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Catecholamine binding by adrenal medullary protein can interfere with a sensitive radioenzymatic assay for norepinephrine
|,ire Sciences, Vol. 36, pp. 881-887 Printed in the U.S.A.
Pergamon Press
CATECHOLAMINE BINDING BY ADRENAL MEDULLARY PROTEIN CAN INTERFERE WITH A SENSITIVE RADIOENZYMATIC ASSAY FOR NOREPINEPHRINE D.R. Studelska, N.R.C. Campbell and W.S. Brimijoin Department of Pharmacology, Mayo Foundation, Rochester, MN 55905 USA (Received in final form December 20, 1984)
SUMMARY Norepinephrine (NE) binds extensively to protein that copurifies with phenylethanolamine-N-methyltransferase (PNMT) prepared from bovine adrenal medulla. This binding i n t e r f e r e s with a NE assay that employs PNMT to catalyze the t r a n s f e r of a t r i t i a t e d methyl group from S-adenosyl-L-methionine to the amine group of NE. It was discovered that the protein binding of endogenous NE is reversed by d i a l y s i s at pH 6.0. Preparations of PNMT intended for use in radioenzymatic assays should involve one or more p u r i f i c a t i o n steps at pH 6.0 Several years ago in t h i s j o u r n a l , Henry et al. (i) described an assay capable of detecting picogram quantities of norepinephrine (NE) in biological samples. In t h i s assay, phenylethanolamine-N-methyltransferase (PNMT), p a r t i a l l y p u r i f i e d from bovine adrenal glands, is used to convert NE to t r i t i a t e d epinephrine (E) via a labeled methyl group donated by S-adenosyl-L-methionine (SAM). The PNMT-based assay offers certain advantages over high performance l i q u i d chromatography (HPLC) in regard to s e n s i t i v i t y (detection threshold for
NE about 2-fold lower) and convenience for multiple determinations (daily throughput about 2-fold higher). Unfortunately, we have recently discovered that NE bound to protein that copurifies with PNMT can severely reduce the signal-to-noise r a t i o of the assay by increasing the a c t i v i t y assayed in blank samples. Below we describe experiments that define the problem and show how to remove i t . METHODS PNMT p u r i f i c a t i o n : Whole frozen bovine adrenal glands or medullas (100-200 g) were thawed and homogenized with a Waring blender in two volumes of 1.15% KCI. The homogenate was centrifuged at I0,000 x g f o r 30 minutes in a r e f r i g e r a t e d centrifuge at 4°C. The supernatant was then centrifuged at lO0,OOO x g in an u l t r a c e n t r i f u g e at 4°C for I hour. The high-speed supernatant was gradually brought to 30% saturation with ammonium s u l f a t e while constantly s t i r r e d on ice. After 30 minutes, the mixture was centrifuged at I0,000 x g f o r 30 minutes to remove p r e c i p i t a t e d protein. The supernatant was then brought to 50% saturation with ammonium s u l f a t e . After 30 minutes of stirring, the p r e c i p i t a t e was collected by c e n t r i f u g a t i o n and was gently resuspended in 0.01 M Tris-HCI, pH 7.4, added to a volume one-tenth of the volume of the high-speed supernatant. This suspension was dialyzed at 4°C against 20 L of 0 . 0 1 M Tris-HCl, pH 7.4, for 18 hours with one change. The dialysate (20-30 ml) was then loaded onto a 5 x 1NO cm gel f i l t r a t i o n column (Biogel A 1.5 M) that was e q u i l i b r a t e d with 0.01M Tris-HCl, pH 7.4, at 4°C. 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
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Fractions (18.5 ml) were c o l l e c t e d and assayed f o r PNMT a c t i v i t y with the s o l v e n t - e x t r a c t i o n radioenzymatic assay of Axelrod (2), using phenylethanolamine as a substrate. Fractions of high a c t i v i t y were pooled and concentrated with PMIO f i l t e r s (Amicon, mw c u t o f f I 0 , 0 0 0 ) . Norepinephrine assa~: Each assay tube contained 20 ul of a PNMT preparation to which optional c o n s t i t u e n t s were added in I0 ul increments before the volume was brought to I00 ~I with 0.01M T r i s - H C l , pH 7.0. The concentrations of the optional a d d i t i o n s at the f i n a l reaction volume of 200 ~I were: 50 or i00 uM EDTA t h a t sometimes contained 30 nM NE; 20 uM LY-134046; or 200 ~M S-adenosylL-homocysteine (SAH). The reaction was i n i t i a t e d with the a d d i t i o n of I00 ~I of a c o c k t a i l c o n t a i n i n g : 77.5 ul of 2 M T r i s - H C l , pH 9.2, 2.5 ~I S(methyl-3H)-adenosyl-L-methionine (80 Ci/mmol, New England Nuclear); and 20 ul 0.01 M T r i s - H C l , pH 7.0. The tubes were incubated in a shaking water bath at 37°C f o r I hour. The reaction was terminated with the a d d i t i o n of i00 mg alumina and 2 ml of 2 M T r i s - H C l , 5% EDTA, pH 8.6. Following v o r t e x i n g and c e n t r i f u g a t i o n , the supernatant was aspirated from each tube and the alumina was vortexed in 3 ml glass d i s t i l l e d water. The water was aspirated and t h i s water wash step was repeated twice before the catecholamine bound to the alumina was eluted in I ml of i c e - c o l d 0 . I N HCIO3. After c e n t r i f u g a t i o n , 0.8 ml of the e l u t a n t was transfered to a tube c h i l l e d on i c e . One hundred ~I of a mixture of SAM ( I . 