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Preparation and characterization of monoclonal antibodies against 4-aminobenzoate hydroxylase from Agaricus bisporus T a d a s h i O g a w a , H i d c a k i Tsuji. M a s u m i K i m o t o a n d Kei S a s a o k a I~'part:ucnt o t Nntriri~m. .%cit~Ndof M~'d~in:t'. ltu" Umrl'r~il~" of Tcd~u~l~:n:a. T¢&ushmia ¢]al~i~ (Rci.~¢d

II I

JUlyItJtgl)

Key ,,xt~rd~: -t-Ammobcnzoatchydn~xila~:;FAD-binding&lmain:tMoralchmalant~lt~l A monoclonal antibody tmAb, At recognizing the FAD-binding domain of 4-aminobenzoute h)-drox)-Iase (4-aminobenzoate, NAD(P)H:ox~gen oxidoreductase I I-h)dro.~.lating, decarboxylatingJ, EC 1.14.13.27~ from b i s p e r u s , a common edible mushroom, had been produced ¢Tsuji, H., Oga~a, T., Bando, N~ Kimoto, M. and Sasaoka, K. 11990) J. Biol. Chem. 265, 16064-16067L In the present study, three other mAbs (Bp B 2 and B3) against the enz~'me have been further prepared in order to facilitate the structural characterization of the enzyme. The three new mAbs immunobiotted the enz)'me. The four mAbs, including A, were specific for different epitopcs on the enzyme. B t and B: immunoprecipitated the apoenz)me and the immunoprecipitation was inhibited in the p ~ s e u r e ofFAl), whereas B.~ failed to immunoprecipitate the apoenz~ne in the absence or presence ofFAl). B t and B. competed with FAD for the binding to the a p o e n z ~ . These findings show that B t and B z recognize the FAD-binding domain of the enz~'me in analog- with A. The immunobiotting analyses of the peptides obtained from the enzyme by digestion with l y s y l endopeptid:ise {EC 3.4.21.50~ provided useful knowledge as to the location of the epitopes to the mAbs on the enz)me, suggesting that the FAD-binding domain of the enzyme can be located and characterized b) detailed investigations on the location of the epitopes. Introduction 4-Aminobenzoate hydroxylase is a FAD-dependent monooxygenase, which belongs to the cla~s called external monoox'v,genases [!.2]. The en~'me catalyzes the dccarbox'ylative hydrox),|ation of 4-aminobenzoate to form 4-hydroxyaniline, which is the aromatic component of N-(~/-t.-glutamyl)-4-hydrox)'aniline, a characteristic constituent of the mushrt~3m [!-4]. Among flavoproteins, two FAD-dependent cnzymcs. glutathione reductase (EC 1.6.4.2) [5] and p-hydroxybenzoate hydroxylase (EC 1.14.13.2) [6] and a FMNcontaining protein, flavodoxin [7] have been actively investigated with respect to the three-dimensional structures of the flavin-binding proteins using X-ray crystallography. However, studies on the threc-dimen-

Abbreviations: ELISA. cno'me-linkcdimmunosorbentassay; mAb. monoclonal antibody; PAGE. pillyacp,'lamid¢gel electrophoresis: SDS. ~dium dodec~"l sulfate. Correspondence: T. Oga~a. Department of Nutrition. Sehotll of Medicine.The Uni,..e~it.vof Tukushima. Kuramoto-cho.Tokushima 770. Japan.

