Biochimwa et Bioph)'sica Acta, 11181) ( 1991 ) 103 - 1I)t) (t. ltP.)l Elsevier Science Publishers B.V. All rights resep, cd (11~17-483~/91/$1~3.511 ADONIS 016748389100309Y

1I13

B B A P R O 341117

Immunoaffinity purification and partial amino acid sequence analysis of catechol-O-methyltransferase from pig liver B a r b a r a B e r t o c c i i, G i a n n i G a r o t t a 2, M o s 6 D a P r a d a i, H a n s - W e r n e r L a h m -~, G e r h a r d Z i i r c h e r ~, G i u s e p p e V i r g a l l i t a 2 a n d V i n c e n z o M i g g i a n o 2 I Pharma Research, F. ttoffm~mn-La Roche Ltd. Basel (SwitzerlandJ and e Central Research Units, F. Hoffmann-La R~'he Ltd.. Basel (Switz,'rland)

(Received I February 19'41)

Key words: Immunoaffinity chromatography; Amino acid sequencc parlial; CatechoI-O-melhyltranslerase: (Pig liver)

Monoclonal zntibodies (mAbs) against the soluble form (S-COMT) of catechoI-O-methyltJansferasc (COMT, EC 2.1.1.6) were produced using a purified preparation of the enzyme from pig liver as antigen. The selected monoclonai antibodies recognized the enzyme with different capacities. One of them (Co60-1B/7) showed a significant cross reaction with S-COMT from rat and human liver. A proteil~ band of 23 kDa was recognized by the mAbs on Western blots of the soluble fraction of pig liver. The mAbs ~vere also a~qe to recognize the membrane-bound form of the enzyme, which was found to be mainly localized in the microsomal fraction o." pig and rat liver as well as of the human hepatoma cell line Hep G2. The protein bands detected in microsomes had a molecular mass of 26 kDa in pig and rat liver and displayed a slightly higher molecular mass (29 kDa) in the Hep G2 cell line. A single step method for the immunoafl~nity purification of pig liver S-COMT was developed by using a Sepharose 4B column to which the mAb Co54-5F/8 was covalently coupled. Acid elution conditions were optimized to obtain the enzyme in active form with a good yield. SDS-PAGE analysis of the purified preparation revealed a single protein band with a molecular mass of 23 kDa with 154-fold enrichment in enzyme activity over the starting material. Since the N-terminus was blocked, purified enzyme preparations were cleaved with trypsin. Two fragments of 22 and 33 amino acids in length could be sequenced by Edman degradation.

Introduction

CatechoI-O-methyltransferase (COMT, EC. 2.1.1.6) is an Mg2+-dependent enzyme, which catalyzes the transfer of a methyl group from S-adenosyI-Lmethionine to a hydroxy group on a catechol ring. This enzyme, widely distributed in several tissues of all mammalian species [1], has a broad substrate specificity, O-methylating not only catecholamine neurotransmitters (dopamine, noradrenaline, adrenaline), their catecholic metabolites and L-DOPA but also some steroids, alpha-methyl DOPA, isoproterenol and apomorphine [2-4]. COMT plays a major role in the inactivation of catecholamines both in the CNS and peripheral tissues [5,6], its inhibition inducing a potentiation of the biological effects of catecholamine neurotransmitters [7,8]. COMT exists in a soluble cytosolic

Correspondence: M. Da Prada, Pharma Research CNS, F. Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland.

(S-COMT) and in a particulate membrane-bound form (MB-COMT) [9,10]. S-COMT has been isolated from different tissues and studied more extensively than MB-COMT [11-16]. In rat and pig, S-COMT has a molecular mass of about 23-24 kDa but it is not clear whether it exists in multiple isoforms. In the soluble fraction of rat liver, rabbit anti-COMT sera detect three polypeptides that differ by their relative molecular mass and charge [17]. According to Grossman et ai. [17] rat liver MBCOMT exists as a single form of molecular mass 26 kDa. This form of the enzyme shows a higher affinity for substrates [18,191 than S-COMT, is mainly localized in the microsomal fraction of different tissues and appears to be an intrinsic membrane protein [20]. In addition, MB-COMT seems to represent the predominant form in neurons, whereas S-COMT is the major form of the enzyme in non neuronal tissues [21]. Recently the COMT cDNA from rat liver was isolated and sequenced [22]. The open reading frame consisted of 663 nucleotides coding for a 221-amino