0 mg/ml) and E (0.5 mg/ml) in 0.2 N a c e t i c acid was added followed by 200 ~I of 25% phosphotungstic acid. The tubes were vortexed b r i e f l y and allowed to stand 5 minutes before the p r e c i p i t a t e was removed by c e n t r i f u g a t i o n at 4,000 x g f o r 15 minutes. 0.8 ml of each supernatant was mixed with I0 ml of s c i n t i l l a t i o n f l u i d (3a70, Research Products I n t e r n a t i o n a l ) f o r the detection of t r i t i u m . Product i d e n t i f i c a t i o n : The t r i t i u m labeled products of the NE assay were i d e n t i f i e d by high performance l i q u i d chromatography (HPLC) with amperometric d e t e c t i o n . The system employed included a Waters 6000 pump, a Whatman P a r t i s i l PXS 10/25 ODS 3 column and a BSA LC-4 electrochemical detector set at 0.6 volts. Two solvent systems were used. The f i r s t consisted of 8% a c e t o n i t r i l e , 5 mM l-heptane s u l f o n i c acid, 0 . I mM EDTA, and 70 mM phosphoric acid pH 3.5. With t h i s system NE eluted at 5'5" and E eluted at 7'20" when the flow rate was I cc/minute. The second solvent system was 10% methanol; 4 mM l-heptane s u l f o n i c acid; 0 . I M EDTA; and 70 mM phosphoric acid, pH 5.2. At a flow rate of 1.5 cc/minute, NE eluted at 4'27" and E eluted at 5'07". For the product i d e n t i f i c a t i o n runs, 50 ul of the supernatant produced by phosphotungstic acid precipitation was i n j e c t e d and 1 minute f r a c t i o n s were c o l l e c t e d for s c i n t i l l a t i o n counting. The secono solvent system was also used to d i r e c t l y q u a n t i t a t e NE and E in enzyme preparations. NE and E standard curves were l i n e a r from 7.8 to 2,000 ng/ml and 3.9 t o 500 ng/ml, r e s p e c t i v e l y . The l i m i t s of detection with a s i g n a l - t o - n o i s e r a t i o of 2 and 20 ~I i n j e c t i o n volume were 7.8 ng/ml f o r NE and 3.9 ng/ml f o r E. RESULTS Our o b j e c t i v e was to i d e n t i f y the source of the high blanks in a NE assay that had worked before in our hands. I n i t i a l experiments demonstrated that the problem was not due to i m p u r i t i e s in the batch of t r i t i a t e d SAM or to the contamination of a commerical reagent with NE or a s i m i l a r methyl acceptor. Our f i r s t clues were t h a t the i n t e r f e r i n g a c t i v i t y was more abundant in the presence of EDTA and was more evident in some preparations of PNMT than in others. The p o s s i b i l i t i e s included the presence of a substrate f o r
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N-methylation or the existence of another kind of methyltransferase in our PNMT preparation. Because the PNMT p u r i f i c a t i o n entailed extensive d i a l y s i s of protein p r e c i p i t a t e d by ammonium s u l f a t e , followed by chromatography on a large gel f i l t r a t i o n column, we f e l t that potential substrates for PNMT ( i . e . , small molecules), were not l i k e l y to be present. In an attempt to remove i n t e r f e r i n g methyltransferases, we selected a batch of PNMT (ru20 ml) that was high in the i n t e r f e r i n g a c t i v i t y and subjected i t to a second gel f i l t r a t i o n . This re-chromatography revealed a minor peak of PNMT a c t i v i t y ( f r a c t i o n s 16-31) preceding a major peak of PNMT a c t i v i t y ( f r a c t i o n s 32-37). The f r a c t i o n s containing the two enzyme peaks were separately pooled and concentrated to volumes with s i m i l a r levels of PNMT a c t i v i t y and were d i r e c t l y compared in a NE assay. To test for the possible presence of another methyltransferase in these preparations we employed S-adenosyl-L-homocysteine (SAH), an i n h i b i t o r of most methyltransferases, and LY-134046, a potent i n h i b i t o r of PNMT (3). NE was not added to the assay tubes, but some contained EDTA, which had been shown to f a c i l i t a t e the i n t e r f e r i n g a c t i v i t y . TABLE 1 Re-Chromatographed PNMT
FRACTIONS 16-31 10 ~M SAH
None
i ~M LY-134046
Inhibitor CPM
CPM
% Inhibition
CPM
% Inhibition
50 ~M EDTA
4156
2090
49.7
1628
60.8
H20 blank
1072
527
50.9
391
63.6
FRACTIONS 32-37 I0 uM SAH
None
1 ~M LY-134046
Inhibitor
50 ~M EDTA H20 blank
CPM
CPM
% Inhibition
CPM
% Inhibition
1576
518
67.1
496
68.6
175
131
25.3
106
39.3
Effect of enzyme i n h i b i t o r s on the no substrate blank a c t i v i t y of rechromatographed PNMT in the NE assay.
