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Direct assay method for guanosine 5′-monophosphate reductase activity
ANALYTICALBIOCHEMISTRY 2 0 6 , 115 118 (1992)
Direct Assay Method for Guanosine 5'-Monophosphate Reductase Activity Hiroyuki Nakamura, Yutaka Natsumeda, Masami Nagai, Taiichi Shiotani, and George Weber 1 Laboratory for Experimental Oncology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5200
Received April 28, 1992
A s e n s i t i v e a n d s i m p l e m i c r o m e t h o d for t h e a c c u r a t e m e a s u r e m e n t o f G M P r e d u c t a s e (EC 1 . 6 . 6 . 8 ) a c t i v i t y i n c r u d e e x t r a c t s is d e s c r i b e d . T h e r e a c t i o n p r o d u c t o f [814C]IMP w a s s e p a r a t e d f r o m t h e s u b s t r a t e [8-14C]GMP by descending chromatography on Whatman DE81 ionexchange paper. This separation method provides an a n a l y s i s o f t h e p o s s i b l e i n t e r f e r i n g r e a c t i o n s , s u c h as t h e m e t a b o l i c c o n v e r s i o n o f t h e s u b s t r a t e G M P to G D P , GTP, and/or guanosine, and guanine and the loss of the p r o d u c t I M P to i n o s i n e , h y p o x a n t h i n e , a n d o t h e r m e t a b olites. Low blank values (70-90 cpm) were obtained consistently with this assay because the IMP spot moves f a s t e r t h a n t h e G M P spot. T h e m a j o r a d v a n t a g e s o f t h i s method are direct measurement of GMP reductase activity in crude extracts, high sensitivity (with a limit of detection of less than 10 pmol of IMP production), high r e p r o d u c i b i l i t y (< _+5%), a n d c a p a b i l i t y to m e a s u r e act i v i t y i n s m a l l s a m p l e s (9 # g p r o t e i n ) . ~ 1992 A c a d e m i c Press, Inc.
During the course of trials with tiazofurin (NSC 286193, 2-fl-D-ribofuranosylthiazole-4-carboxamide) in patients with chronic myelogenous leukemia in blast crisis at Indiana University School of Medicine, there was a 70 to 80% clinical response rate (1-3). These patients received repeated cycles of daily tiazofurin infusions to contain the leukemic cell proliferation and some of the patients became clinically resistant to the drug. T h e target of tiazofurin is IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of de novo G T P biosynthesis, which governs the first step converting I M P to XMP. T h r o u g h the subsequent step, catalyzed by G M P synthase (EC 6.3.5.2), G M P is produced. G M P reductase (EC 1.6.6.8) in effect opposes the synthetic action of IMP dehydrogenase because it catalyzes the
1To whom correspondenceshould be addressed. 0003-2697/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
essentially irreversible reductive deamination of G M P to IMP. T h e increased activity of G M P reductase may explain, in part at least, the emergence of resistant cells. Therefore, to underst and the mechanism of resistance, it was i m port ant to determine G M P reductase activity in leukemic cells of patients undergoing tiazofurin therapy. Our interest was to develop a quick and sensitive method for measuring the enzyme activity in samples of less t han a million cells from leukemic patients in blast crisis and in other tissues. G M P reductase was first dem onst rat ed in extracts of bacteria, including Escherichia coli (4). G M P reductase has been purified from mammalian sources, e.g., calf thymus (5), hum an erythrocytes (6), and the assay procedures for the activity of these purified enzymes were reported. However, difficulties were encountered in measuring the enzyme activities with precision in crude extracts of rat or hum an tissue or tissue culture samples because of some of the side products formed during the G M P reductase reaction. T he present report describes a direct assay method of G M P reductase activity in crude extracts which provides greater accuracy and decreases the am o u n t of tissue required. For solving these problems we investigated in crude extracts all possible interfering reactions such as conversion of G M P to GDP, G T P , guanosine, and guanine and the loss of IMP to inosine and hypoxanthine.
MATERIALS AND METHODS Materials
[8-14C]GMP am m oni um salt (55 mCi/mmol) was purchased from Moravek Biochemicals, Inc. (Brea, CA), and prior to use the specific activity was diluted to 20 mCi/mmol with cold GMP. R P M I 1640 culture medium and dialyzed fetal bovine serum were from GIBCO (Grand Island, NY). All other biochemicals were also 115
116
NAKAMURA ET AL.
r e a g e n t grade a n d were o b t a i n e d f r o m S i g m a C h e m i c a l Co. (St. Louis, MO).
