyllysine; methylarginine; DMA, NO, No- and (overN’,N”-dimethylarginine lapped as described in text).
3
2
1
o
100
50
Elution
volume
( ml
)
Measurement of Protein Methylases Protein rnethylase was measured with a 100,000 g supernatant solution of tissue homogenate with a sucrose solution, 0.32 M for brain and 0.25 M for other tissues. Enzyme activity was measured with the method described before” using histone and myelin basic protein as substrates.
RESULTS
Methylamino Acid Residues of Tissue Proteins After Injection of (Methyl-“C) Methionine (Methyl-14C) methionine (10 &i/mouse) was injected intraperitoneally. Mice were killed by decapitation 20, 40, 60, 120, and 240 min after injection. The methyl-amino acid residues of proteins of liver, kidney, spleen, brain, and striate muscle were analyzed. The results are shown in Fig. 2. In every tissue, N’-mono-, di-, and trimethyllysine and NC-monoand NG-dimethylarginine residues were found to contain radioactivity. The radioactivities and their time courses are different for organs. An unknown compound disappeared rapidly in every tissue. This compound seems to be a degradation product of methionine during acid hydrolysis. Correlation Between Protein Synthesis and Methylation Activities Radioactivities of neutral amino acid residues, mainly of methionine, were measured at 20 and 40 min after the intraperitoneal injection of 10 &i of (methyl-14C) methionine. The radioactivity was incorporated in increasing order in muscle, brain, liver, and kidney (Fig. 3A). Total radioactivity of the methylamino acid residues are in the same order as that of methionine (Fig. 3B). The activity of protein methylase was highest in the brain and lowest in the liver (Fig. 3E), while in vivo methylation of tissue proteins was highest in kidney and liver and lowest in brain and muscle (Fig. 3B). Radioactivities of free methionine and S-adenosylmethionine in these periods were not proportional either to protein synthetic rates or to methylating activities (Fig. 3C and D). The rate of protein methylation was considered to be determined by the rate of protein synthesis rather than the activity of protein
MIYAKE AND
KAHIMOTO
Kidney
Liver 5-
TML TML
/
DMA
.0
DML
.5-
I I I I 0- 204060
I
120
Ill
I
I
120 204060 14 Time after injection of [methyl- c]methion ine (min.) 240
Brain 5-
I 240
Muscle 30-
TML
.o-
zo-
5-
lo-
1 0:
Time after injection
1 -0; 104060
120
of [methyl- 14C]methionine
240 (min.)
Fig. 2. Chonge in the rodieoctivities of the mathylamino acids in protein fractions of mouse orgons ofter injection of (methyl-“C) methionine. (Methyl-“C) methionine wos injected introperitonaally to mice, and the mice were sacrificed in different intervals. Pmtein fractions of organs were hydrolyzed, and the basic amino acid fraction was separated. The fmction wos chmmotogmphed OS in Fig. 1. Abbreviations of the omino acids ore the same OS in Fig. 1. Each dot represents the mean value of four mice.
METHYLATED
PROTEINS
889
Methionine
Free
Residue
M&hionine
S-Adenosylmethionine
Methylamino Residue
Acid
Methylase
Activity
I
x
Kidney
Liver
Muscle
Brain
afi
0
Kidney
Liver
Muscle
Brain
Fig. 3. Radioactivities of methionine and methylamino acid residues and of free methionine and S-adenosylmethionine in mouse organs and activities of protein methylares. (Methyl-“C) methionine was iniected intraperitoneally to two groups of four mice, and the mice were sacrificed 20 and 40 min later. Organs were homogenized with trichloroacetic acid. Protein fraction WCIS processed as described in text. Radioactivities of free methionine and S-adenosylmethionine were measured by passing the trichloroacetic acid-soluble fmction of tissue through 1 x 3 cm column of Amberlite IR 120, H+ form. The column was washed with water. Methionine was eluted with 15 ml of 1 M pyridine, and S-adenosylmethionine was then eluted with 20 ml of 3 M ammonia. Paper electrophonsis revealed that the radioactivity in this fraction represents that of S-adenosylmethionine and its degradation product formed in alkaline condition. Aliquots were applied to the measurement of radioactivity. Activity of protein methylases was measured as previously reported.20 iI, 20 min. 40 min, 0, histone methylating activity; m myelin basic protein methyloting activity;l, standard error.