sional structures of other flavoproteins have .scarcely been reported. Recently, we produced a monoclonal antibody (mAb, A) against the FAD-binding domain of 4-aminobenzoate hydroxylase in order to carry out the structural charactcrization of the enz~tne [8]. Interestingly, thc mAb was also shown to recognize the FADbinding domains of other FAD-dependent enzymes, saiio'late hydroxylase (EC !. 14. ! 3. ! ) and o-amino acid oxidase (EC 1.4.3.3). These findings are of great intercst in connection with the hypothesis that similar structures may exist in the FAD- a n d / o r NADlP)H-binding domains of FAD- a n d / o r NAIMP)H-rcquiring enzymes [9,10]. Jn general, a mAb can serve as a po~'erful tool for the structural characterization of the functional domains of enzymes. If several mAbs recognizing distinct epiiopes on a domain of an enzyme can be obtained, the mAbs should facilitate the structural characterization of the domain. From this point of view, we attempted to produce mAbs recognizing epitopes, which were discrete from the epitope to A, on the FAD-binding domain of 4-aminobenzoate hydroxylase. in the present paper, wc describe the preparation of three new mAbs against 4-aminobenzoatc hydroxylase and their immunological properties. O f the mAbs. two

221 were shown to recognize the FAD-binding domain of the cnz~rne. The lca.~tion of the epitopes on the enzyme was also examined using peptide mapping and immunoblotting techniques. Materials and Methods

Materials The materials used were obtained from the sources indicated: Mouse myeloma cell line, P 3 × 6 3 A g 8 U I (P3UI) (Shino Test Institute, Sagamihara, Japan), B A L B / c mice (SLC, Shizuoka, Japan), fetal bovine serum, sterilized L-glutamine and streptomycinpenicillin mixture (MA Bioproducts, CA, U.S.A.L RPM! 1640 (Nissui Pharmaceutical, Tokyo, .lapanL G I T medium (Nihon Pharmaceutical, Tokyo, Japan), isotyping mouse monoclonal antibody kit (Amersham International PLC, Amersham, U.K.), aminopterin (Sigma, MO, U.S.A.), pols~ethylene glycol) 4000 (Merck. Darmstadt. Germany), prepacked protein A column (Pierce Chemicals, I L U.S.A.L nitrocellulose membrane (Bio-Rad, CA, U.S.A.), StaphylococaLs aureus Cowan ! (Seikagaku Kogs'o, Tokyo, Japan), lys~l endopeptidase (Achromobacter protease I) from Adzromobacter lyticus M497-1 (Wako Pure Chemical Industries, Osaka, Japan), mouse igG (Zymed Laboratories, CA, U.S.A.), peroxidase-conjugated sheep antimouse lgG (Organon Teknika, West Chester, PA, U.S.A.) and electrophoresis calibration kit for mole~:ular weight determination of low molecular weight proteins (Pharmacia, Uppsala, Sweden). 4-Aminobenzoate hydroxylase and its apocnzyme were prepared as described earlier [2]. The mAb. A, was prepared as described in the previous paper [8].

in the absence or presence of FAD in the same manner as described previously [8]. Briefly, the apoenzyme (0.2 /ag) of 4-aminobenzoate hydroxylase was incubated with varying amounts of the mAbs in 1.2 ml of 50 mM potassium phosphate buffer (pH 7.5) containing 0.02% bovine serum albumin in an ice bath for 8 h, after which 100/~l of a 10% (w/v) cell suspension of Staphylococcus aureus in 50 mM potassium phosphate buffer (pH 7.5) was added to the mixtures. After a 3-h incubation in an ice bath, the mixtures were centrifuged at 1600 x g for 10 min. The remaining enzyme activities in the sul:ernatants were assayed as described below, in the case of the experiments with FAD, immunoprccipitation was done as described above, except that the apoenzyme was incubated with 50 mM potassium phosphate buffer (pH 7.5) containing 0.02% bovine .serum albumin and 50/,tM FAD in an ice bath for 20 min before the mAbs were added to the mixtures.

Enzyme assay 4-Aminobenzoate hydroxylase activity was assayed by determining the amount of 4-hydroxyaniline formed from 4-aminobenzoate as described in a previous paper [2].

