Combined biochemical and immunochemical comparison of peptidylarginine deiminases present in various tissues

Biochimica et Biophysica Acta, 966 (1988) 375-383 Elsevier

375

BBA 22963

Combined biochemical and immunochemical comparison of peptidylarginine deiminases present in various tissues

Kazutada Watanabe, Kyoichi Akiyama, Kiyoko Hikichi, Rika Ohtsuka, Ayumi Okuyama and Tatsuo Senshu Tokyo Metropolitan Institute of Gerontology, Tokyo (Japan) (Received 14 October 1987) (Revised manuscript received 9 February 1988)

Key words: Peptidylarginine deiminase; Protein-arginine deiminase; Tissue distribution; (Rat)

We have performed a combined biochemical and immunochemical study on the identity of peptidylarginine deiminases (EC 3.5.3.15) present in various mammalian tissues. First, we purified peptidylarginine deiminase from rat skeletal muscle. It gave a single band of molecular weight 83000 in sodium dodecyl sulfate polyacrylamide gel electrophoresis. Next we immunized rabbits with the purified enzyme. The resulting antibodies reacted specifically with the antigen in Western blot assay. Most of the enzyme activities present in rat skeletal muscle, brain, spinal cord, submaxillary gland and spleen could be characterized as the same muscle-type enzyme by immunoprecipitation and Western blot assay. The antibodies did not react with enzyme samples obtained from rat hair follicles and bovine epidermis. The lack of immunoreactivity of the epidermal enzyme could not be accounted for by the species difference, since the antibodies reacted with a 83 kDa polypeptide of bovine brain, which was thought to represent a bovine counterpart of the muscle-type enzyme. The epidermal enzyme could be distinguished from the other enzyme samples by its high activity towards benzoylarginine. These data suggest the existence of at least three types of peptidylarginine deiminase in mammalian tissues, i.e., a muscle type, a hair follicle type, and an epidermal type.

Introduction Citrulline residues in proteins were first reported in inner root sheath cells of guinea-pig hair follicles [1]. Subsequently, it has been shown that these citrulline residues are in the normal peptide bonds [2], despite their inability to be incorpo-

Abbreviations: SDS, sodium dodecyl sulfate; PMSF, phenylmethanesulfonyl fluoride; DTT, dithiotbreitol; BAEE, benzoyl-L-arginineethyl ester. Correspondence: K. Watanabe, Tokyo Metropolitan Institute of Gerontology, 35-2, Sakae-cho, Itabashi-ku, Tokyo 173, Japan.

rated into proteins through the ordinary translational pathway. In fact, the citrulline residues in the peptide bonds are formed by posttranslational deimination of arginine residues by enzymatic activities called peptidylarginine deiminase (EC 3.5.3.15) [3]. The enzyme activity was first detected in guinea-pig hair follicles [4]. Such activities were then extracted from bovine epidermis [5], newborn rat epidermis [3], rabbit skeletal muscle [6], and bovine brain [7]. The enzymes in the latter two tissues have been purified to apparent homogeneity. Although all of these enzymes were shown to convert arginine residues to citrulline residues in proteins, earlier reports from other laboratories presented fragmentary evidence suggesting dissimilarity of enzyme samples derived from different

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376 sources in terms of their molecular weights, activities towards various substrates, and antigenic properties [3,5,7,9,10]. Consequently we are now facing a somewhat confused idea about the identity of peptidylarginine deiminases present in different tissues. In order to resolve the confusion, we have taken combined biochemical and immunochemical approaches using rabbit antibodies elicited with an enzyme sample purified from rat skeletal muscle. Here we present evidence suggesting that there are at least three types of peptidylarginine deiminase in mammalian tissues, i.e., a muscle type, a hair follicle type, and an epidermal type. The muscle-type enzyme appeared to represent most of the enzyme activities found in the brain, spinal cord, submaxillary gland and spleen. Materials and Methods

