Substrate specificity for myelin basic protein-specific protein methylase I

142

Biochimica et Biophysica Acta, 1039 (1990) 142-148 Elsevier

BBAPRO 33669

Substrate specificity for myelin basic protein-specific protein methylase I Subrata K. Ghosh, Samiuddin K. Syed, Serena Jung, Woon Ki Paik and Sangduk Kim Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA (U.S.A.) (Received 13 February 1990)

Key words: Myelin basic protein; N°-Methylarginine; Myelin basic protein-specific protein methylase I; S-Adenosyl-L-methionine

The substrate specificity of bovine brain myelin basic protein (MBP)-specific protein methylase ! (S-adenosyI-Lmethionine:protein-L-arginine N-methyltransferase, EC 2.1.1.23), which methylates arginine residues of protein, has been studied using various MBPs, several synthetic peptides and heterogeneous nuclear ribonucleoprotein complex protein (hnRNP). (1) Among MBPs from different species of brain, the carp MBP was found to be the best substrate for MBP-specific protein methylase I. This high degree of methyl acceptability is most likely due to the fact that carp MBP is not in vivo methylated at the arginine residue (Deibler, G.E. and Martenson, R.E. (1973) J. Biol. Chem. 248, 2387-2391) and that the methylatable amino acid sequence is present in this protein. (2) In order to study the minimum chain length of MBP polypeptide which functions as the methyl acceptor, several synthetic polypeptides whose sequences are identical to the region surrounding the residue 107 of bovine MBP (the in vivo methylation site) were synthesized. It was found that the hexapeptide, Gly-Lys-Gly-Arg-Gly-Leu (corresponding to residues 104-109 of bovine MBP), was the shortest methyl accepting peptide, while the tetrapeptide, Gly-Arg-Gly-Leu (corresponding to residues 106-109) was inactive as a substrate. (3) hnRNP protein is known to contain methylarginine at residue 193 (Williams, K.R., Stone, K.L., LoPresti, M.B., Merrill, B.M. and Plank, S.R. (1985) Proc. Natl. Acad. Sci. USA 82, 5666-5670) which is post-translationally modified. Thus, the RNP protein overproduced in Escherichia coil and therefore did not contain methylarginine was examined for its methyl acceptability. It was found that neither MBP-specific nor histone-specific protein methylase I could methylate this methylarginine-less RNP protein. This suggests a possible existence of a distinct protein methylase I specific for this nuclear protein.

Introduction Protein methylase I (PMI) (S-adenosyl-L-methionine :protein-L-arginine N-methyltransferase, EC 2.1.1.23) from bovine brain, which methylates arginine residues of protein substrate, can be classified into two subtypes of enzymes; myelin basic protein(MBP)-specific and histone-specific protein methylase I [1,2]. The former enzyme methylates residue Arg-107 of MBP yielding both N°-monomethyl - and NG, N'G(symmetric)-di -

Abbreviations: AdoMet, S=adenosyI-L-methionine; MBP, myelin basic protein; PMI, protein methylase I; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; MMeArg, NG-monomethylarginine; DiMeArg, NG-dimethylarginine; HPLC, high-performance liquid chromatography; hnRNP, heterogenous nuclear ribonucleoprotein complex. Correspondence: S. Kim, Fels Research Institute, Temple University School of Medicine, Philadelphia, PA 19140, USA.

methylarginine * [3,4]. Recently, we have purified both enzymes to near homogeneity and their molecular identities were compared [1]. The native molecular mass of the former enzyme was shown to be about 500 kDa, consisting of two unidentical subunits of 100 and 72 kDa, while the latter enzyme of about 275 kDa consists of 110 and 75 kDa subunits. Furthermore, patterns of biogenesis of these enzymes have been shown to be quite distinct in developing and in dysmyelinating rodent brains [5-8] as well as in cultures of myelinogenic brain cells [9]. Studies using highly purified PMIs indicated that MBP is a high affinity substrate for MBP-specific enzyme ( K m = 2 . 1 0 -7 M), while histone functions as a much inferior substrate for this PMI ( K m = 1 • 10 -4 M).

* There are three kinds of NG-methylarginines found in protein molecules. These are, NG-monomethylarginine, N °, N '°(symmetric)-dimethylarginine, and N°,N°(asymmetric)-dimethylarginine, and are formed in vivo and in vitro by protein methylase I.