1

2

3

4

5

kDfl 94--

Preparation of mAb The immunization of mice and the preparation of three hybridoma cells producing mAbs against 4aminobenzoate hydroxylase were performed in the same manner as described previously [8]. The three mAbs produced lay the hybridoma cells are designated B I, B 2 and B 3. The mAbs Were prepared from a serum-free medium, G I T medium, where the hybridoma cells were cultured, or the ascites of mice intraperitoneaily injected with the bybridoma cells by ammonium sulfate fractionation. B n and B: were stored in a refrigerator, whereas B 3 was stored at -20°C.

67~

4 3 - -

30 B

:



:•• •

i

Enz3"me-linked bnmanosorbent assay (ELISA) and immanoblotting ELISA and immunoblotting were done in the same manner as described previously [8].

lmmunoprecipitation The immunoprecipitation of 4-aminobenzoate hydroxylase with the mAbs was done using its apoenzyme

Fig. I. Immunoblot of 4-aminobenzoate hydroxylase. 4-Aminobenzoate hydroxylase(0.5 p.g per lane) was eleetrophoresedon 12% slab polyacrylamide gels and then was electrotransferred to nitrocellulose membranes.The protein on the nitrocellulosemembranes was either stained with Am/do black 10B (lane 1) or immunoblotted with nonimmunemouse IgG (lane 2), Bt (lane 3), Bz (lane 4) and B3 (lane 5).

222

Proteo~'sis o f the en..'3"me T h e enzyme was incubated at 3 T C in 50 mM TfisHC! buffer ( p H 8.0) in the presence of | y ~ l endopeptidase. T h e ratio o f the enzyme to the protease was 1000: I ( w / w ) . After incubat/on for a g b e n time, the reaction was s t o p p e d by add(thin o f the sample buffer for sodium dod¢cyl sulfate-po|yacrylamide gel clectrophoresis ( S D S - P A G E ) and heating in a boiling water bath for 5 min. T h e p r o t e o b l k : products were subjected to S D S - P A G E on 17% slab polyacrylamide gels and stained with Coomassie brilliant blue R250. For the experiments investigating the cro.~-reacti~t o f the peptidcs with the mAbs, the peptidcs separated by S D S - P A G E were clcctmtransferrcd o n t o a nitrocellulose m e m b r a n e . T h e m e m b r a n e ~ a s blocked at r o o m t e m p e r a t u r e for 1 h with I G bovine s e r u m albumin in 20 m M Tris-HCi buffer ( p H 7.3) containing 0.05% T w e e n 20. After the m e m b r a n e was washed with the same buffer, it was p r o b e d with the mAbs. The concentrations o f the m A b s u ~ d ~vcrc 0.5 ,~tg/ml. Nonim-

A

0oot \

"-

'°[

-04

-02

0

02

04

06

FAD -~( p M ~ }

Fi~ 3. Effect of BI and B, on the b/nding of FAD to the agmenzymc of 4-aminobenzoate hvdru~-lase. The agmen~.'me {0.4 ~ug) of 4a m i ~ n z o a t e hydras'last was p~Teincubaled vigh va~3ing ccm~nti'a|~"ig of FAD in an ice bath for ~ rain and then lh¢ nfixluresv¢re incubated w/th BI (o) or B: {e). As a conlroL the mixtures were incubated vdth 50 mM polassium phosphate buffer (pH 7.5) inslea~d of the mAbs ( A ). The conc~nt~t~as ~" B~ and B : ¢,~:re 4.6-10 -~ and 6.2-10 7 M. respectk-eK-. Alter a 8-h incubat~'l in an ice bath. the mixlur~ were subjected to tim determinathm of 4-amivot~nzoate hydro~-lase actiniC" as descn'bcd under Materials and Methods. The results obtained are represented as ¢hmb~-ree~arocal ph~Ls.

!

m u n c mouse lgG (!.5 / t g / m l ) was used as a control instead of the mAbs. Bound antibodies were detected by reaction with peroxidase-conjugated s h e e p antim o u s e lgG (1 :_2000 dilution). T h e nitrocellulose-bouod complex was visualized by reaction with 4-chloro-lnaphthol as a substrate.

