PNAS-1986-Lee-6790-4
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7/28/2019 PNAS-1986-Lee-6790-4 http://slidepdf.com/reader/full/pnas-1986-lee-6790-4 1/5 Proc. Nati. Acad. Sci. USA Vol. 83, pp. 6790-6794, September 1986 Biochemistry Purification, biochemical characterization, and biological function of human esterase D (genetic marker/esterase antibodies/detoxification/phenobarbital Induction) WEN-HWA LEE*t, WILLIAMWHEATLEY*, WILLIAM F. BENEDICTt, CHUN-MING HUANG§, AND EVA Y.-H. P. LEE* *Department of Pathology, University of California, San Diego, La Jolla, CA 92093; *Clayton Molecular Biology Program and Division of Hematology-Oncology, Childrens Hospital of Los Angeles, Los Angeles, CA 90027; and §Institute of Clinical Medicine, School of Medicine, National Taiwan University, Taipei, Taiwan Communicated by Harvey A. Itano, June 9, 1986 ABSTRACT Human esterase D (carboxylesterase; carbox- ylic-ester hydrolase, EC 3.1.1.1), a genetic marker of retinoblastoma, was purified to biochemical homogeneity from erythrocytes. The purification scheme including carboxy- methylcellulose, phenyl-Sepharose, chromatofocusing, and hy- droxylapatite chromatographies resulted in a 10,000-fold pu- rification of the enzyme with 15% recovery of total activity. The Km of esterase D was estimated to be 10 x 10-6 M using 4-methylumbelliferyl acetate as substrate. The enzymatic ac- tivity was inhibited by p-chloromercuribenzoate and HgCI2, suggesting an important role of SH group(s) in enzyme func- tion. Specific rabbit polyclonal and mouse monoclonal anti- bodies against esterase D were prepared and recognized either denatured or native human esterase D protein. Moreover, the polyconal antibodies immunoprecipitated a polypeptide with a molecular mass of about 33-34 kDa from various cell lines of different mammalian species, indicating that the esterase D protein is highly conserved. The highest levels of this enzyme werefound in liver and kidney. Furthermore, the expression of esterase D was enhanced 3-fold in a promonocytic cell line treated with phenobarbital but not with phorbol myristate acetate, suggesting that esterase D may have a role in detoxi- fication. The availability of the homogeneous protein and its specific antibodies allows for cloning of the esterase D gene and facilitates studies of retinoblastomas. Human esterase D (carboxylesterase; carboxylic-ester hy- drolase, EC 3.1.1.1) is one member of the nonspecific esterase family defined by their reaction with synthetic o-acyl ester substrates (1, 2). This enzyme is distinguishable from other esterases by its electrophoretic mobility and its relative specificity for methylumbelliferyl esters as substrate (2). The dimeric enzyme displays several phenotypes as a result of the expression of codominant autosomal alleles, primarily ESDI and ESD2. This polymorphic nature of esterase D provides a valuable genetic marker for studies in population genetics (2, 3). Since the esterase D gene has been mapped to the chromosome 13q14.11 region where the putative retinoblas- toma gene is also located (4-7), the polymorphic nature of the esterase D protein becomes a very useful genetic marker for the diagnosis of hereditary retinoblastomas (8, 9). In addition, the defective gene of Wilson disease was found to be linked to the esterase D gene (10). Thus, it is important to investigate the nature of the esterase D protein and its gene so as to understand these inherited disorders. Esterase D has been found in most tissues, but not much is known about its structure and function. A protocol for partial purification of this enzyme has been described (11). However, it remains difficult to obtain a sufficient amount of the homogeneous enzyme and to generate specific antibod- ies. In this communication, we describe the purification and characterization of this enzyme as well as the preparation of esterase D-specific polyclonal and monoclonal antibodies. Furthermore, we observed that this enzyme was mainly distributed in liver and kidney and could be induced by treatment with phenobarbital but not with phorbol ester. MATERIALS AND METHODS Esterase D Enzymatic Assays. (i) Quantitative assay. Esterase D activity was determined essentially as described (11). The reaction was started by adding the enzyme into 1 ml of reactionbuffer containing 50 mM potassium phosphate, 1 mM EDTA (pH 6.0), with 0.1 mM 4-methylumbelliferyl acetate as substrate. The increase in absorbance at 340 nm was recorded. A unit of activity was defined as the amount of enzyme that hydrolyzed 1 /.kmol of substrate per min at 230C using 7.27 mM-1 cm-1 as the coefficient of absorption. (ii) Spot test. A rapid semiquantitative test was developed by us for assaying enzyme activity in column fractions. Briefly, 5 jul of 4-methylumbelliferyl acetate (20 Ag/ml) in sodium acetate (pH 5.2) was spotted onto a piece of SaranWrap over a UV transilluminator together with 5 A.l of enzyme samples from each column fraction. The intensity of fluorescence emitted corresponded with the enzyme activity. (iii) Qualitative assay. Electrophoresis in 1% agarose gels was performed as described (12) with slight modifications. The agarose gel buffer was a 1:5 dilution of running buffer that contained 62 mMTris, 15.5 mMcitric acid, 18 mMboric acid, and 1.65 mM lithium hydroxide (pH 7.5). Electrophoresis was performed at 40C for 90 min at 20 V/cm. The gel was stained in 50 ml of 50 mM sodium acetate (pH 5.2) containing 1.0 ml of 4-methylumbelliferyl acetate (1 mg/ml in acetone) for 5 min and then photographed under UV light. Purification of Human Esterase D. Step 1. Outdated human erythrocytes were washed once in phosphate-buffered saline (PBS) with 1 mM EDTA. A precooled (-20°C) mixture of chloroform and butanol was added to the packed cells to concentrations of 6% and 15%, respectively (13). The lysed cells were centrifuged at 23,000 x g for 20 min and >95% of the hemoglobin was pelleted at this step. Buffers used in the subsequent steps all contained 1 mM EDTA, 0.1% 2- mercaptoethanol, and 0.05 mM phenylmethylsulfonyl fluo- ride (PhMeSO2F). The supernatant was then dialyzed over- night against several changes of 10 mM potassium phosphate buffer (pH 6.5). Step 2. Carboxymethylcellulose C52 equilibrated with 10 mM potassium phosphate buffer (pH 6.5) was added to the Abbreviations: PhMeSO2F, phenylmethylsulfonyl fluoride; PMA, phorbol 12-myristate 13-acetate. tTo whom reprint requests should be addressed. 6790 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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Transcript of PNAS-1986-Lee-6790-4
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P r o c . N a t i . A c a d . S c i . USAV o l . 8 3 , p p . 6 7 9 0 - 6 7 9 4 , S e p t e m b e r 1 9 8 6B i o c h e m i s t r y
P u r i f i c a t i o n , b i o c h e m i c a l c h a r a c t e r i z a t i o n , a n d b i o l o g i c a l f u nc t i o n o fhuman e s t e r a s e D
( g e n e t i c m a r k e r / e s t e r a s e a n t i b o d i e s / d e t o x i f i c a t i o n / p h e n o b a r b i t a l I n d u c t i o n )
WEN-HWA L E E * t , W I L L I A M W H E A T L E Y * , W I L L I A M F . B E N E D I C T t , CHUN-MING H U A N G § ,AND EVA Y . - H . P . L E E *
* D e p a r t m e n t o f P a t h o l o g y , U n i v e r s i t y o f C a l i f o r n i a , S a n D i e g o , L a J o l l a , C A 9 2 0 9 3 ; * C l a y t o n M o l e c u l a r B i o l o g y P r o g r a m a n d D i v i s i o n o fH e m a t o l o g y - O n c o l o g y , C h i l d r e n s H o s p i t a l o f L o s A n g e l e s , L o s A n g e l e s , C A 9 0 0 2 7 ; a n d § I n s t i t u t e o f C l i n i c a l M e d i c i n e ,S c h o o l o f M e d i c i n e , N a t i o n a l T a i w a n U n i v e r s i t y , T a i p e i , T a i w a n
C o m m u n i c a t e d b y H a r v e y A . I t a n o , J u n e 9 , 1 9 8 6
ABSTRACT Human e s t e r a s e D ( c a r b o x y l e s t e r a s e ; c a r b o x -y l i c - e s t e r h y d r o l a s e , EC 3 . 1 . 1 . 1 ) , a g e n e t i c m a r k e r o fr e t i n o b l a s t o m a , w a s p u r i f i e d t o b i o c h e m i c a l h o m o g e n e i t y f r o m
e r y t h r o c y t e s . T h e p u r i f i c a t i o n s c h e m e i n c l u d i n g c a r b o x y -m e t h y l c e l l u l o s e , p h e n y l - S e p h a r o s e , c h r o m a t o f o c u s i n g , a n d h y -d r o x y l a p a t i t e c h r o m a t o g r a p h i e s r e s u l t e d i n a 1 0 , 0 0 0 - f o l d p u -r i f i c a t i o n o f t h e e n z y m e w i t h 1 5 % r e c o v e r y o f t o t a l a c t i v i t y .
