Journal oJ Nrurochrrnrsfry. 1975. Vol. 24. pp. 909-915. Pergamon Press. Printed in Great Britain
METHYLASES OF MYELIN BASIC PROTEIN A N D HISTONE I N RAT BRAIN M. MIYAKE Department of Neurology, The Institute of Higher Nervous Activity, Osaka University Medical School, Fukushima-ku, Osaka, Japan (Receiued 6 M a y 1974. Accepted 16 October 1974)
Abstract-The two enzymes methylating myelin basic protein and histone were purified 170- and 250-fold respectively from the cell sap fraction of rat brain. These enzymes methylated only arginine residues of the two proteins. The enzyme activities were present in all organs tested. Testis has the highest, brain a moderate and liver the lowest activity. Most of the activities were present in the cell sap fraction in brain, liver and testis. Methylation of myelin basic protein and histone was examined in both the cell sap and solubilized nuclear fraction of rat brain during life span after birth. The myelin basic protein methylating activity in the cell sap fraction increased during myelination. Histone methylase from the nuclear fraction was highest at birth and dropped rapidly thereafter. The other activities remained unchanged. The natural occurrence of NG-mono- and NG,NG-dimethylarginineresidues in histones prepared from rabbit liver was demonstrated.
p, NG- AND NG,NG-dimethylargininea n d NG-monomethylarginine have been identified as natural constituents of various proteins (NAUAJIMA et al., 1971; REPORIER& CORBIN,1971; BROSTOFF et a!., 1972;
METHODS AND MATERIALS
S-Adenosyl [Me-'4]-~-methionine, 52 mCi/mmol was purchased from New England Nuclear Corp., histone (calf thymus type 11) from Sigma and Bio-Gel A 0.5-m resins MATSUOKA, 1972; DEIBLER & MARTENSON, 1973; KAK- from Bio-Rad Laboratories, Richmond, CA. Myelin basic protein was prepared from bovine, rabbit and human brain IMOTO et al., 1975).These are formed by methylation of (1970). arginine residues, and protein methylases catalyse the according to OSHIRO& EYLAR Preparation of histones from rabbit liver and measurement transfer of the methyl group of S-adenosylmethionine to lysine and arginine residues of proteins as reported ofmethylated amino acids in each histone. Histones were prepared from rabbit liver (320g) by the procedure of DUERRE by PAIK& KIM(1969, 197Oa, 1973), PAIKet al. (1972a, & GAITONDE (1971). Histones were extracted from the chrob), KAYE& SHERATSKY (1969) and BURWN& GARVEN matin with O.~M-HCIand purified by gel filtration on a (1971). These authors have used histones as substrate Sephadex G-100 column. Histones were separated by CMprotein. We have reported on the multiplicity of the cellulose column chromatography (PHILLIPS& JOHNS, methylating enzymes and substrate proteins (KAKI- 1959). Methylamino acids in each histone were measured MOTO, 1971; MIYAKE & KAKIMOTO, 1973). The enzyme with an amino acid analyser under the conditions reported & AKAZAWA, 1970; KAKIMOTO, 1971). methylating arginine was separated from those methyl- earlier (KAKIMOTO ating lysine by ammonium sulphate fractionation of Protein concentration was determined by the method of et al. (1951). bovine brain extract. There were enzymes having dif- LOWRY Subfractionation oftissues. Rat brain was fractionated into ferent substrate specificities for myelin basic protein subcellular fractions by the method of WHITTAKER (1959) and histone respectively in rat brain. The arginine resiand testis and liver according to SCHNEIDER & HOGEBOOM due at position 107 of myelin basic protein is mono(1 950). or dimethylated in nature (BROSTOFF & EYLAR,1971). Enzyme assay. The activity of protein methylase was BALDWIN& CARNECIE (1971) showed an enzymic determined as reported previously (MIYAKE & KAKIMOTO, methylation of human myelin basic protein and sug- 1973). Radioactivity was measured in 10 ml of Bray's solugested that the methylation of this protein might be tion (BRAY,1960)with a liquid scintillation counter, Nuclear et al. Chicago, Mark I. The counting efficiency was between 60 important for myelin formation. KAKIMOTO (1975) demonstrated that most of the NG-mono- and and 70 per cent with external standard (channels ratio) NGJV"'-dimethylarginine residues in the brain protein method. Large scale reaction mixtures consisted of 1.52.5 ml ofenzyme solution, 1-2 mg of myelin basic protein or are derived from myelin basic protein. In the present study, the organ- a n d subcellular dis- histone, 0.25 pCi of S-adenosyl [~bfe-'~C]-~-methionine, 1 ml of 0.2 M-phosphate buffer, pH 6.7, or 0.2 M-Tris-HCI tribution, age-dependency and species specificity of buffer, pH 8.0,both containing 0.01% 2-mercaptoethanol in myelin basic protein methylase and histone methylase a total volume of 4 mi. This was incubated for 4 h a t 37°C. are reported. Histone was used as the sole substrate to The reaction was terminated by the addition of an equal classify and characterize the methylases in the previous volume of 20% TCA. The proteins were washed and hydrostudy, but a different enzyme was found when myelin lysed. Basic aliphatic amino acid fractions containing the methylarginines and methyl-lysines were prepared from basic protein was used as a substrate.
M. MIYAKE
910
& protein hydrolysates as described previously (MIYAKE KAKIMOTO, 1973). Radioactivity was measured with a sample of the aqueous solution of this fraction.
Chromatography of the methyl-lysines and methylarginines.
The basic aliphatic amino acid fractions were chromatographed on a 1 x 20 or 1 x 15 cm column of Amberlite IR120, NH: form, 20&400 mesh with 75 or 40 ml of 0.5 M and then 1.0 M-ammonium hydroxide. Fractions of the eluate (3 or 2 rnl) were collected and the radioactivity was measured using 2 or 1.5 ml of the eluate in 10 ml of Bray's solution (BRAY,1960) with a liquid scintillation counter, Nuclear Chicago, Mark I. A shortcoming of the method is that N",NG- and NC,N'G-dimethylarginineswere not resolved. The ratio of these dimethylarginines was determined by paper chromatography (pyridine:acetone:3~-ammonium hydroxide = 50: 30:25) to measure their relative radioactivities.
,
fiyelin b a s i c protein
0
X
s
A!.