104

acid polypcptidc with a predicted molecular mass of 24.7 kDa. To further elucidate the biochemistry of COMT, wc have raised monoclonal antibodies (mAbs) against the soluble form of porcine enzyme and developed a straightforward immunoaffinity purification procedure. The amino acid sequence was determined from two tryptic fragments of the enzyme. Materials and Methods

Materials. S-adenosyI-L-[methyl-3H] methionine (15 Ci/mmol), IZSl sheep anti-mouse Ig (750 Ci/mmol), Na~251 (9 m C i / # g of iodine) and radiolabeled markers for protein size were purchased from Amersham (Little Chalfont, U.K.). Peroxidase-conjugated rabbit lg anti.mouse lgG, hypoxanthine, aminopterine, thvmidine, bovine serum albumin (BSA), benzamidine and NP-40 were from Sigma (St Louis, MO, U.S.A.); Pansorbin from Calbiochem Corp. (La Julia, CA. U.S.A.). Sephadex G75 and CNBr-activated Sepharose 4B from Pharmacia Fine Chemicals (Uppsala. Sweden); DEAE Bio-gel agarose and reagents for electrophoresis flora Bio-Rad (Richmond, CA, U.S.A.); RPMI 1640 medium. HAT, L-glutamine, sodium pyruvate, penicillin, streptomycin from Gibco Laboratories (Paisley, U.K.); fetal bovine serum (FBS) from Amined AG (Muttenz, CH); complete and incomplete Freund's adjuvants (CFA, IFA) from DIFCO (Detroit, MI, U,SA.); Trypsin-TPCK from Worthington (Freehold, NJ, U.S.A.), Diaflo UM 10 membrane from Amicon (Denvers, MA, U.S.A.); aprotinin (Trasylol R) from Bayer (Leverkusen, F.R.G.). S-COMT preparation. Pig liver S-CGMT was partially purified by ammonium sulfate fractionation and size exclusion chromatography according to Ziircher and Da Prada [23] and further purified by ion-exchange chromatography on a DEAE Bio-gel Agarose column ~7 cm × 1.8 cm). After extensive washing with 0.01 M sodium phosphate buffer (pH 7.0) containing 0.1 mM EDTA, 0.1 mM MgCI 2 and 0.02 mM dithiothreitol (Dq~F), the column was eluted stepwise with increasing concentrations of KCI in 0.01 M phosphate buffer (pH 7.0). The active S-COMT fractions were eluted at 50 mM KCI, pooled, concentrated on a Diaflo UM-10 membrane and finally stored at -80°C. This enzyme preparation was used for immunize mice. The COMT activity was measured radioenzymatically using pyrocatechol (2.5 mM) as substrate and S-adenosyI-L-(3H-methyl) as methyl donor [24]. Preparation of monoclonal antibodies (rrL4bs). Female Balb/cJ mice were immunized with S-COMT preparation, emulsified in CFA and boosted three times, at 3-week intervals, with the same preparation in IFA. After the final boost, the splenocytes were fused with azaguanin-resistant PAI-O myeloma cells [25],

re-suspended in HAT-medium and distributed in microtiter plates [26]. Hybridoraa supernatants were screened by radioimmune assay (RIA) [27]. Mierotiter plates were coated overnight at 4°C with 50 p.I of purified S-COMT (10 # g protein/ml). After saturation of the plastic binding sites with 3% BSA in 10 mM phosphate buffer (pH 7.2), 0.9% NaCI (PBS), 50 p.I of the hybridoma supernatants were distributed in each well and the plates were incubated for 2 h at room temperature. After extensive washings, the presence of antibody bcmnd to the antigen was detected by incubating with 50/xl of 1251-sheep anti-mouse lgG (106 c p m / m l ) and counting the samples by a y-counter. The selected hybridomas were cloned twice by limiting dilution. The culture supernatants were collected for mAb purification on Scpharose 4B coupled with affinity purified rabbit anti-mouse lg [28]. The aJltibody isotype was determined by the immuno-diffusion technique using a mouse monoclonal typing kit (The Binding Site, Birmirlgham, U.K.). lmmunopreeipitation tests were performed according to Denney et al. [29] and the precipitated enzymatic a, fivity was measured as described.

