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Purification of Adenine Phosphoribosyltransferase by Affinity Chromatography a
Howard V. Hershey & Milton W. Taylor
a
a
Dept. of Biology , Indiana University , Bloomington, IN, 47401 Published online: 05 Dec 2006.
To cite this article: Howard V. Hershey & Milton W. Taylor (1978) Purification of Adenine Phosphoribosyltransferase by Affinity Chromatography, Preparative Biochemistry, 8:6, 453-462, DOI: 10.1080/00327487808061662 To link to this article: http://dx.doi.org/10.1080/00327487808061662
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PREPARATIVE BIOCHEMISTRY, 8 ( 6 ) , 453-462 (1978)
PURIFICATION OF ADENINE PHOSPHORIBOSYLTRANSFEMSE BY AFFINITY CHROMATOGRAPHY Howard V. Hershey and M i l t o n W. T a y l o r Dept. of Biology, I n d i a n a U n i v e r s i t y , Bloomington, I N
47401
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ABSTRACT The p u r i n e s a l v a g e pathway enzyme a d e n i n e phosphoribosyltransferase (AMP:
p y r o p h o s p h a t e phosphoribosyltransferase EC 2.42.7)
h a s been p u r i -
f i e d t o g r e a t e r t h a n 85% homogeneity from c r u d e r a t l i v e r 100,000 x g s u p e r n a t a n t i n one s t e p by a f f i n i t y chromatography.
The enzyme b i n d s t o
an AMP-agarose column and i s e l u t e d o f f t h e column by 1 mM 5-phosphorib o s y l p y r o p h o s p h a t e w i t h a 50 t o 80% r e c o v e r y .
Enzyme k i n e t i c s i n d i c a t e
t h a t t h e mechanism of t h e s p e c i f i c e l u t i o n is due t o c o m p e t i t i o n o f t h e p r o d u c t AMP and s u b s t r a t e 5 - p h o s p h o r i b o s y l p y r o p h o s p h a t e f o r t h e same s i t e on t h e enzyme.
INTRODUCTION
Adenine phosphoribosyltransferase (APRT, AMP: p y r o p h o s p h a t e p h o s p h o r i b o s y l t r a n s f e r a s e , EC 2 . 4 2 . 7 )
i s t h e o n l y known mechanism by which d i e t a r y
a d e n i n e can be u t i l i z e d i n mammalian c e l l s , and may p l a y a n i m p o r t a n t r o l e i n m a t u r e r e d c e l l s which have no p u r i n e b i o s y n t h e t i c pathway and i n r a p i d l y growing t i s s u e s ( 1 , 2 ) .
The enzyme i s p r e s e n t i n a wide phylo-
g e n e t i c s p e c t r u m of organisms and may w e l l be u b i q u i t o u s (3-8).
Indi-
v i d u a l s w i t h e i t h e r a p a r t i a l ( 9 , l O ) o r complete ( 1 1 ) a b s e n c e of APRT a c t i v i t y have been o b s e r v e d , and t h e s e d e f e c t s are c o r r e l a t e d w i t h hyperu r i c e m i a , gouty a r t h r i t i s , and u r o l i t h i a s i s ( 1 1 , 1 2 ) , a l t h o u g h some i n d i -
453 Copyright 0 1979 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may he reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming. and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
454
HERSHEY AND TAYLOR
v i d u a l s a r e asymptomatic ( 1 1 ) .
However, t h e f a m i l y o f t h e two i n d i v i d u a l s
documented t o have complete d e f i c i e n c y d i d e x h i b i t f o u r s p o n t a n e o u s a b o r t i o n s (11). which may i n d i c a t e t h e p o s s i b i l i t y of l e t h a l developmental forms of t h e d e f e c t .
D e s p i t e t h e a p p a r e n t l a c k of s e r i o u s medi-
c a l problems a s s o c i a t e d w i t h d e f e c t s i n t h e enzyme, t h e r e a p p e a r s t o be l i t t l e g e n e t i c v a r i a b i l i t y a s d e t e c t e d by e l e c t r o p h o r e t i c methods ( 1 3 ) .
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Moreover, t h e r e i s some e v i d e n c e f o r u n u s u a l and i n c r e a s e d APRT a c t i v i t y i n i n d i v i d u a l s w i t h Lesch-Nyhan
syndrome ( 1 4 ) .
In c e l l c u l t u r e , APRT i s n e c e s s a r y f o r t h e c o n v e r s i o n of diaminop u r i n e and 8-azaadenine i n t o t h e i r c y t o t o x i c r i b o t i d e s , and c e l l s w i t h d e f e c t i v e APRT a r e r e f r a c t o r y t o t h e s e d r u g s (15-17).
Thus, i t i s pos-
s i b l e t o d e t e c t and i s o l a t e APRT m u t a n t c e l l l i n e s by l o o k i n g f o r drugr e s i s t a n t clones.
In t h i s l a b o r a t o r y a series o f s u c h m u t a n t s have been
i s o l a t e d (17).
I n humans, APRT i s composed o f i d e n t i c a l s u b u n i t s of 10,000 d a l t o n s (about 100 amino a c i d s ) ( 3 ) ; t h e a c t i v e enzyme i s a dimer of 20,000 d a l t o n s (13).
In r a t l i v e r . t h e enzyme i s a p p a r e n t l y a n e n z y m a t i c a l l y
a c t i v e monomer o f 20,000 d a l t o n s ( 4 ) .
In e i t h e r c a s e , APRT i s a
remarkably s m a l l enzyme, which s h o u l d make t h i s p r o t e i n p a r t i c u l a r l y amenable t o d e t a i l e d mapping of g e n e t i c and e v o l u t i o n a r y c h a n g e s . However, APRT h a s p r o v e n t o be a d i f f i c u l t enzyme t o p u r i f y by s t a n d a r d techniques, with e x t e n s i v e l o s s of a c t i v i t y a t e v e r y s t e p i n t h e p u r i f i c a t i o n procedure ( 3 , 4 ) .