I
5
L
1
2
3
J
5
1
-
Tube number
FIG. 2. Chromatography of products of myelin basic protein and histone methylated with partially purified enzymes. RESULTS Partial purification of methytases of myelin basic pro- Volumes of 1.5 ml of Enzyme A or 2.5 ml of Enzyme B and tein and histones. The enzyme methylating myelin basic 2 mg of bovine myelin basic protein or histone were incuprotein (Enzyme A) and that of methylating histone bated in the presence of 0.25pCi of S-adenosyl [ M e ''C]methionine at 37°C for 4 h, and products of these pro(Enzyme B) were partially purified from the cell sap teins were analysed as described in Methods. The dotted fraction of rat brain as described previously (MIYAKE line indicates the location of authentic methyl-lysine deri& UKIMOTO, 1973). Elution patterns of both enzymes vatives and -0, radioactivity ofeach fraction. Peaks 1on a 2 x 110 cm column of Bio-Gel A 0.5-m are shown 5 correspond to N'-dimethyl-lysine, N%nonomethyl-lysine, in Fig. 1. Enzyme A (from 145 to 170 ml of the elution N'-trimethyl-lysine, a mixture of NG,NC- and N C , N G volume in Fig. 1) was purified approx. 170 times and dimethylarginine and NG-monomethylargininerespectively. enzyme B (from 155 to 185 ml of the elution volume) Peak 4 in myelin basic protein was identified as NG,N'Gabout 250 times in comparison with the homogenate. dimethylarginine and one in histone as NG,NG-dimethylarginine by paper chromatography. Enzyme A preparation is free from endogenous substrate proteins. Products of myelin basic protein and histone methyl- adenosyl [Me-14C]methionine at 37°C for 4 h, and ated with partially purified enzymes. Bovine myelin products of these proteins were analysed (Fig. 2a,b). basic protein (2 mg) or histone (2 mg) was incubated Myelin basic protein was methylated by Enzyme A to with Enzyme A or B in presence of 0.25pCi of S- form N",NG-dimethylarginine and p-monomethylarginine. Histone was methylated by Enzyme B to form NG,NG-dimethylarginineand NG-monomethylarginine. The incorporation of radioactivity into NfN 0.06 monomethyl-lysine was due to the methylation of the 10 '2 endogenous substrate protein in the Enzyme B prepX 0.04 aration since the same amount of N'-monomethyl;5 lysine was formed in absence of histones. Both enzyme 5 a 5 0.02 A and B methylated only arginine residues of both m N 10 myelin basic protein and histone. Thus there are at ; c 0 3 least two different arginine residue methylases having > rl U 0 30 3 different protein substrate specificities. There remains U 5U 1 . c a possibility that different enzymes methylate proteins 2 w 20 7 to produce mono- and dimethylarginine residues, but U several trials to resolve the monomethylating and 0.1 e! 10 2 dimethylating enzymes have not been successful. 3 The occurrence of methylarginine in histone isolated C 0 from rat liver nuclei was reported by PAIK& KIM 120 160 200 24 0 Elution volume, ml (197Ob), but their identification of the methylarginines 1971) and the conwas not correct (KAKIMOTO, FIG. 1. Purification of methylase A and B by gel filtration centration of the amino acids was not measured accuon Bio-Gel A 0.5-m. The enzyme activitiesof each tube were rately. Since the amounts of the methylarginines were measured using 100 pg of myelin basic protein and histone much lower than the arginine residues, preliminary as substrate under the conditions reported previously (MIYAKE & KAKIMOTO, 1973).Protein (-0) was deter- purification of the methylarginines was neccessary mined by extinction at 280 nm. Methylating acti- prior to amino acid analysis (KAKIMOTO,1971). Hisvity of myelin basic protein; A-A, methylating activity tones were prepared from rabbit liver, and methylamino acids in each histone fraction was measured by of histone.
+.,
1.5
I40
7.8 N.D. 1.2 8.3
16.0
9.1
1.7 0.4 I .3
1.6
0.2 N.D. 0.2 N.D.
Mcthylatcd amino acids (nip rnol/rng protein) N'-DiniethylN", Nr'-Dimet hylNG1 MG-Dime thyllysine arginine arginine
0.3 N.D.
0.3 0.5
N"-Monomethylargin ine
N.D.. not detectable. Histones were prepared from the chromatin of rabbit liver (320g) by the procedure of DUERRE & GAITONDE (1971). Each histone was separated by CM-cellulose column chromatography (PHILLIPS & JOHNS, 1959).Methylamino acids in each histone (6-50mg) were measured with an amino acid analyser under the conditions reported by KAKI~om(1971).
F, F3
F,* F,,
Histonr fractions
NE-Trimethyllysine
TABLE 1. AMOUNTSOF METHYLARGININES AND METHYL-LYSINES IN RABBIT LIVER HISTONES
E.
2-
5' E -I
y, m D
w5.
M. MIYAKE
912
TABLE 2. SUBCELLULAR DISTRIBUTION OF MYELIN BASIC PROTEIN A N D HISTONE METHYLATING Histone
Supernatant fractlon Myelin basic protein mrthylattnp :ic!i\it) Sp. Act.* Total Ac1.t
methylating : c l ~ v s t v Sp Act.' Total Act.+
ACTIVITIES I N RAT BRAIN
Precipitate fraction Histonr Myelin bdW protcln methykiting .ILII\ it! mrthylatinp . i c l i \ t l \ Sp. Act.* Total Ac1.t Sp. Act.* Tolal Act t ~
Cell sap Mitochondria1 Microsomal Nuclear Myelin Synaptosomal ~~~
3.18 0.43 0.13 0.7 I 0.44 0.59
341.5
3.80
8.3
0.90 0.35 0.64 0.49 I.IR
1.5
x.2 8.3 12.1
40x.7 17.5
0.12
3-8
oin
7-4 9.3 24.2
0.05
0.19 0.15
23 1.3 23 0.9 3.0
0.00 0.07
17 0.9
0.06 0-02
0.4
0.09
1.8
0.7
* Specific enzyme activity, [c.p.m./mg protein] x w3. t Total enzyme activity, c.p.m. x
Subcellular fractionswere obtained from pooled brains of two rats. Reaction mixtures consisted of 0.2 ml of enzyme solution (6CL900pg proteins), 25 nCi of S-adenosyl [M~-'~C]methionine, 100pg of bovine myelin basic protein or histone as substrate,0.1 ml of 0.2 M-phosphate buffer, pH 6.7. or 0.2 M-Tris-HC1 buffer, pH 8.0, both containing 0.01% 2-mercaptoethanol. in a total volume of 0.45 ml. For each assay a blank tube was placed in the absence of myelin basic protein or histone. This was incubated for 1 h at 37°C. See the earlier report (MIYAKE & KAKIMOTO, 1973) for details of the enzyme assay.
fractions. It was considered that the methylase of subcellular fractions other than the cell sap fraction is in an inactive form or absent, or that substrate proteins did not gain access to the enzyme. More than 95 per cent of two enzyme activities was also concentrated in the cell sap fraction of testis and liver. Table 3 lists the distribution of these enzyme activities in the cell sap fraction of rat organs. Both enzyme activities were present in all organs tested. Testis had the highest, brain a moderate, and liver the lowest activity. The result agrees well with the organdistribution of protein methylase I reported by PAIK& KIM(1971). Distribution of the two enzyme activities was somewhat different. Distribution of the two enzyme activities among cell sap fractions of various regions in the brain of a man was also examined (Table 4). Caudate nucleus and cerebellar grey matter have the highest enzyme activities and medulla oblongata the lowest. Enzyme activities were present in all regions tested and the activities of the two enzymes were parallel to each other. Age dependency of myelin basic protein and histone methylating activities in rat brain. Myelin basic protein and histone methylating activities were present almost exclusively in the cell sap fraction in brain, liver and testis of rat. Histone methylating activity has also been demonstrated in the nuclear fraction of calf (PAIK& KIM, 1 9 7 0 ~ )and rat (GALLWITZ,1971) thymus and TABLE3. ORGANDISTRIBUTION OF MYELIN BASIC PROTMN Krebs 2 ascites tumour cell (BURDON& GARVEN, AND HISTONE METHYLATING ACTIVITIES IN THE RAT 1971).The specific activity of histone methylase in the Specific enzyme activity nuclear fraction of rat brain was second to that of the [c.p.m./mgprotein] x cell sap fraction (Table 2). Soluble methylase was preOrgans Myelin basic protein Histone pared by extraction of chromatin isolated from the nuclear fraction ofrat brain with 1.5 M - N ~ Cin I 0.02 Mmale female male female Tris-HCI buffer, pH 7 8 ,followed by dialysis against 3.17 Testis 8.62 1.28 1.38 Brain 1.23 1.03 002 M-Tris-HCl buffer and removal of precipitate by 015 040 Spleen 1.06 0.95 (1971). centrifugation according to BURDON& GARVEN 0.40 Heart 0.8 I The level of myelin basic protein and histone methylatPancreas 0.50 0.18 ing activities was examined in both the cell sap and Muscle 0.44 0.14 0.2 I 0.23 0.1 7 0.55 Kidney 0.26 0.16 solubilized nuclear fraction of rat brain during the life 0.10 0.33 Liver 0.09 0.03 span after birth (Fig. 3). Myelin basic protein methylating activity in the cell sap fraction decreased for the Enzyme assay carried out as described in Table 2. The amount of protein in the enzyme solution used first 10 days of life, and thereafter increased a t the ranged from 185 to 1080pg. period of formation of myelin and reached the adult
our method (Table 1). Purity of chromatographically purified histones was confirmed with acrylamide gel electrophoresis. All histones contained N"-mono- and NG,NG-dimethylarginine residues although the amounts were less than a tenth of methyl-lysines. N",N'"-Dimethylarginine was barely detectable, which is in clear contrast to myelin basic protein. Distribution of myelin basic protein and histone methyl ating activities in subcellular ,fractions and organs of rat. The subcellular distribution of myelin basic protein and histone methylating activities in rat brain was studied. In preliminary studies, most enzyme activity was found in cell sap and the rest in nuclear, mitochondrial and synaptosomal fractions in duplicate determinations. Each subcellular fraction of rat brains was sonicated in cold 0.05 M-phosphate buffer, pH 7.0, three times for 30 s at 40 W and centrifuged for 20 min at 11,000g. The precipitates were washed with the same buffer, centrifuged and suspended in the same volume of buffer as the supernatants. Enzyme activities in the precipitate and supernatant fraction of each subcellular fraction were measured (Table 2). Activities of the two enzyme were concentrated in the cell sap fraction and were low both in the supernatant and precipitate fractions of the other subcellular
Protein methylases in rat brain m
Body weight, 9
40
Days after birth
FIG.3. Enzyme activities methylating myelin basic protein and histone in rat brain during the life span. Brains were removed from 3 or 4 rats a t each stage after birth and fractionated into the cell sap and nuclear fractions. Methylating activities were solubilized from pooled nuclear fractions (1971). according to the method of BURWN& GARVEN Myelin basic protein and histone methylating activities in both cell sap and solubilized nuclear fractions were determined as described in the legends to Table 2. The amount of protein in the enzyme solution of cell sap ranged from 530 to 730 pg, and that of nuclear from 93 to 188 pg. +a, Myelin basic protein methylating activity in the cell sap; -0, histone methylating activity in the cell sap; A-A, histone methylating activity in the nuclear; A-A, myelin basic protein methylating activity in the nuclear.