0167-4838/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

143 However, histone-specific PMI methylates only histone, and not MBP [10]. Although NC-methylarginines are also known to be present in nuclear/nucleolar acidic proteins [11-13], heat shock protein [14] and muscle protein [15], the methyl accepting capacity of these proteins toward these PMIs has not been investigated. In view of the fine differences in substrate specificity of these two methylases, it is quite reasonable to assume that the specificity lies in the total substrate structure as well as the amino acid sequences with proper chain length proximity to the methylatable arginine residue. In the present study, we attempted to answer some of these questions with highly purified PMIs using several synthetic polypeptides and methylarginine-deficient hnRNP protein [16].

Experimental procedures

Materials S-Adenosyl-t-[methyl-laC]methionine (specific radioactivity, 50-52 mCi/mmol) was purchased from ICN Radiochemicals. Sephadex G-25, G-10 and histones (IIAS) were obtained from Sigma Chemical Co. MBPs from bovine, human, rat, chicken, guinea pig and carp were purified from freshly frozen brains according to the method previously described [7,17,18]. Rabbit MBP, however, was obtained from Calbiochemicals (No. 475917) and further purified by CM-52 chromatography as described above. Mammalian hnRNP protein A-l, which has been overproduced in Escherichia coli and purified [16], was a generous gift from Dr. Kenneth R. Williams (Yale University). Amino acid analysis of this protein was shown to contain no methylarginine [16]. Peptides were synthesized at Temple University Protein Chemistry Laboratory using an Applied Biosystem Model 430A peptide synthesizer according to the standard solid-phase procedure [19].

Purification of protein methylase I The purification of PMI (both MBP-specific and histone-specific) was carried out essentially as described previously [1].

Enzymatic assay for protein methylase I The incubation condition for PMI was essentially the same as previously described [1,20], except that the reaction was carried out at 4 5 ° C for MBP-specific PMI, and at 35 ° C for histone-specific PMI, for incubation periods as indicated. These incubation temperatures were specifically chosen because the two PMIs exhibit marked differences in their sensitivities toward the temperatures, and therefore, a maximum amount of methyl groups can be incorporated in the respective reactions. It should be mentioned that since M B P - P M I reaction was linear at 4 5 ° C for 60 rain, a possible conformational effect on the protein, if any at this

temperature, will be minimum. Two different methods were used to measure the methyl incorporation into substrate; the conventional CC13COOH precipitation [20] and a newly developed gel-filtration method. For the latter, in the case of protein substrate, the reaction mixture was added to 5 #1 of 3 M HC1 and applied to a column of Sephadex G-25 (0.7 × 85 cm) which was preequilibrated in 0.01 M HC1. Elution was carried out with 0.01 M HC1 at room temperature, and the collected fractions were analyzed for proteins and radioactivity incorporation. In the case of synthetic peptides, however, the pH of the reaction mixture was adjusted to 4.0 with sodium citrate and heated at 100 ° C for 25 min prior to chromatographic separation of the mixture by a Sephadex G-10 column (0.5 × 100 cm). This acid treatment was shown to cleave Ado[methyl-14C]Met to yield 5'-[methyl-14C]thioadenosine [21], and consequently, methyl-14C-labeled peptides could be separated from the bulk of unreacted radioactivity by a single step gel filtration (Fig. 2). This method was shown to be very reproducible with 5% variation.

Polyacrylamide gel electrophoresis SDS-PAGE was carried out according to the method of Laemmli [22] with 15% acrylamide in the running gel at a constant current of 8 mA per slab. Gels were stained with Coomassie brilliant blue R-250 and destained by diffusion.

Protein estimation Protein concentration was estimated by the Coomassie blue method of Bradford [23] modified by Pierce Chemical Co. (Cat. No. 23200) using bovine serum albumin as the standard, or by measuring the absorbance at 280 or 276 nm.

Amino acid analysis The methyl-14C-labeled polypeptides were hydrolyzed in 6 M HC1 at l l 0 ° C in vacuo for 24 h. Amino acid analysis of the hydrolysates was performed on a Resolve C18 column (3.9 mm × 15 cm; Waters Associates) by a Waters HPLC apparatus equipped with a model 721 programmable system controller, data module and an automatic sampler (WISP model 710B), using Model 1 analysis which utilizes o-phthaldialdehyde derivatization [24]. Solvent A was m e t h a n o l / tetrahydrofuran/aq. 50 mM sodium acetate, 50 mM sodium phosphate (pH 7.5) ( 2 : 2 : 9 6 , v/v) and solvent B was methanol/water (65:35, v/v), Fractions (0.5 min) were collected at a rate of 1 m l / m i n and counted for radioactivity.