T h e Km o f e s t e r a s e D w a s e s t i m a t e d t o b e 1 0 x 1 0 - 6 M u s i n g4 - m e t h y l u m b e l l i f e r y l a c e t a t e a s s u b s t r a t e . T h e e n z y m a t i c a c -t i v i t y w a s i n h i b i t e d b y p - c h l o r o m e r c u r i b e n z o a t e a n d H g C I 2 ,
s u g g e s t i n g a n i m p o r t a n t r o l e o f SH g r o u p ( s ) i n e n z y m e f u n c -t i o n . S p e c i f i c r a b b i t p o l y c l o n a l a n d m o u s e m o n o c l o n a l a n t i -b o d i e s a g a i n s t e s t e r a s e D w e r e p r e p a r e d a n d r e c o g n i z e d e i t h e rd e n a t u r e d o r n a t i v e human e s t e r a s e D p r o t e i n . M o r e o v e r , t h ep o l y c o n a l a n t i b o d i e s i m m u n o p r e c i p i t a t e d a p o l y p e p t i d e w i t h a
m o l e c u l a r m a s s o f a b o u t 3 3 - 3 4 k D a f r o m v a r i o u s c e l l l i n e s o fd i f f e r e n t m a m m a l i a n s p e c i e s , i n d i c a t i n g t h a t t h e e s t e r a s e Dp r o t e i n i s h i g h l y c o n s e r v e d . T h e h i g h e s t l e v e l s o f t h i s e n z y m ew e r e f o u n d i n l i v e r a n d k i d n e y . F u r t h e r m o r e , t h e e x p r e s s i o n o fe s t e r a s e D w a s e n h a n c e d 3 - f o l d i n a p r o m o n o c y t i c c e l l l i n et r e a t e d w i t h p h e n o b a r b i t a l b u t n o t w i t h p h o r b o l m y r i s t a t ea c e t a t e , s u g g e s t i n g t h a t e s t e r a s e D m a y h a v e a r o l e i n d e t o x i -f i c a t i o n . T h e a v a i l a b i l i t y o f t h e h o m o g e n e o u s p r o t e i n a n d i t s
s p e c i f i c a n t i b o d i e s a l l o w s f o r c l o n i n g o f t h e e s t e r a s e D g e n e a n df a c i l i t a t e s s t u d i e s o f r e t i n o b l a s t o m a s .
Human e s t e r a s e D ( c a r b o x y l e s t e r a s e ; c a r b o x y l i c - e s t e r h y -d r o l a s e , EC 3 . 1 . 1 . 1 ) i s o n e m e m b e r o f t h e n o n s p e c i f i ce s t e r a s e f a m i l y d e f i n e d b y t h e i r r e a c t i o n w i t h s y n t h e t i c o - a c y le s t e r s u b s t r a t e s ( 1 , 2 ) . T h i s e n z y m e i s d i s t i n g u i s h a b l e f r o mo t h e r e s t e r a s e s b y i t s e l e c t r o p h o r e t i c m o b i l i t y a n d i t s r e l a t i v es p e c i f i c i t y f o r m e t h y l u m b e l l i f e r y l e s t e r s a s s u b s t r a t e ( 2 ) . T h ed i m e r i c e n z y m e d i s p l a y s s e v e r a l p h e n o t y p e s a s a r e s u l t o f t h ee x p r e s s i o n o f c o d o m i n a n t a u t o s o m a l a l l e l e s , p r i m a r i l y E S D Ia n d E S D 2 . T h i s p o l y m o r p h i c n a t u r e o f e s t e r a s e D p r o v i d e s a
v a l u a b l e g e n e t i c m a r k e r f o r s t u d i e s i n p o p u l a t i o n g e n e t i c s ( 2 ,3 ) . S i n c e t h e e s t e r a s e D g e n e h a s b e e n m a p p e d t o t h ec h r o m o s o m e 1 3 q 1 4 . 1 1 r e g i o n w h e r e t h e p u t a t i v e r e t i n o b l a s -
t o m a g e n e i s a l s o l o c a t e d ( 4 - 7 ) , t h e p o l y m o r p h i c n a t u r e o f t h ee s t e r a s e D p r o t e i n b e c o m e s a v e r y u s e f u l g e n e t i c m a r k e r f o rt h e d i a g n o s i s o f h e r e d i t a r y r e t i n o b l a s t o m a s ( 8 , 9 ) . I n a d d i t i o n ,t h e d e f e c t i v e g e n e o f W i l s o n d i s e a s e w a s f o u n d t o b e l i n k e dt o t h e e s t e r a s e D g e n e ( 1 0 ) . T h u s , i t i s i m p o r t a n t t o i n v e s t i g a t et h e n a t u r e o f t h e e s t e r a s e D p r o t e i n a n d i t s g e n e s o a s t ou n d e r s t a n d t h e s e i n h e r i t e d d i s o r d e r s .
E s t e r a s e D h a s b e e n f o u n d i n m o s t t i s s u e s , b u t n o t m u c hi s k n o w n a b o u t i t s s t r u c t u r e a n d f u n c t i o n . A p r o t o c o l f o rp a r t i a l p u r i f i c a t i o n o f t h i s e n z y m e h a s b e e n d e s c r i b e d ( 1 1 ) .H o w e v e r , i t r e m a i n s d i f f i c u l t t o o b t a i n a s u f f i c i e n t a m o u n t o f
t h e h o m o g e n e o u s e n z y m e a n d t o g e n e r a t e s p e c i f i c a n t i b o d -i e s . I n t h i s c o m m u n i c a t i o n , w e d e s c r i b e t h e p u r i f i c a t i o n a n dc h a r a c t e r i z a t i o n o f t h i s e n z y m e a s w e l l a s t h e p r e p a r a t i o n o fe s t e r a s e D - s p e c i f i c p o l y c l o n a l a n d m o no c l o n a l a n t i b o d i e s .F u r t h e r m o r e , we o b s e r v e d t h a t t h i s e n z y m e w a s m a i n l yd i s t r i b u t e d i n l i v e r a n d k i d n e y a n d c o u l d b e i n d u c e d b yt r e a t m e n t w i t h p h e n o b a r b i t a l b u t n o t w i t h p h o r b o l e s t e r .
MATERIALS A ND METHODS
E s t e r a s e D E n z y m a t i c A s s a y s . ( i ) Q u a n t i t a t i v e a s s a y .E s t e r a s e D a c t i v i t y w a s d e t e r m i n e d e s s e n t i a l l y a s d e s c r i b e d( 1 1 ) . T h e r e a c t i o n w a s s t a r t e d b y a d d i n g t h e e n z y m e i n t o 1 m lo f r e a c t i o n b u f f e r c o n t a i n i n g 5 0 mM p o t a s s i u m p h o s p h a t e , 1mM EDTA ( p H 6 . 0 ) , w i t h 0 . 1 mM 4 - m e t h y l u m b e l l i f e r y la c e t a t e a s s u b s t r a t e . T h e i n c r e a s e i n a b s o r b a n c e a t 3 4 0 n mw a s r e c o r d e d . A u n i t o f a c t i v i t y w a s d e f i n e d a s t h e a m o u n to f e n z y m e t h a t h y d r o l y z e d 1 / . k m o l o f s u b s t r a t e p e r m i n a t2 3 0 C u s i n g 7 . 2 7 mM-1 c m - 1 a s t h e c o e f f i c i e n t o f a b s o r p t i o n .
( i i ) S p o t t e s t . A r a p i d s e m i q u a n t i t a t i v e t e s t w a s d e v e l o p e db y u s f o r a s s a y i n g e n z y m e a c t i v i t y i n c o l u m n f r a c t i o n s .B r i e f l y , 5 j u l o f 4 - m e t h y l u m b e l l i f e r y l a c e t a t e ( 2 0 A g / m l ) i ns o d i u m a c e t a t e ( p H 5 . 2 ) w a s s p o t t e d o n t o a p i e c e o fS a r a n W r a p o v e r a U V t r a n s i l l u m i n a t o r t o g e t h e r w i t h 5 A . l o fe n z y m e s a m p l e s f r o m e a c h c o l u m n f r a c t i o n . T h e i n t e n s i t y o ff l u o r e s c e n c e e m i t t e d c o r r e s p o n d e d w i t h t h e e n z y m e a c t i v i t y .