level within 30 days after birth. The enzyme activity in the nuclear fraction compared to the cell sap fraction remained unchanged at a low level during the life span. Histone methylating activity in the cell sap fraction decreased slightly during the life span, while the activity in the nuclear fraction was high immediately after birth and then decreased markedly and reached the adult level within 30 days. Products of methylation of arginine and lysine residues in myelin basic protein and histone were examined at the first, 30th and 60th d a y after birth. Relative amounts of products in myelin basic protein methylation with the cell sap enzyme did not change and the ratio of N",N'"-dimethylarginine to NG-monomethylarginine was 1 : 3-4 (Fig. 4).Figure 5a,b illustrates the change in the products of histone with the cell sap and nuclear enzymes on the first and 60th days after birth. Lysine residues of histones were methylated more than arginine residues with the nuclear methylases on the first day after birth while arginine residues were methvlated more than lysine residue on the 60th day (Fig.
4! 10
30 Tube number
20
40
5(
FIG. 4. Products of myelin basic protein methylation with the cell sap enzymes on days 1, 30 and 60 after birth. Samples (2 ml) of the cell sap fraction from 3 or 4 rats brain on first, 30th and 60th day after birth and 1 mg of bovine myelin basic protein were used for myelin basic protein methylation. Analysis of the products was carried out as described in Methods. Peaks 4 and 5 correspond to NC,N'"-dimethylarginine and NG-monomethylarginine.
5b). However, the pattern of histone methylation with the cell sap enzymes remained unchanged (Fig. 5a). Species specijicity of myelin basic protein methylation. Although the amino acid sequences of myelin
Tube number
FIG.5. Changes in the products of histone methylation with the enzymes of the cell sap or nuclear fraction on the first and sixtieth day after birth. Samples (2 ml) of the cell sap (5.9-63 mg) and solubilized nuclear fraction (46S-940 pg) and I mg of histone were used for histone methylation. Analysis of the products was carried out as described in the legends of Fig. 2. Peaks 1-5 correspond to N'-dimethylNG,NGlysine, N'-monomethyl-lysine, N'-trimethyl-lysine. . . . . dimethylarginine, Nc-monomethylarginine respectively.
M. MIYAKE
914
TABLE4. ENZYMEACTIVITY
METHYLATING MYELIN BASIC PROTEIN AND HISTONE IN VARIOUS REGIONS OF HUMAN BRAIN
Regions of human brain
Specific enzyme activity [c.p.m./mg protein] x Myelin basic protein Histone
Caudate nucleus Cerebellar grey Thalamus Hypothalamus Cerebellar white Callosal body Cortical white Cortical grey Pons Medulla oblongata
6.4 4.5 4.4 3.7 3.0 2.9 2.8 2.7 2.4 1.4
6.2 7.6 5.6 4.2 3.5 3.3 3.8 4.7 3.5 2.2
Enzyme assay was carried out as described in Table 2. The amount of protein in enzyme solution used ranged from 400 to 624 pg.
basic proteins of bovine (EYLARet al., 1971), human (CARNEGIE, 1971) and rabbit brain were different, they were methylated by the cell sap methylase of rat and rabbit brain (Table 5). Substrate activity of bovine myelin basic protein was high, human myelin basic protein middle and rabbit myelin basic protein low for either rat or rabbit enzyme. Differences in the substrate activity among species seem to be due to the proportion of unmethylated arginine residue a t position 107 of the myelin basic protein. The ratio of methylated arginine residue at position 107 in bovine and human myelin basic protein was determined by the conditions reported earlier (KAKIMOM & AKAZAWA, 1970) (Table 6*). The ratio of the arginine residue shown in the table was calculated by subtracting those of mono- and dimethylarginine from 1. Bovine myelin bitsic protein possesses more unmethylated arginine residue than human myelin basic protein. Substrate activity of myelin basic protein seems to be determined by the amount of non- or mono-methylated arginine (arginine and monomethylarginine at position 107 of the molecule). Incorporation of ['4C]methyl groups into arginine and monomethylarginine results in the formation of [14C]NC-monomethyland ['4C]N",N'G-dimethylarginine respectively. The
TABLE5. METHYLATION OF BOVINE,
HUMAN AND RABBIT MYELIN BASIC PROTEIN WITH THE CELL SAP METHYLASES OF RAT AND RABBIT BRAIN
Specific enzyme activity [c.p.m./mg protein] x 1O-j Myelin basic protein Bovine Human Rabbit
Rat enzyme
Rabbit enzyme
1.21 0.79 0.29
0.86 068 0.28
Enzyme assay was carried out using 0.2 ml samples of rat brain cell sap (650pg protein) and rabbit brain cell sap (630 pg protein) and 100 pg of bovine, human and rabbit myelin basic protein as described in the legends to Table 2.
ratios of ['4C]mono- and ['4C]dimethylarginine in bovine and human myelin basic protein methylation with rat and rabbit enzymes were examined in this way (Table 67). The ratio of dimethylarginine to monomethylarginine is about 1:4 in bovine myelin basic protein and 1 :2 in human myelin basic protein respectively and those ratios are similar to the ratio of monomethylarginine to arginine residues in bovine and human myelin basic protein (Table 6*).