Electrophoretic

and immunoblotting techniques.

SDS-PAGE was performed on 12.5% or 15% linear gradient gels [30]. Gel s!ahs were stained with silver or Coomassie blue. After SDS-PAGE, proteins were electroblotted to nitrocellulose paper according to Towbin et al. [3!]. The nitrocellulose membrane was incubated with anti-COMT mAbs and bound immunoglobulins were detected by incubation for 2 h wi:h 12Sl-sheep anti-mouse lg ( = 10 ~ cpm/mD. The radioactivity on nitrocellulose sheets was visualized by exposure to Kodak X-ray film.

Preparation of subcelhdar fractions from rat and pig liter and from Hep G2 cell line. Liver from pig and rat was homogenized in a glass-Teflon homogenizer in 5 vols. of 10 mM potassium-phosphate buffer (pH 7.5) containing 5 mM 2-mercaptoethanoi. The homogenate was centrifuged for 20 rain at 600 × g and the resulting supernatant was centrifuged at 11 000 × g for 20 min to obtain a mitochondrial pellet. The microsomal fraction was then prepared by ultracentrifugation (10O000 x g for 60 rain) of the 11 000 × g supernatant. The mitochondria and the microsomal pellets were washed twice and re-suspended in potassium-phosphate buffer. Soluble and microsomal fractions from Hep G2 human hepatoma epithelial-like cell line (ATCC HB 8065), we:e obtained essentially as above.

lmmunoaffinity chromatography of pig liver S-COMT. The purified mAb Co54 (24 rag) was coupled to 8 ml cyanogen bromide-activated Sepharose 4B (8 ml) according to Axen et al. [32]. Pig liver was homogenized in isotonic KCI containing 10 mM 2-mercaptoethanol and fractionated with ammonium sulfate (35-55% of

105 saturation). The preparation obtained was diluted in PBS containing 10 mM benzamidine, 5 t.tg/ml Trasy1olR and loaded on the mAb Sepharose column. After rinsing with 100 ml of buffer, the enzyme was eluted with 0.2 M glycine (pH 2.8). Fractions were immediately neutralized with 1 M Tris-base and the enzymatic activity determined. The active fractions were dialyzed against PBS and stored at - 8 0 ° C for several months with negligible loss of activity. Protein concentration. The concentrations of purified mAbs were estimated spectrophotometrically at 280 nm (1.4 A for 1 m g / m l standard IgG). During the enzyme purification steps, the protein concentration was measured according to Ix)wry et al. [33] or Bradford [34]. Amino acid sequencing of pig licer S-COMT. The affinity-purified S-COMT was desalted on Aquapore BU-300 (30 x 2.1 mm; C 4, Brownlee) and the proteinase inhibitor (Trasysol R) removed simultaneously. The enzyme was reduced with D'lq" and either Scarboxvmethylated [34] or S-pyridylethylated [36], and then digested with trypsin (enzyme:substrate ratio = 1 : 5 0 ) in 50 mM NH4HCO3/2 M guanidine (pH 8.0) at 37°C for 16 h. The resulting peptide fragments were separated by reverse-phase HPLC (model 1090/1040, Hewlett Packard, Waidbronn, F.R.G.) either on an Aquapore RP-300 column (100 × 2.1 ram, C m Brownlee) for the S-carboxymethylated material or on a Vydac column (250 x 4 mm C~s) for the S-pyridylethylated sample. The eluate was monitored by UV absorption at 214 nm. Amino acid sequence analyses were performed using an AB! 475A protein sequencer equipped with an on-line phenylthiohydantoin (PTH) amino acid analyzer model 120 (Applied Biosystems; Foster City, CA, U.S.A.).

12] E c

A

0.8

e~

,," ~

g 0.4 u2 o

,,,,

/

10 2

nnAb ng/rnl

] re

08

/

~-"

~ .....

JD

]

104

~

i r

..g

,/

A

/

,'

04 02

10 3

/

i' t ,+

, 06.

