20
Biochimica et Biophysica Acta, 544 (1978) 20--28
© Elsevier/North-Holland Biomedical Press
BBA 28699 SEPARATION OF CYCLIC AMP AND CYCLIC GMP FROM THYMIDINE, E L E C T R O L Y T E S AND P O L Y V A L E N T NUCLEOTIDES IN TISSUE SAMPLES
GLENDA M. ROBERSON and LARRY D. BARNES Department o f Biochemistry, University o f Texas Health Science Center, San A ntonio, Texas 78284 (U.S.A.)
(Received February 22nd, 1978) (Revised manuscript received May llth, 1978)
Summary Cyclic AMP and cyclic GMP can be separated from thymidine and its possible metabolites, electrolytes, and polyvalent nucleotides using columns of acidic alumina. Electrolytes and thymidine are not adsorbed on acidic alumina at pH 4.4 while cyclic nucleotides and polyvalent nucleotides are adsorbed at this pH. Cyclic AMP and cyclic GMP are eluted together from acidic alumina with 0.2 M ammonium formate (pH 6.0) and the polyvalent nucleotides remain adsorbed. The cyclic nucleotides are separated by chromatography on Dowex AG 1 X 8 resin. Recovery is 60--64% for cyclic AMP and cyclic GMP isolated from renal tissue samples. This m e t h o d o l o g y permits the separation of tritiated thymidine from cyclic nucleotides which are present in tissue preparations used in studies on the role of cyclic nucleotides in cellular growth.
Introduction Studies on the roles of adenosine 3',5'-cyclic monophosphate (cyclic AMP) and guanosine 3',5'-cyclic monophosphate (cyclic GMP) in cellular functions have generated different techniques for isolating these cyclic nucleotides. These techniques include ion-exchange column chromatography [1--3], thin-layer chromatography [4--8], electrophoresis [9,10], and adsorption chromatography using alumina [11--14]. Although these methods separate the cyclic nucleotides from other nucleotides, they are often inadequate to separate completely cyclic AMP and cyclic GMP from each other and from both nucleosides and polyvalent nucleotides. In addition, excess electrolytes often interfere with A b b r e v i a t i o n : bis-MSB, 1 , 4 - d i - ( 2 - m e t h y l s t y r y l ) - b e n z e n e .
21 ion-exchange chromatography [3]. Consequently, a combination of methods is often required to achieve the desired separation [15,16]. Our studies on the possible roles of cyclic AMP and cyclic GMP in renal hyperplasia necessitated separation of three tritiated compounds. Rats were injected with [3H] thymidine prior to killing to measure incorporation of [ 3H]thymidine into DNA. Subsequently, renal tissue was homogenized in trichloroacetic acid containing cyclic [3H]AMP and cyclic [3H]GMP to monitor recoveries of endogenous cyclic nucleotides. Thus, the supernatant fraction processed for cyclic nucleotide analyses contained unincorporated [3H]thymidine and its possible metabolites as well as the tritiated cyclic nucleotides. One could use separate animals for measurements of [3H]thymidine incorporation and endogenous cyclic nucleotides and obviate the problem. However, this is impracticable when large numbers of animals are used. Also, one can more reliably correlate changes in metabolite levels with a cellular process if these determinations are made on the same animal. Ion-exchange chromatography alone was inadequate to separate cyclic AMP and cyclic GMP from each other and from thymidine and its possible metabolites. The high electrolyte content of renal tissue was probably one reason for such chromatography being unsuccessful. We have isolated cyclic AMP and cyclic GMP from renal tissue using columns of acidic alumina plus columns of Dowex 1 X 8 resin. Quantitation of recoveries and amounts of endogenous cyclic nucleotides can be determined after removal of thymidine and its metabolites, electrolytes and polyvalent nucleotides by this chromatographic procedure. Methods
Materials. [Me-3H]Thymidine; [8-3H]cyclic AMP; and [8-3H]cyclic GMP were purchased from Amersham/Searle. Cyclic AMP, cyclic GMP, AMP, ADP, ATP, GMP, GDP, GTP, thymine, thymidine, TMP, TDP, TTP and beef heart cyclic nucleotide phosphodiesterase were purchased from Sigma Chemical Company. Radioimmunoassay kits for measurement of cyclic AMP and cyclic GMP were from Schwarz/Mann. Acidic alumina was purchased from Fisher (Brockman activity I, 80--200 mesh) and Dowex AG 1 X 8 (200--400 mesh, formate form) from Bio-Rad. PEI-cellulose and cellulose thin-layer sheets were from EM Laboratories, Inc. Plastic columns (8 × 200 mm) were supplied by Kontes. Male, Sprague-Dawley rats (150--200 g) were purchased from BioLabs, Inc. Scintillation cocktail was composed of 7.5 g PPO and 0.1 g bis-MSB per 667 ml toluene and 333 ml Triton X-100 [17]. Counting efficiency was 33--37% for tritiated compounds as determined by the channels-ratio method in a Beckman LS-230 scintillation counter. Triton X-100 was purchased from Research Products, Inc., and scintillation-grade fluors and toluene were from Fisher. Preparation of tissue samples. Rats were injected intraperitoneally with [Me-3H]thymidine in saline (0.4 ~Ci per g body weight). 1 h later we anesthesized the rats with ether, withdrew a sample of blood from the jugular vein, cut the adominal wall, exposed the abdominal cavity and removed one kidney from each rat. Each kidney was immediately cut into coronal sections weighing
22 approx. 200--300 mg (wet wt.). One section from each kidney was rinsed in saline and homogenized in a Potter-Elvehjem tissue grinder containing 0.5 pmol cyclic [3H]AMP (11 560 cpm) and 0.5 pmol cyclic [3H]GMP (7980 cpm) in 4 ml of cold, 5% (w/v) trichloroacetic acid. The homogenate was centrifuged at 5900 × g for 20 min at 2°C. HC1 was added to the supernatant fraction to a final concentration of 0.1 M. Trichloroacetic acid was extracted from the supernatant fraction with 12 ml of water-saturated ether per extraction, and the extraction was repeated four additional times. The aqueous sample was heated at 85°C in a water bath for 5 min to remove residual ether. The sample was stored at --75°C until chromatographed. (Other sections from each kidney were used for histology and autoradiography.) Test solutions. We tested individual solutions to determine the conditions necessary to achieve chromatographic separation of electrolytes, thymine, thymidine, cyclic nucleotides and polyvalent nucleotides. The electrolyte solution was 0.18 M NaC1, 0.12 M KC1, and 0.02 M NH4C1 in 0.1 M HC1. The measured osmolality of this solution was 802 mOsm/kg H20. Solutions of thymine, TMP, TDP, TTP, AMP, ADP, ATP, GMP, GDP, and GTP contained 3--4 ~mol of a single c o m p o u n d in 4 ml of 0.1 M HC1. The other test solutions were 1 ~mol [3H]thymidine (39 800 cpm); 775 pmol cyclic [3H]AMP (12 400 cpm); and 98 pmol cyclic [3H]GMP (8360 cpm) each in 4 ml of 0.1 M HC1. Column chromatography. Acidic alumina was washed with several changes of distilled H20 to remove fines and dried at 110°C for 16 h. It can be stored at least 6 months at room temperature in a tightly-closed bottle w i t h o u t changes in chromatographic properties. Dowex AG 1 X 8 resin was washed with 2 M HCOOH and then H20 until the pH was 4.2--4.4 for an aqueous suspension. This resin was stored at 4°C as a 50% (v/v) aqueous suspension. Approx. 