DE 81 O~G~
0
GTP GDP
o
GMP
o
IMP
o o
Guanine Hypoxanthine
Enzyme Extracts H L - 6 0 h u m a n p r o m y e l o c y t i c l e u k e m i a cells were p u r c h a s e d f r o m A m e r i c a n T y p e C u l t u r e Collection (Rockville, M D ) a n d m a i n t a i n e d in R P M I 1640 m e d i u m cont a i n i n g 10% dialyzed fetal b o v i n e serum, s u p p l e m e n t e d with penicillin (100 U / m l ) a n d s t r e p t o m y c i n (100 ~g/ ml) at 37°C in a humidified a t m o s p h e r e with 5% CO2. E n z y m e e x t r a c t s were p r e p a r e d by h o m o g e n i z a t i o n in 40 mM T r i s - H C 1 ( p H 7.4) c o n t a i n i n g I mM E D T A a n d 1 mM dithiothreitol, a n d c e n t r i f u g e d at 10,000g for 10 min. T h e s u p e r n a t a n t fluid was u s e d for t h e G M P red u c t a s e assay.
Standard Assay Method T h e s t a n d a r d a s s a y m i x t u r e was a modification of t h a t for t h e h u m a n e r y t h r o c y t e e n z y m e (6). T h e reaction m i x t u r e c o n t a i n e d 100 mM T r i s - H C 1 ( p H 7.8), 1 mM E D T A , 6.6 mM N A D P H , 4 mM dithiothreitol, 0.2 mM [8-14C]GMP, a n d e n z y m e p r e p a r a t i o n in a final volu m e of 25 ttl. T h e m i x t u r e was p r e i n c u b a t e d w i t h o u t e n z y m e for 3 m i n a n d the r e a c t i o n was s t a r t e d by adding 5 #1 of e n z y m e . At 10 a n d 20 min, the reaction was s t o p p e d b y i m m e r s i n g the r e a c t i o n t u b e in a boiling wat e r b a t h for 30 s, a n d t h e n the t u b e was placed on ice. A f t e r c e n t r i f u g a t i o n at 1000g for 10 min, 15 #l of clear s u p e r n a t a n t was s p o t t e d with 50 n m o l of a u t h e n t i c I M P a n d G M P as c a r r i e r s u b s t a n c e s on W h a t m a n DE81 ione x c h a n g e c h r o m a t o g r a p h y p a p e r , which was subjected to d e s c e n d i n g c h r o m a t o g r a p h y in 95% ethanol: 1 M a m m o n i u m a c e t a t e ( p H 5) (70:30, v / v ) c o n t a i n i n g 3.3 mM E D T A . T h e m a r k e r s p o t s were located u n d e r uv light, cut out, a n d p l a c e d in scintillation vials with 10 ml of scintillation fluid (OCS f r o m A m e r s h a m ) . T h e radioactivities were c o u n t e d in a B e c k m a n liquid scintillation counter, M o d e l 544. All radioactivities were c o r r e c t e d b y s u b t r a c t i n g the 0-time b l a n k values.
Protein Determination T h e p r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d by a BioR a d p r o t e i n a s s a y kit ( R i c h m o n d , CA) with bovine s e r u m a l b u m i n as s t a n d a r d (7). RESULTS AND DISCUSSION
Products of G M P Reductase Assay in Crude Extract T h e p a t t e r n of s e p a r a t i o n of d e s c e n d i n g c h r o m a t o g r a p h y is s h o w n in Fig. 1. In this G M P r e d u c t a s e a s s a y in t h e c r u d e extract, G M P m u s t be s e p a r a t e d n o t only f r o m I M P b u t also f r o m G D P , G T P , guanosine, guanine, inosine, a n d h y p o x a n t h i n e , since G M P c a n be c o n v e r t e d to t h e s e m e t a b o l i t e s . W e s e p a r a t e d the p r o d -
FRONT FIG. 1. Descending chromatographic pattern of the substrate, GMP, the product, IMP, and other reaction products with DE81 ionexchange chromatography paper. (See also Materials and Methods.)
uct, I M P , f r o m the s u b s t r a t e , G M P , a n d f r o m G D P , G T P , guanosine, guanine, inosine, a n d h y p o x a n t h i n e (R r = 0.42, 0.29, 0.10, 0.08, 0.76, 0.72, 0.84, a n d 0.85, respectively) with W h a t m a n DE81 i o n - e x c h a n g e p a p e r (Fig. 1). T h e 14C radioactivity i n c r e a s e d in a t i m e - d e p e n d e n t f a s h i o n for all t h e s e s p o t s e x c e p t for inosine a n d h y p o x a n t h i n e , which was negligible. T h e ~4C radioactivity applied on W h a t m a n DE81 p a p e r was acc o u n t e d for in the s u m of radioactivity in the s p o t s of I M P , G M P , G D P , G T P , guanosine, a n d guanine.
GMP Reductase Activity in Crude Extract from HL-60 Leukemia Cells Studies were carried out to elucidate o p t i m a l kinetic conditions of G M P r e d u c t a s e in the crude e x t r a c t in order to develop a s t a n d a r d a s s a y w h e r e linear kinetics were obtained.