( i i i ) Q u a l i t a t i v e a s s a y . E l e c t r o p h o r e s i s i n 1% a g a r o s e g e l sw a s p e r f o r m e d a s d e s c r i b e d ( 1 2 ) w i t h s l i g h t m o d i f i c a t i o n s .T h e a g a r o s e g e l b u f f e r w a s a 1 : 5 d i l u t i o n o f r u n n i n g b u f f e r t h a tc o n t a i n e d 6 2 mMT r i s , 1 5 . 5 mM c i t r i c a c i d , 1 8 mMb o r i c a c i d ,a n d 1 . 6 5 mM l i t h i u m h y d r o x i d e ( p H 7 . 5 ) . E l e c t r o p h o r e s i sw a s p e r f o r m e d a t 4 0 C f o r 9 0 m i n a t 2 0 V / c m . T h e g e l w a ss t a i n e d i n 50 m l o f 5 0 mM s o d i u m a c e t a t e ( p H 5 . 2 ) c o n t a i n i n g1 . 0 m l o f 4 - m e t h y l u m b e l l i f e r y l a c e t a t e ( 1 m g / m l i n a c e t o n e )f o r 5 m i n a n d t h e n p h o t o g r a p h e d u n d e r U V l i g h t .
P u r i f i c a t i o n o f Human E s t e r a s e D . S t e p 1 . O u t d a t e d h u m a ne r y t h r o c y t e s w e r e w a s h e d o n c e i n p h o s p h a t e - b u f f e r e d s a l i n e( P B S ) w i t h 1 mM EDTA. A p r e c o o l e d ( - 2 0 ° C ) m i x t u r e o fc h l o r o f o r m
a n db u t a n o l
w a s a d d e d t o t h e p a c k e d c e l l s t oc o n c e n t r a t i o n s o f 6% a n d 1 5 % , r e s p e c t i v e l y ( 1 3 ) . T h e l y s e dc e l l s w e r e c e n t r i f u g e d a t 2 3 , 0 0 0 x g f o r 2 0 m i n a n d >95% o f
t h e h e m o g l o b i n w a s p e l l e t e d a t t h i s s t e p . B u f f e r s u s e d i n t h es u b s e q u e n t s t e p s a l l c o n t a i n e d 1 mM EDTA, 0 . 1 % 2 -m e r c a p t o e t h a n o l , a n d 0 . 0 5 mM p h e n y l m e t h y l s u l f o n y l f l u o -r i d e ( P h M e S O 2 F ) . T h e s u p e r n a t a n t w a s t h e n d i a l y z e d o v e r -
n i g h t a g a i n s t s e v e r a l c h a n g e s o f 1 0 mM p o t a s s i u m p h o s p h a t eb u f f e r ( p H 6 . 5 ) .
S t e p 2 . C a r b o x y m e t h y l c e l l u l o s e C 5 2 e q u i l i b r a t e d w i t h 1 0
mM p o t a s s i u m p h o s p h a t e b u f f e r ( p H 6 . 5 ) w a s a d d e d t o t h e
A b b r e v i a t i o n s : PhMeSO2F, p h e n y l m e t h y l s u l f o n y l f l u o r i d e ; P M A ,p h o r b o l 1 2 - m y r i s t a t e 1 3 - a c e t a t e .
t T o w ho m r e p r i n t r e q u e s t s s h o u l d b e a d d r e s s e d .
6 7 9 0
T h e p u b l i c a t i o n c o s t s o f t h i s a r t i c l e w e r e d e f r a y e d i n p a r t b y p a g e c h a r g ep a y m e n t . T h i s a r t i c l e m u s t t h e r e f o r e b e h e r e b y m a r k e d " a d v e r t i s e m e n t "
i n a c c o r d a n c e w i t h 1 8 U . S . C . § 1 7 3 4 s o l e l y t o i n d i c a t e t h i s f a c t .
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P r o c . N a t l . A c a d . S c i . U SA 8 3 ( 1 9 8 6 ) 6 7 9 1
d i a l y z a t e t o a b s o r b t h e r e s i d u a l h e m o g l o b i n . T h e w h o l e
m i x t u r e was f i l t e r e d t h r o u g h a s i n t e r e d g l a s s f u n n e l a n d t h ef i l t r a t e was c o n c e n t r a t e d b y p r e c i p i t a t i o n w i t h 90% s a t u r a t e dammonium s u l f a t e .
S t e p 3 . T h e p r o t e i n p r e c i p i t a t e was c o l l e c t e d b y c e n t r i f u -g a t i o n a t 1 0 , 0 0 0 x g f o r 1 0 m i n a n d r e s u s p e n d e d i n a n d
d i a l y z e d a g a i n s t c o l d d i s t i l l e d w a t e r u n t i l j u s t c l a r i f i e d . T h e
p r o t e i n s a m p l e was l o a d e d o n t o a p h e n y l - S e p h a r o s e C L - 4 B
c o l u m n ( 5 x 2 7 . 5 c m ) e q u i l i b r a t e d w i t h 5 0 mM T r i s HC l
b u f f e r ( p H 8 . 0 ) a n d 1 M ammonium s u l f a t e . T h e c o l u m n was
w a s h e d e x t e n s i v e l y w i t h t h e same b u f f e r a n d t h e n e l u t e d w i t h
a d e c r e a s i n g g r a d i e n t o f ammonium s u l f a t e ( 1 . 0 - 0 M ) i n 5 0
mM T r i s HCl b u f f e r ( p H 8 . 0 ) .S t e p 4 . Th e f r a c t i o n s w i t h e s t e r a s e a c t i v i t y f r o m s t e p 3
w e r e c o n c e n t r a t e d b y p r e c i p i t a t i o n w i t h 90o s a t u r a t e d a m -
monium s u l f a t e , r e s u s p e n d e d i n 2 5 mM i m i d a z o l e b u f f e r ( p H
7 . 4 ) , a n d t h e n d i a l y z e d a g a i n s t t h e s a m e b u f f e r . T h e s a m p l e
was l o a d e d o n t o a c h r o m a t o f o c u s i n g c o l u m n ( 1 . 0 x 6 2 c m ) o f
P h a r m a c i a P o l y b u f f e r E x c h a n g e r 9 4 g e l e q u i l i b r a t e d w i t h t h e
sam e b u f f e r a n d e l u t e d w i t h P o ly b u f f e r 7 4 ( p H 4 . 0 ) .S t e p 5 . T h e f r a c t i o n s w i t h e s t e r a s e D a c t i v i t y f r o m t h e
c h r o m a t o f o c u s i n g c o l u m n w ere p o o l e d , c o n c e n t r a t e d w i t h
ammonium s u l f a t e , d i a l y z e d a g a i n s t 1 0 mM p o t a s s i u m p h o s -
p h a t e b u f f e r ( p H 7 . 0 ) , a n d l o a d e d o n t o a c o l u m n o f h y d r o x -
y l a p a t i t e ( 1x
9 . 5 c m ) e q u i l i b r a t e dw i t h
t h es a m e b u f f e r . T h e
e nzym e was t h e n e l u t e d w i t h a g r a d i e n t o f ammonium s u l f a t e( 0 - 0 . 4 M) i n t h e same b u f f e r .
P u r i t y o f e s t e r a s e D was m o n i t o r e d b y N a D o d S O 4 / p o l y -
a c r y l a m i d e g e l e l e c t r o p h o r e s i s a c c o r d i n g t o t h e p r o c e d u r e s o f
L a e m m l i ( 1 4 ) . P r o t e i n s w er e s t a i n e d w i t h e i t h e r C o o m a s s i e
b r i l l i a n t b l u e or s i l v e r . P r o t e i n was q u a n t i t a t e d b y t h e m e t h o d
o f Lowry e t a l . ( 1 5 ) w i t h b o v i n e serum a l b u m i n as t h es t a n d a r d .
P r e p a r a t i o n o f A n t i bo d i e s A g a i n s t E s t e r a s e D . ( i ) P o l y c l o n a l
a n t i b o d i e s . Ne w Z e a l a n d Re d r a b b i t s w er e i n j e c t e d w i t h 1 0
j i g o f p u r i f i e d e s t e r a s e D p r o t e i n m i x e d w i t h c o m p l e t e
F r e u n d ' s a d j u v a n t a n d t h e n g i v e n b o o s t e r i n j e c t i o n s o f 1 0 , u go f e s t e r a s e D p r o t e i n i n i n c o m p l e t e F r e u n d ' s a d j u v a n t u n t i lh i g h t i t e r s o f a n t i b o d y w e r e d e t e c t e d b y i m m u n o b l o t t i n g
a n a l y s i s ( 1 6 ) .
( i i ) M o n o c l o n a l a n t i b o d i e s . A n t i b o d y - p r o d u c i n g h y b r i d o -ma c e l l l i n e s w ere g e n e r a t e d as d e s c r i b e d ( 1 7 ) . Tw o h y b r i d -oma c l o n e s , T D - 1 a n d T D - 2 , w e r e i s o l a t e d , b o t h o f w h i c h
s e c r e t e d h i g h t i t e r s o f a n t i b o d i e s a g a i n s t e s t e r a s e D .
I m m u n o p r e c i p i t a t i o n . A s t a n d a r d p r o t o c o l was f o l l o w e d as
d e s c r i b e d ( 1 8 ) .