2 g acidic alumina were added with a glass measuring scoop to individual columns filled with H20. The alumina rapidly settled as the H20 drained to yield uniformly packed columns. Dowex columns were prepared by adding 3.5 ml of the 50% (v/v) aqueous suspension to individual columns. The pH of the aqueous effluents from both the acidic alumina and Dowex columns was 4.2--4.4. Columns were placed in Plexiglas racks designed to hold 100 columns. These racks could be placed on top of one another or over a box of scintillation vials. Initially the test solutions were applied to individual acidic alumina columns and the elution profiles determined by successive 1-ml additions of eluting solutions. Electrolytes, t h y m i n e and thymidine were eluted with H20; cyclic nucleotides were eluted together with 0.2 M a m m o n i u m formate (NH4CHO2), pH 6.0. TMP, TDP, TTP, AMP, ADP, ATP, GMP, GDP and GTP remained adsorbed to acidic alumina even in the presence of 1 M NH4CHO2, pH 6.0. NH4CHO2 eluates containing cyclic nucleotides were applied to Dowex columns, and elution profiles of cyclic AMP and cyclic GMP determined by successive 1-ml additions of eluting solutions. Eluates of [3H]thymidine, cyclic [3H]AMP, and cyclic [3H]GMP solutions were counted in a liquid scintillation counter. Absorbances of eluates from the t h y m i n e test solution were measured at 264 nm, and eluates from the polyvalent nucleotide solutions of thymidine, adenosine and guanosine were measured at 267 rim, 259 nm and 252 nm, respectively. Osmolalities were deter-
23 mined for eluates of the electrolyte test solution by freezing point depression using an Advanced Instrument Osmometer. Based on the results of elution profiles of test solutions, the following procedure was used for chromatography of tissue extracts. Supernatant fractions were applied to acidic alumina columns and washed with 10 ml of H20. The sample plus water eluates were discarded. The rack of alumina columns was then placed on top of the rack of Dowex columns. Cyclic nucleotides were eluted from the acidiC alumina columns onto the Dowex columns with 11 ml of 0.2 M NH4CHO2, pH 6.0. Dowex columns were washed with 10 ml of H20 and the eluates discarded. Cyclic AMP was eluted with 6 ml of 2 M HCOOH. Columns were washed with 3 ml of 5 M HCOOH and the elutes discarded. Cyclic GMP was eluted with an additional 8 ml of 5 M HCOOH. Eluates containing the cyclic nucleotides were quick-frozen in a solid CO2/acetone bath and lyophilized. Lyophilized samples were dissolved in 0.05 M sodium acetate, pH 6.2. 25--50 ~1 of each sample was counted to determine the percent of recovery, and the amounts of cyclic AMP and cyclic GMP were measured by radioimmunoassay [ 18]. Other methods. Cyclic nucleotide samples were hydrolyzed by incubating 300 pl of the isolated fraction (lyophilized column eluates dissolved in 0.05 M sodium acetate, pH 6.2) with 30 ~1 of buffer containing 0.02 unit cyclic nucleotide phosphodiesterase and 40 mM MgC12. Samples were incubated for 16 h at 25°C and the reaction was stopped by boiling for 5 min. Standard solutions of cyclic AMP and cyclic GMP, untreated and treated with phosphodiesterase, served as controls for the hydrolysis conditions. PEI-cellulose sheets developed in 1 M LiC1 [19] and cellulose sheets developed in n-butanol/acetic acid/H20 (12 : 3 : 5) [18] were used for thin-layer chromatography. Amounts of proteins were determined according to Lowry et al. [20] after dissolving samples in 1% sodium dodecyl sulfate. Results Individual solutions of electrolytes, thymine, thymidine, cyclic AMP, cyclic GMP, TMP, TDP, TTP, AMP, ADP, ATP, GMP, GDP and GTP were prepared to simulate the volume and HC1 concentration of the supernatant fractions of tissue samples. These solutions were individually chromatographed on columns of acidic alumina and Dowex AG 1 X 8. Electrolytes, thymine and thymidine were eluted from acidic alumina with H20, and cyclic AMP and cyclic GMP were eluted with 0.2 M NH4CHO2, pH 6.0 {Fig. 1). The elution profiles of thymine and thymidine are similar. Less than 1% of the polyvalent nucleotides of thymidine, adenosine and guanosine are eluted from acidic alumina with 0.2 M NH4CHO2 or 10 ml of 1 M NH4CHO2, pH 6.0. Cyclic AMP and cyclic GMP were separated on D o w e x AG 1 X 8 columns where cyclic AMP was eluted with 2 M HCOOH and cyclic GMP with 5 M HCOOH {Fig. 2). Each of the tritiated solutions which eluted from the columns was quantitatively recovered. Approx. 70% of the electrolytes and 100% of the thymine eluted from acidic alumina with H20. Elution profiles were unchanged when all of the test solutes were combined in 4 ml 0.1 N HC1 and chromatographed together. Based on results with test solutions, the chromatographic procedure
24 Ck
SAMPLE i t ELUATE
ck
)
HzO
//-4
02M NH4COOH,pH 60
i
i
~ 53
E3 ~
4S
c~
3O
II
Electrolytes.
-1-
4oo ~
~_c
~" I0 3
5
7
9
14 15
~7
~9
21
23
z5
Fractions ( I ml)
Fig. 1. C o m p o s i t e e l u t i o n p r o f i l e of [ 3 H ] t h y m i d i n e , cyclic [ 3 H ] A M P , cyclic [ 3 H ] G M P a n d e l e c t r o l y t e s f r o m acidic a l u m i n a . 2 g o f acidic a l u m i n a w e r e p o u r e d i n t o an 8 - r a m d i a m e t e r c o l u m n a n d r i n s e d w i t h H 2 0 . T h e f o l l o w i n g c o m p o u n d s in 4 m l 0.1 M HC1 w e r e a p p l i e d to i n d i v i d u a l c o l u m n s : (a) e l e c t r o l y t e s o l u t i o n o f 0 . 1 8 M NaC1, 0 . 1 2 M KC1, a n d 0 . 0 2 M NH4C1; (b) 1 ]~nol [ M e - 3 H ] t h y m i d i n e , 39 8 0 0 c p m ; (c) 7 7 5 p m o l cyclic [ 3 H ] A M P , 12 4 0 0 c p m ; a n d (d) 9 8 p m o l cyclic [ 3 H ] G M P , 8 3 6 0 c p m . C o m p o u n d s w e r e e l u t e d b y 10 m l H 2 0 a n d 11 m l 0.2 M N H 4 C H O 2, p H 6.0, a p p l i e d in 1-ml p o r t i o n s . 1-ml f r a c t i o n s w e r e c o l l e c t e d . O s m o l a l i t e s o f f r a c t i o n s c o n t a i n i n g e l e c t r o l y t e s w e r e d e t e r m i n e d b y f r e e z i n g - p o i n t depression. F r a c t i o n s of t r i t i a t e d c o m p o u n d s w e r e c o l l e c t e d d i r e c t l y in s c i n t i l l a t i o n vials a n d c o u n t e d a f t e r addit i o n of 10 m l s c i n t i l l a t i o n s o l u t i o n . T h e left o r d i n a t e expresses t h e p e r c e n t of t h e a p p l i e d r a d i o a c t i v i t y r e c o v e r e d in e a c h f r a c t i o n , a n d t h e right o r d i n a t e e x p r e s s e s t h e o s m o l a l i t y o f f r a c t i o n s c o n t a i n i n g e l e c t r o lytes. [ M e - 3 H ] t h y m i d i n e is a b b r e v i a t e d [ 3 H ] T d R ; cyclic [ 3 H ] A M P , [ 3 H ] c A M P ; c y c l i c [ 3 H ] G M P , [ 3 H ] cGMP.
70
&= Go
'~
'5o
4o
NH4COOH' pH6.0
II I/
1P"tcAMP
§ io
Fraction ( I m l )
Fig. 2. C o m p o s i t e e l u t i o n profile of cyclic [ 3 H ] A M P a n d cyclic [ 3 H ] G M P f r o m D o w e x A G 1 X 8 (form a t e ) resin. T h e 8 - m m d i a m e t e r c o l u m n c o n t a i n e d 3.5 m l 50% ( v / v ) a q u e o u s s u s p e n s i o n o f D o w e x A G l X 8 ( f o r m a t e ) resin. 4 m l o f the cyclic [ 3 H ] A M P a n d cyclic [ 3 H ] G M P s o l u t i o n s w e r e a p p l i e d t o indiv i d u a l acidic a l u m i n a c o l u m n s as d e s c r i b e d in Fig. 1. A f t e r w a s h i n g t h e a l u m i n a c o l u m n s w i t h 10 m l H 2 0 , t h e y w e r e p l a c e d a b o v e t h e D o w e x c o l u m n s . Cyclic n u e l e o t i d e s e l u t e d f r o m t h e a l u m i n a w i t h 0.2 M N H 4 C H O 2, p H 6.0, w e r e d r a i n e d directly i n t o t h e D o w e x c o l u m n s . Cyclic [ 3 H ] A M P a n d cyclic [ 3 H ] GMP w e r e e l u t e d f r o m t h e D o w e x resin b y 10 m l H 2 0 , ' 1 0 m l 2 M H C O O H , a n d 15 m l 5 M H C O O H , e a c h a p p l i e d in 1 m l p o r t i o n s . F r a c t i o n s w e r e c o l l e c t e d , c o u n t e d a n d t h e results e x p r e s s e d as d e s c r i b e d in Fig. 1. A b b r e v i a t i o n s as p e r Fig. 1.