Requirement for GMP W h e n t h e G M P c o n c e n t r a t i o n was increased, the enz y m e activity in H L - 6 0 cells showed a s a t u r a b l e kinetic p a t t e r n with a m a x i m u m velocity o b t a i n e d at 50 ttM with a K~ of 12.5 ttM (Fig. 2). M i c h a e l i s - M e n t e n c o n s t a n t s for G M P in c a l f t h y m u s a n d h u m a n e r y t h r o c y t e s were 1.4 to 4.9 #M, respectively (5,8); t h e s e were n e a r t h e r a n g e o b s e r v e d in this study. In the routine e n z y m e assay, 200 #M [14C]GMP was used. T h e e n z y m e activity was n o t inhibited by G M P u p to 300 #M.
Requirement for N A D P H T h e influence of c h a n g i n g the c o n c e n t r a t i o n of t h e cofactor N A D P H in H L - 6 0 cells yielded s a t u r a b l e kinetics with a m a x i m u m velocity a t t a i n e d at 1.65 mM with a K~ of 244 ~M (Fig. 3). T h i s value is 8- to 30-fold higher t h a n t h o s e r e p o r t e d in purified calf t h y m u s a n d h u m a n e r y t h r o c y t e p r e p a r a t i o n s w h e r e the K~ was 31 to 8.5 #M. T h e higher K~ o b s e r v e d in the crude p r e p a r a t i o n
ENZYMATIC ASSAY IN CRUDE EXTRACTS
117 TABLE 1
G M P Reductase Activity in Various Rat Tissues a 0.8
~
Protein
06
Enzyme activity
Tissues
mg/g
% of liver
nmol/h/mg protein
% of liver
Heart Brain Muscle Bone marrow Thymus Kidney Liver Spleen Lung Testis
45.8 +_0.9 40.5 _+ 1.8 50.2 ___1.5 29.4 _+ 1.7 48.0 + 1.0 65.0 _+ 1.8 79.3 _+ 1.4 76.7 _+2.2 72.5 _+ 1.6 49,0 _+ 1.7
58* 58* 63* 37* 61" 82* 100 97 91 62*
25.7 _+0.8b 12.9 -_+0.5 12.1 _+0.8 9.4 - 0.6 7.5 - 0.5 7.1 -+ 0.1 6.8 -+ 0.3 6.4 _+0.2 5.5 _ 0.3 2.2 _+0.1
378* 190" 178" 138" 110 104 100 94 81 32*
02
0
. . . . 0.2 0.3 0.4 l]S (o.M"1)
0.1 i
0
50
,
100
,
i
150
,
200
0
,
250
0.' i
300
GMP (~M)
FIG. 2. Effect of GMP concentration on GMP reduetase activity in HL-60 cells. Conditions for the assay are given under Materials and Methods. The GMP concentration varied from 2.5 to 300 #M with an NADPH concentration of 3.3 mM.
is a t t r i b u t e d t o t h e p r e s e n c e of h i g h N A D P H o x i d a s e a c t i v i t y . I n o u r s t a n d a r d a s s a y s 3.3 m M N A D P H w a s used. T h e e n z y m e activity was not i n h i b i t e d by N A D P H u p to 6.6 mM. V a r i a t i o n s for Km's we o b s e r v e d w e r e < ___5%.
Linearity of Assay with Time and Amount of Protein Added As a result of d e t e r m i n i n g the o p t i m u m c o n c e n t r a t i o n s for t h e s u b s t r a t e a n d c o f a c t o r , G M P a n d N A D P H , I M P p r o d u c t i o n w a s p r o p o r t i o n a t e a n d l i n e a r w i t h react i o n t i m e u p t o 40 m i n a n d w i t h a m o u n t o f e n z y m e a d d e d f r o m 9 t o 45 #g p r o t e i n . T h e m i n i m a l a m o u n t o f t h e p r o d u c t d e t e c t e d w a s 10 p m o l . T h e r a d i o a c t i v i t y o f I M P at 0 time was 70-90 cpm. T h e e n z y m e activity in H L - 6 0 cells (log p h a s e , 72 h a f t e r s e e d i n g 1 x 105 c e l l s / ml) w a s 8.3 + 0.1 n m o l / h / m g p r o t e i n .
~.~
/
8"
~
~
["N
~
a
•
.20
4.o,
2~
0
0
1
2
3
1
4
2 3 I/S (ram-~ ) 5
4
5
6
N A D P H (raM)
FIG. 3. Influence of NADPH concentration on GMP reductase activity from HL-60 cells. Conditions for the assay are described under Materials and Methods, except for the concentration of NADPH which varied from 0.10 to 6.6 mM, with a GMP concentration of 200 #M.
a Tissue samples were obtained from male Wistar rats (200 g) and assayed as described under Materials and Methods. b Means _+SE of three or more determinations. * Significantly different from values of liver, which were taken as 100% (P < 0.05).