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As shown in Table 1, the i n t e r f e r i n g NE assay a c t i v i t y could not be completely separated from PNMT a c t i v i t y by gel f i l t r a t i o n . However, there was less interference when enzyme concentrated from the most active f r a c t i o n s (32-37) was employed. SAH and LY-134046 both markedly reduced a c t i v i t y in every instance of t h e i r use, suggesting that the high blank a c t i v i t y was dependent on a methyltransferase, most probably PNMT i t s e l f . Another batch of PNMT was prepared using frozen beef adrenals from the same source and shipment as before. Ten gel f i l t r a t i o n f r a c t i o n s e l u t i n g p r i o r to the f r a c t i o n s containing the highest PNMT a c t i v i t y and 10 f r a c t i o n s collected afterwards were pooled and concentrated to y i e l d s i m i l a r PNMT a c t i v i t y per unit volume. These preparations were dubbed Pre-PNMT, PNMT, and Post-PNMT. The r a t i o s of t h e i r NE assay blank a c t i v i t y (in the presence of I00 ~M EDTA) to t h e i r PNMT a c t i v i t y were as follows: Pre-PNMT, 2.33; PNMT, 0.39; and Post-PNMT, 0.17. These results indicated that the factor necessary for the i n t e r f e r i n g a c t i v i t y was dissociable from PNMT and was more abundant in Pre-PNMT ( i . e . , the higher molecular weight preparation). In an i n i t i a l attempt to characterize t h i s f a c t o r , an aliquot of Pre-PNMT was heated to 95°C f o r 5 minutes. Twenty ul of t h i s m a t e r i a l , when added to 20 ~I of PNMT in the presence of I00 ~M EDTA, increased the a c t i v i t y of NE assay blanks 1000%. This increase was comparable to the 880% increase obtained when 20 ~1 of nonheated Pre-PNMT was added, i n d i c a t i n g that the f a c t o r was heat stable and, t h e r e f o r e , probably not a protein. Other experiments performed at t h i s point made i t clear that the a c t i v i t y was dependent on a viable enzyme (heated preparations alone produced no a c t i v i t y ) and that the enhancing e f f e c t of EDTA was probably due to the c h e l a t o r ' s a b i l i t y to protect a putative substrate against metal catalyzed oxidation (not a l l of the EDTA-dependent a c t i v i t y was recovered when EDTA was added a f t e r the reaction had s t a r t e d ) . These r e s u l t s led us to suspect that the inter--e-r-f-ering a c t i v i t y might well be endogenous NE that had survived the steps of PNMT p u r i f i c a t i o n by binding to PNMT or to proteins that were not separated from PNMT. Using HPLC with an amperometric detector and d i f f e r e n t solvent systems, one containing a c e t o n i t r i l e , the other methyl alcohol, we found that the radioactive product produced in the NE assay blank had the same retention times as genuine epinephrine. This was the f i n d i n g with Pre-PNMT, PNMT, and Post-PNMT in the presence and in the absence of EDTA. As anticipated, the amount of radioactive E generated by the Pre-PNMT preparation was greatest, while that generated by the Post-PNMT preparation was l e a s t . An attempt to quantify the catecholamine l e v e l s of the three enzyme preparations d i r e c t l y via HPLC led to results that suggested the protein binding was s e l e c t i v e f o r NE. In an i n i t i a l experiment an aliquot of Pre-PNMT was adjusted to 0 . i N with 2 N HClO3 and then the sample was spun at 4,000 x g f o r 15 minutes. The c e n t r i f u g a t i o n did not clear a l l of the supernatant of p r e c i p i t a t e d p r o t e i n , but 20 ~I of clear supernatant was injected nevertheless into the HPLC column (which was e q u i l i b r a t e d with the methanol solvent system). The amperometric signal indicated NE at a concentration of 1,510 ng/ml with E present at 136 ng/ml. Subsequently, aliquots of Pre-PNMT, PNMT and Post-PNMT were s i m i l a r l y treated with HCIO3 but were centrifuged at higher speed (16,000 x g f o r 15 minutes). This produced supernatants that were e n t i r e l y clear. In the c l a r i f i e d Pre-PNMT preparation, NE was reduced to 220 ng/ml while E remained much the same as before (140 ng/ml). NE could not be detected in the other preparations but E was found in both (14.8 ng/ml PNMT; 11.6 ng/ml Post-PNMT). T h u s not only did denaturation seem to have l i t t l e e f f e c t on protein binding of NE, but t h i s binding seemed to be s e l e c t i v e for NE, e s p e c i a l l y since the level of E in the adrenal gland is three times higher than that of NE ( P f e i f f e r , unpublished r e s u l t s ) .