25 described in Methods was used to separate [3H]thymidine, cyclic [3H]AMP and cyclic [3H]GMP present in a single renal tissue extract. Table I shows the elution scheme and distribution of labelled compounds in column eluates from renal tissue samples. [3H]Thymidine was present in the sample plus water eluate from the acidic alumina column. If thymine, TMP, TDP and TTP were metabolic products of the [3H]thymidine injected into the rat, [3H]thymine would elute from acidic alumina with H20 and the tritiated polyvalent nucleotides would remain adsorbed. Although the total counts of [3H]thymidine present in the renal tissue were considerably larger than the counts due to cyclic [3H]AMP and cyclic [3H]GMP, there was no carry-over of [3H]thymidine or its metabolic products into the water eluate from the Dowex column. Identity of the cyclic nucleotide fractions isolated from tissue samples and validity of their measurement by radioimmunoassay were confirmed by the following results. The radioactivity in the presumed cyclic AMP and cyclic GMP eluates comigrated with their respective standards on PEI-cellulose (in 1 M LiC1) and cellulose (in n-butanol/acetic acid/H20 (12 : 3 : 5) thin-layer sheets. Phosphodiesterase hydrolyzed 90--100% of both the cyclic AMP and cyclic GMP fractions. Amounts of cyclic nucleotides measured were linear with the volume of sample added to their respective radioimmunoassay. Cyclic AMP and cyclic GMP concentrations in rat renal tissue were 9.7 _+0.3 pmol/mg prorein and 0.51 _+0.03 pmol/mg protein (mean -+ S.E. for 42 rats), respectively. Recoveries of cyclic AMP and cyclic GMP from the point of homogenization of tissue to elution from Dowex AG 1 X 8 columns were 68 -+ 2% and 74 +_4%, respectively (Table I). Overall recoveries from homogenization to dissolution of lyophilized eluates were 61 + 2% for cyclic AMP (mean _+S.E. for 73 rats) and 64 + 2% for cyclic GMP (mean + S.E. for 71 rats} for animals which received different treatments in various experiments. Analyses of recoveries at each step TABLE I CHROMATOGRAPHY OF RENAL DOWEX AG 1 × 8 (FORMATE)
TISSUE SAMPLES ON COLUMNS
OF ACIDIC ALUMINA AND
Separation of [3H]thymidine, cyclic [3H]AMP, and cyclic [3H]GMP on acidic alumina and Dowex AG 1 × 8 ( f o r m a t e ) c o l u m n s f r o m r e n a l tissue h o m o g e n a t e s . R a t s w e r e i n j e c t e d i n t r a p e r i t o n e a l l y w i t h [ 3 H ] t h y m i d i n e ( 0 . 4 ~Ci p e r g b o d y w t . ) . 1 h l a t e r a 2 0 0 - - 3 0 0 m g s e c t i o n o f k i d n e y w a s r e m o v e d (as d e s c r i b e d in M e t h o d s ) , a n d h o m o g e n i z e d i n 4 m l c o l d 5% t r i c h l o r o a c e t i c a c i d c o n t a i n i n g 0 . 5 p m o l c y c l i c [ 3 H ] A M P (11 1 0 0 e p m ) a n d 0 . 5 p m o l c y c l i c [ 3 H ] G M P ( 7 4 3 0 c p m ) . H o m o g e n a t e s w e r e e x t r a c t e d a n d c h r o m a t o g r a p h e d o n a c i d i c a l u m i n a a n d D o w e x A G 1 X 8 c o l u m n s as d e s c r i b e d in M e t h o d s . T h e f o l l o w i n g data illus t r a t e the e l u t i o n s c h e m e u s e d and t h e m e a n r e c o v e r i e s +_ S.E. f o r c y c l i c [ 3 H ] A M P a n d c y c l i c [ 3 H ] G M P f r o m k i d n e y h o m o g e n a t e s f r o m f o u r rats. Elution Scheme
T o t a l C P M in eluate
1 2
4 5 5 1 4 -+ 2 2 5 4
3 4 5 6 7
Sample plus 10 ml H20 through AI203 11 m l 0 . 2 M N H 4 C H O 2, p H 6 . 0 , t h r o u g h AI203 and Dowex 10 ml H20 through Dowex 6 ml 2 M HCOOH through Dowex 3 ml 5 M HCOOH through Dowex 8 ml 5 M HCOOH through Dowex 4 ml 5 M HCOOH through Dowex
767 82 7 585 324 5 554 118
_+ 9 5 + 14 -+ 3 0 9 -+ 45 + 237 -+ 16
P e r c e n t r e c o v e r y of c y c l i c [ 3 H ] AMP and cyclic [3H]GMP from initial h o m o g e n a t e
6 8 -+ 2 ( c y c l i c [ 3 H ] A M P ) 7 4 -+ 4 ( c y c l i c [ 3 H ] G M P )
26 of the procedure indicated that no one process was particularly detrimental, but t h a t small quantities of cyclic nucleotides were lost at each step. Comments on the chromatographic procedure. The column chromatographic system described in Methods and Table I was based on extensive preliminary studies in addition to the results obtained with the test solutions. Preparations of acidic alumina from Fisher (catalog No. A-948); Sigma (Type WA-1, catalog No. A-8753) and EM Laboratories (catalog No. 1078) were compared. 50 mM NHaCHO:, pH 6.0, eluted cyclic nucleotides from acidic alumina obtained from EM Laboratories and the reproducibility was poor. Reproducibility was better using acidic alumina from Sigma, but 15--20% of the cyclic nucleotides was eluted with H20 and 0.05 M NH4CHO2 while 0.2 M NH4CHO2 completely eluted the cyclic nucleotides. Partial elution with H20 prevented clean separation from thymidine. 50 mM NH4CHO2 eluted 2--3% of the total a m o u n t of cyclic nucleotides from acidic alumina obtained from Fisher and 0.2 M NH4CHO: completely eluted cyclic AMP and cyclic GMP (Fig. 1). Reproducibility with acidic alumina from Fisher has been excellent for several batches prepared from different lots. A m m o n i u m formate solutions were prepared fresh for best reproducibility, and the pH was adjusted to 6.0 with formic acid. Use of NHaCHO2 for elution of cyclic nucleotides from acidic alumina onto Dowex AG 1X8 is compatible with the formate form of the latter resin. The amounts of acidic alumina and Dowex AG 1X8 resins used per column were based on our particular renal tissue samples. These amounts of resins and the elution scheme should be directly applicable to other types of tissue samples in which the amounts of cyclic AMP and cyclic GMP do not exceed 800 pmol and 100 pmol, respectively (as based on the test solutions). The adsorption capacity of 2 g acidic alumina for polyvalent thymidine, adenosine and guanosine nucleotides was n o t determined, but these test solutions contained three to four orders of magnitude more solute than the cyclic nucleotide test solutions. Discussion White and Zenser [11] reported that cyclic nucleotides could be separated from polyvalent nucleotides on different types of alumina in the neutral pH range. However, nucleosides, purines and pyrimidines also eluted with cyclic nucleotides at neutral pH. Their data demonstrated t h a t cyclic nucleotides firmly adsorbed to neutral alumina at pH 4.1 and eluted at pH 6.3 while polyvalent nucleotides remained adsorbed. They also reported that xanthine was not adsorbed at pH 5 on neutral alumina which suggested that cyclic nucleotides might be separated from bases, such as thymine, under acidic conditions. Ramanchandran [ 12] also reported t h a t cyclic AMP was unadsorbed on neutral alumina at pH 7.4 while AMP, ADP and ATP were tightly adsorbed and t h a t 0.2 M Na2HPO4 was required for elution of the polyvalent nucleotides. Filburn and Karn [13] described an assay for cyclic nucleotide phosphodiesterase based on the separation of purine bases and nucleosides from cyclic nucleotides on neutral alumina at pH 4.0. Acidic alumina has also been used in a similar assay [21]. Acidic alumina has been used in isolating cyclic AMP formed in assays of adenylate cyclase activity [22]. Our data demonstrate that cyclic AMP and cyclic GMP are separated from