GMP Reductase Activity in Various Rat Tissues GMP reductase activity was compared in extracts f r o m v a r i o u s t i s s u e s o f W i s t a r r a t s ( T a b l e 1). I n n o r m a l r a t l i v e r c y t o s o l p r e p a r a t i o n s t h e specific a c t i v i t y o f G M P r e d u c t a s e w a s ( m e a n + S E ) 6.8 + 0.3 n m o l / h / m g p r o t e i n . H e a r t h a d t h e h i g h e s t a c t i v i t i e s (378% o f liver), a n d a c t i v i t i e s of b r a i n a n d m u s c l e w e r e also g r e a t e r (190 a n d 178%), r e s p e c t i v e l y , t h a n t h a t of t h e liver. A c t i v i t y i n t e s t i s w a s t h e l o w e s t a m o n g all t h e t i s s u e s e x a m i n e d .
Assay of GMP Reductase in Crude Extracts Spectrophotometric assay methods measuring GMP reductase activity are u n a v o i d a b l y inexact due to the low specific a c t i v i t y o f t h i s e n z y m e a n d t h e n o n s p e c i f i c a b s o r p t i o n change caused by i n t e r f e r i n g e n z y m e s (911). T h e r e f o r e , r a d i o m e t r i c a s s a y s h a v e b e e n d e v e l o p e d where thin-layer chromatography on polyethylenimine p l a t e s (8) o r a s c e n d i n g c h r o m a t o g r a p h y w i t h W h a t m a n 3 M M p a p e r (5) h a v e b e e n u s e d t o s e p a r a t e [14C]IMP from [14C]GMP and [14C]guanine f r o m [14C]h y p o x a n t h i n e , w h i c h w e r e h y d r o l y z e d f r o m [14C]IMP a n d [14C]GMP b y b o i l i n g t h e acidic m i x t u r e for 30 m i n . However, these methods yielded high and inconsistent 0-time values a n d r e s u l t e d in poorer s e p a r a t i o n of I M P , GMP, and other reaction products than with Whatman DE81 paper. T h e sensitivity a n d reproducibility of these e a r l i e r a s s a y s w e r e n o t specified, b u t i n o u r i m p r o v e d m e t h o d h i g h s e n s i t i v i t y ( w i t h a l i m i t o f d e t e c t i o n o f 10 p m o l o f [14C]IMP p r o d u c t s ) is c o u p l e d w i t h h i g h r e p r o d u c i b i l i t y ( S E < + 5 % ) . M o r e o v e r , a l t h o u g h a l m o s t all the previously used methods need partial purification, l a r g e s a m p l e s , a n d a l o n g p e r i o d o f t i m e to p r o c e s s , o u r
118
NAKAMURA ET AL.
d i r e c t m e t h o d r e q u i r e s l e s s t h a n 100 #g p r o t e i n a n d 10 a n d 20 m i n a s s a y . Novel Aspects of This Study
T h e a d v a n t a g e s o f o u r G M P r e d u c t a s e a s s a y a r e : (i) H i g h s e n s i t i v i t y is a c h i e v e d e v e n i n t h e l o w G M P r e d u c t a s e a c t i v i t y r a n g e , w i t h a l i m i t o f d e t e c t i o n o f 10 p m o l o f I M P p r o d u c t i o n . (ii) T h e a s s a y h a s h i g h r e p r o d u c i b i l i t y w i t h l o w S E o f < 5 % . (iii) I t is m o r e c o n v e n i e n t t o operate with Whatman DE81 paper than TLC sheets. (iv) T h e d i r e c t a s s a y m e t h o d i n c r u d e t i s s u e e x t r a c t a n d t h e s m a l l t i s s u e s a m p l e size r e q u i r e d p e r m i t a s s a y in clinical biopsy material and blood samples. In conclusion, the present method using Whatman DE81 ion-exchange chromatography paper proves to be highly sensitive, c o n v e n i e n t , a n d v a l i d i n c r u d e t i s s u e e x t r a c t s . ACKNOWLEDGMENTS This work was supported by United States Public Health Service Outstanding Investigator Grant CA-42510 to G. Weber and by American Cancer Society Institutional Grant IN-161-D to H. Nakamura.