A c c o r d i n g l y , w e have d e v i s e d a r a p i d ,
h i g h l y s p e c i f i c , o n e - s t e p p u r i f i c a t i o n of APRT w i t h h i g h r e c o v e r y o f a c t i v i t y by t a k i n g a d v a n t a g e of t h e c o m p e t i t i o n o f t h e p r o d u c t , AMP, f o r t h e enzyme s i t e s p e c i f i c f o r 5 - p h o s p h o r i b o s y l p y r o p h o s p h a t e (P-ribose-PP).
MATERIALS AND METHODS
100,000 x g Rat L i v e r S u p e r n a t a n t :
Rat l i v e r was homogenized w i t h a n
e q u a l w e i g h t of 50 mM T r i s , pH 7 ; 30 mM KC1; 10 mM MgS04 ( B u f f e r A) i n a
455
ADENINE PHOSPHORIBOSYLTRANSFERASE
Waring B l e n d e r a t 4'C.
The homogenate was c e n t r i f u g e d t h r e e t i m e s a t
4°C i n a S o r v a l l r e f r i g e r a t e d c e n t r i f u g e f o r 30 min a t 27,000 x g w i t h t h e p e l l e t d i s c a r d e d i n o r d e r t o remove l a r g e p a r t i c l e s . was t h e n c e n t r i f u g e d f o r 2 h r a t 4'C
The s u p e r n a t a n t
i n a T i 6 0 r o t o r a t 100,000 x g i n a
Beckman u l t r a c e n t r i f u g e , and t h e s u p e r n a t a n t r e t a i n e d . 3 APRT a c t i v i t y was d e t e r m i n e d by t h e c o n v e r s i o n of [ HI-
Enzyme Assays:
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a d e n i n e (New England N u c l e a r ; 3 . 3 Ci/mmol) o r [ S e a r l e ; 61 mCi/mmol) to l a b e l l e d AMP. c o n t a i n e d 10 mM P-ribose-PP;
14 C l a d e n i n e (Amersham -
The s t a n d a r d r e a c t i o n m i x t u r e
0.36 mM [ I 4 C ] a d e n i n e ; 1 0 0 mM T r i s , pH 7 ; 1 6
mM MgSOq; and 2 mg/ml b o v i n e serum albumin.
To 50 111 of t h i s r e a c t i o n
m i x t u r e , 50 111 of t h e sample t o be t e s t e d was added, a l l a t 2OC. were t h e n t r a n s f e r r e d t o 37*C f o r 2-5 min.
Samples
A f t e r i n c u b a t i o n a t 37OC, t h e
samples were t r a n s f e r r e d back t o t h e ice b a t h and r a d i o l a b e l l e d AMP measured by e i t h e r DEAE-filter d i s k b i n d i n g (17) o r LaCl p r e c i p i t a t i o n
(18). I n t h e DEAE-filter d i s k method, 1 0 ~1 of 8N f o r m i c a c i d w a s added t o t h e sample, and t h e sample and a 50 u 1 wash of t h e sample t u b e w i t h 1 mM Tris, 1 mM MgCl
2
were t r a n s f e r r e d t o DEAE-filter d i s k s .
The d i s k s were
t h e n s u c c e s s i v e l y washed w i t h a t l e a s t 20 m l / d i s k of 2 mM sodium f o r m a t e f o r 10 min, 1:l methanol:H20 f o r 5 , 10 and 1 5 min, 95% e t h a n o l f o r 10 and 10 m i n u t e s .
A f t e r d r y i n g , d i s k s were p u t i n s c i n t i l l a t i o n v i a l s c o n t a i n i n s
1 m l 0.1 N H C 1 , 0 . 1 M KC1.
A f t e r a d d i n g 10 m l Aquasol (New England N u c l e a r ) ,
t h e r a d i o a c t i v i t y was c o u n t e d . I n t h e LaCl p r e c i p i t a t i o n method, 1 m l of 50 mM sodium a c e t a t e , 2 mM sodium p h o s p h a t e , pH 6 was added t o t h e sample, f o l l o w e d by 0 . 2 m l 0 . 5 M LaC1.
A f t e r 1 5 min a t 2'C,
t h e s a m p l e s were f i l t e r e d t h r o u g h Whatman
GF/C g l a s s f i b e r f i l t e r s and washed w i t h a t l e a s t 2 5 m l c o l d H20. were t h e n d r i e d and c o u n t e d i n toluene-PPO-POPOP
P r o t e i n Determination:
Filters
scintillation fluid.
P r o t e i n d e t e r m i n a t i o n was by a d y e - b i n d i n g method
( 1 9 ) u s i n g dye r e a g e n t from Bio-Rad.
456
HERSHEY AND TAYLOR
Gel E l e c t r o p h o r e s i s :
Sodium dodecylsulfate-polyacrylamide g e l e l e c t r o -
p h o r e s i s was performed i n a 1 2 % g e l a s d e s c r i b e d by Weber and Osborn ( 2 0 ) . Gels were run a t 3 mamp/gel, s t a i n e d w i t h 0.25% Coomasie Blue R-250
(Bio-
Rad) i n 45% m e t h a n o l , 9.2% g l a c i a l a c e t i c a c i d , d e s t a i n e d i n 5% m e t h a n o l , 7.5% g l a c i a l a c e t i c a c i d , and scanned i n a G i l f o r d s p e c t r o p h o t o m e t e r . Nondenaturing p o l y a c r y l a m i d e s l a b g e l e l e c t r o p h o r e s i s was performed
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w i t h a 10%r u n n i n g g e l (0.375 M T r i s , pH 8 . 9 ) , a 3% s t a c k i n g g e l (0.125 M T r i s , pH 6. 8) w i t h a n e l e c t r o p h o r e s i s b u f f e r o f 0.025 M T r i s , 0.192 M g l y c i n e , pH 8.3.