27 thymine, thymidine and polyvalent nucleotides on columns of acidic alumina. Thus, [3H] thymidine and its metabolites, which would retain the tritium label, were completely separated from cyclic [3H]AMP and cyclic [3H]GMP used to monitor recoveries of endogenous cyclic nucleotides. Tritiated compounds were used instead of a combination of 14C- or 32P-labelled compounds because of economy and the specific activities available for the tritiated compounds. The relative elution pattern (Fig. 1) obtained on acidic alumina agrees with previous reports [11--13] on the relative strength of binding of nucleosides, cyclic nucleotides and polyvaient nucleotides to neutral alumina at pH 4--8. Use of acidic alumina rather than neutral alumina obviates equilibrium of the latter with acidic buffers. Although we did not study the chromatography of bases and nucleosides, other than thymine and thymidine, on acidic alumina, such compounds that have negligible anionic charge at pH 4.3--4.4 probably do not absorb. This chromatographic system may be applicable to studies on RNA synthesis and cyclic nucleotides in which both [3H]uridine and 3H-labelled cyclic nucleotides are used. Excess electrolytes in tissue extracts should be removed prior to chromatography on anion-exchange resins to obtain reproducible separation of the cyclic nucleotides. Adsorption of nucleotides onto charcoal and elution with ammonia/alcohol or homogenization of tissue in zinc acetate/alcohol followed by chromatography on QAE-Sephadex are procedures to overcome interference by electrolytes [3]. However, the former method involves an additional step with potential loss of recovery, and only small amounts of tissue are readily chromatographed on QAE-Sephadex. Cation-exchange resins more readily accept excess electrolytes, but sample volumes must be small [3]. Excess electrolytes are eluted from acidic alumina with H20 and are separated from the cyclic nucleotides (Fig. 1). In addition, relatively large sample volumes (4 ml) can be directly applied to acidic alumina columns. Cyclic AMP and cyclic GMP are separated from each other on Dowex AG 1X8 (formate) resin using 2 M HCOOH and 5 M HCOOH, respectively (Fig. 2). Murad et al. [2] initially described the differential elution of the cyclic nucleotides from this resin. Use of 5 M HCOOH rather than 4 M HCOOH [2] decreased the volume required to completely elute cyclic GMP. The chromatographic separation of cyclic AMP and cyclic GMP on acidic alumina and Dowex AG 1X8 (formate) is relatively rapid and one hundred samples can be processed in a working day using the appropriate Plexiglass column racks. The overall recoveries of 60-65% for cyclic AMP and cyclic GMP are comparable to those reported for other multistep isolations [15,16]. Kimura et al. [23] reported renal tissue levels of 0.94 + 0.24 pmol cyclic GMP/mg protein and 10.08 + 1.15 pmol cyclic AMP/mg protein (mean _+S.E. for 12 rats) for rats anesthetized with ether. Our value of 0.51 + 0.03 pmol cyclic GMP/mg protein is lower, but the value of 9.7 + 0.3 pmol cyclic AMP/mg protein is in excellent agreement with their value [23]. These determinations of the cyclic nucleotides may not reflect the actual cellular levels during homeostasis because of the changes which may occur during anesthetization and removal of renal tissue. However, these determinations do reflect control values for comparison with values from experimentally treated animals in which the procedures for processing the tissue are identical.
28
Addendum Jakobs et al. reported at a NATO conference that cyclic nucleotides could be purified by applying perchloric acid extracts to neutral alumina, eluting the cyclic nucleotides with ammonium formate, and separating cyclic AMP and cyclic GMP on Dowex-50 W-X8. This work was published [24] while we were doing this research. Asakawa et al. [25] reported the isolation of cyclic GMP from tissue samples using columns of alumina and Dowex 1X8. They applied acidic extracts to neutral alumina and eluted cyclic nucleotides with ammonium formate. Cyclic nucleotides were succinylated and subsequently separated from one another on Dowex 1X8 resin.
Acknowledgments Ms. Janice Menefee kindly measured the osmolalities. Encouragement in this study by Dr. Thomas P. Dousa was appreciated. Support was provided to Dr. Larry D. Barnes from National Institute of Health General Research Support Grant 5 S01-RR05654-06 to The University of Texas Health Science Center at San Antonio. Dr. Jay H. Stein generously provided support from National Institutes of Health Grant AM 17387-04.
References i H a r d m a n , J.G., David, J.W. a n d S u t h e r l a n d , E.W. ( 1 9 6 9 ) J. Biol. Chem. 2 4 4 , 6 3 5 4 - - 6 3 6 2
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
M u r a d , F., Manganiello, V. a n d V a u g h a n , M. ( 1 9 7 1 ) Proc. Natl. A c a d . Sci. U.S. 6 8 , 7 3 6 - - 7 3 9 Schultz, G., B S h m e , E. a n d H a r d m a n , J.G. ( 1 9 7 4 ) M e t h o d s E n z y m o l . 38, P a r t C, 9 - - 2 0 F l o u r e t , G. a n d H e c h t e r , O. ( 1 9 7 4 ) Anal. B i o c h e m . 5 8 , 2 7 6 - - 2 8 5 B S h m e , E. a n d S c h u l t z , G. ( 1 9 7 4 ) M e t h o d s E n z y m o l . 38, p a r t C, 2 7 - - 3 8 White, A.A. a n d A u r b a c h , G.D. ( 1 9 6 9 ) B i o c h i m . B i o p h y s . A e t a 1 9 1 , 6 8 6 - - 6 9 7 Woods, W.D. a n d W a i t z m a n , M.B. ( 1 9 7 0 ) J. C h r o m a t o g r . 47, 5 3 6 - - 5 4 2 Keirns, J.J., Wheeler, M.A. a n d B i t e n s k y , M.W. ( 1 9 7 4 ) Anal. B i o c h e m . 6 1 , 3 3 6 - - 3 4 8 S a n d h u , R.S. a n d H o k i n , M.R. ( 1 9 6 8 ) J. C h r o m a t o g r . 35, 1 3 6 - - 1 3 7 V e r b e r t , A. a n d C a c a n , R. ( 1 9 7 2 ) Anal. B i o c h e m . 54, 1 4 9 1 - - 1 4 9 2 White, A.A. a n d Zenzer, T.V. ( 1 9 7 1 ) Anal. B i o c h e m . 4 1 , 3 7 2 - - 3 9 6 R a m a c h a n d r a n , J. ( 1 9 7 1 ) Anal. B i o c h e m . 4 3 , 2 2 7 - - 2 3 9 F f l b u r n , C.R. a n d K a r n , J. ( 1 9 7 3 ) Anal. B i o c h e m . 52, 5 0 5 - - 5 1 6 White, A.A. ( 1 9 7 4 ) M e t h o d s E n z y m o l . 38, Part C, 4 1 - - 4 6 Mao, C.C. a n d G u i d o t t i , A. ( 1 9 7 4 ) Anal. B i o c h e m . 59, 6 3 - - 6 8 M a t s u z a w a , H. a n d N i r e n b e r g , M. ( 1 9 7 5 ) Proc. Natl. A c a d . Sci. U.S. 72, 3 4 7 2 - - 3 4 7 6 P a t t e r s o n , M.S. a n d Greene, R.C. ( 1 9 6 5 ) Anal. C h e m . 3 7 , 8 5 4 - - 8 5 7 Stelner, A.L., Parker, C.W. a n d Kipnis, D.M. ( 1 9 7 2 ) J. Biol. Chem. 247, 1 1 0 6 - - 1 1 1 3 R a n d e r a t h , K. ( 1 9 6 6 ) T h i n - L a y e r C h r o m a t o g r a p h y , p. 2 1 9 , A c a d e m i c Press, New Y o r k L o w r y , O.H., R o s e b r o u g h , N.J., Fal~, A.L. a n d R a n d a l l , R . J . ( 1 9 5 1 ) J. Biol. C h e m . 1 9 3 , 2 6 5 - - 2 7 5 B o u d r e a u , R.J. a n d D r u m m o n d , G.L ( 1 9 7 5 ) Anal. B i o c h e m . 6 3 , 3 8 8 - - 3 9 9 Garbers, D.L. a n d H a r d m a n , J.G. ( 1 9 7 6 ) J. Cyclic N u c l e o t i d e Res. 2, 5 9 - - 7 0 K i m u r a , H., T h o m a s , E. a n d M u r a d , F. ( 1 9 7 4 ) Biochim. Biophys. A c t a 3 4 3 , 5 1 9 - - 5 2 8 J a k o b s , K.H., B S h m e , E. a n d S c h u l t z , G. ( 1 9 7 6 ) in E u k a r y o t i c Cell F u n c t i o n a n d G r o w t h : R e g u l a t i o n b y I n t r a c e U u l a r Cyclic N u c l e o t i d e s ( D u m o n t , J.E., B r o w n , B.L. a n d Marshall, N.J., eds.), N A T O Adv a n c e d S t u d y I n s t i t u t e s Series, Vol. 9, p p . 2 9 5 - - 3 1 1 , P l e n u m Press, New Y o r k 25 A s a k a w a , T., Russell, T.R. a n d H o , R. ( 1 9 7 6 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 68, 682---690
Biochimica et Biophysica Acta, 544 (1978) 20--28
© Elsevier/North-Holland Biomedical Press
BBA 28699 SEPARATION OF CYCLIC AMP AND CYCLIC GMP FROM THYMIDINE, E L E C T R O L Y T E S AND P O L Y V A L E N T NUCLEOTIDES IN TISSUE SAMPLES
GLENDA M. ROBERSON and LARRY D. BARNES Department o f Biochemistry, University o f Texas Health Science Center, San A ntonio, Texas 78284 (U.S.A.)