REFERENCES
1. Tricot, G., Jayaram, H. N., Lapis, E., Natsumeda, Y., Nichols, C. R., Kneebone, P., Heerema, N., Weber, G., and Hoffman, R. (1989) Cancer Res. 49, 3696-3701. 2. Weber, G., Jayaram, H. N., Lapis, E., Natsumeda, Y., Yamada, Y., Yamaji, Y., Tricot, G. J., and Hoffman, R. (1988) Adv. Enzyme Regul. 27,405-433. 3. Tricot, G., Jayaram, H. N., Weber, G., and Hoffman, R. (1990) Int. J. Cell Cloning 8, 161-170. 4. Mager, J., and Magasanik, B. (1960) J. Biol. Chem. 235, 14741478. 5. Stephens, R. W., and Whittaker, V. K. (1973) Biochem. Biophys. Res. Commun. 53,975-981. 6. Spector, T., Jones, T. E., and Miller, R. L. (1979) J. Biol. Chem. 254, 2308-2315. 7. Bradford, M. (1976) Anal. Biochem. 72, 248-252. 8. MacKenzie, J. J., and Sorensen, L. B. (1973) Biochim. Biophys. Acta 327,282-294. 9. Nijkamp, H. J., and De Haan, P. G. (1967) Biochim. Biophys. Acta 145, 31-40. 10. Benson, C. E., Brehmeyer, B. A., and Gots, J. S. (1971) Biochem. Biophys. Res. Commun. 43, 1089-1094. 11. Garber, B. B., Jochimsen, B. U., and Gots, J. S. (1980) J. Bacteriol. 143, 105-111.
Direct Assay Method for Guanosine 5'-Monophosphate Reductase Activity Hiroyuki Nakamura, Yutaka Natsumeda, Masami Nagai, Taiichi Shiotani, and George Weber 1 Laboratory for Experimental Oncology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5200
Received April 28, 1992
A s e n s i t i v e a n d s i m p l e m i c r o m e t h o d for t h e a c c u r a t e m e a s u r e m e n t o f G M P r e d u c t a s e (EC 1 . 6 . 6 . 8 ) a c t i v i t y i n c r u d e e x t r a c t s is d e s c r i b e d . T h e r e a c t i o n p r o d u c t o f [814C]IMP w a s s e p a r a t e d f r o m t h e s u b s t r a t e [8-14C]GMP by descending chromatography on Whatman DE81 ionexchange paper. This separation method provides an a n a l y s i s o f t h e p o s s i b l e i n t e r f e r i n g r e a c t i o n s , s u c h as t h e m e t a b o l i c c o n v e r s i o n o f t h e s u b s t r a t e G M P to G D P , GTP, and/or guanosine, and guanine and the loss of the p r o d u c t I M P to i n o s i n e , h y p o x a n t h i n e , a n d o t h e r m e t a b olites. Low blank values (70-90 cpm) were obtained consistently with this assay because the IMP spot moves f a s t e r t h a n t h e G M P spot. T h e m a j o r a d v a n t a g e s o f t h i s method are direct measurement of GMP reductase activity in crude extracts, high sensitivity (with a limit of detection of less than 10 pmol of IMP production), high r e p r o d u c i b i l i t y (< _+5%), a n d c a p a b i l i t y to m e a s u r e act i v i t y i n s m a l l s a m p l e s (9 # g p r o t e i n ) . ~ 1992 A c a d e m i c Press, Inc.
During the course of trials with tiazofurin (NSC 286193, 2-fl-D-ribofuranosylthiazole-4-carboxamide) in patients with chronic myelogenous leukemia in blast crisis at Indiana University School of Medicine, there was a 70 to 80% clinical response rate (1-3). These patients received repeated cycles of daily tiazofurin infusions to contain the leukemic cell proliferation and some of the patients became clinically resistant to the drug. T h e target of tiazofurin is IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of de novo G T P biosynthesis, which governs the first step converting I M P to XMP. T h r o u g h the subsequent step, catalyzed by G M P synthase (EC 6.3.5.2), G M P is produced. G M P reductase (EC 1.6.6.8) in effect opposes the synthetic action of IMP dehydrogenase because it catalyzes the
1To whom correspondenceshould be addressed. 0003-2697/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
essentially irreversible reductive deamination of G M P to IMP. T h e increased activity of G M P reductase may explain, in part at least, the emergence of resistant cells. Therefore, to underst and the mechanism of resistance, it was i m port ant to determine G M P reductase activity in leukemic cells of patients undergoing tiazofurin therapy. Our interest was to develop a quick and sensitive method for measuring the enzyme activity in samples of less t han a million cells from leukemic patients in blast crisis and in other tissues. G M P reductase was first dem onst rat ed in extracts of bacteria, including Escherichia coli (4). G M P reductase has been purified from mammalian sources, e.g., calf thymus (5), hum an erythrocytes (6), and the assay procedures for the activity of these purified enzymes were reported. However, difficulties were encountered in measuring the enzyme activities with precision in crude extracts of rat or hum an tissue or tissue culture samples because of some of the side products formed during the G M P reductase reaction. T he present report describes a direct assay method of G M P reductase activity in crude extracts which provides greater accuracy and decreases the am o u n t of tissue required. For solving these problems we investigated in crude extracts all possible interfering reactions such as conversion of G M P to GDP, G T P , guanosine, and guanine and the loss of IMP to inosine and hypoxanthine.