Results and Discussion

Methylation of MBPs from different species of animal The initial discovery of the occurrence of NG-meth ylarginine in human and rabbit MBPs [3,4] was fol-

144

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a r g i n i n e v a r i e d w i d e l y d e p e n d i n g o n the species, a n d t h a t n o N G - m e t h y l a r g i n i n e was f o u n d i n c a r p M B P [25]. W e have, therefore, i s o l a t e d M B P s f r o m several species a n d s t u d i e d their m e t h y l a c c e p t a b i l i t i e s t o w a r d MBP-specific PMI. Fig. 1 i l l u s t r a t e s S D S - P A G E a n a l y s e s of several purified MBPs presently employed. Three Coomassies t a i n e d b a n d s are seen i n rat p r e p a r a t i o n , f o u r i n c h i c k e n , a n d o n e p r e d o m i n a n t b a n d each i n b o v i n e , g u i n e a p i g a n d r a b b i t p r e p a r a t i o n s w i t h several m i n o r bands. Molecular heterogeneity of MBPs from rodent b r a i n s h a s b e e n r e p o r t e d [26,27]; these are the p r o d u c t s f r o m a single gene, b u t arose via d i f f e r e n t R N A s p l i c i n g p a t h w a y s [27,28]. O n the o t h e r h a n d , several m i n o r b a n d s s h o w n i n o t h e r p r e p a r a t i o n s c o u l d m o s t likely b e

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Fig. 1. SDS-PAGE of MBPs from different species of animal. MBPs prepared from various species of brains were applied on a SDS-polyacrylamide gel and subjected to electrophoresis as described in the text. Symbols indicate: A, MBP from rat (40/Lg); B, from chicken (20 ~g); C, from bovine (40 ~g); D, from guinea pig (25 #g); and E, from rabbit (20 ~g). The molecular marker proteins used were phosphorylase b (Mr 97400), bovine serum albumin (66200), ovalbumin (42700), carbonic anhydrase (31000), soybean trypsin inhibitor (21 500) and lysozyme (14400).

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l o w e d b y the i n v e s t i g a t i o n o n t h e relative c o n t e n t of this m e t h y l a t e d a m i n o acid i n M B P s i s o l a t e d f r o m b r a i n s o f d i f f e r e n t species. D e i b l e r a n d M a r t e n s o n [25] r e p o r t e d that the f r a c t i o n of N G - m e t h y l a r g i n i n e to the t o t a l

TABLE I

Relative substrate efficiency of various MBPs for MBP-specific protein methylase I The methylation reaction was carried out with 6/~g of MBP-specific PMI using 0.5 mg each of MBP at 45°C for 60 rain. The methyl-14C incorporation into MBP substrate was analyzed by the gel-filtration method on Sephadex (3-25 cohmm (0.7 x 85 cm) as described in the Experimental procedures.

"~ ~ 40

50

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60

70

8O

Frecl;ion number

MBP from brains of:

14CH3 incorporated (pmol) (%)

Methylarginine in MBP a (% total Arg)

Carp Rat Chicken Bovine Guinea pig Human Rabbit

147.0 b 43.0 34.9 30.4 13.8 12.5 8.8

0.0 1.52 2.00 c 1.76; 1.23 2.26; 1.79 2.60; 2.80 2.59; 3.00

(484) (141) (115) (100) (45) (41) (29)

a Data taken from Ref. 25. Methylarginine is the sum of both NG-MMeArg and NG-DiMeArg. b Values are average of two independent determinations. Data obtained from this laboratory.

Fig. 2. Isolation of methylated hexapeptide by Sephadex G-10 chromatography. Hexapeptide (350/~g; residues 104-109 of bovine MBP) was incubated with Ado[methylJ4C]Met as described in the text in the presence of (A) or in the absence of MBP-specific PMI (B). After the reaction, the mixture was adjusted to pH 4.0 and heated for 25 min to hydrolyze unreacted AdoMet [21]. It was then subjected to chromatography on a column of Sephadex G-10 (0.5 x 100 cm) which had been equilibrated with 0.01 M HC1. Fractions (0.25 ml) were collected, and aliquots from each fraction were counted for radioactivity and assayed for Ninhydrin reaction [34] to analyze the peptide elution profile. Symbols indicate; • . . . . . . •, Ninhydrin at Ass0nm; • • , radioactivity incorporation per 0.25 ml fraction after pH 4 hydrolysis; o o, Ado[methylJ4C]Met which had not been subjected to pH 4 hydrolysis.