methylase. This may be explained proteins are methylated in situ.
by postulating
the only newly
synthesized
Correlation Between the Radioactivities of Methionine and of Methyl Group of Methylamino Acid Residues of Proteins of Subcellular Fractions If proteins are methylated shortly after protein synthesis and then transported to intracellular structures, the methionine and methylamino acid residues are expected to be labeled proportionally in different subcellular fractions.
MIYAKE
AND
KAHIMOTO
4 hr
‘“Jr
1C Liver
Brain 6 _Methionine OMethylamino
Acids
4
lic.Mit.Nuc.
Fig. 4. Distribution of radioactivities of methionine and the methylamino acids in subcellular fractions of mouse liver and brain. Four hours after the injection of ( methyl-14C) methionine to mice, liver and brain were homogenized in sucrose and separated into subcellular fractions. The radioactivities of the methionine and the methylamino acid residues of the protein were measured. Distribution of the radioactivity of each fraction was expressed as percentage of those of whole organs. Sup., supernatant fraction; Mic., microsome; Mt., mitochondria; NW., nuclei; Mye., myelin; Syn., synaptosome.
Ill
L ll-ln-
up.Mlc.Mit.Nuc.Mye.
syn.
If all tissue proteins are methylated nonselectively and continuously after protein synthesis, the previously stated proportionality would not be observed. Four hours after intraperitoneal injection of (methyLL4C) methionine (20 &i/ mouse) to four mice, liver and brain were dissected and subjected to subcellular fractionation. Both the radioactivities of methionine and the methylamino acid residues were roughly proportionally distributed in subcellular fractions of the liver and brain, except nuclear fractions, as shown in Fig. 4. Although the correlation is only approximate as stated, distribution of radioactivities of the methylamino acids is quite different from distribution of protein methylase which is almost exclusively localized in the cytoplasmic fraction.20 This finding is also supportive of the hypothesis that newly synthesized, and not preexisting, proteins are mainly methylated. Cell nuclei contained, however, a relatively high proportion of labeled methylamino acid residues. This may be explained by abundance of methylated proteins.8 Another explanation that methylation of the nuclear proteins is continuing for some time after protein synthesis can not be excluded. Simultaneous Inhibition of Protein Synthesis and Protein Methyiation by Puromyrin If the rate of protein synthesis determines the rate of protein methylation, inhibition of the protein synthesis should decrease protein methylation simultaneously. Puromycin dihydrochloride (0.3 mg/g body weight) was injected to two groups of four mice intraperitoneally 1 and 2 hr prior to (methyl-‘4C) methionine (10 jKi/mouse) injection, and mice were killed 20 min after the injection of the methionine. The radioactivity of the methylamino acid and methionine residues of proteins of kidney, spleen, liver, testis, and brain were measured to obtain the results shown in Table 1. Saline was injected to the control group instead of puromycin. Puromycin inhibited both the rates of protein synthesis and methylation of lysine and arginine residues in each organ.