MATERIALS AND METHODS Materials
[8-14C]GMP am m oni um salt (55 mCi/mmol) was purchased from Moravek Biochemicals, Inc. (Brea, CA), and prior to use the specific activity was diluted to 20 mCi/mmol with cold GMP. R P M I 1640 culture medium and dialyzed fetal bovine serum were from GIBCO (Grand Island, NY). All other biochemicals were also 115
116
NAKAMURA ET AL.
r e a g e n t grade a n d were o b t a i n e d f r o m S i g m a C h e m i c a l Co. (St. Louis, MO).
DE 81 O~G~
0
GTP GDP
o
GMP
o
IMP
o o
Guanine Hypoxanthine
Enzyme Extracts H L - 6 0 h u m a n p r o m y e l o c y t i c l e u k e m i a cells were p u r c h a s e d f r o m A m e r i c a n T y p e C u l t u r e Collection (Rockville, M D ) a n d m a i n t a i n e d in R P M I 1640 m e d i u m cont a i n i n g 10% dialyzed fetal b o v i n e serum, s u p p l e m e n t e d with penicillin (100 U / m l ) a n d s t r e p t o m y c i n (100 ~g/ ml) at 37°C in a humidified a t m o s p h e r e with 5% CO2. E n z y m e e x t r a c t s were p r e p a r e d by h o m o g e n i z a t i o n in 40 mM T r i s - H C 1 ( p H 7.4) c o n t a i n i n g I mM E D T A a n d 1 mM dithiothreitol, a n d c e n t r i f u g e d at 10,000g for 10 min. T h e s u p e r n a t a n t fluid was u s e d for t h e G M P red u c t a s e assay.
Standard Assay Method T h e s t a n d a r d a s s a y m i x t u r e was a modification of t h a t for t h e h u m a n e r y t h r o c y t e e n z y m e (6). T h e reaction m i x t u r e c o n t a i n e d 100 mM T r i s - H C 1 ( p H 7.8), 1 mM E D T A , 6.6 mM N A D P H , 4 mM dithiothreitol, 0.2 mM [8-14C]GMP, a n d e n z y m e p r e p a r a t i o n in a final volu m e of 25 ttl. T h e m i x t u r e was p r e i n c u b a t e d w i t h o u t e n z y m e for 3 m i n a n d the r e a c t i o n was s t a r t e d by adding 5 #1 of e n z y m e . At 10 a n d 20 min, the reaction was s t o p p e d b y i m m e r s i n g the r e a c t i o n t u b e in a boiling wat e r b a t h for 30 s, a n d t h e n the t u b e was placed on ice. A f t e r c e n t r i f u g a t i o n at 1000g for 10 min, 15 #l of clear s u p e r n a t a n t was s p o t t e d with 50 n m o l of a u t h e n t i c I M P a n d G M P as c a r r i e r s u b s t a n c e s on W h a t m a n DE81 ione x c h a n g e c h r o m a t o g r a p h y p a p e r , which was subjected to d e s c e n d i n g c h r o m a t o g r a p h y in 95% ethanol: 1 M a m m o n i u m a c e t a t e ( p H 5) (70:30, v / v ) c o n t a i n i n g 3.3 mM E D T A . T h e m a r k e r s p o t s were located u n d e r uv light, cut out, a n d p l a c e d in scintillation vials with 10 ml of scintillation fluid (OCS f r o m A m e r s h a m ) . T h e radioactivities were c o u n t e d in a B e c k m a n liquid scintillation counter, M o d e l 544. All radioactivities were c o r r e c t e d b y s u b t r a c t i n g the 0-time b l a n k values.
Protein Determination T h e p r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d by a BioR a d p r o t e i n a s s a y kit ( R i c h m o n d , CA) with bovine s e r u m a l b u m i n as s t a n d a r d (7). RESULTS AND DISCUSSION
Products of G M P Reductase Assay in Crude Extract T h e p a t t e r n of s e p a r a t i o n of d e s c e n d i n g c h r o m a t o g r a p h y is s h o w n in Fig. 1. In this G M P r e d u c t a s e a s s a y in t h e c r u d e extract, G M P m u s t be s e p a r a t e d n o t only f r o m I M P b u t also f r o m G D P , G T P , guanosine, guanine, inosine, a n d h y p o x a n t h i n e , since G M P c a n be c o n v e r t e d to t h e s e m e t a b o l i t e s . W e s e p a r a t e d the p r o d -
FRONT FIG. 1. Descending chromatographic pattern of the substrate, GMP, the product, IMP, and other reaction products with DE81 ionexchange chromatography paper. (See also Materials and Methods.)
uct, I M P , f r o m the s u b s t r a t e , G M P , a n d f r o m G D P , G T P , guanosine, guanine, inosine, a n d h y p o x a n t h i n e (R r = 0.42, 0.29, 0.10, 0.08, 0.76, 0.72, 0.84, a n d 0.85, respectively) with W h a t m a n DE81 i o n - e x c h a n g e p a p e r (Fig. 1). T h e 14C radioactivity i n c r e a s e d in a t i m e - d e p e n d e n t f a s h i o n for all t h e s e s p o t s e x c e p t for inosine a n d h y p o x a n t h i n e , which was negligible. T h e ~4C radioactivity applied on W h a t m a n DE81 p a p e r was acc o u n t e d for in the s u m of radioactivity in the s p o t s of I M P , G M P , G D P , G T P , guanosine, a n d guanine.