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Catecholamine Binding by PNMT
885
Another PNMT p u r i f i c a t i o n was i n i t i a t e d using medullas dissected from bovine adrenal glands obtained from a local s u p p l i e r . This time t r i t i a t e d NE (3H-NE) was added t o the b u f f e r in which the enzyme was resuspended a f t e r ammonium s u l f a t e p r e c i p i t a t i o n . The resuspended enzyme was then dialyzed as usual, but samples of the d i a l y s i s medium and the d i a l y z a t e were counted f o r 3H-NE a c t i v i t y . Over 99% of the a c t i v i t y present in equal volumes of d i a l y s i s medium and d i a l y s a t e was found to be bound to non-dialyzable material a f t e r both the f i r s t and the second d i a l y s i s . The proportion of t o t a l a c t i v i t y outside the d i a l y s i s tubing a f t e r the second d i a l y s i s demonstrated t h a t the binding was r e v e r s i b l e . Of greater i n t e r e s t was the pattern of 3H-NE a c t i v i t y present in f r a c t i o n s c o l l e c t e d from the gel f i l t r a t i o n column. This was assayed by l i q u i d s c i n t i l l a t i o n spectroscopy a f t e r mixing 200 ~I a l i q u o t s of each 8.5 ml f r a c t i o n with 10 ml of s c i n t i l l a t i o n fluid. The r e l a t i v e p r o t e i n concentration was also determined by absorbance at 280 nm of a l i q u o t s d i l u t e d 1:9. Figure I shows these r e s u l t s in comparison to the PNMT a c t i v i t y assayed in these column f r a c t i o n s .
LU Z 300 I "l-
n~
0
250 o •
Z LI.J I-- 2 0 0 0 nr" (3. L=_
0
PROTEIN PNMT
l-]> --I 300
I'q 3>
150
--I 200
<
-.I .<
I00
I00 Ld
:;0
rrl
aH-NE
I-Z 0
400
50
0 -n "1] Z
I-
--I 0
50
I00
FRACTION
150
NUMBER
FIGURE 1 D i s t r i b u t i o n of p r o t e i n , PNMT a c t i v i t y , and 3H-NE in the Biogel A 1.5 M gel f i l t r a t i o n eluate (void volume = f r a c t i o n s 8-12).
886
Catecholamine Binding by PNMT
Vol. 36, No. 9, 1985
Evidently, 3H-NE binding closely corresponded to the relative protein concentration except in the fractions highest in PNMTa c t i v i t y , where the ratio of 3H-NE to relative protein concentration was increased. T h i s suggests that PNMT under these conditions may have NE specifically bound at its active site plus more NE bound by whatever forces are responsible for the extensive NE-protein binding evident in preceding fractions. The cause of the problem with the NE assay now seemed to be clearly delineated. The purification procedure for PNMTallowed and perhaps f a c i l i t a t e d the binding of liberated NE to protein, including PNMT i t s e l f . Our next goal was to find conditions that would reduce this binding to yield PNMT suitable for the NE assay. The solution to this problem proved to be surprisingly simple: dialysis at a somewhat lower pH. To test for the effect of pH, small microconcentrators with a molecular weight cutoff of IO,ONO were employed (centricon 10, Amicon Corp.). One ml of 0.05 M Tris buffer, pH 9.2, 8.6, 7.4, or 6.0 was combined with a 250 ul aliquot of Pre-PNMT that had been mixed with 3H-NE. These mixtures were then separated by f i l t r a t i o n under centrifugal force. Retained protein was rinsed by several steps of dilution with 1 ml of buffer at the appropriate pH and re-centrifugation. At the end of each centrifugation, aliquots of the f i l t r a t e from each of the four concentrators were assayed for 3H-NE activity by liquid s c i n t i l l a tion counting. The results indicated that during the f i r s t three cycles of dilution and u l t r a f i l t r a t i o n over 200% more ~H-NE was dissociated from protein at pH 6.0 than at the other hydrogen ion concentrations tested. Three cycles of dilution and u l t r a f i l t r a t i o n of PNMT with a pH 6.0 buffer increased the signal-to-noise ratio for 30 nM NE in the NE assay from 3.1 (11855/3879) to 14.8 (8176/553). DISCUSSION These experiments leave l i t t l e doubt that NE endogenous to the adrenal medulla can bind avidly and reversibly to protein that copurifies with PNMT and can thereby interfere with assays based upon this enzyme. The characteristics of the NE binding are intriguing. Because NE is a phenolic amine with two overlapping deprotonations (pK1 = 8.73, pK2 = 9.84), i t exists in equilibrium between cationic, anionic, neutral, and zwitterionic forms. According to Martin (4), the ratio of zwitterionic to neutral forms of the two groups is about 3 for NE and 4 for E. While the zwitterionic form would not predominate at neutral pH, i f this form favored binding to protein, return towards equilibrium would readily generate more NE that was suitable for binding. Such effects might explain why protein binding of NE becomes prevalent at pH 7. Our results support the conclusion that at an intravesicular pH of 5.6 (5), NE is free (6). But they imply that intracellular NE not sequestered in aminergic vesicles is bound to protein. It is possible that protein binding of NE has physiologically important consequences. For example, i t seems unlikely that the methylation of NE in adrenal chromaffin cells is a solution-phase reaction. This line of reasoning suggests that further study of the subcellular localization of catecholamine synthesis would prove rewarding. As a practical matter, the binding of NE to protein at neutral pH can be a nuisance to the investigator. For example, a biochemist interested in the catalytic properties of PNMT, per se, must be aware of potential artifacts from endogenous substrate in supposedly pure preparations of the enzyme. In a study e x p l i c i t l y focussed on the binding of catecholamine to dialyzed chromaffin granule material, Uvn~s and Eborg (7) were forced to work at acid pH because NE binding at neutrality was non-saturable. In our experiments, NE binding
Vol. 36, No. 9, 1985
Catecholamine Binding by PNMT
887
rendered standard preparations of PNMT useless for the purpose of enzymatic NE assays. We have no ready explanation for the unexpected exacerbation of the l a t t e r problem in our recent work. Perhaps i t had been avoided previously by chance, owing to unintentional drops in pH during the ammonium s u l f a t e f r a c t i o n a t i o n of PNMT. Of course, a moderate degree of NE contamination may be t o l e r a b l e since i t merely reduces assay s e n s i t i v i t y without introducing systematic errors of measurement, so long as approximate blanks are used. In any event, since the PNMT p u r i f i c a t i o n e n t a i l s both d i a l y s i s and gel f i l t r a t i o n , performing these steps at pH 6.0 o f f e r s an easy way to avoid such contamination and enhance assay s e n s i t i v i t y . REFERENCES 1. 2. 3. 4. 5. 6. 7.