RE S U L T S
P u r i f i c a t i o n o f E s t e r a s e D . T h e p u r i f i c a t i o n s c h e m e f o re s t e r a s e D f r o m human e r y t h r o c y t e s i s s u m m a r i z e d i n T a b l e
1 . A t e a c h p u r i f i c a t i o n s t e p e s t e r a s e D was m o n i t o r e d b y b o t h
agarose g e l a n d N a D o d S O 4 / p o l y a c r y l a m i d e g e l e l e c t r o p h o -r e s i s ( F i g . 1 ) . Tw o s t e p s , c h l o r o f o r m / b u t a n o l e x t r a c t i o n a n d
c a r b o x y m e t h y l - c e l l u l o s e a b s o r p t i o n , w er e necessary t o r e -
move a l l h e m o g l o b i n f r o m t h e p r e p a r a t i o n . T h e r e m a i n i n g
p r o t e i n s w ere s e p a r a t e d b a s e d on t h e i r h y d r o p h o b i c n a t u r eb y p h e n y l - S e p h a r o s e c h r o m a t o g r a p h y . P e a k a c t i v i t y o f
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t i a l p u r i f i c a t i o n s t e p s o f e s t e r a s e D . L a n e s : 1 , e r y t h r o c y t e l y s a t e ( 2 5, u g ) ; 2 , s u p e r n a t a n t o f c h l o r o f o r m / b u t a n o l e x t r a c t i o n ( 1 0 , u g ) ; 3 ,p r o t e i n s n o t b o u n d t o C M - c e l l u l o s e ( 5 , u g ) ; 4 , p o o l e d f r a c t i o n s w i t h
enzyme a c t i v i t y a f t e r p h e n y l - S e p h a r o s e c o l u m n ( 2 . 5 , . t g ) ; 5 , p e a k
f r a c t i o n s o f c h r o m a t o f o c u s i n g c o l u m n ( 1 , u g ) ; 6 , p e a k f r a c t i o n s o f
h y d r o x y l a p a t i t e c o l u m n ( 1 j g ) . B o v i n e serum a l b u m i n , o v a l b u m i n ,
g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e , c a r b o n i c a n h y d r a s e ,
t r y p s i n o g e n , s o y b e a n t r y p s i n i n h i b i t o r , a n d l a c t a l b u m i n w er e u s e d as
m o l e c u l a r s i z e m a r k e r s . ( B ) A g a r o s e g e l e l e c t r o p h o r e s i s t o d i s t i n -g u i s h e s t e r a s e D f r o m o t h e r e s t e r a s e s . L a n e s : 1 , s u p e r n a t a n t o f
c h l o r o f o r m / b u t a n o l e x t r a c t i o n ; 2 , f r a c t i o n n o t b o u n d t o C M - c e l l u -
l o s e ; 3 , f r a c t i o n s f r o m t h e p h e n y l - S e p h a r o s e c o l u m n ; 4 , f r a c t i o n sf r o m t h e c h r o m a t o f o c u s i n g c o l u m n ; 5 - 7 , f r a c t i o n s o f e s t e r a s e D a f t e r
h y d r o x y l a p a t i t e ; 8 a n d 9 , f r a c t i o n s o f e s t e r a s e X a f t e r h y d r o x y l a p -a t i t e . A r r o w s i n d i c a t e t h e m i g r a t i o n o f e s t e r a s e s D a n d X i n t h i s g e l .
e s t e r a s e D was e l u t e d i n t h e range o f 0 . 5 M ammonium s u l f a t ewhen a n ammonium s u l f a t e g r a d i e n t ( 1 - 0 M ) was u s e d ( F i g .LA, l a n e 4 ) . T h e s p o t t e s t was u s e d t o q u i c k l y d e t e c t f r a c t i o n sc o n t a i n i n g e s t e r a s e a c t i v i t y . T h e p o s i t i v e f r a c t i o n s w ere
f u r t h e r s u b j e c t e d t o agarose g e l e l e c t r o p h o r e s i s . Tw o f o r m s
o f e s t e r a s e s w i t h d i f f e r e n t m o b i l i t i e s w ere f o u n d ( F i g . 1 B ) .F u r t h e r p u r i f i c a t i o n b y c h r o m a t o f o c u s i n g c h r o m a t o g r a p h y
s e p a r a t e d t h e two a c t i v i t i e s f r o m e a c h o t h e r ( F i g . 2 A ) . Th e
f a s t e r m i g r a t i n g f o r m h a d a m o l e c u l a r mass o f 3 0 k Da i n
NaDodSO4/PAGE. T h i s p r ev i o u s l y unknown e s t e r a s e was
d e s i g n a t e d e s t e r a s e X . T h e s l o w e r m i g r a t i n g f o r m w i t h a
m o l e c u l a r mass o f 3 4 kDa was e s t e r a s e D , w h i c h was
c o n f i r m e d b y e l e c t r o p h o r e s i s i na
s t a r c h g e l ( d a t an o t
s h o w n ) .To remove m i n o r c o n t a m i n a t i ng p r o t e i n s i n t h e e s t e r a s e D
p o o l , s a m p l e s w er e f u r t h e r s u b j e c t e d t o h y d r o x y l a p a t i t e
c h r o m a t o g r a p h y a n d e l u t e d w i t h a g r a d i e n t o f 0 - 0 . 4 M
ammonium s u l f a t e ( F i g . 2 B ) . E s t e r a s e D e l u t e d as a s i n g l ep e a k a n d a p p e a r e d t o b e h o m o g e n e o u s as j u d g e d b y
NaDodSO4/PAGE ( F i g . 1 , l a n e s 6 ) . T h e p u r i t y o f t h i s
p r e p a r a t i o n was f u r t h e r c o n f i r m e d b y sequence a n a l y s i s ( d a t an o t s h o w n ) . U s i n g t h e p r e s e n t p r o c e d u r e s , we a c h i e v e d a
1 0 , 6 5 3 - f o l d p u r i f i c a t i o n o f e s t e r a s e D w i t h 1 3 % recovery. Th e
t o t a l e s t e r a s e D p r o t e i n i n e r y t h r o c y t e s i s = = 0 . 0 1 % .
B i o c h e m i c a l P r o p e r t i e s o f t h e P u r i f i e d E n z y m e . ( i ) Enzymek i n e t i c s . To f u r t h e r c h a r a c t e r i z e e s t e r a s e D , we m e a s u r e d t h e
Km o f t h i s p u r i f i e d e s t e r a s e D enzyme. T h e Km o f e s t e r a s e D
was d e t e r m i n e d t o b e 1 0 x 1 0 - 6 M b y L i n e w e a v e r - B u r k p l o t su s i n g 4 - m e t h y l u m b e l l i f e r y l a c e t a t e as t h e s u b s t r a t e . I n con-
t r a s t , t h e Km was 1 . 7 mM i f n a p h t h a l a c e t a t e was u s e d as t h es u b s t r a t e . T h i s 1 0 0 - t o 2 0 0 - f o l d d i f f e r e n c e i n s u b s t r a t e a f f i n i t y
T a b l e 1 . P u r i f i c a t i o n o f h u m a n e s t e r a s e D t y p e I
E s t e r a s e T o t a l S p e c i f i ca c t i v i t y , p r o t e i n , a c t i v i t y , P u r i f i c a t i o n ,
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E r y t h r o c y t e l y s a t e 3 6 1 . 4 2 1 1 , 2 0 0 0 . 0 0 1 7 1 0 0 1
C h l o r o f o r m / b u t a n o l e x t r a c t 3 1 2 . 6 1 1 , 4 3 1 0 . 0 2 7 3 8 6 . 5 1 6
C a r b o x y m e t h y l c e l l u l o s e 2 5 0 . 6 2 , 8 3 5 0 . 0 8 8 4 6 9 5 2
P h e n y l - S e p h a r o s e 8 4 . 2 4 4 0 . 8 0 . 1 9 1 0 2 3 1 1 2 . 4
C h r o m a t o f o c u s i n g 7 0 . 8 3 2 . 9 8 2 . 1 5 1 9 . 6 1 , 2 6 5H y d r o x y l a p a t i t e 4 7 . 2 2 . 6 0 1 8 . 1 1 1 3 1 0 , 5 6 3
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F I G . 2 . C o l u m n c h r o m a t o g r a p h y p r o f i l e s o f e s t e r a s e D . ( A ) F r a c t i o n s f r o m t h e p h e n y l - S e p h a r o s e c o l u m n c o n t a i n i n g e n z y m a t i c a c t i v i t y werec o n c e n t r a t e d a n d c h r o m a t o g r a p h e d on a c h r o m a t o f o c u s i n g c o l u m n ( 1 . 0 x 6 2 c m ) i n 2 5 mM i m i d a z o l e b u f f e r ( p H 7 . 4 ) a n d e l u t e d w i t h p o l y b u f f e r
7 4 ( p H 4 . 0 ) . E a c h f r a c t i o n c o n t a i n i n g 3 . 7 m l w a s c o l l e c t e d . ( B ) P r o t e i n s w i t h e nzym e a c t i v i t y f r o m A w er e s e p a r a t e d on a h y d r o x y l a p a t i t e c o l u m ne q u i l i b r a t e d w i t h 1 0 mM p o t a s si u m p h o s p h at e b u f f e r ( p H 7 . 0 ) a n d e l u t e d w i t h a 0 - 0 . 4 M ammonium s u l f a t e g r a d i e n t ; 2 m l pe r f r a c t i o n wasc o l l e c t e d . T h e a c t i v e enzyme p e a k w a s d e t e c t e d b y s p o t t e s t a n d q u a n t i t a t i v e assay as d e s c r i b e d i n t h e t e x t .
i s c o n s i s t e n t w i t h t h e o r i g i n a l o b s e rv a t i o n o f H o p k i n s o n e t a l .( 1 ) , d e m o n s t r a t i n g t h a t e s t e r a s e D i s r a t h e r s p e c i f i c f o ru m b e l l i f e r y l a c e t a t e e s t e r s .