DISCUSSION Many protein methylases have been found in both cell sap and nuclear fraction of various organs. The methylases reported in the nuclear fraction of calf thymus by PAIK& KIM(197Oa) and Krebs 2 ascites tumour cell by BURBON & GARVEN (1971) methylated almost exclusively the lysine residue of histone. On the other hand the methylase (histone methyltransferase IIN) in the nuclear fraction of adult rat thymus was demonstrated by GALLWITZ (1971) to methylate predominantly the arginine residue of histone FZal.In the present study it has been shown that the main products of histone methylation with nuclear enzyme changed from methyl-lysine to methylarginine derivatives during growth (Fig. 4b) and that methylarginine as well as methyl-lysine residues occur naturally in histones isolated from rabbit liver chromatin (Table 1). It may be considered that there are different enzymes methylating lysine and arginine residues of histone respectively in the nuclear fraction and the amount of these enzymes changed during growth. All methylases which have been isolated from the cell sap fraction of calf thymus by PAIK& KIM(1968), rat spleen by KAY& SHERATZKY (1969) and rat brain by us (Fig. 2) methylated only the arginine residue of histone and myelin basic protein, though direct comparison of the characteristics of these enzymes has not been made. In the present study we showed that there are at least two different methylases acting on arginine residues. A question is whether methylases of histones in the cell sap and nuclear fraction are different or identical. From the differences in the product patterns (Fig. 5) and in changes in enzyme activity during growth (Fig. 3), it seems likely that histone methylases in the cell sap and nuclear fraction are different. PAIK& KIM (1973) suggested that methylation of arginine residues of protein in rat brain might not be related to the formation of myelin because the activity of protein methylase I (arginine residue methylase) in the cell sap fraction during the development of rat brain did not correlate with the formation of myelin, the methylase activity was very low in myelin and there were no significant changes in the enzyme activity of cell sap between Jimpy and normal mice. They used only histone as the methyl acceptor of the protein methylases. However, we demonstrated that the methylase methylating myelin basic protein is different from the one which methylates histone in the cell sap fraction of rat brain (MIYAKE & KAKIMOTO, 1973). Myelin basic protein had to be used a s substrate protein for
Protein methylases in rat brain TABLE6. RELATIONSHIP BETWEEN
915
AMOUNTS OF METHYLARGININES AT POSITION 107 AND INCORPORATION OF GROUP INTO METHYLARGININES IN HUMAN AND BOVINE MYELIN BASIC PROTEIN
Myelin basic protein Human Bovine
Ratio of amino* acids residue at 107 NG-monomethylarginine arginine 0,221047 0.1 810.75
[ ‘ 4 C ] ~ ~ ~ ~
Ratio of ‘‘C-amino acid residuest NGJ”’-dimethylarginine Nu-monomethylarginine Rat enzyme . Rabbit enzyme 51417.99 7.69127.32
3.751673 1.87110.63
~
* Mol/mol of myelin basic protein. Bovine and human myelin basic protein (5-10 mg) were hydrolysed in 1 ml of 6 M-HCIfor about 20 h at 105°Cin a sealed & KAKIMOTO tube. The basic aliphatic amino acid fraction was prepared from the hydrolysates as described by MIYAKE (1973). Methylarginines in the fractions were measured with an amino acid analyser under the condition reported earlier & AKAZAWA, 1970). (KAKIMOTO t p.m. x 1 0 P . Bovine and human myelin basic protein (500 p g ) were incubated with rat brain (6.5 mg proteins) and rabbit (6.3 mg proteins) cell sap enzyme in the presence of 0.25 pCi of S-adenosyl [Me-’4C]methionine at 37°C for 3 h, and products of these proteins were analysed as described in Methods. DUERRE J. A. & GAITONDE M. K. (1971) J. Neurochem. 18, 1921-1929. EYLARE. H., BROSXIFF S., HASHIM G., CACCAM J. & BURNETT P. (1971) J. bid. Chem. 246, 577&5784. GALLWITZ D. (1971) Archs Biochem. Biophys. 145,65&657. KAKIMOTO Y. & AKAZAWAS. (1970) J. biol. Chem. 245, 5751-5758. KAKIMOTO Y. (1971) Biochim. biophys. Acta 243, 31-37. KAKIMOTO Y., MATSUOKA Y., MIYAKEM. & KONISHIH. (1975) J. Neurochem. 24, in press. KAYEA. M. & SHERATZKY D. (1969) Biochim. biophys. Acta 190, 527-538. LQWRY0.H., ROSEBROUGH N. J., FARR A. L. & RANDALL R. J. (1951) J. b i d . Chem. 193, 265-275. MATSUOKA Y. (1972) Seikagaku 44,364-370. MIYAKE M. & KAKIMOXI Y.(1973) J. Neurochem. 20, 859871. Y. & KAKIMOTOY.(1971)Biochim. NAKAJIMAT., MATSUOKA biphys. Acta 230, 212-222. OGAWAY., QUAGLIAROTTI G., JORDANJ., TAYLOR C. W., W. C. & BUSHH. (1969) J. bid. Chem. 244, STARBUCK 4387-4392. OSHIROY. & EYLARE. H. (1970) Archs Biochem. Biophys. 138, 392-396. PAIKW. K. & KIMS. (1968) J. biol. Chem.243, 2108-21 14. PAIKW. K. & KIMS. (1969) A r c h Biochem. Biophys. 134, 632-637. REFERENCES PAIKW. K. & KIMS. (1970~)J. biol. Chem. 245,601ft-6015. BALDWIN G. S. & CARNEGE P. R. (1971) Science, N.Y. 171, PAIKW. K. & KIMS . (19706) Biochem. Biophys. Res. Com579-581. mun. 40, 224229. BRAYG. A. (1960) Anulyt. Biochem. 1, 279-285. PAIKW. K. & KIM S. (1971) Science, N.Y. 174, 114-119. BROSXIFFS. & E n A R E. H. (1971) Proc. natn. Acad. Sci., PAIKW. K., LEEH. W. & MORRISH. P. (1972~)Cancer Res. U.S.A. 68, 765-769. 32, 37-40. BROSTOFF S . W., ROSEGAY A. B VANDENHEUVELW. J. A. PAIKW. K., KIMS. & LEEH. W. (1972b) Biochem. Biophys. (1972) Archs Biochem. Biophys. 148, 156-160. Rrs. Commun. 46,933-941. BURWNR. H. & GARVENE. V. (1971) Biochim. biophys. PAIKW. K. & KIM S. (1973) Biochim. biophys. Acta 313, Acta 232, 371-378. 181-189. CARNEGIE P. R. (1971) Biochem. J. 123, 57-67. PHILLIPS D. M. P. & JOHNSE. W. (1959) Biochem. J. 72, DEIBLER G. E. & MARTENSON R. E. (1973)J. biol. Chem. 248, 538-544. 2387-2391. J. L. (1971) Biochim. biophys. Res. REPORTER M. & CORBIN DELANGE R. J., FAMBROUGH D. M., SMITHE. L. & BONNER Commun. 43, 644650. J. (1969) J. biol. Chem. 244, 5669-5679. SCHNEIDER W. C. & HOGEBOOM G. H. (1950) J. biol. Chem. DELANGE R. J., HOOPERJ. A. & SMITHE. L. (1972) Proc. 183, 123-128. nutn Acad. Sci., U.S.A. 69, 882-884. WHITTAKER V. P. (1959) Biochem. J. 72, 694-706.
the assay of protein methylases to clarify the correlation between methylation of myelin basic protein and the formation of myelin. In the present study, the levels of myelin basic protein and histone methylating activities were examined in both cell sap and nuclear fraction of rat brain during the life span after birth. Only myelin basic protein methylating activity in the cell sap fraction increased concomitantly with the formation of myelin (from 20 to 30 days after birth) while all the other activities did not. KAKIMOTO et al. (1975) demonstrated that most of the NG-mono- and N G , N G dimethylarginine residues in the rat brain protein are derived from myelin basic protein and that amounts of these methylarginines during growth of rat brain increase for 2@30 days after birth. These observations suggest that methylation of myelin basic protein in rat brain plays an important role in the myelination process. Our results suggest that most proteins are methylated immediately after protein synthesis, and not after being incorporated into cell structure. This information supports the postulate that myelin basic protein is methylated in the cytoplasm before it is transported to myelin.