D.P. HENRY, B.J. STARMAN, D.G. JOHNSON AND R.H. WILLIAMS, L i f e . Sci. 16 375-384 (1975). J~AXELROD, J. Biol. Chem. 237 1657-1660 (1962). R.W. FULLER, S. HEMRICK-LUECKE, R.E. LOOMEY, J. HORNG, R.R. RUFFALO and B.B. MOLLOY, Biochem. Pharmacol. 30 1345-1352 (1981). R.B. MARTIN, J. Phys. Chem. 75 2657-2661 (1971). R.G. JOHNSON AND A. SCARPA, ~ Biol. Chem. 251 2189-2191 (1976). P.R. SHARP AND E.P. RICHARDS, Biochim. Biophys. Acta 497 14-28 (1977). B. UVN~S and C.-H. ~BORG, Acta Physiol. S c a n t 109 345-354 (1980).
Pergamon Press
CATECHOLAMINE BINDING BY ADRENAL MEDULLARY PROTEIN CAN INTERFERE WITH A SENSITIVE RADIOENZYMATIC ASSAY FOR NOREPINEPHRINE D.R. Studelska, N.R.C. Campbell and W.S. Brimijoin Department of Pharmacology, Mayo Foundation, Rochester, MN 55905 USA (Received in final form December 20, 1984)
SUMMARY Norepinephrine (NE) binds extensively to protein that copurifies with phenylethanolamine-N-methyltransferase (PNMT) prepared from bovine adrenal medulla. This binding i n t e r f e r e s with a NE assay that employs PNMT to catalyze the t r a n s f e r of a t r i t i a t e d methyl group from S-adenosyl-L-methionine to the amine group of NE. It was discovered that the protein binding of endogenous NE is reversed by d i a l y s i s at pH 6.0. Preparations of PNMT intended for use in radioenzymatic assays should involve one or more p u r i f i c a t i o n steps at pH 6.0 Several years ago in t h i s j o u r n a l , Henry et al. (i) described an assay capable of detecting picogram quantities of norepinephrine (NE) in biological samples. In t h i s assay, phenylethanolamine-N-methyltransferase (PNMT), p a r t i a l l y p u r i f i e d from bovine adrenal glands, is used to convert NE to t r i t i a t e d epinephrine (E) via a labeled methyl group donated by S-adenosyl-L-methionine (SAM). The PNMT-based assay offers certain advantages over high performance l i q u i d chromatography (HPLC) in regard to s e n s i t i v i t y (detection threshold for
NE about 2-fold lower) and convenience for multiple determinations (daily throughput about 2-fold higher). Unfortunately, we have recently discovered that NE bound to protein that copurifies with PNMT can severely reduce the signal-to-noise r a t i o of the assay by increasing the a c t i v i t y assayed in blank samples. Below we describe experiments that define the problem and show how to remove i t . METHODS PNMT p u r i f i c a t i o n : Whole frozen bovine adrenal glands or medullas (100-200 g) were thawed and homogenized with a Waring blender in two volumes of 1.15% KCI. The homogenate was centrifuged at I0,000 x g f o r 30 minutes in a r e f r i g e r a t e d centrifuge at 4°C. The supernatant was then centrifuged at lO0,OOO x g in an u l t r a c e n t r i f u g e at 4°C for I hour. The high-speed supernatant was gradually brought to 30% saturation with ammonium s u l f a t e while constantly s t i r r e d on ice. After 30 minutes, the mixture was centrifuged at I0,000 x g f o r 30 minutes to remove p r e c i p i t a t e d protein. The supernatant was then brought to 50% saturation with ammonium s u l f a t e . After 30 minutes of stirring, the p r e c i p i t a t e was collected by c e n t r i f u g a t i o n and was gently resuspended in 0.01 M Tris-HCI, pH 7.4, added to a volume one-tenth of the volume of the high-speed supernatant. This suspension was dialyzed at 4°C against 20 L of 0 . 0 1 M Tris-HCl, pH 7.4, for 18 hours with one change. The dialysate (20-30 ml) was then loaded onto a 5 x 1NO cm gel f i l t r a t i o n column (Biogel A 1.5 M) that was e q u i l i b r a t e d with 0.01M Tris-HCl, pH 7.4, at 4°C. 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
882
Catecholamine Binding by PNMT
Vol. 36, No. 9, 1985