METHYLATED
PROTEINS
891
Table 1.Inhibition of Protein Synthesis and Protein Methylation by Puromycin* Radioactivity Monomethyl-
Liver
4
None
Puromycin Brain
Trimethyl-
Monomethyl-
Dimethyl-
lyrine
lysine
arginine
Spleen
argininet
207
5.1
12.8
7.5
5.5
Kidney
210+
61
1.3
4.1
1.6
2.8
3.3
2 hr
4
476 i-
91
2.2
4.9
2.3
3.7
4.4 2.9
5
7BOzt
131
1.5
4.7
1.8
1.6
1 hr
4
562i
118
1.1
2.5
1.5
1.2
I .3
2 hr
4
495 +
157
1.1
t
1.1
1 .o
1.2
5
9604
i
1894
199.7
154.6
78.8
30.9
i hr
4
1171 i
366
28.6
22.0
5.6
4.6
12.9
2 hr
4
2521
i
338
6.4
21.1
6.9
5.0
21.0 15.6
Testis
5
4710
*
1100
7.5
32.8
18.3
6.7
4
2352
*
842
3.2
21.4
5.6
3.7
5.6
2 hr
4
1515 zt
348
2.4
13.6
3.9
2.8
5.6
4
1687 f
326
9.3
13.3
8.5
5.2
15.0
40
5.4
4.9
2.2
4.0
7.0
151
4.6
5.6
4.3
4.3
6.5
NCVle
Puromycin
1 hr
4
245 zt
2 hr
4
579i
*(Methyl-‘4C)-L-methionine they
war
injected to two
groups
of four or five mice 1 and 2 hr after
were killed 20 min later. Tissue proteins were hydrolyzed
methionine.
and
neutral
Basic amino acid fractions of each group were combined
each methylamino
101.7
1 hr
None
Puromycin
9.1
4
None
Puromycin
f
I hr
NOW
Puromycin
3007
protein)
Dimethyl-
Methionine
animals
Treatment
SE/mg
lysine
Number of Orqanr
(dpm f
amino
and
injection of puromycin,
acid fraction
chromotographed
to
was
separated
measure
to
and count
radioactivity
of
acid.
tSum of NG,NG-and
NG,N’
G
-dtmethylarginine.
$An accurate value was not obtained
because of poor resolution from an unknown peak.
Before concluding that the decrease in protein methylation results from inhibition of protein synthesis, other possibilities must be excluded. (1) Protein methylases might be inhibited directly by puromycin. As shown in Table 2, protein methylase activities of the cytosole fractions of liver and brain were not inhibited by 127 pg/ml of puromycin. (2) If the turnover rates of protein methylases are rapid, amounts of tissue protein methylases are reduced by puromycin. Protein methylase activities, in the liver, brain, spleen, and kidney of mice injected with puromycin or saline were not different as shown in Table 3. (3) Formation of S-adenosyl(methyl-r4C) methionine may be reduced by the injection of puromycin. Radioactivity of S-adenosyl-methionine was not decreased in the brain, liver, spleen, kidney, and testis 20 min after the injection of (methyl-%) methionine as shown in Table 4. These observations are consonant with the hypothesis that puromycin decreased the rate of protein methylation by inhibiting protein synthesis, thereby reducing the amount of newly synthesized proteins. Newly synthesized proteins are probably the substrates of protein methylases. Table 2. Effect of Puromycin on the Activity of Protein Methylares Histone Methylore
Number of Puromycin
Experiments
+
3
0.87 f 0.24 0.14
_
3
0.83&0.10
The with as
cytoplasmic calf
reported
tration
was
thymus
Brain
fractions histones
previously’9 127 pg/ml.
of
brains, bovine
ztO.05
presence
The activity
livers, myelin ond
is expressed
Brain
Spleen 1.65
0.12ztO.02
and
m
Myelin Basic Protein Methylase
Liver
ho.28
1.74
2.23zkO.40 and
spleens
basic absence as (dpm
of
+
three as
mice substrates.
puromycin SE)
ztO.08
0.06
1.87&0.10
protein of
Liver
x
ho.04
0.10~1~0.03 were
used
The
dihydrochloride
10m3/hr/mg
Spleen
protein.
as
activity of
4.65
f
0.64
3.60+0.39 enzyme was which
sources measured concen-
892
MIYAKE AND
Table 3. Activities of Protein Methylares Number Treatment
Saline
Histone
of
Animals
Brain
0.87
3
ho.24 Puromycin
4
After the Injection of Puromycin
Methylose
Liver
My&n
Spleen
Brain
Kidney
0.14
1.65
0.39
hO.05
l o.za
l O.lO
0.86
0.14
+O.lO
zto.04
KAHIMOTO
1.61 ho.56
Cytoplasmic fractions of mouse orgons were obtained
1.74 ko.08
0.40
2.90
ho.08
zto.45
Basic Protein Liver
Methylare
Spleen
Kidney
0.06
4.65
0.64
*to.04
zt0.64
hO.21
0.04
4.82
*0.02
*0.22
from mice killed 1 hr after
0.65 ho.24
the injection of
puromycin (0.3 mg/g body weight) or saline, and the activity of protein methyloses were measured.”