Gels were r u n a t 1 5 mamp.
After electrophoresis, the
s l a b was covered w i t h Whatman f i l t e r p a p e r soaked i n a s s a y m i x t u r e as d e s c r i b e d f o r t h e LaCl a s s a y (18) w i t h t h e a d d i t i o n of 1 mM NaHP04.
After
30 min, t h e p a p e r w a s added t o sodium a c e t a t e (50 mM), pH 6 b u f f e r cont a i n i n g 0.1 M LaCl f o r 1 h r a t 2'C.
A f t e r washing o v e r n i g h t i n d i s t i l l e d
H 0 , t h e p a p e r w a s s l i c e d i n t o 2 . 5 mm s e c t i o n s and counted i n t o l u e n e -
2
PPO-POPOP.
The g e l was s t a i n e d w i t h Coomassie Blue and t h e m i g r a t i o n of
t h e p r o t e i n band was d e t e r m i n e d . Materials:
AMP-agaroses were p u r c h a s e d from P.-L.
Biochemicals.
P-
ribose-PP was from Sigma.
RESULTS AND DISCUSSION
APRT was p u r i f i e d by r u n n i n g 150-200 m l o f 100,000 x g r a t l i v e r s u p e r n a t a n t t h r o u g h a 3 ml column of AMP-agarose
(P.-L.
Biochemicals,
Type 3 , AMP l i n k e d v i a a 6-carbon s p a c e r t h r o u g h t h e 8-carbon o f t h e a d e n i n e m o i e t y , 6.7 mol AMP/ml).
There is a small amount of l e a k a g e of
APRT t h r o u g h t h e column, r a n g i n g from 3-4% i n i t i a l l y t o a b o u t 20% as t h e
column a p p r o a c h e s s a t u r a t i o n of AMP b i n d i n g p r o t e i n s .
Maximal l o a d i n g
c a p a c i t y w i t h e v e n t u a l b r e a k t h r o u g h of enzyme a c t i v i t y occ'Jrs a t around 150 m l ( a b o u t 8 g p r o t e i n ) of t h e 100,000 x g s u p e r n a t a n t ( F i g . 1). Because o f t h e h i g h p r o t e i n c o n c e n t r a t i o n , f r a c t i o n s i z e d u r i n g l o a d i n g of t h e s u p e r n a t a n t i s 3 m l .
Subsequent f r a c t i o n s are 5 ml.
457
ADENINE PHOSPHORIBOSYLTRANSFERASE
2.0
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1.6
0.4
Fr8ction FIGURE 1
P u r i f i c a t i o n of APRT on AMP-agarose column. 100,000 x g s u p e r n a t a n t o f r a t l i v e r is p a s s e d t h r o u g h a 3 m l AMP-agarose column, and a t t h e approp r i a t e p o i n t s , t h e column i s e l u t e d w i t h t h e f o l l o w i n g b u f f e r s : A , 0.5 M B u f f e r A (50 mM T r i s , pH 7 ; 30 mM K C 1 ; 10 mM MgS04); B, B u f f e r A K C 1 ; C , B u f f e r A ; D , B u f f e r A + 1 mM P-ribose-PP; E , B u f f e r A + 10 mM AMP. APRT a c t i v i t y r e l a t i v e t o a c t i v i t y i n t h e 100,000 x g s u p e r n a t a n t 0 ) ;mg/ml p r o t e i n (0). F r a c t i o n s p r i o r t o f r a c t i o n 60 c o n t a i n 70 mg/ml Due t o p r o t e i n and are o f f s c a l e . 200 d r o p f r a c t i o n s were c o l l e c t e d . s u r f a c e t e n s i o n c h a n g e s , f r a c t i o n s 1-60 c o n t a i n e d 3 m l s and s u b s e q u e n t f r a c t i o n s contained 5 m l s .
+
A f t e r 50 m l of B u f f e r A t o wash t h e column, t h e column i s e l u t e d w i t h B u f f e r A p l u s 0.5 M K C 1 , which removes loosely-bound p r o t e i n s ( s e e F i g . 1). APRT a c t i v i t y remains bound t o t h e column. m l of B u f f e r mM P-ribose-PP
A f t e r a f u r t h e r 50
A, AF'RT a c t i v i t y i s e l u t e d o f f w i t h 50 m l B u f f e r A p l u s 1
( F i g . 1).
The f r a c t i o n c o n t a i n i n g t h e APRT a c t i v i t y h a s
a b o u t 0.06% of t h e p r o t e i n which was l o a d e d on t h e column b e f o r e t h e b r e a k t h r o u g h of APRT a c t i v i t y .
T h i s f r a c t i o n c o n t a i n e d 50-80% of t h e
APRT a c t i v i t y l o a d e d on t h e column.
Subsequent washing of t h e column
458
HERSHEY AND TAYLOR
w i t h B u f f e r A p l u s 10 mM AMP d e m o n s t r a t e s t h a t t h e P-ribose-PP
wash had
s e l e c t i v e l y removed APRT a c t i v i t y , s i n c e a s i g n i f i c a n t amount of p r o t e i n had remained bound t o t h e AMP-agarose d u r i n g t h e P-ribose-PP
wash.
S i m i l a r p u r i f i c a t i o n c a n be o b t a i n e d w i t h AMP-agarose on which t h e AMP is l i n k e d v i a a 6-carbon c h a i n e i t h e r t h r o u g h t h e 6 - n i t r o g e n of t h e
a d e n i n e moiety (P.-L.
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Yls
(P.-L.
B i o c h e m i c a l s , Type 2 ) o r t h r o u g h t h e r i b o s e hydrox-
B i o c h e m i c a l s , Type 4 ) .