B

Results

mAb { pg) Fig. 2. Immunoprecipitation of 4-aminobenzoate h)'dro~'las¢ with the new mAbs in the absence (A) or presence (B) of FAD. The apocnzyme (0.2 /zg) of 4-amino~nzoate hydro~'lase was incubated with va~'ing amounts of B= (o). B 2 (e) and B3 ( A ) in ~ mM potassium phosphate buffer (pH 7~) containing 0.02% bovine serum

albumin in a total volume of 1.2 ml and then the complexes were incubated and precipitated with a 10% (v-/v) cell suspension of Staphyloeoccus aureu.s as described under Materials and Methods. In order to investigate the effect of FAD on the immu,,mprecipitatiou. the apnenzyme (0.2 gg) was preincubated with 5{)/zM FAD in 1.2 ml of 50 mM potassium phosphate buffer (pH 7.5) containing 0.02% bovine serum albumin and the hotocnz3.'me reconstituted v-as subjeered to the abcwe-mentioned immuuoprecipitationv.ith Bt (o). B2 (o) and B3 ( A). The values represent the residual en~Tne activities in the supernatants expressed as a perceutage of enz~ll¢ actb,i~" assa.vcd in the absence of any antibody.

Preparation o f m A b in the present study, we used as the antigen the enzyme d e n a t u r e d by dialysis against phosphatebuffered saline containing 0.1% SDS as described previously [8]. Mice were effectively immunized with the denatured en .zyme. T h e spleen cells obtained from the mice were fused with P3UI myeloma cells by the conventional method [11]. Finally, three h vbridoma cells producing m A b s against the enzyme have been established. O f the mAbs, B~ and B 2 lost their antibody activities by freezing and thawing, whereas A and B 3 were very stable for the treatments. Therefore, the two former m A b s which were p r e p a r e d from a serumfree medium or ascites of mice as described u n d e r Materials and Methods have never been frozen in the present study.

223

Characterization of mAb T h e isotyping o f the newly p r e p a r e d m A b s was performed using a mouse monoclonal antibody isolyping kit. All three m A b s had ~ light chains. The heavy chains of B t were Yt and those o f B2 and B 3 were y ~ . All o f the newly p r e p a r e d m A b s bound to the enzyme after S D S - P A G E and electrotransfer to nitrocellulose m e m b r a n e s (Fig. !). T h e findings suggest that these m A b s recognize sequential epitopes. T h e mAb, A, which had been prev/ously prepared [8], recognizes the FAD-binding domain o f the enzyme. Therefore, it was interesting to examine w h e t b e r the newly p r e p a r e d m A b s are specific for the FAD-binding domain. As shown in Fig, 2, BI and B 2 immunoprecipRated the apoenzyme and the immunoprecipitation was inhibited in the presence o f FAD. O n the o t h e r hand, B 3 could not immunoprecipitate the apoenz3ane

A 1

2

3

4

5

6

7

g~-,O

O

D94

O

--e7

either in the absence or the presence of FAD. These findings suggest that B I and B, specifically recognize the FAD-binding domain of the enzyme, but B.~ is not specific for it. In order to confirm the above findings, the effect of B I and B2 on the binding of F A D to the apoenzyme was examined. Fig. 3 clearly demonstrates that both of B~ and B2 compete with F A D for the binding to the apoenzyme. The Ki values of B I and B 2 were 1.2- 10 -~ and 5 . 3 . 1 0 -7 M, respectively. These findings show that B I and B z are specific for the FAD-binding domain of the enzyme.