(Received February 22nd, 1978) (Revised manuscript received May llth, 1978)
Summary Cyclic AMP and cyclic GMP can be separated from thymidine and its possible metabolites, electrolytes, and polyvalent nucleotides using columns of acidic alumina. Electrolytes and thymidine are not adsorbed on acidic alumina at pH 4.4 while cyclic nucleotides and polyvalent nucleotides are adsorbed at this pH. Cyclic AMP and cyclic GMP are eluted together from acidic alumina with 0.2 M ammonium formate (pH 6.0) and the polyvalent nucleotides remain adsorbed. The cyclic nucleotides are separated by chromatography on Dowex AG 1 X 8 resin. Recovery is 60--64% for cyclic AMP and cyclic GMP isolated from renal tissue samples. This m e t h o d o l o g y permits the separation of tritiated thymidine from cyclic nucleotides which are present in tissue preparations used in studies on the role of cyclic nucleotides in cellular growth.
Introduction Studies on the roles of adenosine 3',5'-cyclic monophosphate (cyclic AMP) and guanosine 3',5'-cyclic monophosphate (cyclic GMP) in cellular functions have generated different techniques for isolating these cyclic nucleotides. These techniques include ion-exchange column chromatography [1--3], thin-layer chromatography [4--8], electrophoresis [9,10], and adsorption chromatography using alumina [11--14]. Although these methods separate the cyclic nucleotides from other nucleotides, they are often inadequate to separate completely cyclic AMP and cyclic GMP from each other and from both nucleosides and polyvalent nucleotides. In addition, excess electrolytes often interfere with A b b r e v i a t i o n : bis-MSB, 1 , 4 - d i - ( 2 - m e t h y l s t y r y l ) - b e n z e n e .
21 ion-exchange chromatography [3]. Consequently, a combination of methods is often required to achieve the desired separation [15,16]. Our studies on the possible roles of cyclic AMP and cyclic GMP in renal hyperplasia necessitated separation of three tritiated compounds. Rats were injected with [3H] thymidine prior to killing to measure incorporation of [ 3H]thymidine into DNA. Subsequently, renal tissue was homogenized in trichloroacetic acid containing cyclic [3H]AMP and cyclic [3H]GMP to monitor recoveries of endogenous cyclic nucleotides. Thus, the supernatant fraction processed for cyclic nucleotide analyses contained unincorporated [3H]thymidine and its possible metabolites as well as the tritiated cyclic nucleotides. One could use separate animals for measurements of [3H]thymidine incorporation and endogenous cyclic nucleotides and obviate the problem. However, this is impracticable when large numbers of animals are used. Also, one can more reliably correlate changes in metabolite levels with a cellular process if these determinations are made on the same animal. Ion-exchange chromatography alone was inadequate to separate cyclic AMP and cyclic GMP from each other and from thymidine and its possible metabolites. The high electrolyte content of renal tissue was probably one reason for such chromatography being unsuccessful. We have isolated cyclic AMP and cyclic GMP from renal tissue using columns of acidic alumina plus columns of Dowex 1 X 8 resin. Quantitation of recoveries and amounts of endogenous cyclic nucleotides can be determined after removal of thymidine and its metabolites, electrolytes and polyvalent nucleotides by this chromatographic procedure. Methods
Materials. [Me-3H]Thymidine; [8-3H]cyclic AMP; and [8-3H]cyclic GMP were purchased from Amersham/Searle. Cyclic AMP, cyclic GMP, AMP, ADP, ATP, GMP, GDP, GTP, thymine, thymidine, TMP, TDP, TTP and beef heart cyclic nucleotide phosphodiesterase were purchased from Sigma Chemical Company. Radioimmunoassay kits for measurement of cyclic AMP and cyclic GMP were from Schwarz/Mann. Acidic alumina was purchased from Fisher (Brockman activity I, 80--200 mesh) and Dowex AG 1 X 8 (200--400 mesh, formate form) from Bio-Rad. PEI-cellulose and cellulose thin-layer sheets were from EM Laboratories, Inc. Plastic columns (8 × 200 mm) were supplied by Kontes. Male, Sprague-Dawley rats (150--200 g) were purchased from BioLabs, Inc. Scintillation cocktail was composed of 7.5 g PPO and 0.1 g bis-MSB per 667 ml toluene and 333 ml Triton X-100 [17]. Counting efficiency was 33--37% for tritiated compounds as determined by the channels-ratio method in a Beckman LS-230 scintillation counter. Triton X-100 was purchased from Research Products, Inc., and scintillation-grade fluors and toluene were from Fisher. Preparation of tissue samples. Rats were injected intraperitoneally with [Me-3H]thymidine in saline (0.4 ~Ci per g body weight). 1 h later we anesthesized the rats with ether, withdrew a sample of blood from the jugular vein, cut the adominal wall, exposed the abdominal cavity and removed one kidney from each rat. Each kidney was immediately cut into coronal sections weighing
22 approx. 200--300 mg (wet wt.). One section from each kidney was rinsed in saline and homogenized in a Potter-Elvehjem tissue grinder containing 0.5 pmol cyclic [3H]AMP (11 560 cpm) and 0.5 pmol cyclic [3H]GMP (7980 cpm) in 4 ml of cold, 5% (w/v) trichloroacetic acid. The homogenate was centrifuged at 5900 × g for 20 min at 2°C. HC1 was added to the supernatant fraction to a final concentration of 0.1 M. Trichloroacetic acid was extracted from the supernatant fraction with 12 ml of water-saturated ether per extraction, and the extraction was repeated four additional times. The aqueous sample was heated at 85°C in a water bath for 5 min to remove residual ether. The sample was stored at --75°C until chromatographed. (Other sections from each kidney were used for histology and autoradiography.) Test solutions. We tested individual solutions to determine the conditions necessary to achieve chromatographic separation of electrolytes, thymine, thymidine, cyclic nucleotides and polyvalent nucleotides. The electrolyte solution was 0.18 M NaC1, 0.12 M KC1, and 0.02 M NH4C1 in 0.1 M HC1. The measured osmolality of this solution was 802 mOsm/kg H20. Solutions of thymine, TMP, TDP, TTP, AMP, ADP, ATP, GMP, GDP, and GTP contained 3--4 ~mol of a single c o m p o u n d in 4 ml of 0.1 M HC1. The other test solutions were 1 ~mol [3H]thymidine (39 800 cpm); 775 pmol cyclic [3H]AMP (12 400 cpm); and 98 pmol cyclic [3H]GMP (8360 cpm) each in 4 ml of 0.1 M HC1. Column chromatography. Acidic alumina was washed with several changes of distilled H20 to remove fines and dried at 110°C for 16 h. It can be stored at least 6 months at room temperature in a tightly-closed bottle w i t h o u t changes in chromatographic properties. Dowex AG 1 X 8 resin was washed with 2 M HCOOH and then H20 until the pH was 4.2--4.4 for an aqueous suspension. This resin was stored at 4°C as a 50% (v/v) aqueous suspension. Approx. 2 g acidic alumina were added with a glass measuring scoop to individual columns filled with H20. The alumina rapidly settled as the H20 drained to yield uniformly packed columns. Dowex columns were prepared by adding 3.5 ml of the 50% (v/v) aqueous suspension to individual columns. The pH of the aqueous effluents from both the acidic alumina and Dowex columns was 4.2--4.4. Columns were placed in Plexiglas racks designed to hold 100 columns. These racks could be placed on top of one another or over a box of scintillation vials. Initially the test solutions were applied to individual acidic alumina columns and the elution profiles determined by successive 1-ml additions of eluting solutions. Electrolytes, t h y m i n e and thymidine were eluted with H20; cyclic nucleotides were eluted together with 0.2 M a m m o n i u m formate (NH4CHO2), pH 6.0. TMP, TDP, TTP, AMP, ADP, ATP, GMP, GDP and GTP remained adsorbed to acidic alumina even in the presence of 1 M NH4CHO2, pH 6.0. NH4CHO2 eluates containing cyclic nucleotides were applied to Dowex columns, and elution profiles of cyclic AMP and cyclic GMP determined by successive 1-ml additions of eluting solutions. Eluates of [3H]thymidine, cyclic [3H]AMP, and cyclic [3H]GMP solutions were counted in a liquid scintillation counter. Absorbances of eluates from the t h y m i n e test solution were measured at 264 nm, and eluates from the polyvalent nucleotide solutions of thymidine, adenosine and guanosine were measured at 267 rim, 259 nm and 252 nm, respectively. Osmolalities were deter-