Materials BAEE was purchased from Nakarai Chemicals, DEAE-Sephacel and AH-Sepharose 4B were obtained from Pharmacia Fine Chemicals, and BioGel A-0.5m and Bio-Gel HTP, DEAE Affi-Gel Blue and horseradish peroxidase-conjugated goat anti-rabbit IgG from Bio-Rad Laboratories. Pansorbin was supplied by Calbiochem, and RIBI adjuvant system by Immunochem Research. All the rat tissues used were collected from 5-weeksold male Wistar rats unless otherwise stated. Fresh bovine tissues were obtained from a local slaughterhouse. Purification of peptidylarginine deiminase Rat skeletal muscle (600 g) was minced by repeated passages through an electric meat grinder, and then homogenized with 3 vol. of buffer A (0.1 M Tris-HC1 (pH 7.6)/10 mM 2-mercaptoethanol/1 mM E D T A / 1 mM PMSF), using a Polytron homogenizer. The homogenate was centrifuged at 7000 × g for 20 min, and filtered through two layers of cheesecloth. The filtrate was fractionated by ammonium sulfate precipitation and chromatographed on a DEAE-Sephacel column (2.5 X 31 cm) as described by Takahara et al. [6]. The fractions containing peptidylarginine deiminase activities were pooled and concentrated by a m m o n i u m sulfate precipitation. The con-

centrated solution was subjected to gel filtration on a Bio-Gel A-0.5m column (2.5 x 99 cm) equilibrated with buffer B (10 mM Tris-HC1 (pH 7.6)/10 mM 2-mercaptoethanol/1 mM E D T A / 10% ( w / v ) glycerol/0.2 mM PMSF) containing 0.3 M NaC1. The enzyme fractions were directly applied to an AH-Sepharose 4B column (1 × 19 cm) and eluted with a linear gradient of 0.3-1.0 M NaC1 in 150 ml of buffer B. The enzyme fractions were pooled and dialyzed against buffer C (50 mM Tris-HC1 (pH 6.9), 3 mM MgCI 2, 10 mM 2-mercaptoethanol, 10% ( w / v ) glycerol, 0.2 mM PMSF) containing 100 mM KC1 and applied to a Bio-Gel H T P column (1 × 7.5 cm) equilibrated with the same buffer. The column was first washed with buffer C containing 100 mM KH2PO 4 (adjusted to pH 6.9 with NaOH). Peptidylarginine deiminase was then eluted with buffer C containing 200 mM K H z P O 4 (adjusted to pH 6.9 with NaOH). The enzyme fractions were dialyzed against buffer B containing 0.1 M NaC1, and stored at - 7 0 ° C as purified peptidylarginine deiminase.

Preparation of extracts from rat tissues for enzyme assay and Western blot assay Tissues were homogenized with 9 vol. of buffer A. Homogenizations of cerebrum, cerebellum, submaxillary gland, thymus, lung, heart, liver, pancreas, spleen, kidney, and testis were performed in a Potter-Elvejem homogenizer with an electrically rotated Teflon pestle. For homogenization of skeletal muscle, stomach and intestine, a Polytron homogenizer was used. Pituitary and adrenal were homogenized in a microcentrifuge tube with a pellet pestle (Kontes Scientific). The homogenates were centrifuged at 10000 × g for 10 min. The supernatants were subjected to the enzyme assay, and Western blot assay. Preparation of crude enzyme fraction from hair follicles Root regions of vibrissae plucked from 20 adult (3- to 10-months-old) Wistar rats of both sexes were extracted with 4 ml of buffer A by repeated grinding in a Dounce homogenizer fitted with a loose pestle (Kontes Scientific). The mixture was centrifuged at 10000 x g for 10 min, and the supernatant was brought to 50% saturation in

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ammonium sulfate. The precipitate collected by centrifugation was suspended in 300/~1 of buffer B containing 0.1 M NaC1, and dialyzed against the same buffer. Insoluble materials were removed by centrifugation, and the clear supernatant was used as a crude enzyme fraction of hair follicles.