( i i ) E n z y m e i n h i b i t o r s . S e v er a l c h e m i c a l s s u c h as m e r c u r i c
c h l o r i d e , p - c h l o r o m e r c u r i b e n z o a t e , s o d i u m f l u o r i d e , a n dPhMeSO2F as w e l l as s p e c i f i c a n t i b o d i e s w er e u s e d t o t e s t f o ri n t e r f e r e n c e w i t h t h e e n z y m a t i c a c t i v i t y o f p u r i f i e d e s t e r a s e
D ( s e e T a b l e 2 ) . T h e e n z y m a t i c a c t i v i t y w a s i n h i b i t e dc o m p l e t e l y b y H g C l 2 a n d p c m b a t very l o w c o n c e n t r a t i o n s ,s u g g e s t i n g t h a t a n S H group i s e s s e n t i a l t o t h e e n z y m a t i cr e a c t i o n . H o w e v e r , o n l y a t h i g h c o nc e nt r a t i o ns c o u l d NaF
a n d PhMeSO2F i n t e r f e r e w i t h t h e enzyme a c t i v i t y . T h ea n t i b o d i e s we p r e p a r e d d i d n o t a f f e c t t h e e n z y m a t i c a c t i v i t yeven a t h i g h m o l a r r a t i o s o f a n t i b o d y t o enzyme.
P o l y c l o n a l a n d M o n o c l o n a l A n t i b o d i e s A g a i n s t E s t e r a s e D .R a b b i t a n t i - e s t e r a s e D I g G w a s p u r i f i e d b y e s t e r a s e D a f f i n i t yc o l u m n c h r o m a t o g r a p h y or p r o t e i n A - S e p h a r o s e c o l u m n
c h r o m a t o g r a p h y a n d u s e d i n s u b s e q u e n t e x p e r i m e n t s . T h e
I g G r e c o g n i z e d t h e d e n a t u r e d e s t e r a s e D p r o t e i n i n i m m u -
n o b l o t t i n g ( F i g . 3 B ) as w e l l as t h e n a t i v e p r o t e i n f o l l o w i n gi m m u n o p r e c i p i t a t i o n ( F i g . 3 D ) . T h e i m m u n o c o m p l e x o f t h i sI g G a n d t h e e s t e r a s e D enzyme r e m a i n s a c t i v e i n h y d r o l y s i so f 4 - m e t h y l u m b e l l i f e r y l a c e t a t e , w h i c h f u r t h e r d e m o n s t r a t e s
t h e s p e c i f i c i t y o f t h e I g G ( F i g . 3 E ) . Tw o h y b r i d o m a c e l l l i n e s ,T D - 1 a n d T D - 2 , w h i c h p r o d u c e d h i g h t i t e r s o f a n t i b o d i e s
a g a i n s t human e s t e r a s e D p r o t e i n , w er e a l s o s p e c i f i c a l l yr e c o g n i z e d b y d e n a t u r e d human e s t e r a s e D p r o t e i n i n i m m u -
n o b l o t t i n g ( F i g . 3 C ) .C o n s e r v a t i o n o f E s t e r a s e D . T h e presence o f e s t e r a s e D
p r o t e i n w a s e x a m i n e d i n c e l l s f r o m d i f f e r e n t s p e c i e s o f
m a m m a l s . M o u s e NI H 3 T 6 f i b r o b l a s t s , r a t - 2 f i b r o b l a s t s ,C h i n e s e h a m s t e r o v a r i a n c e l l s , t h e C OS c e l l l i n e o f m o n k e yf i b r o b l a s t s , a n d a human p r e m o n o c y t i c c e l l l i n e ( U 9 3 7 ) w er e
l a b e l e d w i t h [ 3 5 S ] m e t h i o n i n e a n d i m m u n o p r e c i p i t a t e d w i t h
t h e r a b b i t p o l y c l o n a l I g G . As s h o w n i n F i g . 3 D , a s i n g l ep o l y p e p t i d e w i t h a m o l e c u l a r m ass o f 3 3 - 3 4 kDa was f o u n di n a l l s p e c i e s . M o r e o v e r , e n z y m a t i c a c t i v i t y a s s o c i a t e d w i t ht h e i m m u n o p r e c i p i t a t e s d e r i v e d f r o m t h e s e c e l l l i n e s was a l s oo b s e r v e d b y t h e s p o t t e s t ( d a t a n o t s h o w n ) , i n d i c a t i n g t h a te s t e r a s e D enzyme i s c o n s e r v e d i n m a m m a l s .
T i s s u e D i s t r i b u t i o n o f E s t e r a s e D . A l t h o u g h e s t e r a s e D i sknown t o b e d i s t r i b u t e d w i d e l y i n d i f f e r e n t t i s s u e s ( 1 ) ,
d e t a i l e d q u a n t i t a t i v e d i s t r i b u t i o n h a s y e t t o b e s h o w n . Wep e r f o r m e d q u a n t i t a t i v e c o m p a r i s o n s b y t w o d i f f e r e n t m e a -
s u r e m e n t s . F i r s t , a l i q u o t s o f d i f f e r e n t r a t t i s s u e e x t r a c t s w i t h
e q u a l a m o u n t s o f p r o t e i n w e r e a s s a y e d f o r e s t e r a s e De n z y m a t i c a c t i v i t y u s i n g 4 - m e t h y l u m b e l l i f e r y l a c e t a t e a ss u b s t r a t e . A s s h o w n i n T a b l e 3 , l i v e r a n d k i d n e y h a d 1 0 - t o6 - f o l d h i g h e r e n z y m a t i c a c t i v i t y , r e s p e c t i v e l y , t h a n o t h e r
T a b l e 2 . E f f e c t o f a n t i b o d i e s * a n d i n h i b i t o r s t o n e s t e r a s eD a c t i v i t y
A n t i b o d y / % r e m a i n i n g a c t i v i t ye n z y m e R a b b i t T D - 1 T D - 2
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* P u r i f i e d e s t e r a s e D ( 0 . 5 4 A g ) was m i x e d w i t h a n t i b o d y a t v a r i o u sm o l a r r a t i o s i n a t o t a l v o l o f 1 1 0 A l . A f te r 4 5 m i n i n c u b a t i o n o n i c e ,d u p l i c a t e 5 0 - M l s a m p l e s w e r e a s s a y e d .
t p - C h l o r o m e r c u r i b e n z o a t e ( P C M B ) a n d P h M e S O 2 F w e r e m i x e dw i t h 0 . 5 4 M x g o f p u r i f i e d e s t e r a s e D i n a t o t a l v o l o f 1 1 0 M l ( 1 0 mMp o t a s s i u m p h o s p h a t e , pH 7 . 0 / 1 mM E D T A / 0 . 1 % m e r c a p t o e t h a n o l )a n d t h e n i n c u b a t e d f o r 1 5 m i n a t 3 7 ° C b e f o r e a s s a y i n g . F o r t h e s ec o m p o u n d s , v a l u e s a r e e x p r e s s e d r e l a t i v e t o e n z y m e a l o n e i n c u -b a t e d a t 3 7 ° C f o r 1 5 m i n . H g C l 2 a n d Na F w e r e i nc u ba t ed i n t h es t a n d a r d a s s a y m i x t u r e a n d a c t i v i t y w a s m o n i t o r e d i m m e d i a t e l ya f t e r a d d i n g e n z y m e . D u p l i c a t e m e a s u r e m e n t s w e r e m a d e f o r e a c hp o i n t . N D , n o t d o n e .
6 7 9 2 B i o c h e m i s t r y : L e e e t a l .