23 mined for eluates of the electrolyte test solution by freezing point depression using an Advanced Instrument Osmometer. Based on the results of elution profiles of test solutions, the following procedure was used for chromatography of tissue extracts. Supernatant fractions were applied to acidic alumina columns and washed with 10 ml of H20. The sample plus water eluates were discarded. The rack of alumina columns was then placed on top of the rack of Dowex columns. Cyclic nucleotides were eluted from the acidiC alumina columns onto the Dowex columns with 11 ml of 0.2 M NH4CHO2, pH 6.0. Dowex columns were washed with 10 ml of H20 and the eluates discarded. Cyclic AMP was eluted with 6 ml of 2 M HCOOH. Columns were washed with 3 ml of 5 M HCOOH and the elutes discarded. Cyclic GMP was eluted with an additional 8 ml of 5 M HCOOH. Eluates containing the cyclic nucleotides were quick-frozen in a solid CO2/acetone bath and lyophilized. Lyophilized samples were dissolved in 0.05 M sodium acetate, pH 6.2. 25--50 ~1 of each sample was counted to determine the percent of recovery, and the amounts of cyclic AMP and cyclic GMP were measured by radioimmunoassay [ 18]. Other methods. Cyclic nucleotide samples were hydrolyzed by incubating 300 pl of the isolated fraction (lyophilized column eluates dissolved in 0.05 M sodium acetate, pH 6.2) with 30 ~1 of buffer containing 0.02 unit cyclic nucleotide phosphodiesterase and 40 mM MgC12. Samples were incubated for 16 h at 25°C and the reaction was stopped by boiling for 5 min. Standard solutions of cyclic AMP and cyclic GMP, untreated and treated with phosphodiesterase, served as controls for the hydrolysis conditions. PEI-cellulose sheets developed in 1 M LiC1 [19] and cellulose sheets developed in n-butanol/acetic acid/H20 (12 : 3 : 5) [18] were used for thin-layer chromatography. Amounts of proteins were determined according to Lowry et al. [20] after dissolving samples in 1% sodium dodecyl sulfate. Results Individual solutions of electrolytes, thymine, thymidine, cyclic AMP, cyclic GMP, TMP, TDP, TTP, AMP, ADP, ATP, GMP, GDP and GTP were prepared to simulate the volume and HC1 concentration of the supernatant fractions of tissue samples. These solutions were individually chromatographed on columns of acidic alumina and Dowex AG 1 X 8. Electrolytes, thymine and thymidine were eluted from acidic alumina with H20, and cyclic AMP and cyclic GMP were eluted with 0.2 M NH4CHO2, pH 6.0 {Fig. 1). The elution profiles of thymine and thymidine are similar. Less than 1% of the polyvalent nucleotides of thymidine, adenosine and guanosine are eluted from acidic alumina with 0.2 M NH4CHO2 or 10 ml of 1 M NH4CHO2, pH 6.0. Cyclic AMP and cyclic GMP were separated on D o w e x AG 1 X 8 columns where cyclic AMP was eluted with 2 M HCOOH and cyclic GMP with 5 M HCOOH {Fig. 2). Each of the tritiated solutions which eluted from the columns was quantitatively recovered. Approx. 70% of the electrolytes and 100% of the thymine eluted from acidic alumina with H20. Elution profiles were unchanged when all of the test solutes were combined in 4 ml 0.1 N HC1 and chromatographed together. Based on results with test solutions, the chromatographic procedure
24 Ck
SAMPLE i t ELUATE
ck
)
HzO
//-4
02M NH4COOH,pH 60
i
i
~ 53
E3 ~
4S
c~
3O
II
Electrolytes.
-1-
4oo ~
~_c
~" I0 3
5
7
9
14 15
~7
~9
21
23
z5
Fractions ( I ml)
Fig. 1. C o m p o s i t e e l u t i o n p r o f i l e of [ 3 H ] t h y m i d i n e , cyclic [ 3 H ] A M P , cyclic [ 3 H ] G M P a n d e l e c t r o l y t e s f r o m acidic a l u m i n a . 2 g o f acidic a l u m i n a w e r e p o u r e d i n t o an 8 - r a m d i a m e t e r c o l u m n a n d r i n s e d w i t h H 2 0 . T h e f o l l o w i n g c o m p o u n d s in 4 m l 0.1 M HC1 w e r e a p p l i e d to i n d i v i d u a l c o l u m n s : (a) e l e c t r o l y t e s o l u t i o n o f 0 . 1 8 M NaC1, 0 . 1 2 M KC1, a n d 0 . 0 2 M NH4C1; (b) 1 ]~nol [ M e - 3 H ] t h y m i d i n e , 39 8 0 0 c p m ; (c) 7 7 5 p m o l cyclic [ 3 H ] A M P , 12 4 0 0 c p m ; a n d (d) 9 8 p m o l cyclic [ 3 H ] G M P , 8 3 6 0 c p m . C o m p o u n d s w e r e e l u t e d b y 10 m l H 2 0 a n d 11 m l 0.2 M N H 4 C H O 2, p H 6.0, a p p l i e d in 1-ml p o r t i o n s . 1-ml f r a c t i o n s w e r e c o l l e c t e d . O s m o l a l i t e s o f f r a c t i o n s c o n t a i n i n g e l e c t r o l y t e s w e r e d e t e r m i n e d b y f r e e z i n g - p o i n t depression. F r a c t i o n s of t r i t i a t e d c o m p o u n d s w e r e c o l l e c t e d d i r e c t l y in s c i n t i l l a t i o n vials a n d c o u n t e d a f t e r addit i o n of 10 m l s c i n t i l l a t i o n s o l u t i o n . T h e left o r d i n a t e expresses t h e p e r c e n t of t h e a p p l i e d r a d i o a c t i v i t y r e c o v e r e d in e a c h f r a c t i o n , a n d t h e right o r d i n a t e e x p r e s s e s t h e o s m o l a l i t y o f f r a c t i o n s c o n t a i n i n g e l e c t r o lytes. [ M e - 3 H ] t h y m i d i n e is a b b r e v i a t e d [ 3 H ] T d R ; cyclic [ 3 H ] A M P , [ 3 H ] c A M P ; c y c l i c [ 3 H ] G M P , [ 3 H ] cGMP.
70
&= Go
'~
'5o
4o
NH4COOH' pH6.0
II I/
1P"tcAMP
§ io
Fraction ( I m l )
Fig. 2. C o m p o s i t e e l u t i o n profile of cyclic [ 3 H ] A M P a n d cyclic [ 3 H ] G M P f r o m D o w e x A G 1 X 8 (form a t e ) resin. T h e 8 - m m d i a m e t e r c o l u m n c o n t a i n e d 3.5 m l 50% ( v / v ) a q u e o u s s u s p e n s i o n o f D o w e x A G l X 8 ( f o r m a t e ) resin. 4 m l o f the cyclic [ 3 H ] A M P a n d cyclic [ 3 H ] G M P s o l u t i o n s w e r e a p p l i e d t o indiv i d u a l acidic a l u m i n a c o l u m n s as d e s c r i b e d in Fig. 1. A f t e r w a s h i n g t h e a l u m i n a c o l u m n s w i t h 10 m l H 2 0 , t h e y w e r e p l a c e d a b o v e t h e D o w e x c o l u m n s . Cyclic n u e l e o t i d e s e l u t e d f r o m t h e a l u m i n a w i t h 0.2 M N H 4 C H O 2, p H 6.0, w e r e d r a i n e d directly i n t o t h e D o w e x c o l u m n s . Cyclic [ 3 H ] A M P a n d cyclic [ 3 H ] GMP w e r e e l u t e d f r o m t h e D o w e x resin b y 10 m l H 2 0 , ' 1 0 m l 2 M H C O O H , a n d 15 m l 5 M H C O O H , e a c h a p p l i e d in 1 m l p o r t i o n s . F r a c t i o n s w e r e c o l l e c t e d , c o u n t e d a n d t h e results e x p r e s s e d as d e s c r i b e d in Fig. 1. A b b r e v i a t i o n s as p e r Fig. 1.