GMP Reductase Activity in Crude Extract from HL-60 Leukemia Cells Studies were carried out to elucidate o p t i m a l kinetic conditions of G M P r e d u c t a s e in the crude e x t r a c t in order to develop a s t a n d a r d a s s a y w h e r e linear kinetics were obtained.
Requirement for GMP W h e n t h e G M P c o n c e n t r a t i o n was increased, the enz y m e activity in H L - 6 0 cells showed a s a t u r a b l e kinetic p a t t e r n with a m a x i m u m velocity o b t a i n e d at 50 ttM with a K~ of 12.5 ttM (Fig. 2). M i c h a e l i s - M e n t e n c o n s t a n t s for G M P in c a l f t h y m u s a n d h u m a n e r y t h r o c y t e s were 1.4 to 4.9 #M, respectively (5,8); t h e s e were n e a r t h e r a n g e o b s e r v e d in this study. In the routine e n z y m e assay, 200 #M [14C]GMP was used. T h e e n z y m e activity was n o t inhibited by G M P u p to 300 #M.
Requirement for N A D P H T h e influence of c h a n g i n g the c o n c e n t r a t i o n of t h e cofactor N A D P H in H L - 6 0 cells yielded s a t u r a b l e kinetics with a m a x i m u m velocity a t t a i n e d at 1.65 mM with a K~ of 244 ~M (Fig. 3). T h i s value is 8- to 30-fold higher t h a n t h o s e r e p o r t e d in purified calf t h y m u s a n d h u m a n e r y t h r o c y t e p r e p a r a t i o n s w h e r e the K~ was 31 to 8.5 #M. T h e higher K~ o b s e r v e d in the crude p r e p a r a t i o n
ENZYMATIC ASSAY IN CRUDE EXTRACTS
117 TABLE 1
G M P Reductase Activity in Various Rat Tissues a 0.8
~
Protein
06
Enzyme activity
Tissues
mg/g
% of liver
nmol/h/mg protein
% of liver
Heart Brain Muscle Bone marrow Thymus Kidney Liver Spleen Lung Testis
45.8 +_0.9 40.5 _+ 1.8 50.2 ___1.5 29.4 _+ 1.7 48.0 + 1.0 65.0 _+ 1.8 79.3 _+ 1.4 76.7 _+2.2 72.5 _+ 1.6 49,0 _+ 1.7
58* 58* 63* 37* 61" 82* 100 97 91 62*
25.7 _+0.8b 12.9 -_+0.5 12.1 _+0.8 9.4 - 0.6 7.5 - 0.5 7.1 -+ 0.1 6.8 -+ 0.3 6.4 _+0.2 5.5 _ 0.3 2.2 _+0.1
378* 190" 178" 138" 110 104 100 94 81 32*
02
0
. . . . 0.2 0.3 0.4 l]S (o.M"1)
0.1 i
0
50
,
100
,
i
150
,
200
0
,
250
0.' i
300
GMP (~M)
FIG. 2. Effect of GMP concentration on GMP reduetase activity in HL-60 cells. Conditions for the assay are given under Materials and Methods. The GMP concentration varied from 2.5 to 300 #M with an NADPH concentration of 3.3 mM.
is a t t r i b u t e d t o t h e p r e s e n c e of h i g h N A D P H o x i d a s e a c t i v i t y . I n o u r s t a n d a r d a s s a y s 3.3 m M N A D P H w a s used. T h e e n z y m e activity was not i n h i b i t e d by N A D P H u p to 6.6 mM. V a r i a t i o n s for Km's we o b s e r v e d w e r e < ___5%.
Linearity of Assay with Time and Amount of Protein Added As a result of d e t e r m i n i n g the o p t i m u m c o n c e n t r a t i o n s for t h e s u b s t r a t e a n d c o f a c t o r , G M P a n d N A D P H , I M P p r o d u c t i o n w a s p r o p o r t i o n a t e a n d l i n e a r w i t h react i o n t i m e u p t o 40 m i n a n d w i t h a m o u n t o f e n z y m e a d d e d f r o m 9 t o 45 #g p r o t e i n . T h e m i n i m a l a m o u n t o f t h e p r o d u c t d e t e c t e d w a s 10 p m o l . T h e r a d i o a c t i v i t y o f I M P at 0 time was 70-90 cpm. T h e e n z y m e activity in H L - 6 0 cells (log p h a s e , 72 h a f t e r s e e d i n g 1 x 105 c e l l s / ml) w a s 8.3 + 0.1 n m o l / h / m g p r o t e i n .