No m a j o r d i f f e r e n c e s i n APRT b i n d i n g
a b i l i t y were n o t e d among t h e s e AM€'-agaroses.
However, p r e l i m i n a r y e v i -
dence i n d i c a t e s t h a t t h e Type 4 AMP-agarose is less s t a b l e and c a n l o s e binding capacity.
The o t h e r columns c a n be r e u s e d f o l l o w i n g t r e a t m e n t
w i t h 8M u r e a . The p u r i f i e d APRT h a s a s p e c i f i c a c t i v i t y o f 8 . 1 umoles AMP formed/ T h i s v a l u e i s somewhat h i g h e r t h a n t h e v a l u e
min/mg p r o t e i n (Table I ) .
of 1.1 pmoles AMP/min/mg p r o t e i n r e p o r t e d f o r r a t l i v e r enzyme p u r i f i e d by s t a n d a r d t e c h n i q u e s ( 4 ) .
The d i f f e r e n c e may b e p a r t l y due t o d i s c r e -
p a n c i e s i n t h e assay c o n d i t i o n s used.
However, o u r p u r i f i c a t i o n p r o c e d u r e
i s s h o r t e r and t h e scheme used p r o b a b l y s e l e c t i v e l y b i n d s o n l y a c t i v e enzyme m o l e c u l e s , s i n c e i t r e l i e s on p r o d u c t b i n d i n g . e n r i c h i n g f o r a c t i v e enzyme.
Thus, we may be
Based on a m o l e c u l a r weight of 20,000
d a l t o n s . t h e enzyme t u r n o v e r number i s a b o u t 1 6 0 m o l e s AMP formed/min/ mole APRT.
TABLE I STEP
VOWME
TOTAL PROTEIN mg
SPECIFIC ACTIVITY pmoles AMP/min/ml:
RECOVERY
mls
PURIFICATION f o l d
100,000 x g supt
20
1,500
0.00267
-
-
AMP-agarose p u r i f i e d APRT
11.5
0.32
8.1
65%
3,034
459
ADENINE PHOSPHORIBOSYLTRANSFERASE
When t h e f r a c t i o n c o n t a i n i n g APRT a c t i v i t y i s c o n c e n t r a d e d on a M i l l i p o r e PSC f i l t e r and s u b s e q u e n t l y s u b j e c t e d t o sodium d o d e c y l s u l f a t e polya c r y l a m i d e e l e c t r o p h o r e s i s , o n l y one major p r o t e i n band i s o b s e r v e d ( F i g .
2).
T h i s band c o n t a i n s more t h a n 85% of t h e A565 a b s o r b i n g m a t e r i a l i n
the gel.
The m o l e c u l a r weight of t h i s band i s 20.000, which a g r e e s w i t h t h e
m o l e c u l a r w e i g h t of r a t l i v e r APRT i s o l a t e d by s t a n d a r d p r o t e i n p u r i f i c a t i o n
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techniques ( 4 ) . When t h e APRT-containing
f r a c t i o n i s s u b j e c t e d t o nondenaturing poly-
a c r y l a m i d e e l e c t r o p h o r e s i s , t h e major p r o t e i n band and APRT a c t i v i t y c o i n c i d e (Fig. 3 ) .
Thus, t h e APRT a c t i v i t y and t h e p r o t e i n e l u t e d o f f t h e
AMP-agarose by P-ribose-PP
are i d e n t i c a l .
The r e a s o n f o r t h e h i g h d e g r e e of s p e c i f i c i t y o f t h i s enzyme p u r i f i c a t i o n scheme i s made c l e a r by examining t h e k i n e t i c s of enzyme i n h i b i t i o n
8
4
12
MIGRATION (crn) FIGURE 2
Scan of sodium dodecysulfate-polyacrylamide g e l e l e c t r o p h o r e s i s o f p u r i f i e d APRT s t a i n e d w i t h Coomasie b l u e . M i g r a t i o n o f APRT r e l a t i v e t o i n t e r n a l m a r k e r s i n a n o t h e r g e l is g i v e n i n t h e i n s e r t . Markers are, from l e f t t o r i g h t : heavy c h a i n o f IgG, ovalbumin, DNase I , l i g h t c h a i n of IgG, APRT, myoglobin, lysozyme, and c y t o c h r o m C .
HERSHEY AND TAYLOR
460
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A
B
migratlon (cm)
FIGURE 3
Nondenaturing polyacrylamide gel electrophoresis of purified APRT. A. APRT activity in gel. B. Diagram of Coomasie blue stain of polyacrylamide gel.
by the product, AMP.
Fig. 4 demonstrates that AMP is an uncompetitive
inhibitor of adenine and a competitive inhibitor of P-ribose-PP. This indicates that P-ribose-PP and AMP interact at the same site on the enzyme, whereas adenine binds at a separate site.
Similar kinetics have also
been observed with APRT from every other tested source ( 5 , 6 , 8 , 2 2 , 2 3 ) . Thus, the purification scheme given here for rat liver APRT should work equally well for AF'RT from any source.
461
ADENINE PHOSPHORIBOSYLTRANSFERASE 320
A
x
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r
2
4
l/mM
6
0
1
0
5
adenine
10
2
16
l / m M PRPP
FIGURE 4 Competition of AMP with substrates of AF'RT reaction. A. Competition of AMP with adenine (P-ribose-PP at 0.2 mM; 60 ng APRT/assay); 0 mM AMP ( 0 1 , 0.1 mM AMP ( m ) , 0.2 mM AMP ( O ) , 0.4 mM AMP ( U ) , 1 mM AMP (+). B. Competition of AMP with P-ribose-PP (adenine at 0.18 mM; 30 ng APRT/assay); 0 mMAMP (a), 0.2 mMAMP ( m ) , 0 . 5 mM AMP ( O ) , 1 mMAMP ( 0 ) .