METHYLASES OF MYELIN BASIC PROTEIN A N D HISTONE I N RAT BRAIN M. MIYAKE Department of Neurology, The Institute of Higher Nervous Activity, Osaka University Medical School, Fukushima-ku, Osaka, Japan (Receiued 6 M a y 1974. Accepted 16 October 1974)
Abstract-The two enzymes methylating myelin basic protein and histone were purified 170- and 250-fold respectively from the cell sap fraction of rat brain. These enzymes methylated only arginine residues of the two proteins. The enzyme activities were present in all organs tested. Testis has the highest, brain a moderate and liver the lowest activity. Most of the activities were present in the cell sap fraction in brain, liver and testis. Methylation of myelin basic protein and histone was examined in both the cell sap and solubilized nuclear fraction of rat brain during life span after birth. The myelin basic protein methylating activity in the cell sap fraction increased during myelination. Histone methylase from the nuclear fraction was highest at birth and dropped rapidly thereafter. The other activities remained unchanged. The natural occurrence of NG-mono- and NG,NG-dimethylarginineresidues in histones prepared from rabbit liver was demonstrated.
p, NG- AND NG,NG-dimethylargininea n d NG-monomethylarginine have been identified as natural constituents of various proteins (NAUAJIMA et al., 1971; REPORIER& CORBIN,1971; BROSTOFF et a!., 1972;
METHODS AND MATERIALS
S-Adenosyl [Me-'4]-~-methionine, 52 mCi/mmol was purchased from New England Nuclear Corp., histone (calf thymus type 11) from Sigma and Bio-Gel A 0.5-m resins MATSUOKA, 1972; DEIBLER & MARTENSON, 1973; KAK- from Bio-Rad Laboratories, Richmond, CA. Myelin basic protein was prepared from bovine, rabbit and human brain IMOTO et al., 1975).These are formed by methylation of (1970). arginine residues, and protein methylases catalyse the according to OSHIRO& EYLAR Preparation of histones from rabbit liver and measurement transfer of the methyl group of S-adenosylmethionine to lysine and arginine residues of proteins as reported ofmethylated amino acids in each histone. Histones were prepared from rabbit liver (320g) by the procedure of DUERRE by PAIK& KIM(1969, 197Oa, 1973), PAIKet al. (1972a, & GAITONDE (1971). Histones were extracted from the chrob), KAYE& SHERATSKY (1969) and BURWN& GARVEN matin with O.~M-HCIand purified by gel filtration on a (1971). These authors have used histones as substrate Sephadex G-100 column. Histones were separated by CMprotein. We have reported on the multiplicity of the cellulose column chromatography (PHILLIPS& JOHNS, methylating enzymes and substrate proteins (KAKI- 1959). Methylamino acids in each histone were measured MOTO, 1971; MIYAKE & KAKIMOTO, 1973). The enzyme with an amino acid analyser under the conditions reported & AKAZAWA, 1970; KAKIMOTO, 1971). methylating arginine was separated from those methyl- earlier (KAKIMOTO ating lysine by ammonium sulphate fractionation of Protein concentration was determined by the method of et al. (1951). bovine brain extract. There were enzymes having dif- LOWRY Subfractionation oftissues. Rat brain was fractionated into ferent substrate specificities for myelin basic protein subcellular fractions by the method of WHITTAKER (1959) and histone respectively in rat brain. The arginine resiand testis and liver according to SCHNEIDER & HOGEBOOM due at position 107 of myelin basic protein is mono(1 950). or dimethylated in nature (BROSTOFF & EYLAR,1971). Enzyme assay. The activity of protein methylase was BALDWIN& CARNECIE (1971) showed an enzymic determined as reported previously (MIYAKE & KAKIMOTO, methylation of human myelin basic protein and sug- 1973). Radioactivity was measured in 10 ml of Bray's solugested that the methylation of this protein might be tion (BRAY,1960)with a liquid scintillation counter, Nuclear et al. Chicago, Mark I. The counting efficiency was between 60 important for myelin formation. KAKIMOTO (1975) demonstrated that most of the NG-mono- and and 70 per cent with external standard (channels ratio) NGJV"'-dimethylarginine residues in the brain protein method. Large scale reaction mixtures consisted of 1.52.5 ml ofenzyme solution, 1-2 mg of myelin basic protein or are derived from myelin basic protein. In the present study, the organ- a n d subcellular dis- histone, 0.25 pCi of S-adenosyl [~bfe-'~C]-~-methionine, 1 ml of 0.2 M-phosphate buffer, pH 6.7, or 0.2 M-Tris-HCI tribution, age-dependency and species specificity of buffer, pH 8.0,both containing 0.01% 2-mercaptoethanol in myelin basic protein methylase and histone methylase a total volume of 4 mi. This was incubated for 4 h a t 37°C. are reported. Histone was used as the sole substrate to The reaction was terminated by the addition of an equal classify and characterize the methylases in the previous volume of 20% TCA. The proteins were washed and hydrostudy, but a different enzyme was found when myelin lysed. Basic aliphatic amino acid fractions containing the methylarginines and methyl-lysines were prepared from basic protein was used as a substrate.
M. MIYAKE
910
& protein hydrolysates as described previously (MIYAKE KAKIMOTO, 1973). Radioactivity was measured with a sample of the aqueous solution of this fraction.
Chromatography of the methyl-lysines and methylarginines.
The basic aliphatic amino acid fractions were chromatographed on a 1 x 20 or 1 x 15 cm column of Amberlite IR120, NH: form, 20&400 mesh with 75 or 40 ml of 0.5 M and then 1.0 M-ammonium hydroxide. Fractions of the eluate (3 or 2 rnl) were collected and the radioactivity was measured using 2 or 1.5 ml of the eluate in 10 ml of Bray's solution (BRAY,1960) with a liquid scintillation counter, Nuclear Chicago, Mark I. A shortcoming of the method is that N",NG- and NC,N'G-dimethylarginineswere not resolved. The ratio of these dimethylarginines was determined by paper chromatography (pyridine:acetone:3~-ammonium hydroxide = 50: 30:25) to measure their relative radioactivities.
,
fiyelin b a s i c protein
0
X
s
A!.