145 d e r i v e d from s p o n t a n e o u s d e g r a d a t i o n o f M B P b y p r o teinases i n h e r e n t l y a s s o c i a t e d with myelin [16,29]. T a b l e I shows that the c a r p M B P exhibits 484% of s u b s t r a t e activity o f the b o v i n e MBP. This high m e t h y l a c c e p t a b i l i t y m a y be d u e to the fact that the M B P

TABLE II

Substrate specificity of various MBP°derivedpeptides for MBP-specific protein methylase I Substrate peptides (0.35 mg) were methylated with the purified MBPspecific PMI at 45°C for 2 h. The methyl incorporation into the substrate was analyzed by Sephadex G-10 column (0.5 × 100 cm) after pH 4 treatment of the mixture as described in the Experimental procedures. A representative chromatogram is shown in Fig. 2. Substrate ~

molecular weight

14CH3 incorporation (nmol) (mol%)

Res. 1-170 (MBP, bovine) Res. 98-109 (TPPPSQGKGRGL) Res. 102-109 (SQGKGRGL) Res. 104-109 (GKGRGL) Res. 105-109 (KGRGL) Res. 106-109 (GRGL) GKDRGL GKFRGL GKHRGL GKLRGL

18400

0.097

0.51

0.493 0.791 1.190 inactive b 0.002 0.173 0.041 0 0

0.17 0.18 0.20 _ 0.03 0.008 -

1194 802 587 602 402 645 677 667 643

Single-letter amino acid abbreviations are used. Res. = residues. b Data taken from Schafer and Colman [31]. "

isolated f r o m c a r p is n o t in vivo m e t h y l a t e d [25], a n d yet the p o t e n t i a l m e t h y l a t i o n site is p r e s e n t in this b a s i c protein. It s h o u l d b e n o t e d here t h a t the m e t h y l acceptability o f c a r p b r a i n M B P has p r e v i o u s l y b e e n r e p o r t e d [30]. E m p l o y i n g b o v i n e M B P as the m e t h y l - a c c e p t o r substrate, the c a r p b r a i n was f o u n d to c o n t a i n o n l y 18% of the e n z y m e activity of that o f the r a t b r a i n ( d a t a n o t shown). This low P M I activity in the c a r p b r a i n m i g h t m o s t likely b e a factor for the negligible a m o u n t of N G - m e t h y l a r g i n i n e p r e s e n t in this b r a i n M B P [25].

Methyl acceptability of synthetic peptides N G - M e t h y l a r g i n i n e in b o v i n e M B P occurs n a t u r a l l y o n l y at the residue 107 a r g i n i n e a m o n g the 18 o t h e r arginines in the m o l e c u l e [3,4]. Thus, it is of i n t e r e s t to investigate the m i n i m u m c h a i n length o f p e p t i d e containing the specific a r g i n i n e which f u n c t i o n s as the methyl acceptor. Thus, several p e p t i d e s ( d o d e c a - , octa-, hexa- a n d tetra-) whose a m i n o a c i d sequences o v e r l a p with the residue 107 M B P region (see T a b l e I I I ) were synthesized a n d s t u d i e d for their s u b s t r a t e activity tow a r d MBP-specific P M I . D u r i n g the study, however, we o b s e r v e d that unlike a large p r o t e i n s u b s t r a t e these short length methyl-14C-peptides are n o t well s e p a r a t e d from the u n r e a c t e d Ado[methyl-14C]Met b y gel filtration. Therefore, p r i o r to the s e p a r a t i o n of the m i x t u r e b y S e p h a d e x G-10, the r e a c t i o n m i x t u r e was s u b j e c t e d to the p H 4.0 t r e a t m e n t [21], the c o n d i t i o n k n o w n to h y d r o l y z e A d o M e t into 5 ' - m e t h y l t h i o a d e n o s i n e .