25 described in Methods was used to separate [3H]thymidine, cyclic [3H]AMP and cyclic [3H]GMP present in a single renal tissue extract. Table I shows the elution scheme and distribution of labelled compounds in column eluates from renal tissue samples. [3H]Thymidine was present in the sample plus water eluate from the acidic alumina column. If thymine, TMP, TDP and TTP were metabolic products of the [3H]thymidine injected into the rat, [3H]thymine would elute from acidic alumina with H20 and the tritiated polyvalent nucleotides would remain adsorbed. Although the total counts of [3H]thymidine present in the renal tissue were considerably larger than the counts due to cyclic [3H]AMP and cyclic [3H]GMP, there was no carry-over of [3H]thymidine or its metabolic products into the water eluate from the Dowex column. Identity of the cyclic nucleotide fractions isolated from tissue samples and validity of their measurement by radioimmunoassay were confirmed by the following results. The radioactivity in the presumed cyclic AMP and cyclic GMP eluates comigrated with their respective standards on PEI-cellulose (in 1 M LiC1) and cellulose (in n-butanol/acetic acid/H20 (12 : 3 : 5) thin-layer sheets. Phosphodiesterase hydrolyzed 90--100% of both the cyclic AMP and cyclic GMP fractions. Amounts of cyclic nucleotides measured were linear with the volume of sample added to their respective radioimmunoassay. Cyclic AMP and cyclic GMP concentrations in rat renal tissue were 9.7 _+0.3 pmol/mg prorein and 0.51 _+0.03 pmol/mg protein (mean -+ S.E. for 42 rats), respectively. Recoveries of cyclic AMP and cyclic GMP from the point of homogenization of tissue to elution from Dowex AG 1 X 8 columns were 68 -+ 2% and 74 +_4%, respectively (Table I). Overall recoveries from homogenization to dissolution of lyophilized eluates were 61 + 2% for cyclic AMP (mean _+S.E. for 73 rats) and 64 + 2% for cyclic GMP (mean + S.E. for 71 rats} for animals which received different treatments in various experiments. Analyses of recoveries at each step TABLE I CHROMATOGRAPHY OF RENAL DOWEX AG 1 × 8 (FORMATE)
TISSUE SAMPLES ON COLUMNS
OF ACIDIC ALUMINA AND
Separation of [3H]thymidine, cyclic [3H]AMP, and cyclic [3H]GMP on acidic alumina and Dowex AG 1 × 8 ( f o r m a t e ) c o l u m n s f r o m r e n a l tissue h o m o g e n a t e s . R a t s w e r e i n j e c t e d i n t r a p e r i t o n e a l l y w i t h [ 3 H ] t h y m i d i n e ( 0 . 4 ~Ci p e r g b o d y w t . ) . 1 h l a t e r a 2 0 0 - - 3 0 0 m g s e c t i o n o f k i d n e y w a s r e m o v e d (as d e s c r i b e d in M e t h o d s ) , a n d h o m o g e n i z e d i n 4 m l c o l d 5% t r i c h l o r o a c e t i c a c i d c o n t a i n i n g 0 . 5 p m o l c y c l i c [ 3 H ] A M P (11 1 0 0 e p m ) a n d 0 . 5 p m o l c y c l i c [ 3 H ] G M P ( 7 4 3 0 c p m ) . H o m o g e n a t e s w e r e e x t r a c t e d a n d c h r o m a t o g r a p h e d o n a c i d i c a l u m i n a a n d D o w e x A G 1 X 8 c o l u m n s as d e s c r i b e d in M e t h o d s . T h e f o l l o w i n g data illus t r a t e the e l u t i o n s c h e m e u s e d and t h e m e a n r e c o v e r i e s +_ S.E. f o r c y c l i c [ 3 H ] A M P a n d c y c l i c [ 3 H ] G M P f r o m k i d n e y h o m o g e n a t e s f r o m f o u r rats. Elution Scheme
T o t a l C P M in eluate
1 2
4 5 5 1 4 -+ 2 2 5 4
3 4 5 6 7
Sample plus 10 ml H20 through AI203 11 m l 0 . 2 M N H 4 C H O 2, p H 6 . 0 , t h r o u g h AI203 and Dowex 10 ml H20 through Dowex 6 ml 2 M HCOOH through Dowex 3 ml 5 M HCOOH through Dowex 8 ml 5 M HCOOH through Dowex 4 ml 5 M HCOOH through Dowex
767 82 7 585 324 5 554 118
_+ 9 5 + 14 -+ 3 0 9 -+ 45 + 237 -+ 16
P e r c e n t r e c o v e r y of c y c l i c [ 3 H ] AMP and cyclic [3H]GMP from initial h o m o g e n a t e
6 8 -+ 2 ( c y c l i c [ 3 H ] A M P ) 7 4 -+ 4 ( c y c l i c [ 3 H ] G M P )
26 of the procedure indicated that no one process was particularly detrimental, but t h a t small quantities of cyclic nucleotides were lost at each step. Comments on the chromatographic procedure. The column chromatographic system described in Methods and Table I was based on extensive preliminary studies in addition to the results obtained with the test solutions. Preparations of acidic alumina from Fisher (catalog No. A-948); Sigma (Type WA-1, catalog No. A-8753) and EM Laboratories (catalog No. 1078) were compared. 50 mM NHaCHO:, pH 6.0, eluted cyclic nucleotides from acidic alumina obtained from EM Laboratories and the reproducibility was poor. Reproducibility was better using acidic alumina from Sigma, but 15--20% of the cyclic nucleotides was eluted with H20 and 0.05 M NH4CHO2 while 0.2 M NH4CHO2 completely eluted the cyclic nucleotides. Partial elution with H20 prevented clean separation from thymidine. 50 mM NH4CHO2 eluted 2--3% of the total a m o u n t of cyclic nucleotides from acidic alumina obtained from Fisher and 0.2 M NH4CHO: completely eluted cyclic AMP and cyclic GMP (Fig. 1). Reproducibility with acidic alumina from Fisher has been excellent for several batches prepared from different lots. A m m o n i u m formate solutions were prepared fresh for best reproducibility, and the pH was adjusted to 6.0 with formic acid. Use of NHaCHO2 for elution of cyclic nucleotides from acidic alumina onto Dowex AG 1X8 is compatible with the formate form of the latter resin. The amounts of acidic alumina and Dowex AG 1X8 resins used per column were based on our particular renal tissue samples. These amounts of resins and the elution scheme should be directly applicable to other types of tissue samples in which the amounts of cyclic AMP and cyclic GMP do not exceed 800 pmol and 100 pmol, respectively (as based on the test solutions). The adsorption capacity of 2 g acidic alumina for polyvalent thymidine, adenosine and guanosine nucleotides was n o t determined, but these test solutions contained three to four orders of magnitude more solute than the cyclic nucleotide test solutions. Discussion White and Zenser [11] reported that cyclic nucleotides could be separated from polyvalent nucleotides on different types of alumina in the neutral pH range. However, nucleosides, purines and pyrimidines also eluted with cyclic nucleotides at neutral pH. Their data demonstrated t h a t cyclic nucleotides firmly adsorbed to neutral alumina at pH 4.1 and eluted at pH 6.3 while polyvalent nucleotides remained adsorbed. They also reported that xanthine was not adsorbed at pH 5 on neutral alumina which suggested that cyclic nucleotides might be separated from bases, such as thymine, under acidic conditions. Ramanchandran [ 12] also reported t h a t cyclic AMP was unadsorbed on neutral alumina at pH 7.4 while AMP, ADP and ATP were tightly adsorbed and t h a t 0.2 M Na2HPO4 was required for elution of the polyvalent nucleotides. Filburn and Karn [13] described an assay for cyclic nucleotide phosphodiesterase based on the separation of purine bases and nucleosides from cyclic nucleotides on neutral alumina at pH 4.0. Acidic alumina has also been used in a similar assay [21]. Acidic alumina has been used in isolating cyclic AMP formed in assays of adenylate cyclase activity [22]. Our data demonstrate that cyclic AMP and cyclic GMP are separated from