ACKNOWLEDGMENTS This work was supported by grant 11562 from the National Foundation - The March of Dimes. REFERENCES
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Preparative Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb19
Purification of Adenine Phosphoribosyltransferase by Affinity Chromatography a
Howard V. Hershey & Milton W. Taylor
a
a
Dept. of Biology , Indiana University , Bloomington, IN, 47401 Published online: 05 Dec 2006.
To cite this article: Howard V. Hershey & Milton W. Taylor (1978) Purification of Adenine Phosphoribosyltransferase by Affinity Chromatography, Preparative Biochemistry, 8:6, 453-462, DOI: 10.1080/00327487808061662 To link to this article: http://dx.doi.org/10.1080/00327487808061662
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PREPARATIVE BIOCHEMISTRY, 8 ( 6 ) , 453-462 (1978)
PURIFICATION OF ADENINE PHOSPHORIBOSYLTRANSFEMSE BY AFFINITY CHROMATOGRAPHY Howard V. Hershey and M i l t o n W. T a y l o r Dept. of Biology, I n d i a n a U n i v e r s i t y , Bloomington, I N
47401
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ABSTRACT The p u r i n e s a l v a g e pathway enzyme a d e n i n e phosphoribosyltransferase (AMP:
p y r o p h o s p h a t e phosphoribosyltransferase EC 2.42.7)
h a s been p u r i -
f i e d t o g r e a t e r t h a n 85% homogeneity from c r u d e r a t l i v e r 100,000 x g s u p e r n a t a n t i n one s t e p by a f f i n i t y chromatography.
The enzyme b i n d s t o
an AMP-agarose column and i s e l u t e d o f f t h e column by 1 mM 5-phosphorib o s y l p y r o p h o s p h a t e w i t h a 50 t o 80% r e c o v e r y .
Enzyme k i n e t i c s i n d i c a t e
t h a t t h e mechanism of t h e s p e c i f i c e l u t i o n is due t o c o m p e t i t i o n o f t h e p r o d u c t AMP and s u b s t r a t e 5 - p h o s p h o r i b o s y l p y r o p h o s p h a t e f o r t h e same s i t e on t h e enzyme.
INTRODUCTION
Adenine phosphoribosyltransferase (APRT, AMP: p y r o p h o s p h a t e p h o s p h o r i b o s y l t r a n s f e r a s e , EC 2 . 4 2 . 7 )
i s t h e o n l y known mechanism by which d i e t a r y
a d e n i n e can be u t i l i z e d i n mammalian c e l l s , and may p l a y a n i m p o r t a n t r o l e i n m a t u r e r e d c e l l s which have no p u r i n e b i o s y n t h e t i c pathway and i n r a p i d l y growing t i s s u e s ( 1 , 2 ) .
The enzyme i s p r e s e n t i n a wide phylo-
g e n e t i c s p e c t r u m of organisms and may w e l l be u b i q u i t o u s (3-8).
Indi-
v i d u a l s w i t h e i t h e r a p a r t i a l ( 9 , l O ) o r complete ( 1 1 ) a b s e n c e of APRT a c t i v i t y have been o b s e r v e d , and t h e s e d e f e c t s are c o r r e l a t e d w i t h hyperu r i c e m i a , gouty a r t h r i t i s , and u r o l i t h i a s i s ( 1 1 , 1 2 ) , a l t h o u g h some i n d i -
453 Copyright 0 1979 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may he reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming. and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
454
HERSHEY AND TAYLOR
v i d u a l s a r e asymptomatic ( 1 1 ) .
However, t h e f a m i l y o f t h e two i n d i v i d u a l s
documented t o have complete d e f i c i e n c y d i d e x h i b i t f o u r s p o n t a n e o u s a b o r t i o n s (11). which may i n d i c a t e t h e p o s s i b i l i t y of l e t h a l developmental forms of t h e d e f e c t .
D e s p i t e t h e a p p a r e n t l a c k of s e r i o u s medi-
c a l problems a s s o c i a t e d w i t h d e f e c t s i n t h e enzyme, t h e r e a p p e a r s t o be l i t t l e g e n e t i c v a r i a b i l i t y a s d e t e c t e d by e l e c t r o p h o r e t i c methods ( 1 3 ) .
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Moreover, t h e r e i s some e v i d e n c e f o r u n u s u a l and i n c r e a s e d APRT a c t i v i t y i n i n d i v i d u a l s w i t h Lesch-Nyhan
syndrome ( 1 4 ) .
In c e l l c u l t u r e , APRT i s n e c e s s a r y f o r t h e c o n v e r s i o n of diaminop u r i n e and 8-azaadenine i n t o t h e i r c y t o t o x i c r i b o t i d e s , and c e l l s w i t h d e f e c t i v e APRT a r e r e f r a c t o r y t o t h e s e d r u g s (15-17).
Thus, i t i s pos-
s i b l e t o d e t e c t and i s o l a t e APRT m u t a n t c e l l l i n e s by l o o k i n g f o r drugr e s i s t a n t clones.
In t h i s l a b o r a t o r y a series o f s u c h m u t a n t s have been
i s o l a t e d (17).
I n humans, APRT i s composed o f i d e n t i c a l s u b u n i t s of 10,000 d a l t o n s (about 100 amino a c i d s ) ( 3 ) ; t h e a c t i v e enzyme i s a dimer of 20,000 d a l t o n s (13).
In r a t l i v e r . t h e enzyme i s a p p a r e n t l y a n e n z y m a t i c a l l y
a c t i v e monomer o f 20,000 d a l t o n s ( 4 ) .
In e i t h e r c a s e , APRT i s a
remarkably s m a l l enzyme, which s h o u l d make t h i s p r o t e i n p a r t i c u l a r l y amenable t o d e t a i l e d mapping of g e n e t i c and e v o l u t i o n a r y c h a n g e s . However, APRT h a s p r o v e n t o be a d i f f i c u l t enzyme t o p u r i f y by s t a n d a r d techniques, with e x t e n s i v e l o s s of a c t i v i t y a t e v e r y s t e p i n t h e p u r i f i c a t i o n procedure ( 3 , 4 ) .