Fractions (18.5 ml) were c o l l e c t e d and assayed f o r PNMT a c t i v i t y with the s o l v e n t - e x t r a c t i o n radioenzymatic assay of Axelrod (2), using phenylethanolamine as a substrate. Fractions of high a c t i v i t y were pooled and concentrated with PMIO f i l t e r s (Amicon, mw c u t o f f I 0 , 0 0 0 ) . Norepinephrine assa~: Each assay tube contained 20 ul of a PNMT preparation to which optional c o n s t i t u e n t s were added in I0 ul increments before the volume was brought to I00 ~I with 0.01M T r i s - H C l , pH 7.0. The concentrations of the optional a d d i t i o n s at the f i n a l reaction volume of 200 ~I were: 50 or i00 uM EDTA t h a t sometimes contained 30 nM NE; 20 uM LY-134046; or 200 ~M S-adenosylL-homocysteine (SAH). The reaction was i n i t i a t e d with the a d d i t i o n of I00 ~I of a c o c k t a i l c o n t a i n i n g : 77.5 ul of 2 M T r i s - H C l , pH 9.2, 2.5 ~I S(methyl-3H)-adenosyl-L-methionine (80 Ci/mmol, New England Nuclear); and 20 ul 0.01 M T r i s - H C l , pH 7.0. The tubes were incubated in a shaking water bath at 37°C f o r I hour. The reaction was terminated with the a d d i t i o n of i00 mg alumina and 2 ml of 2 M T r i s - H C l , 5% EDTA, pH 8.6. Following v o r t e x i n g and c e n t r i f u g a t i o n , the supernatant was aspirated from each tube and the alumina was vortexed in 3 ml glass d i s t i l l e d water. The water was aspirated and t h i s water wash step was repeated twice before the catecholamine bound to the alumina was eluted in I ml of i c e - c o l d 0 . I N HCIO3. After c e n t r i f u g a t i o n , 0.8 ml of the e l u t a n t was transfered to a tube c h i l l e d on i c e . One hundred ~I of a mixture of SAM ( I . 0 mg/ml) and E (0.5 mg/ml) in 0.2 N a c e t i c acid was added followed by 200 ~I of 25% phosphotungstic acid. The tubes were vortexed b r i e f l y and allowed to stand 5 minutes before the p r e c i p i t a t e was removed by c e n t r i f u g a t i o n at 4,000 x g f o r 15 minutes. 0.8 ml of each supernatant was mixed with I0 ml of s c i n t i l l a t i o n f l u i d (3a70, Research Products I n t e r n a t i o n a l ) f o r the detection of t r i t i u m . Product i d e n t i f i c a t i o n : The t r i t i u m labeled products of the NE assay were i d e n t i f i e d by high performance l i q u i d chromatography (HPLC) with amperometric d e t e c t i o n . The system employed included a Waters 6000 pump, a Whatman P a r t i s i l PXS 10/25 ODS 3 column and a BSA LC-4 electrochemical detector set at 0.6 volts. Two solvent systems were used. The f i r s t consisted of 8% a c e t o n i t r i l e , 5 mM l-heptane s u l f o n i c acid, 0 . I mM EDTA, and 70 mM phosphoric acid pH 3.5. With t h i s system NE eluted at 5'5" and E eluted at 7'20" when the flow rate was I cc/minute. The second solvent system was 10% methanol; 4 mM l-heptane s u l f o n i c acid; 0 . I M EDTA; and 70 mM phosphoric acid, pH 5.2. At a flow rate of 1.5 cc/minute, NE eluted at 4'27" and E eluted at 5'07". For the product i d e n t i f i c a t i o n runs, 50 ul of the supernatant produced by phosphotungstic acid precipitation was i n j e c t e d and 1 minute f r a c t i o n s were c o l l e c t e d for s c i n t i l l a t i o n counting. The secono solvent system was also used to d i r e c t l y q u a n t i t a t e NE and E in enzyme preparations. NE and E standard curves were l i n e a r from 7.8 to 2,000 ng/ml and 3.9 t o 500 ng/ml, r e s p e c t i v e l y . The l i m i t s of detection with a s i g n a l - t o - n o i s e r a t i o of 2 and 20 ~I i n j e c t i o n volume were 7.8 ng/ml f o r NE and 3.9 ng/ml f o r E. RESULTS Our o b j e c t i v e was to i d e n t i f y the source of the high blanks in a NE assay that had worked before in our hands. I n i t i a l experiments demonstrated that the problem was not due to i m p u r i t i e s in the batch of t r i t i a t e d SAM or to the contamination of a commerical reagent with NE or a s i m i l a r methyl acceptor. Our f i r s t clues were t h a t the i n t e r f e r i n g a c t i v i t y was more abundant in the presence of EDTA and was more evident in some preparations of PNMT than in others. The p o s s i b i l i t i e s included the presence of a substrate f o r
Vol. 36, No. 9, 1985
Catecholamine Binding by PNMT
883