27 thymine, thymidine and polyvalent nucleotides on columns of acidic alumina. Thus, [3H] thymidine and its metabolites, which would retain the tritium label, were completely separated from cyclic [3H]AMP and cyclic [3H]GMP used to monitor recoveries of endogenous cyclic nucleotides. Tritiated compounds were used instead of a combination of 14C- or 32P-labelled compounds because of economy and the specific activities available for the tritiated compounds. The relative elution pattern (Fig. 1) obtained on acidic alumina agrees with previous reports [11--13] on the relative strength of binding of nucleosides, cyclic nucleotides and polyvaient nucleotides to neutral alumina at pH 4--8. Use of acidic alumina rather than neutral alumina obviates equilibrium of the latter with acidic buffers. Although we did not study the chromatography of bases and nucleosides, other than thymine and thymidine, on acidic alumina, such compounds that have negligible anionic charge at pH 4.3--4.4 probably do not absorb. This chromatographic system may be applicable to studies on RNA synthesis and cyclic nucleotides in which both [3H]uridine and 3H-labelled cyclic nucleotides are used. Excess electrolytes in tissue extracts should be removed prior to chromatography on anion-exchange resins to obtain reproducible separation of the cyclic nucleotides. Adsorption of nucleotides onto charcoal and elution with ammonia/alcohol or homogenization of tissue in zinc acetate/alcohol followed by chromatography on QAE-Sephadex are procedures to overcome interference by electrolytes [3]. However, the former method involves an additional step with potential loss of recovery, and only small amounts of tissue are readily chromatographed on QAE-Sephadex. Cation-exchange resins more readily accept excess electrolytes, but sample volumes must be small [3]. Excess electrolytes are eluted from acidic alumina with H20 and are separated from the cyclic nucleotides (Fig. 1). In addition, relatively large sample volumes (4 ml) can be directly applied to acidic alumina columns. Cyclic AMP and cyclic GMP are separated from each other on Dowex AG 1X8 (formate) resin using 2 M HCOOH and 5 M HCOOH, respectively (Fig. 2). Murad et al. [2] initially described the differential elution of the cyclic nucleotides from this resin. Use of 5 M HCOOH rather than 4 M HCOOH [2] decreased the volume required to completely elute cyclic GMP. The chromatographic separation of cyclic AMP and cyclic GMP on acidic alumina and Dowex AG 1X8 (formate) is relatively rapid and one hundred samples can be processed in a working day using the appropriate Plexiglass column racks. The overall recoveries of 60-65% for cyclic AMP and cyclic GMP are comparable to those reported for other multistep isolations [15,16]. Kimura et al. [23] reported renal tissue levels of 0.94 + 0.24 pmol cyclic GMP/mg protein and 10.08 + 1.15 pmol cyclic AMP/mg protein (mean _+S.E. for 12 rats) for rats anesthetized with ether. Our value of 0.51 + 0.03 pmol cyclic GMP/mg protein is lower, but the value of 9.7 + 0.3 pmol cyclic AMP/mg protein is in excellent agreement with their value [23]. These determinations of the cyclic nucleotides may not reflect the actual cellular levels during homeostasis because of the changes which may occur during anesthetization and removal of renal tissue. However, these determinations do reflect control values for comparison with values from experimentally treated animals in which the procedures for processing the tissue are identical.
28
Addendum Jakobs et al. reported at a NATO conference that cyclic nucleotides could be purified by applying perchloric acid extracts to neutral alumina, eluting the cyclic nucleotides with ammonium formate, and separating cyclic AMP and cyclic GMP on Dowex-50 W-X8. This work was published [24] while we were doing this research. Asakawa et al. [25] reported the isolation of cyclic GMP from tissue samples using columns of alumina and Dowex 1X8. They applied acidic extracts to neutral alumina and eluted cyclic nucleotides with ammonium formate. Cyclic nucleotides were succinylated and subsequently separated from one another on Dowex 1X8 resin.
Acknowledgments Ms. Janice Menefee kindly measured the osmolalities. Encouragement in this study by Dr. Thomas P. Dousa was appreciated. Support was provided to Dr. Larry D. Barnes from National Institute of Health General Research Support Grant 5 S01-RR05654-06 to The University of Texas Health Science Center at San Antonio. Dr. Jay H. Stein generously provided support from National Institutes of Health Grant AM 17387-04.
References i H a r d m a n , J.G., David, J.W. a n d S u t h e r l a n d , E.W. ( 1 9 6 9 ) J. Biol. Chem. 2 4 4 , 6 3 5 4 - - 6 3 6 2
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
M u r a d , F., Manganiello, V. a n d V a u g h a n , M. ( 1 9 7 1 ) Proc. Natl. A c a d . Sci. U.S. 6 8 , 7 3 6 - - 7 3 9 Schultz, G., B S h m e , E. a n d H a r d m a n , J.G. ( 1 9 7 4 ) M e t h o d s E n z y m o l . 38, P a r t C, 9 - - 2 0 F l o u r e t , G. a n d H e c h t e r , O. ( 1 9 7 4 ) Anal. B i o c h e m . 5 8 , 2 7 6 - - 2 8 5 B S h m e , E. a n d S c h u l t z , G. ( 1 9 7 4 ) M e t h o d s E n z y m o l . 38, p a r t C, 2 7 - - 3 8 White, A.A. a n d A u r b a c h , G.D. ( 1 9 6 9 ) B i o c h i m . B i o p h y s . A e t a 1 9 1 , 6 8 6 - - 6 9 7 Woods, W.D. a n d W a i t z m a n , M.B. ( 1 9 7 0 ) J. C h r o m a t o g r . 47, 5 3 6 - - 5 4 2 Keirns, J.J., Wheeler, M.A. a n d B i t e n s k y , M.W. ( 1 9 7 4 ) Anal. B i o c h e m . 6 1 , 3 3 6 - - 3 4 8 S a n d h u , R.S. a n d H o k i n , M.R. ( 1 9 6 8 ) J. C h r o m a t o g r . 35, 1 3 6 - - 1 3 7 V e r b e r t , A. a n d C a c a n , R. ( 1 9 7 2 ) Anal. B i o c h e m . 54, 1 4 9 1 - - 1 4 9 2 White, A.A. a n d Zenzer, T.V. ( 1 9 7 1 ) Anal. B i o c h e m . 4 1 , 3 7 2 - - 3 9 6 R a m a c h a n d r a n , J. ( 1 9 7 1 ) Anal. B i o c h e m . 4 3 , 2 2 7 - - 2 3 9 F f l b u r n , C.R. a n d K a r n , J. ( 1 9 7 3 ) Anal. B i o c h e m . 52, 5 0 5 - - 5 1 6 White, A.A. ( 1 9 7 4 ) M e t h o d s E n z y m o l . 38, Part C, 4 1 - - 4 6 Mao, C.C. a n d G u i d o t t i , A. ( 1 9 7 4 ) Anal. B i o c h e m . 59, 6 3 - - 6 8 M a t s u z a w a , H. a n d N i r e n b e r g , M. ( 1 9 7 5 ) Proc. Natl. A c a d . Sci. U.S. 72, 3 4 7 2 - - 3 4 7 6 P a t t e r s o n , M.S. a n d Greene, R.C. ( 1 9 6 5 ) Anal. C h e m . 3 7 , 8 5 4 - - 8 5 7 Stelner, A.L., Parker, C.W. a n d Kipnis, D.M. ( 1 9 7 2 ) J. Biol. Chem. 247, 1 1 0 6 - - 1 1 1 3 R a n d e r a t h , K. ( 1 9 6 6 ) T h i n - L a y e r C h r o m a t o g r a p h y , p. 2 1 9 , A c a d e m i c Press, New Y o r k L o w r y , O.H., R o s e b r o u g h , N.J., Fal~, A.L. a n d R a n d a l l , R . J . ( 1 9 5 1 ) J. Biol. C h e m . 1 9 3 , 2 6 5 - - 2 7 5 B o u d r e a u , R.J. a n d D r u m m o n d , G.L ( 1 9 7 5 ) Anal. B i o c h e m . 6 3 , 3 8 8 - - 3 9 9 Garbers, D.L. a n d H a r d m a n , J.G. ( 1 9 7 6 ) J. Cyclic N u c l e o t i d e Res. 2, 5 9 - - 7 0 K i m u r a , H., T h o m a s , E. a n d M u r a d , F. ( 1 9 7 4 ) Biochim. Biophys. A c t a 3 4 3 , 5 1 9 - - 5 2 8 J a k o b s , K.H., B S h m e , E. a n d S c h u l t z , G. ( 1 9 7 6 ) in E u k a r y o t i c Cell F u n c t i o n a n d G r o w t h : R e g u l a t i o n b y I n t r a c e U u l a r Cyclic N u c l e o t i d e s ( D u m o n t , J.E., B r o w n , B.L. a n d Marshall, N.J., eds.), N A T O Adv a n c e d S t u d y I n s t i t u t e s Series, Vol. 9, p p . 2 9 5 - - 3 1 1 , P l e n u m Press, New Y o r k 25 A s a k a w a , T., Russell, T.R. a n d H o , R. ( 1 9 7 6 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 68, 682---690