A c c o r d i n g l y , w e have d e v i s e d a r a p i d ,
h i g h l y s p e c i f i c , o n e - s t e p p u r i f i c a t i o n of APRT w i t h h i g h r e c o v e r y o f a c t i v i t y by t a k i n g a d v a n t a g e of t h e c o m p e t i t i o n o f t h e p r o d u c t , AMP, f o r t h e enzyme s i t e s p e c i f i c f o r 5 - p h o s p h o r i b o s y l p y r o p h o s p h a t e (P-ribose-PP).
MATERIALS AND METHODS
100,000 x g Rat L i v e r S u p e r n a t a n t :
Rat l i v e r was homogenized w i t h a n
e q u a l w e i g h t of 50 mM T r i s , pH 7 ; 30 mM KC1; 10 mM MgS04 ( B u f f e r A) i n a
455
ADENINE PHOSPHORIBOSYLTRANSFERASE
Waring B l e n d e r a t 4'C.
The homogenate was c e n t r i f u g e d t h r e e t i m e s a t
4°C i n a S o r v a l l r e f r i g e r a t e d c e n t r i f u g e f o r 30 min a t 27,000 x g w i t h t h e p e l l e t d i s c a r d e d i n o r d e r t o remove l a r g e p a r t i c l e s . was t h e n c e n t r i f u g e d f o r 2 h r a t 4'C
The s u p e r n a t a n t
i n a T i 6 0 r o t o r a t 100,000 x g i n a
Beckman u l t r a c e n t r i f u g e , and t h e s u p e r n a t a n t r e t a i n e d . 3 APRT a c t i v i t y was d e t e r m i n e d by t h e c o n v e r s i o n of [ HI-
Enzyme Assays:
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a d e n i n e (New England N u c l e a r ; 3 . 3 Ci/mmol) o r [ S e a r l e ; 61 mCi/mmol) to l a b e l l e d AMP. c o n t a i n e d 10 mM P-ribose-PP;
14 C l a d e n i n e (Amersham -
The s t a n d a r d r e a c t i o n m i x t u r e
0.36 mM [ I 4 C ] a d e n i n e ; 1 0 0 mM T r i s , pH 7 ; 1 6
mM MgSOq; and 2 mg/ml b o v i n e serum albumin.
To 50 111 of t h i s r e a c t i o n
m i x t u r e , 50 111 of t h e sample t o be t e s t e d was added, a l l a t 2OC. were t h e n t r a n s f e r r e d t o 37*C f o r 2-5 min.
Samples
A f t e r i n c u b a t i o n a t 37OC, t h e
samples were t r a n s f e r r e d back t o t h e ice b a t h and r a d i o l a b e l l e d AMP measured by e i t h e r DEAE-filter d i s k b i n d i n g (17) o r LaCl p r e c i p i t a t i o n
(18). I n t h e DEAE-filter d i s k method, 1 0 ~1 of 8N f o r m i c a c i d w a s added t o t h e sample, and t h e sample and a 50 u 1 wash of t h e sample t u b e w i t h 1 mM Tris, 1 mM MgCl
2
were t r a n s f e r r e d t o DEAE-filter d i s k s .
The d i s k s were
t h e n s u c c e s s i v e l y washed w i t h a t l e a s t 20 m l / d i s k of 2 mM sodium f o r m a t e f o r 10 min, 1:l methanol:H20 f o r 5 , 10 and 1 5 min, 95% e t h a n o l f o r 10 and 10 m i n u t e s .
A f t e r d r y i n g , d i s k s were p u t i n s c i n t i l l a t i o n v i a l s c o n t a i n i n s
1 m l 0.1 N H C 1 , 0 . 1 M KC1.
A f t e r a d d i n g 10 m l Aquasol (New England N u c l e a r ) ,
t h e r a d i o a c t i v i t y was c o u n t e d . I n t h e LaCl p r e c i p i t a t i o n method, 1 m l of 50 mM sodium a c e t a t e , 2 mM sodium p h o s p h a t e , pH 6 was added t o t h e sample, f o l l o w e d by 0 . 2 m l 0 . 5 M LaC1.
A f t e r 1 5 min a t 2'C,
t h e s a m p l e s were f i l t e r e d t h r o u g h Whatman
GF/C g l a s s f i b e r f i l t e r s and washed w i t h a t l e a s t 2 5 m l c o l d H20. were t h e n d r i e d and c o u n t e d i n toluene-PPO-POPOP
P r o t e i n Determination:
Filters
scintillation fluid.
P r o t e i n d e t e r m i n a t i o n was by a d y e - b i n d i n g method
( 1 9 ) u s i n g dye r e a g e n t from Bio-Rad.
456
HERSHEY AND TAYLOR
Gel E l e c t r o p h o r e s i s :
Sodium dodecylsulfate-polyacrylamide g e l e l e c t r o -
p h o r e s i s was performed i n a 1 2 % g e l a s d e s c r i b e d by Weber and Osborn ( 2 0 ) . Gels were run a t 3 mamp/gel, s t a i n e d w i t h 0.25% Coomasie Blue R-250
(Bio-
Rad) i n 45% m e t h a n o l , 9.2% g l a c i a l a c e t i c a c i d , d e s t a i n e d i n 5% m e t h a n o l , 7.5% g l a c i a l a c e t i c a c i d , and scanned i n a G i l f o r d s p e c t r o p h o t o m e t e r . Nondenaturing p o l y a c r y l a m i d e s l a b g e l e l e c t r o p h o r e s i s was performed
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w i t h a 10%r u n n i n g g e l (0.375 M T r i s , pH 8 . 9 ) , a 3% s t a c k i n g g e l (0.125 M T r i s , pH 6. 8) w i t h a n e l e c t r o p h o r e s i s b u f f e r o f 0.025 M T r i s , 0.192 M g l y c i n e , pH 8.3.