Preparation of crude enzyme fraction from bovine epidermis Partial purification of bovine epidermal peptidylarginine deiminase was performed by a modification of the method of Kubilus et al. [5]. Minced bovine snout epidermis was homogenized with 4 vol. of buffer A using a Polytron homogenizer. The homogenate was centrifuged at 10 000 x g for 15 min. The supernatant was fractionated by ammonium sulfate precipitation, and the fraction precipitated from 30 to 60% saturation was suspended in buffer D (10 mM Tris-HC1 (pH 7.4)/1 mM E D T A / 1 mM 2-mercaptoethanol, 1 / 4 0 volume of the original homogenate volume), and thoroughly dialyzed against the same buffer. Insoluble materials were removed by centrifugation at 100000 x g for 30 min using a Beckman 50Ti rotor, and the clear supernatant was applied to a DEAE-Sephacel column pre-equilibrated with buffer D. Elution was performed by stepwise increases of NaC1 concentrations. Fractions eluted with 50 mM NaC1 were pooled, concentrated by ammonium sulfate precipitation, thoroughly dialyzed against buffer D, and used as a crude enzyme fraction of bovine epidermis.

Assay of enzyme activity The enzyme activity was estimated by measuring the conversion of BAEE to the corresponding derivative of citrulline [7,9]. The reaction was carried out as described by Sugawara et al. [9], except that the incubation temperature was 5 0 ° C and the concentration of D T T was 5 mM. Briefly, sample extracts (100 ~1) were incubated with equal volumes of 200 mM Tris-HC1, 20 mM CaC12 and 10 mM D T T (pH 7.5) with or without 20 mM BAEE, for 1 h at 50 ° C. The reaction was stopped by the addition of 50 /~1 of 5 M perchloric acid, and the mixture was left for 20 min on ice. After centrifugation at 1000 X g for 10 min at 4 ° C, the supernatants were subjected to colorimetric determination of citrulline by the method of Boyde

and Rahmatullah [11]. The samples incubated without BAEE were used to measure color formation caused by free citrulline in the extracts. Since 2-mercaptoethanol and D T T interfered with the color formation, suitable concentrations of these reagents were included in the standard citrulline solutions for the colorimetry. The unit of enzyme activity was defined according to Kubilus and Baden [7].

Determination of proteins Protein concentrations of samples at various stages of purification were measured by the method of Bradford [12], while those of tissue extracts were determined by the method of Lowry et al. [13]. Bovine serum albumin was used as a protein standard in each case.

Preparation of antibodies Rabbits were immunized by injecting the purified enzyme (250 t~g) intradermally every 2 weeks with RIBI adjuvant system for 3 months. The antibodies were detected by enzyme-linked immunosorbent assay [14]. An IgG fraction was obtained by precipitation with 40% saturated ammonium sulfate, followed by DEAE Affi-Gel blue chromatography, according to the manufacturer's protocol. Normal IgG was obtained from pre-immune rabbit serum in a similar manner.

Immunoprecipitation of enzyme activity Anti-peptidylarginine deiminase IgG or pre-immune IgG (100/~g in 50 ffl) was added to 400/~1 of a tissue extract and incubated at 4 ° C overnight. The mixture was combined with pansorbin (50 fib, suspended in buffer B containing 0.1 M NaC1 and 0.5% ovalbumin (a half the original volume) and left on ice for 1 h with occasional mixing. The mixture was centrifuged at 10 000 x g for 5 min, and the supernatant was used for the measurement of the enzyme activity.

Gel electrophoresis, Western blotting and immunochemical detection SDS-polyacrylamide gel electrophoresis was performed on vertical slab gels (1 mm x 9 cm) containing 10% acrylamide and 0.25% N,N'methylenebisacrylamide, by the method of

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Laemmli [15]. Proteins were stained with Coomassic brilliant blue. For the Western blot assay, proteins in the gel were electrophoretically transferred to nitrocellulose membrane at 10 V / c m for 3 h in 25 mM Tris-HC1 (pH 7.5)/192 mM glycine/20% ( v / v ) methanol/0.025% SDS [16]. The nitrocellulose membrane was incubated successively with 2000fold-diluted antiserum, and then with the peroxidase-conjugated second antibody as described by Senshu et al. [17]. The protein bands, which reacted with antiserum, were visualized by the incubation with 4-chloro-l-naphthol and hydrogen peroxide [18]. In some cases, total proteins on the blotted membrane were stained with Amido black 10B. Results