I
5
L
1
2
3
J
5
1
-
Tube number
FIG. 2. Chromatography of products of myelin basic protein and histone methylated with partially purified enzymes. RESULTS Partial purification of methytases of myelin basic pro- Volumes of 1.5 ml of Enzyme A or 2.5 ml of Enzyme B and tein and histones. The enzyme methylating myelin basic 2 mg of bovine myelin basic protein or histone were incuprotein (Enzyme A) and that of methylating histone bated in the presence of 0.25pCi of S-adenosyl [ M e ''C]methionine at 37°C for 4 h, and products of these pro(Enzyme B) were partially purified from the cell sap teins were analysed as described in Methods. The dotted fraction of rat brain as described previously (MIYAKE line indicates the location of authentic methyl-lysine deri& UKIMOTO, 1973). Elution patterns of both enzymes vatives and -0, radioactivity ofeach fraction. Peaks 1on a 2 x 110 cm column of Bio-Gel A 0.5-m are shown 5 correspond to N'-dimethyl-lysine, N%nonomethyl-lysine, in Fig. 1. Enzyme A (from 145 to 170 ml of the elution N'-trimethyl-lysine, a mixture of NG,NC- and N C , N G volume in Fig. 1) was purified approx. 170 times and dimethylarginine and NG-monomethylargininerespectively. enzyme B (from 155 to 185 ml of the elution volume) Peak 4 in myelin basic protein was identified as NG,N'Gabout 250 times in comparison with the homogenate. dimethylarginine and one in histone as NG,NG-dimethylarginine by paper chromatography. Enzyme A preparation is free from endogenous substrate proteins. Products of myelin basic protein and histone methyl- adenosyl [Me-14C]methionine at 37°C for 4 h, and ated with partially purified enzymes. Bovine myelin products of these proteins were analysed (Fig. 2a,b). basic protein (2 mg) or histone (2 mg) was incubated Myelin basic protein was methylated by Enzyme A to with Enzyme A or B in presence of 0.25pCi of S- form N",NG-dimethylarginine and p-monomethylarginine. Histone was methylated by Enzyme B to form NG,NG-dimethylarginineand NG-monomethylarginine. The incorporation of radioactivity into NfN 0.06 monomethyl-lysine was due to the methylation of the 10 '2 endogenous substrate protein in the Enzyme B prepX 0.04 aration since the same amount of N'-monomethyl;5 lysine was formed in absence of histones. Both enzyme 5 a 5 0.02 A and B methylated only arginine residues of both m N 10 myelin basic protein and histone. Thus there are at ; c 0 3 least two different arginine residue methylases having > rl U 0 30 3 different protein substrate specificities. There remains U 5U 1 . c a possibility that different enzymes methylate proteins 2 w 20 7 to produce mono- and dimethylarginine residues, but U several trials to resolve the monomethylating and 0.1 e! 10 2 dimethylating enzymes have not been successful. 3 The occurrence of methylarginine in histone isolated C 0 from rat liver nuclei was reported by PAIK& KIM 120 160 200 24 0 Elution volume, ml (197Ob), but their identification of the methylarginines 1971) and the conwas not correct (KAKIMOTO, FIG. 1. Purification of methylase A and B by gel filtration centration of the amino acids was not measured accuon Bio-Gel A 0.5-m. The enzyme activitiesof each tube were rately. Since the amounts of the methylarginines were measured using 100 pg of myelin basic protein and histone much lower than the arginine residues, preliminary as substrate under the conditions reported previously (MIYAKE & KAKIMOTO, 1973).Protein (-0) was deter- purification of the methylarginines was neccessary mined by extinction at 280 nm. Methylating acti- prior to amino acid analysis (KAKIMOTO,1971). Hisvity of myelin basic protein; A-A, methylating activity tones were prepared from rabbit liver, and methylamino acids in each histone fraction was measured by of histone.
+.,
1.5
I40
7.8 N.D. 1.2 8.3
16.0
9.1
1.7 0.4 I .3
1.6
0.2 N.D. 0.2 N.D.
Mcthylatcd amino acids (nip rnol/rng protein) N'-DiniethylN", Nr'-Dimet hylNG1 MG-Dime thyllysine arginine arginine
0.3 N.D.
0.3 0.5
N"-Monomethylargin ine
N.D.. not detectable. Histones were prepared from the chromatin of rabbit liver (320g) by the procedure of DUERRE & GAITONDE (1971). Each histone was separated by CM-cellulose column chromatography (PHILLIPS & JOHNS, 1959).Methylamino acids in each histone (6-50mg) were measured with an amino acid analyser under the conditions reported by KAKI~om(1971).
F, F3
F,* F,,
Histonr fractions
NE-Trimethyllysine
TABLE 1. AMOUNTSOF METHYLARGININES AND METHYL-LYSINES IN RABBIT LIVER HISTONES
E.
2-
5' E -I
y, m D
w5.
M. MIYAKE
912
TABLE 2. SUBCELLULAR DISTRIBUTION OF MYELIN BASIC PROTEIN A N D HISTONE METHYLATING Histone
Supernatant fractlon Myelin basic protein mrthylattnp :ic!i\it) Sp. Act.* Total Ac1.t
methylating : c l ~ v s t v Sp Act.' Total Act.+
ACTIVITIES I N RAT BRAIN
Precipitate fraction Histonr Myelin bdW protcln methykiting .ILII\ it! mrthylatinp . i c l i \ t l \ Sp. Act.* Total Ac1.t Sp. Act.* Tolal Act t ~
Cell sap Mitochondria1 Microsomal Nuclear Myelin Synaptosomal ~~~
3.18 0.43 0.13 0.7 I 0.44 0.59
341.5
3.80
8.3
0.90 0.35 0.64 0.49 I.IR
1.5
x.2 8.3 12.1
40x.7 17.5
0.12
3-8
oin
7-4 9.3 24.2
0.05
0.19 0.15
23 1.3 23 0.9 3.0
0.00 0.07
17 0.9
0.06 0-02
0.4
0.09
1.8
0.7
* Specific enzyme activity, [c.p.m./mg protein] x w3. t Total enzyme activity, c.p.m. x
Subcellular fractionswere obtained from pooled brains of two rats. Reaction mixtures consisted of 0.2 ml of enzyme solution (6CL900pg proteins), 25 nCi of S-adenosyl [M~-'~C]methionine, 100pg of bovine myelin basic protein or histone as substrate,0.1 ml of 0.2 M-phosphate buffer, pH 6.7. or 0.2 M-Tris-HC1 buffer, pH 8.0, both containing 0.01% 2-mercaptoethanol. in a total volume of 0.45 ml. For each assay a blank tube was placed in the absence of myelin basic protein or histone. This was incubated for 1 h at 37°C. See the earlier report (MIYAKE & KAKIMOTO, 1973) for details of the enzyme assay.
fractions. It was considered that the methylase of subcellular fractions other than the cell sap fraction is in an inactive form or absent, or that substrate proteins did not gain access to the enzyme. More than 95 per cent of two enzyme activities was also concentrated in the cell sap fraction of testis and liver. Table 3 lists the distribution of these enzyme activities in the cell sap fraction of rat organs. Both enzyme activities were present in all organs tested. Testis had the highest, brain a moderate, and liver the lowest activity. The result agrees well with the organdistribution of protein methylase I reported by PAIK& KIM(1971). Distribution of the two enzyme activities was somewhat different. Distribution of the two enzyme activities among cell sap fractions of various regions in the brain of a man was also examined (Table 4). Caudate nucleus and cerebellar grey matter have the highest enzyme activities and medulla oblongata the lowest. Enzyme activities were present in all regions tested and the activities of the two enzymes were parallel to each other. Age dependency of myelin basic protein and histone methylating activities in rat brain. Myelin basic protein and histone methylating activities were present almost exclusively in the cell sap fraction in brain, liver and testis of rat. Histone methylating activity has also been demonstrated in the nuclear fraction of calf (PAIK& KIM, 1 9 7 0 ~ )and rat (GALLWITZ,1971) thymus and TABLE3. ORGANDISTRIBUTION OF MYELIN BASIC PROTMN Krebs 2 ascites tumour cell (BURDON& GARVEN, AND HISTONE METHYLATING ACTIVITIES IN THE RAT 1971).The specific activity of histone methylase in the Specific enzyme activity nuclear fraction of rat brain was second to that of the [c.p.m./mgprotein] x cell sap fraction (Table 2). Soluble methylase was preOrgans Myelin basic protein Histone pared by extraction of chromatin isolated from the nuclear fraction ofrat brain with 1.5 M - N ~ Cin I 0.02 Mmale female male female Tris-HCI buffer, pH 7 8 ,followed by dialysis against 3.17 Testis 8.62 1.28 1.38 Brain 1.23 1.03 002 M-Tris-HCl buffer and removal of precipitate by 015 040 Spleen 1.06 0.95 (1971). centrifugation according to BURDON& GARVEN 0.40 Heart 0.8 I The level of myelin basic protein and histone methylatPancreas 0.50 0.18 ing activities was examined in both the cell sap and Muscle 0.44 0.14 0.2 I 0.23 0.1 7 0.55 Kidney 0.26 0.16 solubilized nuclear fraction of rat brain during the life 0.10 0.33 Liver 0.09 0.03 span after birth (Fig. 3). Myelin basic protein methylating activity in the cell sap fraction decreased for the Enzyme assay carried out as described in Table 2. The amount of protein in the enzyme solution used first 10 days of life, and thereafter increased a t the ranged from 185 to 1080pg. period of formation of myelin and reached the adult
our method (Table 1). Purity of chromatographically purified histones was confirmed with acrylamide gel electrophoresis. All histones contained N"-mono- and NG,NG-dimethylarginine residues although the amounts were less than a tenth of methyl-lysines. N",N'"-Dimethylarginine was barely detectable, which is in clear contrast to myelin basic protein. Distribution of myelin basic protein and histone methyl ating activities in subcellular ,fractions and organs of rat. The subcellular distribution of myelin basic protein and histone methylating activities in rat brain was studied. In preliminary studies, most enzyme activity was found in cell sap and the rest in nuclear, mitochondrial and synaptosomal fractions in duplicate determinations. Each subcellular fraction of rat brains was sonicated in cold 0.05 M-phosphate buffer, pH 7.0, three times for 30 s at 40 W and centrifuged for 20 min at 11,000g. The precipitates were washed with the same buffer, centrifuged and suspended in the same volume of buffer as the supernatants. Enzyme activities in the precipitate and supernatant fraction of each subcellular fraction were measured (Table 2). Activities of the two enzyme were concentrated in the cell sap fraction and were low both in the supernatant and precipitate fractions of the other subcellular
Protein methylases in rat brain m
Body weight, 9
40
Days after birth
FIG.3. Enzyme activities methylating myelin basic protein and histone in rat brain during the life span. Brains were removed from 3 or 4 rats a t each stage after birth and fractionated into the cell sap and nuclear fractions. Methylating activities were solubilized from pooled nuclear fractions (1971). according to the method of BURWN& GARVEN Myelin basic protein and histone methylating activities in both cell sap and solubilized nuclear fractions were determined as described in the legends to Table 2. The amount of protein in the enzyme solution of cell sap ranged from 530 to 730 pg, and that of nuclear from 93 to 188 pg. +a, Myelin basic protein methylating activity in the cell sap; -0, histone methylating activity in the cell sap; A-A, histone methylating activity in the nuclear; A-A, myelin basic protein methylating activity in the nuclear.