Gels were r u n a t 1 5 mamp.
After electrophoresis, the
s l a b was covered w i t h Whatman f i l t e r p a p e r soaked i n a s s a y m i x t u r e as d e s c r i b e d f o r t h e LaCl a s s a y (18) w i t h t h e a d d i t i o n of 1 mM NaHP04.
After
30 min, t h e p a p e r w a s added t o sodium a c e t a t e (50 mM), pH 6 b u f f e r cont a i n i n g 0.1 M LaCl f o r 1 h r a t 2'C.
A f t e r washing o v e r n i g h t i n d i s t i l l e d
H 0 , t h e p a p e r w a s s l i c e d i n t o 2 . 5 mm s e c t i o n s and counted i n t o l u e n e -
2
PPO-POPOP.
The g e l was s t a i n e d w i t h Coomassie Blue and t h e m i g r a t i o n of
t h e p r o t e i n band was d e t e r m i n e d . Materials:
AMP-agaroses were p u r c h a s e d from P.-L.
Biochemicals.
P-
ribose-PP was from Sigma.
RESULTS AND DISCUSSION
APRT was p u r i f i e d by r u n n i n g 150-200 m l o f 100,000 x g r a t l i v e r s u p e r n a t a n t t h r o u g h a 3 ml column of AMP-agarose
(P.-L.
Biochemicals,
Type 3 , AMP l i n k e d v i a a 6-carbon s p a c e r t h r o u g h t h e 8-carbon o f t h e a d e n i n e m o i e t y , 6.7 mol AMP/ml).
There is a small amount of l e a k a g e of
APRT t h r o u g h t h e column, r a n g i n g from 3-4% i n i t i a l l y t o a b o u t 20% as t h e
column a p p r o a c h e s s a t u r a t i o n of AMP b i n d i n g p r o t e i n s .
Maximal l o a d i n g
c a p a c i t y w i t h e v e n t u a l b r e a k t h r o u g h of enzyme a c t i v i t y occ'Jrs a t around 150 m l ( a b o u t 8 g p r o t e i n ) of t h e 100,000 x g s u p e r n a t a n t ( F i g . 1). Because o f t h e h i g h p r o t e i n c o n c e n t r a t i o n , f r a c t i o n s i z e d u r i n g l o a d i n g of t h e s u p e r n a t a n t i s 3 m l .
Subsequent f r a c t i o n s are 5 ml.
457
ADENINE PHOSPHORIBOSYLTRANSFERASE
2.0
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1.6
0.4
Fr8ction FIGURE 1
P u r i f i c a t i o n of APRT on AMP-agarose column. 100,000 x g s u p e r n a t a n t o f r a t l i v e r is p a s s e d t h r o u g h a 3 m l AMP-agarose column, and a t t h e approp r i a t e p o i n t s , t h e column i s e l u t e d w i t h t h e f o l l o w i n g b u f f e r s : A , 0.5 M B u f f e r A (50 mM T r i s , pH 7 ; 30 mM K C 1 ; 10 mM MgS04); B, B u f f e r A K C 1 ; C , B u f f e r A ; D , B u f f e r A + 1 mM P-ribose-PP; E , B u f f e r A + 10 mM AMP. APRT a c t i v i t y r e l a t i v e t o a c t i v i t y i n t h e 100,000 x g s u p e r n a t a n t 0 ) ;mg/ml p r o t e i n (0). F r a c t i o n s p r i o r t o f r a c t i o n 60 c o n t a i n 70 mg/ml Due t o p r o t e i n and are o f f s c a l e . 200 d r o p f r a c t i o n s were c o l l e c t e d . s u r f a c e t e n s i o n c h a n g e s , f r a c t i o n s 1-60 c o n t a i n e d 3 m l s and s u b s e q u e n t f r a c t i o n s contained 5 m l s .
+
A f t e r 50 m l of B u f f e r A t o wash t h e column, t h e column i s e l u t e d w i t h B u f f e r A p l u s 0.5 M K C 1 , which removes loosely-bound p r o t e i n s ( s e e F i g . 1). APRT a c t i v i t y remains bound t o t h e column. m l of B u f f e r mM P-ribose-PP
A f t e r a f u r t h e r 50
A, AF'RT a c t i v i t y i s e l u t e d o f f w i t h 50 m l B u f f e r A p l u s 1
( F i g . 1).
The f r a c t i o n c o n t a i n i n g t h e APRT a c t i v i t y h a s
a b o u t 0.06% of t h e p r o t e i n which was l o a d e d on t h e column b e f o r e t h e b r e a k t h r o u g h of APRT a c t i v i t y .
T h i s f r a c t i o n c o n t a i n e d 50-80% of t h e
APRT a c t i v i t y l o a d e d on t h e column.
Subsequent washing of t h e column
458
HERSHEY AND TAYLOR
w i t h B u f f e r A p l u s 10 mM AMP d e m o n s t r a t e s t h a t t h e P-ribose-PP
wash had
s e l e c t i v e l y removed APRT a c t i v i t y , s i n c e a s i g n i f i c a n t amount of p r o t e i n had remained bound t o t h e AMP-agarose d u r i n g t h e P-ribose-PP
wash.
S i m i l a r p u r i f i c a t i o n c a n be o b t a i n e d w i t h AMP-agarose on which t h e AMP is l i n k e d v i a a 6-carbon c h a i n e i t h e r t h r o u g h t h e 6 - n i t r o g e n of t h e
a d e n i n e moiety (P.-L.
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Yls
(P.-L.
B i o c h e m i c a l s , Type 2 ) o r t h r o u g h t h e r i b o s e hydrox-
B i o c h e m i c a l s , Type 4 ) .