7/28/2019 PNAS-1986-Lee-6790-4
http://slidepdf.com/reader/full/pnas-1986-lee-6790-4 4/5
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F I G . 3 . S p e c i f i c i t y Q t f r a b b i t p o l y c l o n a l a n d mouse m o n o c l o n a la n t i b o d i e s . T h r e e s e t s o f p a r t i a i l y p u r i f i e d e s t e r a s e D p r e p a r a t i o nwere e l e ' c t r o p h o r e s e d on a 1 2 . 5 % s l a b g e l . , O n e s e t w as s t a i n e d w i t h
C o o m a s s i e b l u e ( A ) . T h e r e s t was e l e c t r o b l o t t e d t o a n i t r o c e l l u l o s ef i l t e r . O n e s t r i p w as i n c u b a t e d w i t h r a b b i t p o l y c l o n a l a n t i b o d i e s ( B )a n d t h e o t h e r w as i n c u b a t e d W i t h t h e mouse m o n o c l o n a l a n t i b o d yT D - 1 ( C ) . B o t h s t r i p s w e r e f u r t h e r i n c u b a t e d w i t h ` I 5 - l a b e l e d p r o t e i nA a n d e x p o s e d on K o d a k XAR5 f i l m . L a n e s : a , p r o t e i n s a f t e rp h e n y l - S e p h a r o s e ; b , a f t e r c h r o m a t o f o c u s i n g ; C , a f t e r h y d r o x y l a p -a t i t e , a s d e s c r i b e d i n F i g . 1 . ( D ) I m m u n o p r e c i p i t a t i o n b y r a b b i tp o l y c l o n a l a n t i b o d i e s o f e s t e r a s e D l a b e l e d w i t h I Y S ' m e t h i o n i n ef r o m d i f f e r e n t c e l l l i n e s o f s e v e r a l m a m m a l i a n s p e c i e s . T h e p r e c i p -i t a t e d p r o t e i n s were a n a l y z e d on a 1 2 . 5 % p o l y a c r y l a m i d e g e l f o l -l o w e d b y f l u o r o g r a p h y . L a n e s : a , C HO c e l l s ( h a m s t e r ) ; b , 3 T 6 c e l l s( m o u s e ) ; c , r a t - ~ 2 c e l l s ( r a t ) ; d , COS c e l l s ( m o n k e y ) ; e , U 9 3 7 c e l l s( h u m a n ) . I n a d d i t i o n t o t h e e s t e r a s e D p r o t e i n o f 3 3 - 3 4 k t b a ( m a r k e dw i t h a r r o w ) , some u n k n o w n p r o t e i n s w e r e a l s o c o p r e c i p i t a t e d . ( E )E n z y m a t i c t a c t i v i t y w i t h i m m u n o c o m p l e ' x o f i m m u n i z e d r a b b i t I g Ga n d e s t e r a s e D ) e n z y m e . An e q u a l a m o u n t o f e s t e r a s e D e n z y m e ( 0 . 5, u & g ) w as i n c u b a t e d w i t h a s e r i e s o f d i f f e r e n t q u a n t i t i e s o f I g G ( a , 2 4
A . g ; b , 1 2 u . g ; c , 6 A . g ; d , 3 A . g ) a n d p r e c i p i t a t e d . w i t h p r o t e i nA - S e p h a r o s e b e a d s . A f t e r s e v e r a l c y c l e ' s o f w a s h i n g , t h e ' S e p h a r o s eb e a d s w e r e s p o t t e d w i t h 4 - m e t h y l u m b e l l i f e r y l a c e t a t e a n d p h o t o -g r a p h e d u n d e r U V l i g h t . T h e f o u r c o n t r o l s w e r e a s f o l l o w s ; e , n o r m a lr a b b i t - I g G ; f , b e a d s a l o n e ; g , b e a d s i n c u b a t e d w i t h e n z y m e w i t h o u tt h e I g G ; h , 0 . 5 A g o f e s t e r a s e D e n z y m e .
t i s s u e s . S e c o n d , t h e same a m o u n t s o f p r o t e i n e x t r a c t were
e l e c t r o p h o r e s e d t h r o u g h 1 % a g a r o ' s e g e l a n d s t a i n e d w i t h t h es u b s t r a t e . B r i l l i a n t s p o t s r e p r e s e n t i n g t h e e s t e r a s e D a c t i v i t ywere o b s e r v e d i n l i v e r a n d k i d n e y e x t r a c t s ( d a t a n o t s h o w n ) .T h e s e r e s u l t s d e m o n s t r a t e d t h a t l i v e r a n d k i d n e y c o n t a i n e d
t h e most e s t e r a s e D .
E s t e r a s e D I n d u c t i o n b y P h e n o b a r b i t a l . E s t e r a s e D ) i s s o f a rn o t known t o h a v e a n y b i o l o g i c a l f u n c t i o n . H o w e v e r , our
r e s u l t s t h a t e s t e r a s e D w as a h i g h l y c o n s e r v e d p r o t e i n a n d
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Sam l e s o f v a r i o u s t i s s u e s w e r e h o m o g e n i z e d o n i c e i n 2 m l o f 2 5mM T r i s - H C I l , p H 7 . 4 / 5 0 mM N a C l / 0 . 2 % N o n i d e t P - 4 0 / 0 . 5 %d e o x y c h o l a t e / 1 mM P h M e S O 2 F . E x t r a c t s w e r e c l a r i f i e d b y c e n t r i -f u g i n g a t 1 0 , 0 0 0 x g f o r 1 5 m i a t 4 0 C , a n d t h e s u p e r n a t a n t s w e r e u s e d
f o r t h e s e e x p e r i m e n t s a s d e s c r i b e d i n t h e t e x t .
d i s t r i b u t e d w i t h t h e h i g h e s t l e v e l i n l i v e r a n d k i d n e y s u g g e s tt h a t i t m a y h a v e a r o l e i n d e t o x i f i c a t i o n . T o t e s t t h i sp o s s i b i l i t y , p h e n o b a r b i t a l w a s u s e d t o i n d u c e e s t e r a s e De x p r e s s i o n i n a p r o m o n o c y t i c c e l l l i n e , U 9 3 7 , w h i c h w a sr e c e n t l y f o u n d t o s y n t h e s i z e l a r g e q u a n t i t i e s . d f e s t e r a s e Dp r o t e i n w h e n c o m p a r e d w i t h s e v e r a l o t h e r c e l l l i n e s , i n c l u d -i n g a h u m a n h e p a t o m a , a r e t i n o b l a s t o m a ( Y 7 9 ) , a n e u r o b l a s -t o m a ( L A - N - 5 ) , a n d f i b r o b l a s t s ( u n p u b l i s h e d o b s e r v a t i o n s ) .A s s h o w n i n F i g . 4 A a n d B , s y n t h e s i s o f e s t e r a s e D . p r o t e i nw a s i n d u c e d 3 - f o l d o v e r b a c k g r o u n d i n c e l l s a f t e r 4 8 h r o ft r e a t m e n t w i t h 0 . 1 mM p h e n o b a r b i t a l . E s t e r a s e D l e v e l sg r a d u a l l y r e t u r n e d t o n o r m a l a f t e r 9 6 h r o f e x p o s u r e t o t h ed r u g . When c e l l s w e r e t r e a t e d w i t h 2 mM p h e n o b a r b i t a l , w ec o u l d n o t o b s e r v e a n y i n d u c t i o n u n t i l 9 6 h r l a t e r , b e c a u s es i g n i f i c a n t c e l l d e a t h o c c u r r e d a t t h i s d o s e . I n a d d i t i o n t o t h ei n d u c t i o n o f t h e s y n t h e s i s o f e s t e r a s e D p r o t e i n , t o t a l e n z y -
m a t i c a c t i v i t y w a s i n c r e a s e d a b o u t 5 0 % a f t e r 0 . 1 mM p h e -n o b a r b i t a l t r e a t m e n t ( d a t a n o t s h o w n ) . To t e s t w h e t h e r t h ee n z y m e w a s a f f e c t e d b y o t h e r b i o l o g i c a l l y a c t i v e c o m p o u n d s ,w e t r e a t e d U 9 3 7 c e l l s w i t h p h o r b o l 1 2 - m y r i s t r a t e 1 3 - a c e t a t e( P M A ) , w h i c h i s k n o w n t o i n d u c e t h e d i f f e r e n t i a t i o n o f U 9 3 7c e l l s i n t o m o n o c y t i c p h a g o c y t e s . A s s h o w n i n F i g . 4 C ,s y n t h e s i s o f e s t e r a s e D p r o t e i n w a s n o t s i g n i f i c a n t l y c h a n g e di n c e l l s t r e a t e d w i t h t w o d i f f e r e n t d o s e s o f PMA f o r 9 6 h r ,
e v e n t h o u g h t h e c e l l s h a d a l r e a d y d i f f e r e n t i a t e d i n t om o n o c y t e s a f t e r a 2 4 - h r t r e a t m e n t . T h e s e r e s u l t s i n d i c a t e dt h a t i n d u c t i o n o f e s t e r a s e D s y n t h e s i s w a s s p e c i f i c , o r l i m i t e dt o , t o x i c c o m p o u n d s s u c h a s p h e n o b a r b i t a l .