TABLE III

Methylarginine containingpeptides a Myelin basic protein (human brain) [3] 107

98

109

---Thr-Pro-Pro-Pro-Ser-Gln-Gly-Lys-Gty- Arg -Gly-Leu-PNP protein A-1 ''b [13,25] 194

---Ser-Ser-Ser-Gln- Arg-Gly-Arg-Ser-Gly-Phe--206

---Gly-Gly-Gly- arg -Gly-Gly-Gly-Phe-Phe-Gly--218

315

--- Arg -Gly-Gly-Asn-Phe-Ser-Gly- Arg -Gly .... Nucleolar scleroderma antigen (34 kOa protein) [12] 5

8

---Phe-Ser-Pro- Arg -G ly-G ly-G ly-Phe-G ly-G ly15

21

Arg -Gly-Gly-Phe-Gly-Asp- A r g 28

24

-Gly-Gly-

Arg

-

31

Gly-Gly-Gly- Arg -Gly-Gly- Arg --Nucleolar protein C23 [11] ---ghe-G ly-G ly- A rg -G ly-G ly- A rg -G ly-G ly-PheGly-Gly- Arg -Gly-Gly-Gly- Arg -Gly-Gly- Arg Gly-Gly-Phe-Gly-Gly- Arg -Gly- Arg -Gly---

a Underlined Arg is the potential sites for methylation. b Numbering of~mino acids is according to Cobianchi et al. [35]. Residue Arg-194 is shown as residue 193 in Ref. 13.

146 Clearly, the enzymaticaUy methylated hexapeptide separated from the unreacted radioactivity (Fig. 2A). As shown in Fig. 2B, the peak radioactivity of the unhydrolyzed AdoMet eluted at fraction number 57, while that of cleaved (5'-methylthioadenosine) eluted at around number 86. As shown in Table II, the hexapeptide (Glys-LysGly-Arg-Gly-Leu) whose sequence corresponds to residues 104-109 of bovine MBP was found to be the shortest methyl acceptor among the peptides tested, and the tetrapeptide of Gly-Arg-Gly-Leu (residues 106-109) exhibited only a negligible activity. The result is consistent with the earlier report that the pentapeptide (residues 105-109) was inactive as a substrate for PMI [31]. The fact that the hexapeptide (residues 104-109 of MBP) serves better than the dodecapeptide (residues 98-109) as a methyl acceptor indicates the non-essentiality of the tri-Pro sequence (residues 99-101) in MBP for the methylation of residue Arg-107. Although the

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Fig. 3. Amino acid analysis of [methyl-]4C]-labeled octapeptide by MBP-specific protein methylaseI. The [methyl-14C]octapeptide prepared by the purifiedMBP-speeificPMI was hydrolyzedin 6 M HC! at ll0°C for 24 h, and the hydrolysatewas analyzedby HPLCusing Method 2 of WatersAssociates[24]. Fractions(0.5 ml) werecollected and counted for radioactivity. The authentic arginine deriVatives eluted; MMArgat 16 rain and both NG, N °- and NG,N'G-DiMArg at 16.5rain.

total number of methyl groups incorporated into the hexapeptide is much higher than that into the intact MBP molecule, mol% incorporation was calculated to be the highest in MBP (0.5 mol%), indicating that the total MBP molecule is needed for the high affinity reaction. Having found the hexapeptide as the minimum length methyl acceptor, several analogs of the hexapeptide (residues 104-109 of MBP) were synthesized to study amino acid sequence specificity. Substitution of the residue Gly-106 with Asp in the peptide retained only 15% of methyl acceptability of the MBP hexapeptide, whereas the substitution with Phe, His and Leu almost completely abolished the methyl acceptability. The presence of Gly next to the methylatable Arg is consistent with the general feature of the amino acid sequence of several naturally occurring methylated nuclear proteins (Table III). Although not shown, none of these synthetic peptides served as the methyl acceptor for histone-specific PMI. In order to confirm the nature of amino acid methylated by MBP-specific PMI, the enzymatically methylated octapeptide was hydrolyzed in 6 M HC1 and the radiomethylated amino acid was analyzed by HPLC. As can be seen in Fig. 3, a single radioactive peak at 16-16.5 min, corresponding to the retention time of standard methylated arglnines was observed, indicating the enzymatic product to be a mixture of NG-mono methylarginine (MMeAxg') and NG-dimethylarginine (DiMeArg). As previously shown [24], this analytical method can not separgte'the two isomers, NG, NG-Di MeArg and NG, N'G-DiMeArg. However, it is known that MBP contains only N,,G, N 'G(symmetric)-DiMeArg, but not NG,NG(asymmetric)-DiMeArg [25]. Methyl acceptability of R N P complex A-1 protein The RNP complex A-1 protein is a single-stranded nucleic acid binding protein with molecular mass of 34 kDa [13], and is characterized by a high content of Gly (about 20%) and the presence of post-translationally modified methylarginine [16,32,331. However, an enzyme responsible for methylating this protein has not yet been reported. With the availability of purified PMI (both MBP-specific and histone-specific), we have systematically investigated the methyl acceptability of the A-1 protein, which has been overproduced in E. coli [16], thus, the methylatable arginine being fully available for in vitro modification. As shown in Fig. 4, however, the A-1 protein was completely inactive as a methyl acceptor for both MBP-specific and histonespecific PMIs (Fig. 4, panels B and D). These results strongly suggest that there should be a PMI in nature which is specific for the RNP A-1 protein. It is of interest to note that the occurrence of DiMeArg is isomer-specific, depending on the proteins. As mentioned earlier, only NG,N'G(symmetric)-DiMeArg