The
activity is expressed os (dpm + SE) x 10m3/hr/mg protein.
Degradation of the Methylated Proteins If protein methylation is reversible and if methylation-demethylation is a cyclic process, methyl-‘4C group on lysine and arginine residues of a protein should be diluted more rapidly than the 14C-methionine residue of the protein. If irreversible, both radioactivities should decrease in parallel. (Methyl-14C) methionine was administered to mice (7.2 &i/mouse) intraperitoneally, and the change in the radioactivities of the tissue proteins was observed for 3 wk. The radioactivity of methionine and methylamino acid residues of tissue protein of brain, liver, and striate muscle decreased in parallel in every organ as shown in Fig. 5. This finding suggests that protein methylation is an irreversible process. In another experiment, proteins from homogenates of rat liver and brain were methylated in vitro with S-adenosyl-(methyl-%) methionine in a manner of 1 g of tissue in 5 ml of 0.1 M reported previously. 2’ Thus the homogenate phosphate buffer, pH 7.4, was incubated with 10 &i of (methyl-‘4C) S-adenosylmethionine for 3 hr at 37”, and 1 ml aliquot was used for the determination of the radioactivity of the methylamino acid residues. The remaining homogenate was dialyzed overnight against 500 ml of the phosphate buffer and was incubated at 37” after the addition of 5 ml of the freshly prepared homogenate. Two milliliter aliquots were taken at 1 hr intervals up to 3 hr, and subjected to analysis of radioactivities of the methylamino acid residues. No decrease in the radioactivities was observed after the addition of the fresh homogenate. Any Table 4.
Effect of Puromycin Injection on the Radioactivity S-Adenorylmethionine
Number Treatment
of
in Mouse Orgons
of Liver
animals
Brain
Spl&?ll
Kidney
Testis
dpm & SE/mg protein Without puromycin
5
29+
5
161 +21
a52 f
137
4
55 f
11
157 f 36
1033 f 279
4
66*
7
145 f 28
710 + 148
658 f
151
444 f
185
699+
80
388+2lo
632 &
97
463 +219
1 hr after puromycin 2 hr after puromycin
One and 2 hr after the injection of puromycin (0.3 mg/g body weight) (methyl-?)
methionine (10 rCi/
mouse) was injected. A fraction containing S-odenosylmethionine was seporoted from trichloroacetic soluble fraction of organs
qs
acid
described under Fig. 3. The radioactivity of S-odenosylmethionine increased
linearly to the highest value at about 20 min after the injection of methionine in every orgon and was taken as on indicator of the rate of formation of S-adenosylmethionine.
METHYLATED
893
PROTEINS
Methionlne
Mechglamino
acids
l/11111111111 0
4
8
12
16
0
20
days
after
injection
of
4
8
12
16
20
Imethyl-14Clmethlonlne
Degradation of proteins and decrease in the radioactivity of methylomino acid residues. Fig. 5. (Methyl-“C) methionine was injected intraperitoneally to mice, and radioactivities of methionine and the methylamino acid residues were measured in the protein fractions of the liver, brain, and muscle from the 1st to the 21st day. Each point represents the value obtained from one mouse.
detectable amounts of radioactive methylamino trichloroacetic acid soluble fraction.