N-methylation or the existence of another kind of methyltransferase in our PNMT preparation. Because the PNMT p u r i f i c a t i o n entailed extensive d i a l y s i s of protein p r e c i p i t a t e d by ammonium s u l f a t e , followed by chromatography on a large gel f i l t r a t i o n column, we f e l t that potential substrates for PNMT ( i . e . , small molecules), were not l i k e l y to be present. In an attempt to remove i n t e r f e r i n g methyltransferases, we selected a batch of PNMT (ru20 ml) that was high in the i n t e r f e r i n g a c t i v i t y and subjected i t to a second gel f i l t r a t i o n . This re-chromatography revealed a minor peak of PNMT a c t i v i t y ( f r a c t i o n s 16-31) preceding a major peak of PNMT a c t i v i t y ( f r a c t i o n s 32-37). The f r a c t i o n s containing the two enzyme peaks were separately pooled and concentrated to volumes with s i m i l a r levels of PNMT a c t i v i t y and were d i r e c t l y compared in a NE assay. To test for the possible presence of another methyltransferase in these preparations we employed S-adenosyl-L-homocysteine (SAH), an i n h i b i t o r of most methyltransferases, and LY-134046, a potent i n h i b i t o r of PNMT (3). NE was not added to the assay tubes, but some contained EDTA, which had been shown to f a c i l i t a t e the i n t e r f e r i n g a c t i v i t y . TABLE 1 Re-Chromatographed PNMT
FRACTIONS 16-31 10 ~M SAH
None
i ~M LY-134046
Inhibitor CPM
CPM
% Inhibition
CPM
% Inhibition
50 ~M EDTA
4156
2090
49.7
1628
60.8
H20 blank
1072
527
50.9
391
63.6
FRACTIONS 32-37 I0 uM SAH
None
1 ~M LY-134046
Inhibitor
50 ~M EDTA H20 blank
CPM
CPM
% Inhibition
CPM
% Inhibition
1576
518
67.1
496
68.6
175
131
25.3
106
39.3
Effect of enzyme i n h i b i t o r s on the no substrate blank a c t i v i t y of rechromatographed PNMT in the NE assay.
884
Catecholamine Binding by PNMT
Vol. 36, No. 9, 1985
As shown in Table 1, the i n t e r f e r i n g NE assay a c t i v i t y could not be completely separated from PNMT a c t i v i t y by gel f i l t r a t i o n . However, there was less interference when enzyme concentrated from the most active f r a c t i o n s (32-37) was employed. SAH and LY-134046 both markedly reduced a c t i v i t y in every instance of t h e i r use, suggesting that the high blank a c t i v i t y was dependent on a methyltransferase, most probably PNMT i t s e l f . Another batch of PNMT was prepared using frozen beef adrenals from the same source and shipment as before. Ten gel f i l t r a t i o n f r a c t i o n s e l u t i n g p r i o r to the f r a c t i o n s containing the highest PNMT a c t i v i t y and 10 f r a c t i o n s collected afterwards were pooled and concentrated to y i e l d s i m i l a r PNMT a c t i v i t y per unit volume. These preparations were dubbed Pre-PNMT, PNMT, and Post-PNMT. The r a t i o s of t h e i r NE assay blank a c t i v i t y (in the presence of I00 ~M EDTA) to t h e i r PNMT a c t i v i t y were as follows: Pre-PNMT, 2.33; PNMT, 0.39; and Post-PNMT, 0.17. These results indicated that the factor necessary for the i n t e r f e r i n g a c t i v i t y was dissociable from PNMT and was more abundant in Pre-PNMT ( i . e . , the higher molecular weight preparation). In an i n i t i a l attempt to characterize t h i s f a c t o r , an aliquot of Pre-PNMT was heated to 95°C f o r 5 minutes. Twenty ul of t h i s m a t e r i a l , when added to 20 ~I of PNMT in the presence of I00 ~M EDTA, increased the a c t i v i t y of NE assay blanks 1000%. This increase was comparable to the 880% increase obtained when 20 ~1 of nonheated Pre-PNMT was added, i n d i c a t i n g that the f a c t o r was heat stable and, t h e r e f o r e , probably not a protein. Other experiments performed at t h i s point made i t clear that the a c t i v i t y was dependent on a viable enzyme (heated preparations alone produced no a c t i v i t y ) and that the enhancing e f f e c t of EDTA was probably due to the c h e l a t o r ' s a b i l i t y to protect a putative substrate against metal catalyzed oxidation (not a l l of the EDTA-dependent a c t i v i t y was recovered when EDTA was added a f t e r the reaction had s t a r t e d ) . These r e s u l t s led us to suspect that the inter--e-r-f-ering a c t i v i t y might well be endogenous NE that had survived the steps of PNMT p u r i f i c a t i o n by binding to PNMT or to proteins that were not separated from PNMT. Using HPLC with an amperometric detector and d i f f e r e n t solvent systems, one containing a c e t o n i t r i l e , the other methyl alcohol, we found that the radioactive product produced in the NE assay blank had the same retention times as genuine epinephrine. This was the f i n d i n g with Pre-PNMT, PNMT, and Post-PNMT in the presence and in the absence of EDTA. As anticipated, the amount of radioactive E generated by the Pre-PNMT preparation was greatest, while that generated by the Post-PNMT preparation was l e a s t . An attempt to