I~-,7:...~-.......o ...... -o........o 10 +

B

/

/

/e

x

• ~_~x/.- ''x 1

Screening and characterization of S-COMT mAbs. Using a solid phase binding assay, 50 hybridoma cultures were identified to produce antibodies which reacted with S-COMT. Five hybridomas, namely Coi62 H / 2 (Co16), Co54-5F/8 (Co54), C o ~ - I B / 7 (Co60), Co34-1C/4 (Co34) and Co51-5F/! (Co51), were selected for further analysis. The clone Co16 produced an lgG2a, Co54 an lgGl, Co60 an IgG2b while Co34 and Co51 produced IgM. Among the lgG, the rnAb Co16 showed the highest capacity to bind the antigen as measured by an ELISA using a peroxidase-conjugated goat anti-mouse lgG [37]. The mAb Co16 detected approx. 05 ng pig liver enzyme whereas in the same conditions the mAb Co54 had the lowest binding capacity (Fig. 1). However, only Co54 was able to form a stable immunocomplex and precipitate S-COMT as detected by measuring the enzymatic activity recovered in the pellet after immunoprecipitation. Although Co16 and Co60 did not efficiently precipitate the enzyme, they inhibited the binding of Co54 to S-COMT. This competition experiment, performed as described by St~ihli et al. [38], demonstrates that t h e ~ mAbs react with spacially related epitopes of the same protein.

1 O"

/

~r"

06

S-COMTpreparation. S-COMT was purified approx. 300-fold from pig liver and used for immunization of mice and screening of the hybridoma supernatants. By SDS-PAGE analysis, the enzyme preparation was found to consist of a single predominant protein band of apparent molecular mass of approx. 23 kDa, which corresponds to the molecular mass previously reported for the enzyme [16], with some impurities of higher molecular weight (not shown).

1 2-

jJ~

1 0t

Results and Discussion

/

/

/"

~/

×

~./'"

0 i

10 ~

1'92

103

10 ~

COMT ng/nnl

Fig. 1. Reactivity of monoclonal autib~xlies with C O M T partially puJifi,;d f, um pi~ '+;vet COMT. (A) Purified antibodies were serially diluted in microtiter wells coated with a constant amount (50 ng) of purified pig liver COMT. (B) A constant amount of purified mAb (i ,ug/ml) was added to the wells coated with 500, 50, 5 and 0.5 ng purified pig liver COMT. The antibodies bound to the enzyme were detected by peroxida.,,e-conjugated rabbit tgG anti-mouse. Absorbance was measured at 492 nm by a Titerlek Multiskan reader after incubation (30 rain at 22°C) with the substrate ~olution. ( • • ) Col6, ( x × ) Co54, (o o) Ct~0, ( o o ) unrelated mAh.

106

1

2 3 4 5

1

1

2

2

kDa kDa 200 •

30

• wammlm,,m~.

92.5• 69

•

46

•

21.5 • 14.3 •i RAT LIVER

30 • 21.5 • 14.3 *

Fig. 2. Detection of COMT present in the subcellular fractions of pig liver. Crude homogenates (I,2L II 000× g pellel~ (3L microsomal pellets (4) and microsomal supernatants (5) were subjected to SDSPAGE (15% acrylamide) and immunoblotted with the anti-COMT mAbs at a concentration of 10 #g/ml.

None of the purified m A b s inhibited S - C O M T activity after incubation (for 2 h at 4°C) of 750 ng of the enzyme with different concentrations (7.5 n g - 7 . 5 p.g) of m A b s (data not shown).

lmmunoblot analysis of soluble and membrane-bound COMT. The three m A b s Co16, Co54, Co60 against S - C O M T were examined by immunoblot analysis for their ability to recognize b o t h S - C O M T a n d M B - C O M T in pig liver. W h e n sufficient amounts of pig liver homogenate were loaded on S D S - P A G E ,two polypeptides were recognized by the different m A b s (Fig. 2, lane 1 for Co16). T h e lower b a n d h a d a molecular mass of 23 kDa, most likely representing S-COMT. whereas the second b a n d displayed a slightly higher molecular mass (26 kDa). T o assess w h e t h e r the ?6 kDa b a n d r e p r e s e n t e d the MB-form of the enzyme, pig liver h o m o g e n a t e s were subjected to subcellular fractionation followed by immunoblot analysis. Since the m A b Co16 a p p e a r e d to detect the 26 kDa b a n d with higher intensity, this m A b was chosen for the experiment. After loading an equal a m o u n t (120 # g of protein) of each fraction, an e n r i c h m e n t of the 26 kDa polypeptide was obcerved in the particulate microsomal fraction (Fig. 2, lane 4), where both the 23 and 26 kDa b a n d s showed the same intensity. In the mitochondrial pellet and the cytosol only the 23 kDa form of S - C O M T was found. Similar results were o b t a i n e d