Purification of peptidylarginine deiminase Table I shows the summary of purification of the enzyme from rat skeletal muscle. Electrophoretic profiles of these fractions are shown in Fig. 1. The peptidylarginine deiminase fraction obtained by Bio-Gel H T P column chromatography gave a single band, and its molecular weight was estimated to be 83 000 from SDS-gel electrophoresis. It was purified about 1900-fold over the crude extract with about 13% recovery. The amino-acid composition of the purified enzyme was shown in Table II, in comparison with the enzyme isolated from rabbit skeletal muscle [6]. The enzyme exhibited about 20% of the maximum activity when D T T was omitted from the incubation mixture. It was stimulated by increasing concentrations of D T T and reached a plateau at

about 2 mM DT-I'. The enzyme activity was not detectable when the concentration of calcium ion was below 3 . 1 0 -5 M, but it increased sharply above 1 0 - 4 M and reached a plateau at around 10 -2 M. The concentration for half-maximum activity was about 3 • 1 0 - 4 M.

Properties of antibodies The antiserum used here showed a highly positive reaction in ELISA at 2000-fold-dilution. At the same dilution of the antiserum, as little as 300 pg of the purified enzyme could be detected by the Western blot assay. In order to examine specificity of the antiserum, equivalent Western blots of total proteins in the crude muscle extract were subjected to different detection procedures. Staining with Amido black 10B visualized a highly heterogeneous pattern of resolved proteins (Fig. 2a). In contrast, immunochemical detection using the antiserum yielded a single band, whose mobility was identical with that of the purified peptidylarginine deiminase (Fig. 2b). When pre-immune serum was used instead of the antiserum, no band became visible, even after prolonged incubation (Fig. 2c). Moreover, the enzyme activity in the crude muscle extract could be quantitatively precipitated by the successive addition of anti-peptidylarginine deiminase IgG and pansorbin. This means that the antibodies reacted specifically with all the peptidylarginine deiminase activities present in the muscle extract.

Detection of muscle-type peptidylarginine deiminase activities in various rat tissues Table III shows the activities of peptidylarginine deiminase in the extracts of various tis-

TABLE I P U R I F I C A T I O N OF P E P T I D Y L A R G I N I N E D E I M I N A S E F R O M R A T S K E L E T A L M U S C L E Purification step

Protein (mg)

Activity (units)

Specific activity (units/mg)

Yield (%)

Relative purity

Crude extract (NH4)2SO 4 DEAE-Sephacel Bio-Gel A-0.5m AH-Sepharose Bio-Gel H T P

33 800 13 800 361 35.4 4.7 2.4

8 760 9410 4 610 2 420 1420 1160

0.256 0.684 12.8 68.3 303 482

100 107 53 28 16 13

1 2.7 50.0 267 1 180 1880

379

931 68

43

30

18 !

2

3

4

5

Fig. 1. Electrophoretic profiles of peptidylarginine deiminase fractions at different steps of purification. Samples were analyzed on an SDS-polyacrylamide slab gel, as described in the text. Rabbit muscle phosphorylase a, bovine serum albumin, ovalbumin, bovine erythrocyte carbonic anhydrase and fl-lactoglobulin, in the order of decreasing molecular weights, were used as markers. The gel was stained with Coomassie brilliant blue. The bands portrayed are: (1) molecular weight markers; (2) DEAE-Sephacel; (3) Bio-Gel A-0.5m; (4) A H Sepharose 4B; (5) Bio-Gel HTP.