No m a j o r d i f f e r e n c e s i n APRT b i n d i n g
a b i l i t y were n o t e d among t h e s e AM€'-agaroses.
However, p r e l i m i n a r y e v i -
dence i n d i c a t e s t h a t t h e Type 4 AMP-agarose is less s t a b l e and c a n l o s e binding capacity.
The o t h e r columns c a n be r e u s e d f o l l o w i n g t r e a t m e n t
w i t h 8M u r e a . The p u r i f i e d APRT h a s a s p e c i f i c a c t i v i t y o f 8 . 1 umoles AMP formed/ T h i s v a l u e i s somewhat h i g h e r t h a n t h e v a l u e
min/mg p r o t e i n (Table I ) .
of 1.1 pmoles AMP/min/mg p r o t e i n r e p o r t e d f o r r a t l i v e r enzyme p u r i f i e d by s t a n d a r d t e c h n i q u e s ( 4 ) .
The d i f f e r e n c e may b e p a r t l y due t o d i s c r e -
p a n c i e s i n t h e assay c o n d i t i o n s used.
However, o u r p u r i f i c a t i o n p r o c e d u r e
i s s h o r t e r and t h e scheme used p r o b a b l y s e l e c t i v e l y b i n d s o n l y a c t i v e enzyme m o l e c u l e s , s i n c e i t r e l i e s on p r o d u c t b i n d i n g . e n r i c h i n g f o r a c t i v e enzyme.
Thus, we may be
Based on a m o l e c u l a r weight of 20,000
d a l t o n s . t h e enzyme t u r n o v e r number i s a b o u t 1 6 0 m o l e s AMP formed/min/ mole APRT.
TABLE I STEP
VOWME
TOTAL PROTEIN mg
SPECIFIC ACTIVITY pmoles AMP/min/ml:
RECOVERY
mls
PURIFICATION f o l d
100,000 x g supt
20
1,500
0.00267
-
-
AMP-agarose p u r i f i e d APRT
11.5
0.32
8.1
65%
3,034
459
ADENINE PHOSPHORIBOSYLTRANSFERASE
When t h e f r a c t i o n c o n t a i n i n g APRT a c t i v i t y i s c o n c e n t r a d e d on a M i l l i p o r e PSC f i l t e r and s u b s e q u e n t l y s u b j e c t e d t o sodium d o d e c y l s u l f a t e polya c r y l a m i d e e l e c t r o p h o r e s i s , o n l y one major p r o t e i n band i s o b s e r v e d ( F i g .
2).
T h i s band c o n t a i n s more t h a n 85% of t h e A565 a b s o r b i n g m a t e r i a l i n
the gel.
The m o l e c u l a r weight of t h i s band i s 20.000, which a g r e e s w i t h t h e
m o l e c u l a r w e i g h t of r a t l i v e r APRT i s o l a t e d by s t a n d a r d p r o t e i n p u r i f i c a t i o n
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techniques ( 4 ) . When t h e APRT-containing
f r a c t i o n i s s u b j e c t e d t o nondenaturing poly-
a c r y l a m i d e e l e c t r o p h o r e s i s , t h e major p r o t e i n band and APRT a c t i v i t y c o i n c i d e (Fig. 3 ) .
Thus, t h e APRT a c t i v i t y and t h e p r o t e i n e l u t e d o f f t h e
AMP-agarose by P-ribose-PP
are i d e n t i c a l .
The r e a s o n f o r t h e h i g h d e g r e e of s p e c i f i c i t y o f t h i s enzyme p u r i f i c a t i o n scheme i s made c l e a r by examining t h e k i n e t i c s of enzyme i n h i b i t i o n
8
4
12
MIGRATION (crn) FIGURE 2
Scan of sodium dodecysulfate-polyacrylamide g e l e l e c t r o p h o r e s i s o f p u r i f i e d APRT s t a i n e d w i t h Coomasie b l u e . M i g r a t i o n o f APRT r e l a t i v e t o i n t e r n a l m a r k e r s i n a n o t h e r g e l is g i v e n i n t h e i n s e r t . Markers are, from l e f t t o r i g h t : heavy c h a i n o f IgG, ovalbumin, DNase I , l i g h t c h a i n of IgG, APRT, myoglobin, lysozyme, and c y t o c h r o m C .
HERSHEY AND TAYLOR
460
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A
B
migratlon (cm)
FIGURE 3
Nondenaturing polyacrylamide gel electrophoresis of purified APRT. A. APRT activity in gel. B. Diagram of Coomasie blue stain of polyacrylamide gel.
by the product, AMP.
Fig. 4 demonstrates that AMP is an uncompetitive
inhibitor of adenine and a competitive inhibitor of P-ribose-PP. This indicates that P-ribose-PP and AMP interact at the same site on the enzyme, whereas adenine binds at a separate site.
Similar kinetics have also
been observed with APRT from every other tested source ( 5 , 6 , 8 , 2 2 , 2 3 ) . Thus, the purification scheme given here for rat liver APRT should work equally well for AF'RT from any source.
461
ADENINE PHOSPHORIBOSYLTRANSFERASE 320
A
x
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r
2
4
l/mM
6
0
1
0
5
adenine
10
2
16
l / m M PRPP
FIGURE 4 Competition of AMP with substrates of AF'RT reaction. A. Competition of AMP with adenine (P-ribose-PP at 0.2 mM; 60 ng APRT/assay); 0 mM AMP ( 0 1 , 0.1 mM AMP ( m ) , 0.2 mM AMP ( O ) , 0.4 mM AMP ( U ) , 1 mM AMP (+). B. Competition of AMP with P-ribose-PP (adenine at 0.18 mM; 30 ng APRT/assay); 0 mMAMP (a), 0.2 mMAMP ( m ) , 0 . 5 mM AMP ( O ) , 1 mMAMP ( 0 ) .
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