HeDG2

Fig. 3. Analysis of soluble and membrane-bound COMT of rat liver and human Hep G2 cell line. Proteins (120 #g/sample) were separated in 15% SDS-PAGE and transferred to nitrocellulose by electroblotting. Nitro~ :llulose transfers were incubated with the antibody Co60. Bound immunoglobulins were detected by incubation with iodinated sheep anti-mouse immunoglobolin. (I) 100000× g supernatant, (2) 100000× g pellet.

with the o t h e r two m A b s (Co54 and Co60, data not shown). In pig liver, w h e r e only the soluble C O M T form h a d b e e n d e t e c t e d to date [39], our m A b s d e t e c t e d two polypeptides that were associated with the m e m b r a n e s of the mierosomal fraction (see Fig. 3). Since the lower b a n d had the same molecular mass as the S - C O M T usually present in the cytosoi, it is not clear w h e t h e r this smaller protein is a c o n t a m i n a t i o n of S - C O M T present in the tissue, or w h e t h e r the M B - C O M T exists as two polypeptides, possibly r e p r e s e n t i n g two subunits or two isoforms of the native m e m b r a n e - b o u n d protein. According to these results, most of the M B - C O M T is localized in microsomes as already r e p o r t e d [20] a n d a smaW! a m o u n t was found in the mitochondrial fraction. I,. confirms the report of G r o s s m a n et al. [17] TABLE I

Solid phase immunosorbentassay Pu./ied antibodies were serially diluted in microtiter plates coated wiret, 500 ng of COMT. The amibodies bound to the enzyme were detected by peroxidase-conjugated sheep IgG anti-mouse immunoglobulins and quantitated at 492 nm by a Titertek Muhiskan reader. The pig, rat and human COMT were purified from liver by ammonium sulfate fractionation (0 55%), gel filtration on Sephadex G75 and chromatography on an anion exchanger DEAE Bio-Gel agarose.

Anti-COMT mA~

Co 16 C,,(,q Co54

Capacity to react with COMT from different species /zg/ml for 50% of maximum binding pig 0.1)09 0.2 3

rat 40 1 60

human 250 I0 158

I()7 TABLE I!

Purification of pig liter l'O; immunoaffinio chromatography Fresh pig liver (4.2 g) was processed as described in Materials and Methods. After Frccipitation ~ith '~. monium ~ullate (',alu!alJorl 5,5'~ ) !he precipitate was dissoh'ed in 2.5 ml PBS, diluted 1 : 1{/ with ?BS and then loaded on the Sepharose-mAb commn. Purification step

Volume (ml)

Proteins (mg/ml)

Activity (nmol/ml per h)

Total nell',it', (nmol)

Crude homogenate (NH4)2SO 4 precipitation lmmunoaffinity

15

64)

I 1,030

165,450

183

I(X)

I

3.761 1,695

94.025 2 l. 187

1.8~0 2&25 :l

50 13

10 154

25 12.5

2.2 0.06

which provided evidence for a small but detectable level of MB-COMT in the rat outer mitochondrial membranes. However, more experiments are needed to establish the proper subeellular Iocaliza6on of MBCOMT.

Detection of COMT in different animal species by rnAbs. The mAbs raised against pig S-COMT recognized S-COMT partially purified from human and rat liver. The mAbs reacted better with rat than with human enzyme (Table I). In both species Co60 showed the h~ghest potency and was used to investigate, by immurm-b~otting analysis, the distribution of COMT in the ~ l u b l e and in the particulate microsomal fraction of rat livec (Fig. 3). The soluble 23 kDa form of the enzyme was predominant in the soluble fraction (Fig. 3, lane l), whereas both a 23 kDa and a 26 kDa polypeptide were present in similar amount in the microsomal fraction (Fig. 3, lane 2). In human Hep G2 cells, both forms of the enzyme are expressed, as shown by immunoreactivity with mAb Co60 (Fig. 3). The subcellular distribution revealed a S-COMT form of 26 kDa (Fig. 3, lane l) whereas the 29 kDa isoenzyme was found to be ass~iated with

2JO0] PBS

Glycme buffer 102M, pM28) t

)0

Specihc ,Joivit~ ( n m o l / m g prot pc, b)

Yiel I {'; )

l-actor ol pu)ilica'ion

microsomes (Fig. 3, lane 2). The molecular mass of Sand MB-COMT seem to be slightly higher in human than in animal tissue.