Peptide mapping and intmunoblotting of the peptides obtained from tile enzyme W h e n the enzyme with the molecular mass of 49 kDa was digested with lysyl endopeptidase (1:1000 lysyl endopeptidase to the enzyme, weight ratio), the enzyme was rapidly proteolyzed into many peptide fragments as shown in Fig. 4A. After a 5-rain incubation, the main peptide bands with molecular masses of 45, 40, 37, 30 and 26.5 kDa were observed (Fig. 4A, lane 2). Most of the peptides were stable for further proteolysis even after a 120-rain incubation and the pepr~des with molecular masses of 20 and 12.5 kDa accumulated (Fig. 4A, lanes 3-6). When the enzyme was incubated with lysyl endopeptidase (1:100 lysyl endopeptidase to the enzyme, weight ratio), the en-

qlP - - 3 0

,~

--20.1

O

-144

B

3

4

5

6

7

k~ 94-67--

kDa

3o- ~ --265 --235

14.4-- ~

.

--15

Fig. 4. A pcptide map and immunoblots of lysyl endopeptidaselimited proteolysis products of 4-aminobenzoate hydroxyla~. 4Aminebenzoate hvdro~lase ( 10 pg) was digested with ly.~l endopeptidase (ll.OI pg) in 50 mM Tris-HCIbuffer (pH 8.l)) at 37°C in a total volume of 40/.d ax described under Materials and Methods. At the indicated limes. 3.5/i.I of the reaction mixture was withdrawn and the digestion was terminated by addition of the sample buffer for SDS-PAGE and heating in a boiling water bath for 5 rain. The mixtures were subjected to SDS-PAGE on a 17% slab polyaerylamid¢ gel and immunoblottedwith nonimmune mouse IgG and the four mAbs as described under Materials and Methods. (A) The peplide map of the ly.~,l eudopcptidase-limited proteolysisproducts. The proteol~,lic products were separated by SDS-PAGE and stained with Coomassiebrilliant blue R.?.50. Lane I. I-rain digestion; lane 2, 5-min digestion: lane 3. 10-rain digestion: lane 4, 30-rain digestion; lane 5, 60-min digestion: lane 6. 120-min digestion; lane 7. standard marker proteins. (B) The immunoblots of the proteolytic products with mAbs. After SDS-PAGE of the proteolytic products obtained by a 30-rain digestion with the proteas¢ in the same manner as described in the above-mentioned peptid¢ mapping, the products were eleclrolransferred to a nitrocellulose membrane. The part of the membrane containing standard marker proteins and the proteolylic produc',swas cut out and stained with Amido black 10B. The other part of the membrane contained the proteolytic products was immunoblotted with the mAbs. Lanes 1 and 2. standard marker proteins and the proteolytie products, respectively, stained with Amido black 10B; lanes 3-7. the proteolylic products immunobloned with nonimmune mouse IgG. A. Bt, B, and B.~, respectively. The standard marker proteins used were as follows: phosphorylaseb (94 kDa). bovine serum albumin (67 kDa). ovalbumin(43 kDa), carbonic anhydrase (30 kDa). ~ybean trypsin inhibitor (20.1 kDa) and alactoglobnlin ( 14.4 kDa).