DISCUSSION
T h e r e a r e a t l e a s t e i g h t o r n i n e d i f f e r e n t n o n s p e c i f i c e s t e r a s e s
i n h u m a n t i s s u e s ( 2 ) . P u r i f i c a t i o n o f a n i n d i v i d u a l s p e c i f i ce s t e r a s e h a s b e e n h a m p e r e d b e c a u s e o f t h e l a c k o f a s p e c i f i ca s s a y . S i n c e e s t e r a s e D h a s b e e n s h o w n t o h y d r o l y z e4 - m e t h y l u m b e l l i f e r y l a c e t a t e i n a s p e c i f i c m a n n e r , a p a r t i a l
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a b c d e f g h i j k I__F I G . 4 . I n d u c t i o n o f e s t e r a s e D i n U 9 3 7 c e l l s t r e a t e d w i t h
p h e n o b a r b i t a l ( A ) o r P M A ( C ) . I m m u n o p r e c i p i t a t e s p r e p a r e d u s i n g
r a b b i t a n t i - e s t e r a s e D a n t i b o d i e s o f 3 5 S - l a b e l e d e s t e r a s e D p r o t e i nf r o m U 9 3 7 c e l l s w e r e a n a l y z e d a n d p r o c e s s e d a s s h o w n i n F i g . 3 C .L a n e s a , d , g , a n d j i n A a n d C w e r e d e r i v e d f r o m c e l l s t r e a t e d w i t h
s o l v e n t a s c o n t r o l f o r 2 4 , 4 8 , 7 2 , a n d 9 6 h r , r e s p e c t i v e l y . L a n e s b , e ,h , a n d k w e r e d e r i v e d f r o m c e l l s t r e a t e d w i t h 0 . 1 mM p h e n o b a r b i t a l( A ) o r PMA a t 0 . 1 u g / m l ( C ) f o r 2 4 , 4 8 , 7 2 , a n d 9 6 h r , r e s p e c t i v e l y .L a n e s c , f , i , a n d 1 w e r e d e r i v e d f r o m c e l l s t r e a t e d w i t h 2 mMp h e n o b a r b i t a l ( A ) o r P M A a t 1 A g / m l ( C ) f o r 2 4 , 4 8 , 7 2 , a n d 9 6 h r ,r e s p e c t i v e l y . T h e 3 5 S - l a b e l e d e s t e r a s e D p r o t e i n s f r o m A were
e x c i s e d , c o u n t e d , a n d p l o t t e d a s p e r c e n t a g e o f c o n t r o l n o r m a l i z e dw i t h t h e c o p r e c i p i t a t e d p r o t e i n o f 4 4 kDa ( B ) . * , C e l l s t r e a t e d w i t h
0 . 1 mM p h e n o b a r b i t a l ; o , c e l l s t r e a t e d w i t h 2 mM p h e n o b a r b i t a l .D o t t e d l i n e i n d i c a t e s c o n t r o l c e l l s .
B i o c h e m i s t r y : L e e e t a l .
7/28/2019 PNAS-1986-Lee-6790-4
http://slidepdf.com/reader/full/pnas-1986-lee-6790-4 5/5
P r o c . N a t l . A c a d . S c i . U S A 8 3 ( 1 9 8 6 )
p u r i f i c a t i o n o f t h i s e n z y m e h a s b e e n r e p o r t e d ( 1 1 ) . H o w e v e r ,a t t e m p t s t o p u r i f y t h i s p r o t e i n f o l l o w i n g t h e p u b l i s h e d p r o -t o c o l o n l y r e s u l t e d i n a n e n z y m e p u r i t y o f 3 0 - 4 0 % . I nc o n t r a s t , o u r c u r r e n t p r o t o c o l f o r p u r i f y i n g h u m a n e s t e r a s e Df r o m o u t d a t e d e r y t h r o c y t e s r e s u l t e d i n a 1 0 , 0 0 0 - f o l d p u r i f i -c a t i o n w i t h a 1 3 % y i e l d o f a c t i v i t y . T h e a c t u a l v a l u e may e v e n
b e h i g h e r , b e c a u s e s a m p l e s o b t a i n e d i n t h e e a r l y s t e p s o f t h ep u r i f i c a t i o n may c o n t a i n o t h e r e s t e r a s e s t h a t a l s o a c t o n
4 - m e t h y l u m b e l l i f e r y l a c e t a t e . T h e p u r i f i e d e s t e r a s e D b e -h a v e d a s a s i n g l e b a n d i n NaDodSO4/PAGE s t a i n e d e i t h e rw i t h C o o m a s s i e b l u e o r s i l v e r , s u g g e s t i n g t h a t o u r p r e p a r a -t i o n o f t h e e n z y m e w a s b i o c h e m i c a l l y h o m o g e n e o u s . Amongt h e l a r g e f a m i l y o f h u m a n e s t e r a s e s , e s t e r a s e D , i n a d d i t i o nt o B 4 ( 2 3 ) , i s t h e s e c o n d e n z y m e t h a t h a s b e e n p u r i f i e d t o t h i sd e g r e e o f p u r i t y . I n t e r e s t i n g l y , a n o t h e r e s t e r a s e , e s t e r a s e X ,
w i t h a m o l e c u l a r m a s s o f 3 0 k D a , w a s p a r t i a l l y p u r i f i e d b y t h i sp r o t o c o l .We e x p e r i e n c e d d i f f i c u l t y i n o b t a i n i n g r a b b i t p o l y c l o n a l
a n t i b o d i e s s p e c i f i c f o r t h e e s t e r a s e . D p r o t e i n . O n l y a f t e rs e v e r a l m o n t h s o f b o o s t e r i n j e c t i o n s o f p u r i f i e d e s t e r a s e Dp r o t e i n w e r e w e a b l e t o g e n e r a t e s p e c i f i c a n t i b o d i e s . U s i n gt h i s n e W l y a v a i l a b l e r a b b i t a n t i - e s t e r a s e D a n t i b o d y , we w e r e
a b l e t o s p e c i f i c a l l y i m m u n o p r e c i p i t a t e a p r o t e i n c o r r e s p o n d -i n g t o h u m a n e s t e r a s e D f r o m m o u s e , r a t , h a m s t e r , a n d
m o n k e y c e l l s , i n d i c a t i n g t h a t e s t e r a s e D i s h i g h l y c o n s e r v e di n e v o l u t i o n a n d may s u b s e r v e a c r i t i c a l p h y s i o l o g i c a l f u n c -t i o n . O u r d a t a t h a t l i v e r a n d k i d n e y c o n t a i n e d 1 0 - f o l d h i g h e re n z y m a t i c a c t i v i t y t h a n o t h e r t i s s u e s i m p l i e d t h a t e s t e r a s e Dm i g h t p l a y a r o l e i n d e t o x i f i c a t i o n . T h e f a c t t h a t a g r o u p o f
n o n s p e c i f i c e s t e r a s e s i n t h e l i v e r s o f r a t s a n d r a b b i t s c o u l d b e
i n d u c e d b y p h e n o b a r b i t a l t r e a t m e n t s u p p o r t s t h i s n o t i o n ( 1 9 ,2 0 ) . O u r o b s e r v a t i o n o f a 3 - f o l d i n c r e a s e o f s y n t h e s i s o fe s t e r a s e D p r o t e i n i n c u l t u r e d U 9 3 7 c e l l s u p o n t r e a t m e n t o f0 . 1 mM p h e n o b a r b i t a l f u r t h e r s u b s t a n t i a t e s t h i s c o n t e n t i o n .I n c o n t r a s t , o n l y a 5 0 % i n c r e a s e i n e n z y m a t i c a c t i v i t y w a s
o b s e r v e d w i t h 4 - m e t h y l u m b e l l i f e r y l a c e t a t e a s s u b s t r a t e .T h i s r e s u l t c a n b e r e c o n c i l e d w i t h o u r o t h e r o b s e r v a t i o n s i nt h a t e s t e r a s e D i s o n l y o n e o f s e v e r a l e s t e r a s e s c a p a b l e o fh y d r o l y z i n g t h i s s u b s t r a t e ; t h u s , a n i n c r e a s e i n e s t e r a s e Da c t i v i t y m i g h t n o t r e s u l t i n a d r a m a t i c i n c r e a s e i n t o t a le s t e r a s e a c t i v i t y . I n t e r e s t i n g l y , w e c o u l d n o t o b s e r v e a n y
c h a n g e i n t h e s y n t h e s i s o f e s t e r a s e D i n U 9 3 7 c e l l s b yt r e a t m e n t w i t h PMA, w h i c h i n d u c e s t h e c e l l s t o d i f f e r e n t i a t ei n t o m o n o c y t e s .