Purification of pig licer COMT by immunoaffinity chromatography. The mAb Co54 was .,c):cted for the affinity purification of pig b~er S-COMT since this mAb formed a stable immunocomplex with COMT. After coupling of the purified mAb to CN-Br activated Sepharose 4B. more than 90% of the antibody was covalently bound to the gel. When the partially purified e n ~ +a: , eparation, obtained after ammonium sulfate fractionation of pig liver, was loaded on the column, the enzyme activity was ,etained virtually quantitatively by the immunosorbent. For the preservation of the enzyme activity, the choice of an appropriate eluting buffer was crucial. Thus, using a glycine buffer (0.2 M, pH 2.8). S-COMT

1

2

3

kDa 200b 92 69

~000

46 _

30 c

-~

o

5 ~0

20

30

40

~0

60

70

80

90

Froct ion (number)

Fig. 4. Purification of C O M T from pig liver by immunoaffini~, chromatography. An affinity co!utah was prepared by coupling the antibody Co54 to Sepharose 4B. A partially purified preparation of pig liver S-COMT (25 ml) was passed through the column at a flow rate of l0 m l / h and unbound proteins were washed with PBS+ S-COMT was eluted with glycine buffer (0.2 M pH 2.8) and 2 ml fractions, immediately neutralized with I M Tris-base to pH 7Jk were collected for en~.'m..atic activity and protein de:e~mhiauons.

21

Fig. 5. SDS-PAGE anaL~is of fractions obtdined at various stages of pit. li:,.r affinity purification. (l) Crude bomogenate, (2) ammonium sulfate fraction and (3) nffinit) purified pig liver S-COMT.

108 was recovered from the column in a catalytically active form (Fig, 4.). As shown in Tablc II, the specific activity of the affinity-purified enzyme was 154-fold higher than that of the crude homogenatc and the recoveD~was 13%. In repeated experiments the capacity of the affinity column was e~timated as 20/zg of pig liver S-COMT/ml of gel. SDS-PAGE analysis of the affinity-purified S-COMT preparation revealed a single protein band with an apparent molecular mass of approx. 23 kDa (Fig. 5). The enzyme was virtually pure and no other protein bands were visualized even after the silver staining technique. In Western blots, the affinity purified material was recognized by all the anti COMT mAbs we have characterized (data not shown). The purification factor, with regard to the enzymatic activity, was relatively low. This is probably due to partial inactivation of the enzyme during the elution step or to the instability of the pure enzyme. We evaluated the effect of several elution buffers on COMT activity: elution with high pH or low ionic strength buffers containing a denaturing agent, such as urea, was precluded since the enzyme was irreversibly denaturated under these conditions. The ..lution of S-COMT

A 200 2201 180! 160~ 140: c

120:

~

100: 80: 6o! 4o:

20 ¸ 40

50

Tithe

60 irn~n )

70

8'3

Yields of PTH-amino acids were no.'. corrected for carryover. Residues in brackets could only be tentatively assigned. Cycle

T:,5

No

amino acid

.,ield (pmol)

T24 amino acid

yield (pmol)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

Leu Leu Thr lie Glu Leu Asn Pro Asp Ash Ala Ala lie Ala Gin Gin Val Val Asp Phe Ala Gly Leu Gin Asp (Trp) Val Thr Val (Val~ (Val) Gly Ala (Set) (Thr)

49.5 45.6 18.4 26.8 9.1 35.2 7.4 8. I 4.7 7.4 12.8 18.8 12.5 16.6 5.6 6.7 7.9 13.8 2.9 5.7 8.9 6.9 8.4 3.0 1.8 2.8 4.4 3. I 5.1 5.7 6.6 3.7 3.9 1.0 1.9

lie Leu Gin Tyr Val Leu Gin Pro Ala Val Ala Gly Asp Pro Glu Set Val Leu Asp Thr lie Gly

18.8 25.9 7.7 6.6 9.7 15.4 5.7 3.2 10.0 8.1 10.1 5.0 5.7 4.9 3.7 1.3 6.5 2.9 3.7 2.2 2.4 1.1