224 zyme was completely digested in 5 rain {Tsuji, H.. Ogawa, T., Kimoto, M. a n d Sasaoka. K., u n p u b l i s h e d data). A f t e r 30 rain, only o n e | 2 . 5 - k D a peptidc h a n d was observed. T h e protcolytic f r a g m e n t s o b t a i n e d by digestion with lysyl e n d o p e p t i d a s e at a 1 : | 0 0 0 ratio o f lye-! e n d o p e p tidase to the e n z y m e were e x a m i n e d with respect to t h e cross-reactivity with t h e m A b s using an i m m u n o blotting technique. T h e i m m u n o b l o t t i n g p a t t e r n s o f t h e peptides o b t a i n e d with t h e m A b s were different {Fig. 4B). All o f t h e m A b s i m m u n o b l o t t e d t h e 40-. 30- a n d 26_5-kDa peptides. A. B , a n d B3 recognized t h e 45-kDa peptide which m i g h t be f o r m e d by t h e removafl o f t h e 4 - k D a peptide f r a g m e n t in t h e N-terminal or C-terminal region o f t h e e n ~ m c , but B~ was not s h o w n to i m m u n o b l o t t h e 45-kDa peptide. B , a n d B; recognized t h e ~;_5-kDa peptide, but the two o t h e r m A b s could not cross-react with it. T h e peptide with a molecular m a s s o f 15 k D a ~ a s very weakly i m m u n o b l o t t e d by only 83 . Discussion W e h a d previously p r e p a r e d a m A b (A) recognizing t h e F A D - b i o d i n g d o m a i n of 4 - a m i n o b e n z o a t e hydroxylas¢ [8]. T h e m A b is a | s o specific for t h e F A D - b i n d i n g d o m a i n s o f D-amino acid oxidase a n d salicylate hydroxylase. Evidence s u p p o r t i n g t h e idea that t h e F A D a n d / o r NALMP)H-binding d o m a i n s o f F A D - a n d / o r N A I M P ) H - r e q u i t i n g e n z y m e s m a y s h a r e similar struct u r e s h a s b e e n a c c u m u l a t e d by X-ray crystallographic studies [5,6] a n d immunological s t u d i e s [12]. O u r finding that t h e F A D - b i n d i n g d o m a i n s o f t h e t h r e e e n z y m e s exhibit immunological similarity is o f great significance in c o n n e c t i o n with t h e a b o v e - m e n t i o n e d hypo.thcsis. In t h e p r e s e n t study, t h r e e m A b s (B t, B , a n d B 3) have b e e n newly p r e p a r e d . O f t h e m A b s , B t a n d B , were s h o w n to i m m u n o p r e c i p i t a t e t h e a p o e n z y m e o f the 4 - a m i n o b e n z o a t e hydroxylase a n d c o m p e t e with F A D for t h e binding to t h e a p o e n z y m e . T h e s e findings show that t h e two m A b s are specific for t h e F A D - b i n d ing d o m a i n o f t h e e n z y m e in analogy with A. T h e above observations s u g g e s t that A, B t a n d B 2 bind to a part o f t h e polypeptide chain which is at t h e surface o f t h e native e n z y m e including t h e F A D - b i n d i n g d o m a i n a n d that B 3 recognizes t h e site which locates in an interior part o f t h e native e n z y m e so that t h e m A b c a n not bind to t h e protein. in o r d e r to elucidate t h e positional relationship b e t w e e n t h e epitopes to t h e four m A b s o n t h e e n z y m e . we e x a m i n e d t h e binding of t h e m A b s to t h e peptides obtained from t h e enz~ane by proteolysis with iysyi endopeptidase. The immunoblotting patterns of the peptides from t h e e n z y m e u s i n g the m A b s were quite different from another. T h i s finding clearly shows that t h e four m A b s are specific for different epitopes on the