sues. Significant activities were easily detected in the extracts of skeletal muscle, spinal cord, submaxillary gland, cerebellum, cerebrum and spleen. We confirmed that the activities in these tissue extracts were quantitatively precipitated by the combined treatments with anti-peptidylarginine deiminase IgG and pansorbin. This suggests that the muscle-type enzyme accounts for most, if not all, of the peptidylarginine deiminase activities observed in these tissues. The other tissue extracts showed barely detectable activities. Fig. 3 shows the results of Western blot assay of various tissue extracts. The extracts of skeletal muscle, spinal cord, submaxillary gland, cerebellum, cerebrum and spleen gave single bands, which showed the same mobilities as that of the purified muscle enzyme. This agrees well with the results obtained by immunoprecipitation described above. When the assay was performed with lower dilution

of the antiserum, faint bands of the same mobilities became visible in the extracts of stomach, thymus, pituitary, and adrenal after prolonged incubation with the peroxidase substrate. Such bands were barely detectable in the extracts of the other tissues examined. Relative intensities of the immunoreactive bands found in the extracts, with the exception of that of submaxillary gland in the Western blot assay, appeared to be consistent with their relative enzyme activities (Table III). The submaxillary extract exhibited higher band intensity than was expected from its peptidylarginine deiminase activity. It was found that the amount of antibodies required to precipitate the enzyme activity in the submaxillary extracts exceeded the amount needed to precipitate comparable activities in other tissues. This means that there were more immunoreactive enzymes in the submaxillary extract than could be estimated from simple enzyme assay. This could be due to inactivation of

T A B L E II C O M P A R I S O N OF A M I N O - A C I D C O M P O S I T I O N S O F PEPTIDYLARGININE DEIMINASES FROM RAT AND RABBIT S K E L E T A L M U S C L E A m i n o acid

Mol% rat

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine

a

6.47 1.82 5.62 9.82 5.55 6.24 12.33 5.93 7.33 5.30 0.80 8.52 2.78 4.93 10.57 3.59 5.42

rabbit b 6.42 1.93 5.34 9.84 5.72 5.89 11.85 4.82 7.74 5.34 1.28 8.81 2.26 4.66 9.77 3.13 5.21

The data in the table are the average values obtained after hydrolysis for 24, 48 and 72 h, except for those of threonine and serine, which were extrapolated to zero time. b The values for the enzyme of the rabbit skeletal muscle were recalculated from the data of Takahara et al. [6], given in mol amino acids per 83 000 g protein.

a

380

a

b

T A B L E III

c

D I S T R I B U T I O N OF P E P T I D Y L A R G I N I N E D E I M I N A S E ACTIVITIES IN V A R I O U S R A T TISSUES. The enzyme activities were measured using BAEE as a substrate by the method described in the text. The data were given as the averaged values of five animals -+ S.D.

1

2

3

I

2

1

2

Fig. 2. Immunochemical detection of muscle peptidylarginine deiminase. The purified enzyme and a crude extract of skeletal muscle were applied to adjacent lanes of SDS-polyacrylamide gels. The resolved protein bands were transferred to nitrocellulose sheets and subjected to three different detection procedures. (a) The bands were visualized by staining with A m i d o black 10B. Lane l and 2 represent purified enzyme 1 ~g and 10 ng, respectively; lane 3, crude extract derived from 500 # g of skeletal muscle; (b) The sheet was subjected to immunochemical detection using 2000-fold-diluted antiserum, as described in the text. lane 1, purified enzyme 10 ng; lane 2, crude extract derived from 500 p,g of skeletal muscle; (c) Another sheet equivalent to (b) was subjected to a similar detection procedure using 2000-fold-diluted pre-immune serum.

Tissue type

Activity ( U n i t s / g of tissue)

Skeletal muscle Spinal cord Submaxillary gland Cerebellum Cerebrum Spleen Liver Testis Lung Pituitary Stomach Thymus Adrenal Small intestine Heart Pancreas Kidney

22.2 _+2.1 10.7 +0.9 6.42 _+0.83 4.45 _+0.35 2.77 + 0.14 1.68 _+0.20 0.83 ± 0.18 0.79-+0.11 0.64 -+ 0.08 0.61 _+0.13 0.61 _+0.11 0.57 -+ 0.14 0.55 + 0.51 0.33 + 0.12 0.19 + 0.22 0.17 _+0.09 0.10 + 0.09

was the best substrate among those tested for muscle and hair follicle enzymes. They showed markedly lower activities towards benzoylarginine,

the enzyme during the incubation period, since high levels of proteinase activities have been identified in rat submaxillary gland [19,20]. We observed that incubation of the purified enzyme with the submaxillary extracts led to reduced intensity of the immunoreactive band. 1 2 3 4 5 6 7 8 9 10111213 14 1516 171819