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Fig. 4. The methyl acceptability of A-1 protein for protein methylases I. Purified protein methylase I (5 /~g of MBP-specific or 8 ttg of histone-specific) was separately incubated with 0.5 mg of the methyl-acceptor substrate (A-1 protein, MBP or historic) in a standard reaction mixture. The reactions were carried out for 2 h at 45 °C for MBP-enzymeand at 35°C for histone-enzyme as described in the Experimental procedures. The reaction was stopped by the addition of 5 #1 of 1 M HC1 and the mixture was applied to a column of Sephadex G-25 (0.7 × 85 cm) which was preequilibrated with 0.01 M HCI. Fractions (1 ml) were collected and 0.1 ml aliquot from each fraction was counted for radioactivity, while the elution of protein (,, . . . . . . ,,) was estimated by absorbance at 276 nm for MBP, and by performing Coomassieblue protein assay [23] for A-1 protein and histone (absorbance read at 595 rim). Panel A indicates MBP-enzymeincubated with MBP as the substrate protein; panel B, MBP-enzyme with A-1 protein; panel C, histone-enzyme with histone; and panel D, histone-enzyrne with A-1 protein. Symbols indicate: O, radioactivity elution profile of the complete reaction mixture, and o, of the blank in which only the enzyme was omitted. Note that large radioactivity peaks emergingaround the fraction No. 30 are the unreacted Ado[methyl-14C]Met.

is present in MBP, while only NC,NC(asymmetric)-Di MeArg is present in the R N P A-1 protein and nucleolar proteins, such as the protein C-23 [11] and scleroderma antigen [12]. Furthermore, these mammalian nucleolar proteins contain clusters of Gly (two or more), and NG, N ° - D i M e A r g interspersed with Phe, whereas MBP contains only one Gly adjacent to the methylatable Arg (residue 107) in both sides and no Phe is present in close proximity to the residue Arg-107 (Table III) [3,4]. It is tempting to speculate that this structural feature might play a role in determining which isomer of N GDiMeArg (symmetric or asymmetric) is formed by the enzymes. In the present study, we have investigated the methyl-acceptor substrate specificity for PMI with synthetic peptides and found that hexapeptide of the proper amino acid sequence is the shortest methyl acceptor and that adjacent Gly to the methylatable Arg is a preferred sequence. Since, the reaction is basically macromolecular modification, it is obvious that in vivo there exists another level of structural requirement, namely the ter-

tiary structure of substrate which enables the accessibility of the methylation site to the enzyme. MBP is one of the major peripheral membrane proteins embedded in the phospholipid bilayer, but the conformation status of the methylation site in vivo is not known at present. It should be noted that the unique and unusual tri-Pro sequence (residues 99-101 in MBP) which has been implicated to form a hair-pin structure of the basic protein [4] precedes a few amino acid from the residue Arg-107. A possible effect of the side chain Arg methylation on the region of this double chain MBP structure in relation to myelin membrane conformation is an intriguing endeavor to merit for further study.

Acknowledgments This work was supported in part by Grants RG1765A-1 from the National Multiple Sclerosis Society, CA 12227 from the National Cancer Institute, and AM09602 from the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases.

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