e n z y m e , s u g g e s t i n g that A. B u a n d B , bind to distinct sites o n the F A D - b i n d i n g d o m a i n o f t h e e n z ~ n e . T h e i m m u n o b l o t s d e m o n s t r a t e d that A. B , a n d B~ b o u n d to t h e 45-kDa peptide, b u t that B e could n o t crogs-react with it. T h i s finding s h o ~ that t h e epitope to B~ locates in t h e 4 - k D a p e p t i d e f r a g m e n t o f t h e N-terminal or C-terminal region o f t h e e n d - m e a n d that t h o s e to t h e t h r e e o t h e r m A b s locate in t h e o t h e r f r a g m e n t {the 45-kDa peptide) o f t h e c .nzyme. T h e 26.5-kDa peptide was t h e smallest peptide o f t h e p e p t i d e s containing t h e e p i t o p e s to t h e four m A b s . T h e peptide a p p e a r s to contain t h e whole o r a part o f t h e F A D binding d o m a i n o f t h e e n d - m e , b e c a u s e A, Bg a n d B_, recognize t h e F A D - b i o d i o g d o m a i n . O n t h e o t h e r h a n d . t h e Z~.5-kDa pepfide c o n t a i n e d only t h e e p i t o p e s to B_, a n d B 3. indicating that t h e c p h o p e s to A a n d Bt d o not locate b e t w e e n t h o s e to B , a n d B 3. T h e p r e s e n t study d o e s not provide f u r t h e r information o n t h e o r d e r o f the e p i t o p e s to t h e m A b s . In o r d e r 1o obtain t h e protcolytic products which g~'e a clue to t h e exact location o f the epitopes, a m o r e e l a b o r a t k e m e t h o d for t h e limited protcolysis o f t h e e n z y m e with lysyi c n d o p e p t i d a s e s h o u l d be developed. T h e limited protcolysis o f t h e e n z y m e with o t h e r protcase s u c h as trypsin a n d Staphylococcus aureus V8 proteinase m a y ~ e l d p e p t i d e s useful for t h e iocadon o f t h e cpitogms to t h e m A b s . T h e N-terminal s e q u e n c e anab.'ses o f t h e peptides o b t a i n e d as described above will definitely locate t h e epitopes to t h e m A b s and, therefore, elucidate t h e p~ition of the FAD-bioding domain on the enzyme. Detailed investigations o n t h e location o f t h e e p i t o p e s to t h e four m A b s on t h e e n z y m e a r e in progress. References I Tsuji. H.. Ogawa. T.. Bando. N. and S~Lsaok~K. {ItJSSI Biochem. Bioph.,,~. Rcs. Commun. I.~IL633-639. 2 Tsuji. IL. ()gawa. T.. Bando. N. and Sit~i;l. K_ {|t~¢.6}J. B/ol. Chem. 261. 133D-133~. 3 Sasaoka. K.. OgawiL1... Tsuji. IL and Bando. N. {I~D B~hirn. Biophys. Acta 630. 137- I-R}. 4 Tsuji. H.. Ogav,a. T_. Bando. N. and S~saoka. K. tiggS) B~chim. Biophys. Acta 840. 287-290. 5 Schulh~.G.E.. Schirmer. R_H.. Sachsenheimcr. W. and Pal. E.F. (1978i Nature 273. 120-124. 6 Wiercnga. R.K.. De Jon~ R.J.. Kalk. K.H_ HoL W.GJ. and Drtmth. J. (1979) J. Mol. Biol. 131.55-73. 7 Bumeu. R.M.. Darlin~ G.D.. Kendall D.S.. LcOucsn¢. M.E.. Ma~,'hew. S.G.. Smith. W.W. and Ludwi~ M.L {19741 J. Biol. Chem. 249. 4383-4392. T~uji. It.. Ogawa. T.. Bando. N.. Kimolo. M and Sasaoka. K. {It)~l) J. Biol. Chem. 265. Ifi(h'~4-16fJ67. Rossmann. M.G.. Moras. D. and Ol~m. K.W. (1974) Nature 250. 194-199. Wcijer. W.J.. Hofstecng¢. J.. Beitcma. JJ.. Wicrenga. R.K. and Drenth. J. (1983) Eur. J. Biochem. 133. 109-118. Gt~ling. J.W. (19811)J. Immunol. Metht~k 39. 285-308. Kativar. S.S. and Porter. J.W. (i9831 Proc. Natl. Acad. ScL USA 8{L 122I- 12!~,.

Preparation and characterization of monoclonal antibodies against 4-aminobenzoate hydroxylase from Agaricus bisporus.

A monoclonal antibody (mAb, A) recognizing the FAD-binding domain of 4-aminobenzoate hydroxylase (4-aminobenzoate, NAD(P)H:oxygen oxidoreductase (1-hy...
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