Comparison of peptidylarginine deiminases from muscle, hair follicle and epidermis In order to check the identity of the purified muscle enzyme with hair follicle and epidermal peptidylarginine deiminases, we prepared crude enzyme samples from roots of rat vibrissae, and bovine snout epidermis. Table IV shows a comparison of the activities of the three enzyme samples towards various synthetic substrates. BAEE

Fig. 3. Western blot assay of extracts obtained from various rat tissues. Extracts derived from 1 mg of tissues were subjected to an SDS-polyacrylamide slab gel for the immunochemical detection, as described in the text. Samples analyzed were; (1) 10 ng of purified peptidylarginine deiminase; (2) skeletal muscle; (3) spinal cord; (4) submaxillary gland; (5) cerebellum; (5) cerebrum; (7) spleen; (8) liver; (9) testis; (10) lung; (11) stomach; (12) thymus; (13) pituitary; (14) adrenal; (15) small intestine; (16) heart; (17) pancreas; (18) kidney; (19) 10 ng of purified peptidylarginine deiminase.

381 a n d further d i m i n i s h e d activities t o w a r d s a r g i n i n e ethyl ester a n d arginine. These i n d i c a t e that b o t h a m i n o - a n d c a r b o x y - g r o u p s of a r g i n i n e need to be b l o c k e d for m a x i m a l activities of the two e n z y m e samples. By contrast, the e p i d e r m a l e n z y m e s h o w e d n e a r l y equal activities t o w a r d s B A E E a n d b e n z o y l a r g i n i n e . W e also f o u n d that gel-filtration profiles of these e n z y m e s differed significantly. T h e e n z y m e s a m p l e f r o m muscle was eluted first, a n d that f r o m e p i d e r m i s was eluted last f r o m a S e p h a d e x G-200 superfine c o l u m n ( d a t a n o t shown). A l l the d a t a d e s c r i b e d a b o v e suggested that the e n z y m e s a m p l e s from muscle, hair follicle a n d e p i d e r m i s were n o t identical to one another. Therefore, we c o m p a r e d i m m u n o c h e m i c a l p r o p e r ties of the three e n z y m e s a m p l e s b y the W e s t e r n b l o t analysis. W e also e x a m i n e d a b o v i n e b r a i n extract as a n o t h e r e n z y m e source. T h e m u s c l e extract y i e l d e d a clear positive b a n d as e x p e c t e d (Fig. 4, l a n e 1). N e i t h e r the hair follicle enzyme, n o r the b o v i n e e p i d e r m a l e n z y m e gave positive b a n d s , a l t h o u g h e n z y m e activities c o m p a r a b l e to that of the m u s c l e extract were i n c l u d e d in the s a m p l e s (Fig. 4, lanes 2 a n d 3). This shows that the m u s c l e - t y p e e n z y m e is a n t i g e n i c a l l y dist i n g u i s h a b l e f r o m b o t h the hair follicle a n d epiderm a l enzymes. It should b e n o t e d that the b o v i n e b r a i n extract gave a b a n d which m i g r a t e d at a rate i n d i s t i n g u i s h a b l e f r o m that of the rat muscle en-

1

2

3

4

5

Fig. 4. Immunochemical differentiation of different types of peptidylarginine deiminase. Enzyme samples from rat skeletal muscle, rat hair follicles, bovine epidermis and bovine brain were compared by the Western blot assay. Bands were visualized by the immunochemical detection, as described in the text. Lane 1 represents rat skeletal muscle extract containing 7.5 mU of peptidylarginine deiminase activity; lane 2, rat hair follicle extract (7.5 mU); lane 3, bovine epidermal enzyme sample (7.5 mU); lane 4, purified muscle peptidylarginine deiminase (1.4 ng) lane 5, bovine brain extract (1.5 mU).