level within 30 days after birth. The enzyme activity in the nuclear fraction compared to the cell sap fraction remained unchanged at a low level during the life span. Histone methylating activity in the cell sap fraction decreased slightly during the life span, while the activity in the nuclear fraction was high immediately after birth and then decreased markedly and reached the adult level within 30 days. Products of methylation of arginine and lysine residues in myelin basic protein and histone were examined at the first, 30th and 60th d a y after birth. Relative amounts of products in myelin basic protein methylation with the cell sap enzyme did not change and the ratio of N",N'"-dimethylarginine to NG-monomethylarginine was 1 : 3-4 (Fig. 4).Figure 5a,b illustrates the change in the products of histone with the cell sap and nuclear enzymes on the first and 60th days after birth. Lysine residues of histones were methylated more than arginine residues with the nuclear methylases on the first day after birth while arginine residues were methvlated more than lysine residue on the 60th day (Fig.
4! 10
30 Tube number
20
40
5(
FIG. 4. Products of myelin basic protein methylation with the cell sap enzymes on days 1, 30 and 60 after birth. Samples (2 ml) of the cell sap fraction from 3 or 4 rats brain on first, 30th and 60th day after birth and 1 mg of bovine myelin basic protein were used for myelin basic protein methylation. Analysis of the products was carried out as described in Methods. Peaks 4 and 5 correspond to NC,N'"-dimethylarginine and NG-monomethylarginine.
5b). However, the pattern of histone methylation with the cell sap enzymes remained unchanged (Fig. 5a). Species specijicity of myelin basic protein methylation. Although the amino acid sequences of myelin
Tube number
FIG.5. Changes in the products of histone methylation with the enzymes of the cell sap or nuclear fraction on the first and sixtieth day after birth. Samples (2 ml) of the cell sap (5.9-63 mg) and solubilized nuclear fraction (46S-940 pg) and I mg of histone were used for histone methylation. Analysis of the products was carried out as described in the legends of Fig. 2. Peaks 1-5 correspond to N'-dimethylNG,NGlysine, N'-monomethyl-lysine, N'-trimethyl-lysine. . . . . dimethylarginine, Nc-monomethylarginine respectively.
M. MIYAKE
914
TABLE4. ENZYMEACTIVITY
METHYLATING MYELIN BASIC PROTEIN AND HISTONE IN VARIOUS REGIONS OF HUMAN BRAIN
Regions of human brain
Specific enzyme activity [c.p.m./mg protein] x Myelin basic protein Histone
Caudate nucleus Cerebellar grey Thalamus Hypothalamus Cerebellar white Callosal body Cortical white Cortical grey Pons Medulla oblongata
6.4 4.5 4.4 3.7 3.0 2.9 2.8 2.7 2.4 1.4
6.2 7.6 5.6 4.2 3.5 3.3 3.8 4.7 3.5 2.2
Enzyme assay was carried out as described in Table 2. The amount of protein in enzyme solution used ranged from 400 to 624 pg.
basic proteins of bovine (EYLARet al., 1971), human (CARNEGIE, 1971) and rabbit brain were different, they were methylated by the cell sap methylase of rat and rabbit brain (Table 5). Substrate activity of bovine myelin basic protein was high, human myelin basic protein middle and rabbit myelin basic protein low for either rat or rabbit enzyme. Differences in the substrate activity among species seem to be due to the proportion of unmethylated arginine residue a t position 107 of the myelin basic protein. The ratio of methylated arginine residue at position 107 in bovine and human myelin basic protein was determined by the conditions reported earlier (KAKIMOM & AKAZAWA, 1970) (Table 6*). The ratio of the arginine residue shown in the table was calculated by subtracting those of mono- and dimethylarginine from 1. Bovine myelin bitsic protein possesses more unmethylated arginine residue than human myelin basic protein. Substrate activity of myelin basic protein seems to be determined by the amount of non- or mono-methylated arginine (arginine and monomethylarginine at position 107 of the molecule). Incorporation of ['4C]methyl groups into arginine and monomethylarginine results in the formation of [14C]NC-monomethyland ['4C]N",N'G-dimethylarginine respectively. The
TABLE5. METHYLATION OF BOVINE,
HUMAN AND RABBIT MYELIN BASIC PROTEIN WITH THE CELL SAP METHYLASES OF RAT AND RABBIT BRAIN
Specific enzyme activity [c.p.m./mg protein] x 1O-j Myelin basic protein Bovine Human Rabbit
Rat enzyme
Rabbit enzyme
1.21 0.79 0.29
0.86 068 0.28
Enzyme assay was carried out using 0.2 ml samples of rat brain cell sap (650pg protein) and rabbit brain cell sap (630 pg protein) and 100 pg of bovine, human and rabbit myelin basic protein as described in the legends to Table 2.
ratios of ['4C]mono- and ['4C]dimethylarginine in bovine and human myelin basic protein methylation with rat and rabbit enzymes were examined in this way (Table 67). The ratio of dimethylarginine to monomethylarginine is about 1:4 in bovine myelin basic protein and 1 :2 in human myelin basic protein respectively and those ratios are similar to the ratio of monomethylarginine to arginine residues in bovine and human myelin basic protein (Table 6*).