R e c e n t l y , V a r k i e t a l . p r o p o s e d t h a t a s i a l i c a c i d - s p e c i f i c0 - a c e t y l e s t e r a s e i n h u m a n e r y t h r o c y t e s w a s i d e n t i c a l t o
e s t e r a s e D ( 2 1 ) . S i n c e t h i s c y t o s o l i c s i a l i c a c i d - s p e c i f i cO - a c e t y l e s t e r a s e a p p e a r s t o b e i n v o l v e d i n t h e " r e c y c l i n g " o fO - a c e t y l a t e d s i a l i c a c i d m o l e c u l e s , a s p e c i f i c b i o l o g i c a l r o l ec o u l d , t h e r e f o r e , b e a s s i g n e d t o e s t e r a s e D e n z y m e . T or e s o l v e t h i s i s s u e , w e h a v e c o l l a b o r a t e d t o c o m p a r e o u r
h o m o g e n e o u s e s t e r a s e D p r o t e i n d e s c r i b e d h e r e w i t h t h e i rp a r t i a l l y p u r i f i e d p r o t e i n s . O u r p r e l i m i n a r y r e s u l t s s u g g e s tt h a t t h e s e t w o e n z y m e s a r e u n l i k e l y t o b e i d e n t i c a l b a s e d o n
b i o c h e m i c a l a n d i m m u n o l o g i c a l d a t a . I t r e m a i n s p o s s i b l e t h a t
a n o t h e r n o n s p e c i f i c e s t e r a s e c o u l d f u n c t i o n a s O - a c e t y l -e s t e r a s e .S i n c e t h e g e n e t i c l o c u s i n v o l v e d i n g e n e s i s o f r e t i n o b l a s -
t o m a i s t i g h t l y l i n k e d t o t h e e s t e r a s e D g e n e , i t i s v e r y c r u c i a l
t o c l o n e t h e e s t e r a s e D g e n e . C o n v e n i e n t a p p r o a c h e s f o rc l o n i n g t h e e s t e r a s e D g e n e a l l r e q u i r e d a s i g n i f i c a n t a m o u n to f t h e p u r i f i e d p r o t e i n . R e c e n t l y , we h a v e o b t a i n e d t h e cDNAc l o n e o f t h e e s t e r a s e D g e n e . D e t a i l e d c h a r a c t e r i z a t i o n o f t h i sg e n e w i l l b e p u b l i s h e d e l s e w h e r e ( 2 2 ) . T h e a v a i l a b i l i t y o f t h i scDNA c l o n e w i l l f a c i l i t a t e f u t u r e r e t i n o b l a s t o m a s t u d i e s ,p a r t i c u l a r l y i n t h e c l o n i n g o f t h e r e t i n o b l a s t o m a g e n e a n d t h ec l i n i c a l d i a g n o s i s o f h e r e d i t a r y r e t i n o b l a s t o m a .
We t h a n k D r . R . S p a r k e s f o r i n d e p e n d e n t c o n f i r m a t i o n o f o u r
e n z y m e e s t e r a s e D ; D r . D . C h e n o w e t h f o r h i s h e l p a n d a d v i c e ; D r s .A . H s u e h a n d H . S . U . a n d S . B a i r d f o r t h e i r c o m m e n t s ; a n d M r s .
V . O t h e n f o r t y p i n g t h e m a n u s c r i p t . T h i s w o r k w a s s u p p o r t e d b y ag r a n t f r o m t h e N a t i o n a l E y e I n s t i t u t e ( E Y - 0 5 7 5 8 ) t o W . - H . L .
1 . H o p k i n s o n , D . A . , M e s t r i n e r , M . A . , C o r t n e r , J . & H a r r i s , H .
( 1 9 7 3 ) A n n . Hum. G e n e t . 3 7 , 1 1 9 - 1 3 7 .2 . C o a t e s , P . M . , M e s t r i n e r , M . A . & H o p k i n s o n ; D . A . ( 1 9 7 5 )
A n n . Hum. G e n e t . 3 9 , 1 - 2 0 .3 . S p a r k e s , R . S . & S p a r k e s , M . C . ( 1 9 8 3 ) i n I s o z y m e s : C u r r e n t
T o p i c s i n B i o l o g i c a l a n d M e d i c a l R e s e a r c h , V o l . 1 1 , M e d i c a l
a n d O t h e r A p p l i c a t i o n s ( L i s s , Ne w Y o r k ) , p p . 1 7 3 - 1 8 2 .4 . C h e n , S . , C r e a g a n , R . P . , N i c h o l s , E . A . & R u d d l e , F . H .
( 1 9 7 5 ) B i r t h D e f e c t s O r i g . A r t i c . S e r . 1 1 , 9 9 - 1 0 2 .
5 . S p a r k e s , R . S . , S p a r k e s , M . C . , W i l s o n , M . G . , T o w n e r ,
J . W . , B e n e d i c t , W . , M u r p h r e e , A . L . & Y u n i s , J . J . ( 1 9 8 0 )S c i e n c e 2 0 8 , 1 0 4 2 - 1 0 4 4 .
6 . W a r d , P . , P a c k m a n , S . , L o u g h m a n , W . , S p a r k e s , M . , S p a r k e s ,R . , M c M a h o n , A . , G r e g o r y , T . & A b l i n , A . ( 1 9 8 4 ) J . M e d .G e n e t . 2 1 , 9 2 - 9 5 .
7 . Y u n i s , J . J . & R a m s a y , N . ( 1 9 7 8 ) A m . J . D i s . C h i l d . 1 3 2 ,1 6 1 - 1 6 3 .
8 . S p a r k e s , R . S . , M u r p h r e e , A . L . , L i n g u a , R . W . , S p a r k e s ,M . E . , F i e l d , L . L . , F u n d e r b u r k , S . J . & B e n e d i c t , W . F .( 1 9 8 3 ) S c i e n c e 2 1 9 , 9 7 1 - 9 7 3 .
9 . G o d b o u t , R . , D r y j a , T . P . , S q u i r e , J . , G a l l i e , B . L . & P h i l l i p s ,R . A . ( 1 9 8 3 ) N a t u r e ( L o n d o n ) 3 0 4 , 4 5 1 - 4 5 3 .
1 0 . F r y d m a n , M . , B o n n e - T a m i r , B . , F a r r e r , L . A . , C o n n e a l l y ,P . M . , M a g a z a n i k , A . , A s h b e l , S . & G o i l d w i t c h , Z . ( i 9 8 5 )P r o c . N a t l . A c a d . S c i . USA 8 2 , 1 8 1 9 - 1 8 2 1 .
1 1 . S c o t t , E . M . & W r i g h t , R . C . ( 1 9 7 8 ) Am. J . - H u m . G e n e t . 3 0 ,1 4 - 1 8 .
1 2 . K o s t e r , B . , L e u p o l d , H . & M a u f f , G . ( 1 9 7 5 ) H u m a n g e n e t i k 2 8 ,
7 5 - 7 8 .1 3 . S c o t t , E . M . ( 1 9 7 6 ) P r e p . B i o c h e m . 6 , 1 4 7 - 1 5 2 .1 4 . L a e m m l i , U . K . ( 1 9 7 0 ) N a t u r e ( L o n d o n ) 2 2 7 , 6 8 0 - 6 8 5 .1 5 . L o w r y , 0 . H . , R o s e b r o u g h , N . J . , F a r r , A . L . & R a n d a l l ,
R . J . ( 1 9 5 1 ) J . B i o l . C h e m . 1 9 3 , 2 6 5 - 2 7 5 .1 6 . T o w b i n , H . , S t a e h e l i n , T . & G o r d o n , J . ( 1 9 7 9 ) P r o c . N a t I .
A c a d . S c i . USA 7 6 , 4 3 5 0 - 4 3 5 4 .
1 7 . H u a n g , C . - M . , P a r s o n s , M . , O i , V . T . , H u a n g , H . - J . S . &H e r z e n b e r g , L . A . ( 1 9 8 3 ) I m m u n o g e n e t i c s ( N . Y . ) 1 8 , 3 1 1 - 3 2 1 .
1 8 . L e e , W . - H . , B i s t e r , K . , M o s c o v i c i , C . & D u e s b e r g , P . H .
( 1 9 8 1 ) J . V i r o l . 3 8 , 1 0 6 4 - 1 0 7 6 .1 9 . K a n e k o , A . , Y o s h i d a , Y . , E n o m o t o , K . , K a k u , T . , H i r a t a , K .
& O n o e , T . ( 1 9 7 9 ) B i o c h i m . B i o p h y s . A c t a 5 8 2 , 1 8 5 - 1 9 5 .2 0 . R a f t e l l , M . , B e r z i n s , K . & B l o m b e r g , F . ( 1 9 7 7 ) A r c h . B i o c h e m .
B i o p h y s . 1 8 1 , 5 3 4 - 5 4 1 .2 1 . V a r k i , A . , M u c h m o r e , E . & D i a z , S . ( 1 9 8 6 ) P r o c . N a t l . A c a d .
S c i . USA 8 3 , 8 8 2 - 8 8 6 .
2 2 . L e e , E . Y . - H . P . & L e e , W . - H . ( 1 9 8 6 ) P r o c . N a t l . A c a d . S c i .USA 8 3 , 6 3 3 7 - 6 3 4 1 .
2 3 . C o a t e s , P . M . , E d w a r d s , Y . H . & H o p k i n s o n , D . A . ( 1 9 7 6 )E u r . J . B i o c h e m . 6 1 , 3 3 1 - 3 3 5 .
6 7 9 4 B i o c h e m i s t r y : L e e e t a l .