~.~
/
8"
~
~
["N
~
a
•
.20
4.o,
2~
0
0
1
2
3
1
4
2 3 I/S (ram-~ ) 5
4
5
6
N A D P H (raM)
FIG. 3. Influence of NADPH concentration on GMP reductase activity from HL-60 cells. Conditions for the assay are described under Materials and Methods, except for the concentration of NADPH which varied from 0.10 to 6.6 mM, with a GMP concentration of 200 #M.
a Tissue samples were obtained from male Wistar rats (200 g) and assayed as described under Materials and Methods. b Means _+SE of three or more determinations. * Significantly different from values of liver, which were taken as 100% (P < 0.05).
GMP Reductase Activity in Various Rat Tissues GMP reductase activity was compared in extracts f r o m v a r i o u s t i s s u e s o f W i s t a r r a t s ( T a b l e 1). I n n o r m a l r a t l i v e r c y t o s o l p r e p a r a t i o n s t h e specific a c t i v i t y o f G M P r e d u c t a s e w a s ( m e a n + S E ) 6.8 + 0.3 n m o l / h / m g p r o t e i n . H e a r t h a d t h e h i g h e s t a c t i v i t i e s (378% o f liver), a n d a c t i v i t i e s of b r a i n a n d m u s c l e w e r e also g r e a t e r (190 a n d 178%), r e s p e c t i v e l y , t h a n t h a t of t h e liver. A c t i v i t y i n t e s t i s w a s t h e l o w e s t a m o n g all t h e t i s s u e s e x a m i n e d .
Assay of GMP Reductase in Crude Extracts Spectrophotometric assay methods measuring GMP reductase activity are u n a v o i d a b l y inexact due to the low specific a c t i v i t y o f t h i s e n z y m e a n d t h e n o n s p e c i f i c a b s o r p t i o n change caused by i n t e r f e r i n g e n z y m e s (911). T h e r e f o r e , r a d i o m e t r i c a s s a y s h a v e b e e n d e v e l o p e d where thin-layer chromatography on polyethylenimine p l a t e s (8) o r a s c e n d i n g c h r o m a t o g r a p h y w i t h W h a t m a n 3 M M p a p e r (5) h a v e b e e n u s e d t o s e p a r a t e [14C]IMP from [14C]GMP and [14C]guanine f r o m [14C]h y p o x a n t h i n e , w h i c h w e r e h y d r o l y z e d f r o m [14C]IMP a n d [14C]GMP b y b o i l i n g t h e acidic m i x t u r e for 30 m i n . However, these methods yielded high and inconsistent 0-time values a n d r e s u l t e d in poorer s e p a r a t i o n of I M P , GMP, and other reaction products than with Whatman DE81 paper. T h e sensitivity a n d reproducibility of these e a r l i e r a s s a y s w e r e n o t specified, b u t i n o u r i m p r o v e d m e t h o d h i g h s e n s i t i v i t y ( w i t h a l i m i t o f d e t e c t i o n o f 10 p m o l o f [14C]IMP p r o d u c t s ) is c o u p l e d w i t h h i g h r e p r o d u c i b i l i t y ( S E < + 5 % ) . M o r e o v e r , a l t h o u g h a l m o s t all the previously used methods need partial purification, l a r g e s a m p l e s , a n d a l o n g p e r i o d o f t i m e to p r o c e s s , o u r
118
NAKAMURA ET AL.
d i r e c t m e t h o d r e q u i r e s l e s s t h a n 100 #g p r o t e i n a n d 10 a n d 20 m i n a s s a y . Novel Aspects of This Study
T h e a d v a n t a g e s o f o u r G M P r e d u c t a s e a s s a y a r e : (i) H i g h s e n s i t i v i t y is a c h i e v e d e v e n i n t h e l o w G M P r e d u c t a s e a c t i v i t y r a n g e , w i t h a l i m i t o f d e t e c t i o n o f 10 p m o l o f I M P p r o d u c t i o n . (ii) T h e a s s a y h a s h i g h r e p r o d u c i b i l i t y w i t h l o w S E o f < 5 % . (iii) I t is m o r e c o n v e n i e n t t o operate with Whatman DE81 paper than TLC sheets. (iv) T h e d i r e c t a s s a y m e t h o d i n c r u d e t i s s u e e x t r a c t a n d t h e s m a l l t i s s u e s a m p l e size r e q u i r e d p e r m i t a s s a y in clinical biopsy material and blood samples. In conclusion, the present method using Whatman DE81 ion-exchange chromatography paper proves to be highly sensitive, c o n v e n i e n t , a n d v a l i d i n c r u d e t i s s u e e x t r a c t s . ACKNOWLEDGMENTS This work was supported by United States Public Health Service Outstanding Investigator Grant CA-42510 to G. Weber and by American Cancer Society Institutional Grant IN-161-D to H. Nakamura.
REFERENCES
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