~

3o

2O

_

I ABL[ 111

Sequen( e analy~t~ of tO'ptic peptide.~ ]?om immunoaffinity purified pig hcer ( OMT

"~ Time

20 (rain)

30

Fig. 6. Separation of tryptic pcptides from S-COMT by RP-HPLC. After digestion, the peptid¢ mapping was accomplished by reversephase H P L C using a Aquapore C ~ or a Vydac Cis column. The eluent was monitored at 214 nm and the peptides 1"35 (panel A) and T24 (panel B) were sequenced.

by PBS containing 4 M KCN was inferior to the elution with low pH buffer since S-COMT eluted from the column in a v e r j broad activity peak resulting in a low specific activity and yield. In conclusion, the conditions chosen for the affinity purification of S-COMT were the most efficient and our method therefore allows one to obtain a pure preparation of pig liver enzyme in a single chromatographic step. The degree of purity of this S-COMT preparation made it feasible to study the primary structure of the enzyme by partial amiro acid analysis.

Partial amino acid sequence analysis of pig liver SCOMT. The purified COMT was not susceptible to direct Edman degradation, although amino acid analysis of the sample confirmed the presence of sufficient

109 a m o u n t o f p r o t e i n . A b l o c k e d N - t e r m i n u s w a s ass u m e d , t h e r e f o r e t h e p r o t e i n w a s s u b j e c t e d to a tryptic t r e a t m e n t a n d t h e r e s u l t i n g p e p t i d e s i s o l a t e d by R P H P L C (Fig. 6). P e p t i d e T 3 5 f r o m t h e S - c a t b o x 3 ' m e t h y l a t e d p~otein ( p a n e l A ) a n d p e p t i d e T 2 4 f r o m t h e S - p y r i d y l e t h y l a t e d C O M T ( p a n e l B) w e r e s u b j e c t e d to automated Edman degradation. The sequencing results a r e s u m m a r i z e d in T a b l e Ill. T h e p o r c i n e tryptic p e p t i d e s s e q u e n c e T 2 4 a n d T 3 5 w e r e f o u n d in t h e p r e d i c t e d p r o t e i n s e q u e n c e o f rat liver e D N A [22]. T 2 4 a n d T 3 5 c o r r e s p o n d to t h e a m i n o acid r e s i d u e s 9 - 3 0 a n d 8 6 - 1 2 0 o n rat s e q u e n c e , respectively; t h e e x t e n t o f t h e s e q u e n c e i d e n t i t y o f t h e p e p t i d e s with r a t is 6 4 % for T 2 4 a n d 6 7 % for T35.

Conclusion This report describes the production of monocional a n t i b o d i e s a g a i n s t t h e s o l u b l e f o r m (S-) o f C O M T f r o m p i g liver. T h e s e m A b s r e c o g n i z e d b o t h t h e S- a n d t h e m e m b r a n e - b o u n d ( M B ) f o r m s o f t h e e n z y m e a n d rea c t e d w i t h pig, rat a n d h u m a n C O M T . T h e u s e o f t h e m A b s f o r a s i m p l e a n d r a p i d affinity p u r i f i c a t i o n r e p r e sents a major improvement over existing conventional p r o c e d u r e s a n d a l l o w e d t h e i s o l a t i o n o f t h e e n z y m e in a high purity state, T w o tryptic p e p t i d e s f r o m t h e h i g h l y p u r i f i e d e n z y m e p r e p a r a t i o n w e r e s e q u e n c e d . T h e availability o f m A b s a n d t h e k n o w l e d g e o f a p a r t i a l a m i n o acid sequence of COMT create new areas of investigation, e.g., c l o n i n g o f e D N A e x p r e s s i n g C O M T in o r d e r to get further information about the primary structure of t h e e n z y m e [40].

Acknowledgements W e a r e g r a t e f u l to P r o f e s s o r W . H a e f e l y , D r s . A . M . C e s u r a , J . G . R i c h a r d s a n d P. S c h o c h for t h e i r critical e v a l u a t i o n o f t h e m a n u s c r i p t , to M. B i i h l e r a n d U. R 6 t h l i s b e r g e r for t e c h n i c a l a s s i s t a n c e a n d to M. W d o n wicki for s e c r e t a r i a l work.

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