acids were not
found
in the
DISCUSSION
Presence of mono-, di-, and trimethyllysine residues22-26 and methylarginine residues20.27 in histones, NG,NrG-dimethyl-, and NG-monomethylarginine28 in myelin basic proteins, methyllysine in flagellin,29 monomethyllysine in S 50 residues in actin, 3’ 3_methylhistidine, microsomal protein, 3o 3-methylhistidine mono- and trimethyllysine and NG,NG-dimethylarginine in myosine,32 and trimethyllysine in bacterial and vegetable cytochrome C33 has been reported. We have found the occurrence of the methylamino acid residues in proteins of bacteria, insects, plants, and animals. Amounts of total methyllysine and methylarginine residues of rat tissue proteins ranged from 3 to 10 pmoles/g protein. The concentrations of methylamino acids in histone or myelin basic protein are 5-15 pmoles/g protein and that of myosin is 2 pmoles/g protein. These concentrations are not very different from the average concentrations of methylamino acid residues of tissue proteins. This means that many other proteins exist in tissue as methylated proteins. Methylamino acid residues of proteins distribute widely in every subcellular component. When (methyl-‘4C) methionine was injected intraperitoneally to rats or mice, radioactivity of methylamino acid residues were one tenth to fiftieth the radioactivity of methionine residues. Postulating the average number of methionine residues in protein being 2-3 of 100 amino acid residues, one of about 500 amino acid residues is calculated to be methylated on the assumption that the
894
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specific radioactivity of free methionine is roughly the same as that of free Sadenosylmethionine. The concentration of S-adenosylmethionine is about 40% of that of methionine in rat brain and liver on molar basis.34 This ratio is in accordance with that shown in Fig. 3C and D. From the above calculation 10 /Imoles of amino acid residues per gram of tissue proteins are the amino acid residues which are acceptable sites for methylation. This also indicates that many proteins are capable of being methylated. In an additional experiment, rats were injected with (methyl-14C) methionine, and microsomal and nuclear fractions of the testis were subjected to SDS-acrylamide gel electrophoresis. Radioactivity of methionine and of the methylamino acid residues of proteins were distributed in parallel on the electropherogram. The previously stated experimental findings indicate that protein methylation is universal for a wide variety of tissue proteins and that our present method of treating tissue proteins as a whole is justified in order to study the relationship between protein metabolism and methylation. Our first conclusion that protein methylation takes place not long after protein synthesis is drawn from the fact that methylation is active in organs having high protein synthetic activity and vice versa and that inhibition of protein synthesis by puromycin inhibited protein methylation. The site of protein methylation is unknown. Cytoplasm contains the highest activity of methylating proto assume that proteins synthesized on teins,‘9,35 so it would be reasonable ribosomes are methylated there or during protein transport. The present findings have been preceeded by other workers using different sources and methods. Reporter’ has demonstrated that the selective protein methylation of nascent myosin begins at ribosome using cultured muscle cells. Duerre et al.’ have injected H3-lysine and (methyl-14C) methionine to rats, and purified four kinds of histones. Specific activities of 3H-lysine and “C-methylamino acid residues of the four histones were the same. This finding indicates histone synthesis and methylation takes place within a short interval. In these two studies the relation of protein methylation to protein synthesis was examined for the specific proteins, myosin and histone, respectively. The hypothesis that nascent proteins are selectively methylated was shown to be applied generally to a wide variety of tissue proteins in our present study. Other