DISCUSSION Many protein methylases have been found in both cell sap and nuclear fraction of various organs. The methylases reported in the nuclear fraction of calf thymus by PAIK& KIM(197Oa) and Krebs 2 ascites tumour cell by BURBON & GARVEN (1971) methylated almost exclusively the lysine residue of histone. On the other hand the methylase (histone methyltransferase IIN) in the nuclear fraction of adult rat thymus was demonstrated by GALLWITZ (1971) to methylate predominantly the arginine residue of histone FZal.In the present study it has been shown that the main products of histone methylation with nuclear enzyme changed from methyl-lysine to methylarginine derivatives during growth (Fig. 4b) and that methylarginine as well as methyl-lysine residues occur naturally in histones isolated from rabbit liver chromatin (Table 1). It may be considered that there are different enzymes methylating lysine and arginine residues of histone respectively in the nuclear fraction and the amount of these enzymes changed during growth. All methylases which have been isolated from the cell sap fraction of calf thymus by PAIK& KIM(1968), rat spleen by KAY& SHERATZKY (1969) and rat brain by us (Fig. 2) methylated only the arginine residue of histone and myelin basic protein, though direct comparison of the characteristics of these enzymes has not been made. In the present study we showed that there are at least two different methylases acting on arginine residues. A question is whether methylases of histones in the cell sap and nuclear fraction are different or identical. From the differences in the product patterns (Fig. 5) and in changes in enzyme activity during growth (Fig. 3), it seems likely that histone methylases in the cell sap and nuclear fraction are different. PAIK& KIM (1973) suggested that methylation of arginine residues of protein in rat brain might not be related to the formation of myelin because the activity of protein methylase I (arginine residue methylase) in the cell sap fraction during the development of rat brain did not correlate with the formation of myelin, the methylase activity was very low in myelin and there were no significant changes in the enzyme activity of cell sap between Jimpy and normal mice. They used only histone as the methyl acceptor of the protein methylases. However, we demonstrated that the methylase methylating myelin basic protein is different from the one which methylates histone in the cell sap fraction of rat brain (MIYAKE & KAKIMOTO, 1973). Myelin basic protein had to be used a s substrate protein for
Protein methylases in rat brain TABLE6. RELATIONSHIP BETWEEN
915
AMOUNTS OF METHYLARGININES AT POSITION 107 AND INCORPORATION OF GROUP INTO METHYLARGININES IN HUMAN AND BOVINE MYELIN BASIC PROTEIN
Myelin basic protein Human Bovine
Ratio of amino* acids residue at 107 NG-monomethylarginine arginine 0,221047 0.1 810.75
[ ‘ 4 C ] ~ ~ ~ ~
Ratio of ‘‘C-amino acid residuest NGJ”’-dimethylarginine Nu-monomethylarginine Rat enzyme . Rabbit enzyme 51417.99 7.69127.32
3.751673 1.87110.63
~
* Mol/mol of myelin basic protein. Bovine and human myelin basic protein (5-10 mg) were hydrolysed in 1 ml of 6 M-HCIfor about 20 h at 105°Cin a sealed & KAKIMOTO tube. The basic aliphatic amino acid fraction was prepared from the hydrolysates as described by MIYAKE (1973). Methylarginines in the fractions were measured with an amino acid analyser under the condition reported earlier & AKAZAWA, 1970). (KAKIMOTO t p.m. x 1 0 P . Bovine and human myelin basic protein (500 p g ) were incubated with rat brain (6.5 mg proteins) and rabbit (6.3 mg proteins) cell sap enzyme in the presence of 0.25 pCi of S-adenosyl [Me-’4C]methionine at 37°C for 3 h, and products of these proteins were analysed as described in Methods. DUERRE J. A. & GAITONDE M. K. (1971) J. Neurochem. 18, 1921-1929. EYLARE. H., BROSXIFF S., HASHIM G., CACCAM J. & BURNETT P. (1971) J. bid. Chem. 246, 577&5784. GALLWITZ D. (1971) Archs Biochem. Biophys. 145,65&657. KAKIMOTO Y. & AKAZAWAS. (1970) J. biol. Chem. 245, 5751-5758. KAKIMOTO Y. (1971) Biochim. biophys. Acta 243, 31-37. KAKIMOTO Y., MATSUOKA Y., MIYAKEM. & KONISHIH. (1975) J. Neurochem. 24, in press. KAYEA. M. & SHERATZKY D. (1969) Biochim. biophys. Acta 190, 527-538. LQWRY0.H., ROSEBROUGH N. J., FARR A. L. & RANDALL R. J. (1951) J. b i d . Chem. 193, 265-275. MATSUOKA Y. (1972) Seikagaku 44,364-370. MIYAKE M. & KAKIMOXI Y.(1973) J. Neurochem. 20, 859871. Y. & KAKIMOTOY.(1971)Biochim. NAKAJIMAT., MATSUOKA biphys. Acta 230, 212-222. OGAWAY., QUAGLIAROTTI G., JORDANJ., TAYLOR C. W., W. C. & BUSHH. (1969) J. bid. Chem. 244, STARBUCK 4387-4392. OSHIROY. & EYLARE. H. (1970) Archs Biochem. Biophys. 138, 392-396. PAIKW. K. & KIMS. (1968) J. biol. Chem.243, 2108-21 14. PAIKW. K. & KIMS. (1969) A r c h Biochem. Biophys. 134, 632-637. REFERENCES PAIKW. K. & KIMS. (1970~)J. biol. Chem. 245,601ft-6015. BALDWIN G. S. & CARNEGE P. R. (1971) Science, N.Y. 171, PAIKW. K. & KIMS . (19706) Biochem. Biophys. Res. Com579-581. mun. 40, 224229. BRAYG. A. (1960) Anulyt. Biochem. 1, 279-285. PAIKW. K. & KIM S. (1971) Science, N.Y. 174, 114-119. BROSXIFFS. & E n A R E. H. (1971) Proc. natn. Acad. Sci., PAIKW. K., LEEH. W. & MORRISH. P. (1972~)Cancer Res. U.S.A. 68, 765-769. 32, 37-40. BROSTOFF S . W., ROSEGAY A. B VANDENHEUVELW. J. A. PAIKW. K., KIMS. & LEEH. W. (1972b) Biochem. Biophys. (1972) Archs Biochem. Biophys. 148, 156-160. Rrs. Commun. 46,933-941. BURWNR. H. & GARVENE. V. (1971) Biochim. biophys. PAIKW. K. & KIM S. (1973) Biochim. biophys. Acta 313, Acta 232, 371-378. 181-189. CARNEGIE P. R. (1971) Biochem. J. 123, 57-67. PHILLIPS D. M. P. & JOHNSE. W. (1959) Biochem. J. 72, DEIBLER G. E. & MARTENSON R. E. (1973)J. biol. Chem. 248, 538-544. 2387-2391. J. L. (1971) Biochim. biophys. Res. REPORTER M. & CORBIN DELANGE R. J., FAMBROUGH D. M., SMITHE. L. & BONNER Commun. 43, 644650. J. (1969) J. biol. Chem. 244, 5669-5679. SCHNEIDER W. C. & HOGEBOOM G. H. (1950) J. biol. Chem. DELANGE R. J., HOOPERJ. A. & SMITHE. L. (1972) Proc. 183, 123-128. nutn Acad. Sci., U.S.A. 69, 882-884. WHITTAKER V. P. (1959) Biochem. J. 72, 694-706.
the assay of protein methylases to clarify the correlation between methylation of myelin basic protein and the formation of myelin. In the present study, the levels of myelin basic protein and histone methylating activities were examined in both cell sap and nuclear fraction of rat brain during the life span after birth. Only myelin basic protein methylating activity in the cell sap fraction increased concomitantly with the formation of myelin (from 20 to 30 days after birth) while all the other activities did not. KAKIMOTO et al. (1975) demonstrated that most of the NG-mono- and N G , N G dimethylarginine residues in the rat brain protein are derived from myelin basic protein and that amounts of these methylarginines during growth of rat brain increase for 2@30 days after birth. These observations suggest that methylation of myelin basic protein in rat brain plays an important role in the myelination process. Our results suggest that most proteins are methylated immediately after protein synthesis, and not after being incorporated into cell structure. This information supports the postulate that myelin basic protein is methylated in the cytoplasm before it is transported to myelin.