TABLE IV COMPARISON OF SUBSTRATE SPECIFICITY OF PEPTIDYLARGININE DEIMINASES FROM DIFFERENT TISSUES

z y m e (Fig. 4, lanes 4 a n d 5). This d e m o n s t r a t e s a n t i g e n i c s i m i l a r i t y b e t w e e n the b o v i n e b r a i n enz y m e a n d the rat m u s c l e enzyme.

Enzyme samples were incubated with 10 mM of any one of the substrates given in the table. The amount formed of citrulline or its derivatives was determined colorimetrically, as described in the text.

Discussion

Substrate Bz-t-Arg-OEt Bz-L-Arg-OMe Bz-L-Arg-NH2 Ac-L-Arg-OMe Bz-e-Arg L-Arg-OEt L-Arg

Relative activity (%) muscle

hair follicle

epidermis

100.0 77.9 51.8 44.8 18.5 2.8 0.84

100.0 18.0 4.1 2.4

100.0 106.0 6.0 7.0

I n the p r e s e n t p a p e r , we have t a k e n c o m b i n e d b i o c h e m i c a l a n d i m m u n o c h e m i c a l a p p r o a c h e s to s t u d y the i d e n t i t y of p e p t i d y l a r g i n i n e d e i m i n a s e s in various m a m m a l i a n tissues. T h e p u r i f i e d rat muscle e n z y m e d e s c r i b e d a b o v e closely r e s e m b l e s the r a b b i t m u s c l e e n z y m e [8] with respect to m o l e c u l a r weight, a m i n o - a c i d c o m p o s i t i o n , relative activity t o w a r d s v a r i o u s s y n t h e t i c substrates, a b s o l u t e r e q u i r e m e n t for C a 2÷, a n d s t i m u l a t i o n b y r e d u c i n g agents such as D T T . T h e p r o p e r t i e s of

382

these muscle enzymes were similar to those of the enzymes detected in brain tissues of various vertebrates [7,8]. The mode of distribution of the enzyme activities observed here was mostly consistent with the observation reported from other laboratories [7,21,22]. All the tissue extracts exhibiting the enzyme activity, except that derived from hair follicles, gave single bands, which showed mobilities indistinguishable from that of the purified muscle enzyme in the Western blot assay. This, and the quantitative immunoprecipitation experiments, suggest that the same type of peptidylarginine deiminase accounts for most of the activities observed in skeletal muscle, spinal cord, submaxillary gland, cerebellum, cerebrum and spleen. The peptidylarginine deiminase activity was first demonstrated in guinea-pig hair follicles [4]. Some preliminary characterization of an enzyme fraction, obtained from guinea-pig hair follicles, was reported using radiolabeled benzoylarginine as a substrate [23]. Peptidylarginine deiminases have also been demonstrated in epidermis. Partial purification of the epidermal enzymes from bovine and rat tissues have been reported [3,5]. Both the epidermal enzyme preparations were characterized by their relatively low molecular weights (69000 and 48 000, respectively) and their high activities towards benzoylarginine, which is a poor substrate for the muscle enzyme. Moreover, the bovine epidermal enzyme was shown to be antigenically different from the enzyme purified from bovine brain [7,10]. However, it is not clear whether or not the hair follicle enzyme differs from peptidylarginine deiminases found in other tissues. The comparison of the purified rat muscle enzyme with the rat hair follicle enzyme described here revealed marked similarity in their relative activities towards various synthetic substrates. However, a clearcut difference has been noted in their antigenic properties. Moreover, their gel-filtration profiles differed significantly. The present study also showed an antigenic difference between the rat muscle enzyme and the crude enzyme sample obtained from bovine epidermis. This is not due to species difference, because the antibodies to the muscle enzyme obviously reacted with the bovine brain enzyme. Therefore, it appears that there are at least three types of peptidylarginine deiminase

in mammalian tissues, i.e., a muscle type, a hair follicle type and an epidermal type. It will be interesting to see what kinds of biological role they play in a wide variety of tissues. Purification and immunochemical characterization of the hair follicle-type and epidermal-type enzymes remain to be accomplished. Further investigation of the problems at gene levels should yield useful information.

Acknowledgments We would like to thank Dr. K. Nomura in our Institute for the analysis of the amino-acid composition of the enzyme. This work was partly supported by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan.

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