indirect evidences are from the studies of protein methylase III which increases in a variety of proliferating cell systems such as hepatoma,3 fetal brain,4 immature rat organs5 regenerating rat liver,6 and HeLa S-3 ce11.36,37Matsuoka’ has shown that an increase in proteins is accompanied by an increased amount of methylated proteins keeping the ratio of the amounts of methylamino acid residues of proteins constant in regenerating rat liver and during growth of bacterial cells. Kakimoto et a1.8 have shown that NC-mono, and NG,N’G-dimethylarginine residues in acid soluble protein fraction of rat brain increase concomitantly with myelination which also provides indirect evidence for the hypothesis that protein methylation takes place mainly on nascent proteins. Allfrey et a1.9 incubated (methyl-14C) methionine with isolated nuclei of calf thymus. Addition of puromycin to the reaction mixture did not inhibit histone methylation. The finding however may not necessarily reflect series of reactions occurring in situ. It is not known whether histones are synthesized and methylated in cell nuclei or not. Recent evidence indicates that synthesis of histone
METHYLATED
PROTEINS
895
occurs predominantly in perikaryon. 38-4oThe products of histone methylation in vitro are mainly methylarginine residues4’ but Allfrey et al. determined the radioactivity of methyllysine residues. The significance of their experiment is not clear. Boyoet” has observed the time course of the radioactivities of methylamino acids and methionine of arginine rich histone from rat spleen Novikoff hepatoma, and hamster’s ovary and concluded that protein methylation is irreversible. Thomas et al.” has reached a similar conclusion by the observation of turnover of the methyl group of histone of Ehrlich ascites tumor cells. These findings are restricted to histone, and our conclusion drawn from the observation of whole tissue proteins of mouse organs is in accordance with theirs. Paik and Kim42 found demethylase of N-alkyllysines and methylated histones, but this enzyme reaction seems to be minor one for the metabolism of the methylated proteins and protein hydrolysis is considered to be a major pathway of breakdown of the methylated proteins. REFERENCES I. Reporter M: Protein synthesis in cultured muscle cells: methylation of nascent proteins. Arch Biochem Biophys 158:577. 1973 2. Duerre JA, Lee CT: In vivo methylation and turnover of rat brain histones. J Neurothem 23:541, 1974 3. Paik WK, Lee HW, Morris HP: Protein methylases in hepatome. Cancer Res 32:37, 1972 4. Pdik WK, Kim S, Lee HW: Protein methylation during the development of rat brain. Biochem Biophys Res Commun 46:933, 1972 5. Paik WK. Lee HW, Lawson D: Age dependency of various protein methylases. Exp Geront 6:271, 1971 6. Lee HW, Paik WK: Histone methylation during hepatic regeneration in rat. Biochim Biophys Acta 277: 107, 1972 7. Matsuoka Y: N’-Methylated lysines and NC;-methylated arginines III. Existence and distribution in nature and mammals. Seikagaku 44~364, 1972 8. Kakimoto Y, Matsuoka Y, Miyake M, Konishi H: Methylated amino acid residues of proteins of brain and other organs. J Neurothem 24:893. 1975 9. Allfrey VG, Faulker R, Mirsky AE: Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis. Proc Nati Acad Sci USA 51:786, 1964 IO. Boyoet P: In vivo turnover and distribution of radio-N-methyl in arginine rich histones from rat tissue. Arch Biochem Biophys 152: 887, 1972 11. Thomas G, Lange HW, Hempel K: Relative Stabilitlt Lysine-gebundener Methylatgruppen bei den argininreichen Histonen und
ihren Unterfraktionen von Ehrlich AscitesTumorzellen in vitro. Hoppe-Seyler’s Z Physiol Chem 353: 1423, 1972 12. Oshiro Y, Eylar EH: Allergic Encephalomyelitis: Preparation of the encephalitogenic basic protein from bovine brain. Arch Biochem Biophys 138:392. 1970 13. Kakimoto Y. Akazawa S: Isolation and identification of NG,NG-and NG,N’G-dimethylarginine, N’-mono, -di, and tri-methyllysine, and glucosylgalactosyland galacotosyl-6hydroxyllysine from human urine. J Biol Chem 245:575 I, 1970 14. Nakajima T, Matsuoka Y, Kakimoto Y: Isolation and identification of NG-monomethyl, NG,NG-dimethyl and NG,NtG-dimethylarginine from the hydrolysate of proteins of bovine brain. Biochim Biophys Acta 230:212, 1971 15. Allfrey tein synthesis Physiol40:45
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16. Lowry OH, Rosebrough NJ, Farr AL. Randall RJ: Protein measurement with the folin phenol reagent. J Biol Chem 193:256, 195 I 17. Whittaker VP: The isolation and acterization of acethylcholine-containing ticles from brain. Biochem J 72:694, 1959
charpar-
18. Schneider WC, Hogeboon GH: Intracellular distribution of Enzymes V. Futher studies on the distribution of cytochrome C in rat liver homogenates. J Biol Chem 183:123. 1950 19. Miyake M, Kakimoto Y: Protein lation by cerebral tissue. J Neurochem 1973 20. Miyake
M: Methylases
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protein and histone in rat brain. J Neurochem 24909, 1975 21. Kakimoto Y: Methylation of arginine and lysine residues of cerebral proteins. Biochim Biophys Acta 243:31, 1971 22. Dalange RJ, Fambrough DM, Smith EL, Bonner J: Complete amino acid sequence of pea seedling histone IV: Comparison with the homologous calf thymus histone. J Biol Chem 2445669, 1969 23. Ogawa Y, Quagliarotti G, Jordan J, Taylor CW, Starbuck WC, Busch M: Structural analysis of the glycine-rich, arginine-rich Histone III Sequence of the amino-terminal half of the molecule containing the modified lysine residues and the total sequence. J Biol Chem 244:4387, 1969 24. Delenge RJ, Hooper JA, Smith EL: Complete amino-acid sequence of calf-thymus histone III. Proc Nat1 Acad Sci USA 69:887, 1972 25. Brandt WF, Holt CV: The complete amino acid sequence of histone F, from chicken erythrocytes. FEBS Letter 23:357, 1972 26. Patthy L, Smith EL: Histone III V. the amino acid sequence of pea embryo histone III. J Biol Chem 248:6834, 1973 27. Paik WK, Kim S: e-N-methylarginine in histones Biochem Biophys Res Commun 40: 224,197O 28. Deibler GE, Martenson RE: Determination of methylated basic amino acids with the amino acid analyzer. J Biol Chem 248:2387, 1973 29. Glazer AN, Delange RJ, Martine RJ: Identification of c-N-methyllysine in spirillum serpens flagella and of t-N-dimethyllysine in salmonella typhimurium flagella. Biochim Biophys Acta 188:164, 1969 30. Alix JH, Hayes D: Properties of ribosomes and RNA synthesized by Escherichia coli grown in the presence of ethionine III, methylated proteins in 50s ribosomes of E cob EA2. J Mol Biol86: 139, 1974
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31. Asatoor AM, Armstrong MD: 3-methylhistidine, a component of actin. Biochem Biophys Res Commun 26: 168,1967 32. Reporter M. Corbin JL: NGNG-dimethylarginine in myosin during muscle development. Biochem Biophys Res Commun 43:644, 1971 33. Delange RJ, Glazer AN, Smith EL: Presence and location of an unusual amino acid, r-N-trimethyllysine, in cytochrome c of wheat germ and neurospora. J Bioi Chem 244: 1385,1969 34. Gaitonde MK: Sulfer amino acids, in Laitha A (ed): Handbook of Neurochem, vol 3. New York. Plenum Press. 1970, p 225 35. Paik WK. Kim S: Protein methylation. Science 174:114, 1971 36. Borun TW, Pearson D, Paik K: Studies of hitsone methylation during HeLa S-3 cell cycle. J Biol Chem 247:4288, 1972 37. Lee HW, Paik WK, Borun T: The periodic synthesis of S-adenosylmethionine: protein methyltransferases during the HeLa S-3 cell cycle. J Biol Chem 248:4194, 1973 38. Gallwitz D, Mueller GC: Histone synthesis in vitro on HeLa cell microsomes. J Biol Chem 24415947, 1969 39. Gallwitz D, Mueller CC: Histone synthesis in vitro by cytoplasmic microsomes from HeLa cells. Science 163:1351, 1969 40. Oliver D, Granner D, Chalkley R: Identification of a distinction between cytoplasmic histone synthesis and subsequent histone deposition. Biochemistry 13:746, 1974 41. Paik WK, Kim S: Enzymatic methylation of protein fractions from calf thymus nuclei. Biochem Biophys Res Commun 29:14, 1967 42. Paik WK, Kim S: c-Alkyllysinase, new assay method, purification, and biological significance. Arch Biochem Biophys 165:369, 1974