quantify the catecholamine l e v e l s of the three enzyme preparations d i r e c t l y via HPLC led to results that suggested the protein binding was s e l e c t i v e f o r NE. In an i n i t i a l experiment an aliquot of Pre-PNMT was adjusted to 0 . i N with 2 N HClO3 and then the sample was spun at 4,000 x g f o r 15 minutes. The c e n t r i f u g a t i o n did not clear a l l of the supernatant of p r e c i p i t a t e d p r o t e i n , but 20 ~I of clear supernatant was injected nevertheless into the HPLC column (which was e q u i l i b r a t e d with the methanol solvent system). The amperometric signal indicated NE at a concentration of 1,510 ng/ml with E present at 136 ng/ml. Subsequently, aliquots of Pre-PNMT, PNMT and Post-PNMT were s i m i l a r l y treated with HCIO3 but were centrifuged at higher speed (16,000 x g f o r 15 minutes). This produced supernatants that were e n t i r e l y clear. In the c l a r i f i e d Pre-PNMT preparation, NE was reduced to 220 ng/ml while E remained much the same as before (140 ng/ml). NE could not be detected in the other preparations but E was found in both (14.8 ng/ml PNMT; 11.6 ng/ml Post-PNMT). T h u s not only did denaturation seem to have l i t t l e e f f e c t on protein binding of NE, but t h i s binding seemed to be s e l e c t i v e for NE, e s p e c i a l l y since the level of E in the adrenal gland is three times higher than that of NE ( P f e i f f e r , unpublished r e s u l t s ) .
Vol. 36, No. 9, 1985
Catecholamine Binding by PNMT
885
Another PNMT p u r i f i c a t i o n was i n i t i a t e d using medullas dissected from bovine adrenal glands obtained from a local s u p p l i e r . This time t r i t i a t e d NE (3H-NE) was added t o the b u f f e r in which the enzyme was resuspended a f t e r ammonium s u l f a t e p r e c i p i t a t i o n . The resuspended enzyme was then dialyzed as usual, but samples of the d i a l y s i s medium and the d i a l y z a t e were counted f o r 3H-NE a c t i v i t y . Over 99% of the a c t i v i t y present in equal volumes of d i a l y s i s medium and d i a l y s a t e was found to be bound to non-dialyzable material a f t e r both the f i r s t and the second d i a l y s i s . The proportion of t o t a l a c t i v i t y outside the d i a l y s i s tubing a f t e r the second d i a l y s i s demonstrated t h a t the binding was r e v e r s i b l e . Of greater i n t e r e s t was the pattern of 3H-NE a c t i v i t y present in f r a c t i o n s c o l l e c t e d from the gel f i l t r a t i o n column. This was assayed by l i q u i d s c i n t i l l a t i o n spectroscopy a f t e r mixing 200 ~I a l i q u o t s of each 8.5 ml f r a c t i o n with 10 ml of s c i n t i l l a t i o n fluid. The r e l a t i v e p r o t e i n concentration was also determined by absorbance at 280 nm of a l i q u o t s d i l u t e d 1:9. Figure I shows these r e s u l t s in comparison to the PNMT a c t i v i t y assayed in these column f r a c t i o n s .
LU Z 300 I "l-
n~
0
250 o •
Z LI.J I-- 2 0 0 0 nr" (3. L=_
0
PROTEIN PNMT
l-]> --I 300
I'q 3>
150
--I 200
<
-.I .<
I00
I00 Ld
:;0
rrl
aH-NE
I-Z 0
400
50
0 -n "1] Z
I-
--I 0
50
I00
FRACTION
150
NUMBER
FIGURE 1 D i s t r i b u t i o n of p r o t e i n , PNMT a c t i v i t y , and 3H-NE in the Biogel A 1.5 M gel f i l t r a t i o n eluate (void volume = f r a c t i o n s 8-12).
886
Catecholamine Binding by PNMT
Vol. 36, No. 9, 1985
Evidently, 3H-NE binding closely corresponded to the relative protein concentration except in the fractions highest in PNMTa c t i v i t y , where the ratio of 3H-NE to relative protein concentration was increased. T h i s suggests that PNMT under these conditions may have NE specifically bound at its active site plus more NE bound by whatever forces are responsible for the extensive NE-protein binding evident in preceding fractions. The cause of the problem with the NE assay now seemed to be clearly delineated. The purification procedure for PNMTallowed and perhaps f a c i l i t a t e d the binding of liberated NE to protein, including PNMT i t s e l f . Our next goal was to find conditions that would reduce this binding to yield PNMT suitable for the NE assay. The solution to this problem proved to be surprisingly simple: dialysis at a somewhat lower pH. To test for the effect of pH, small microconcentrators with a molecular weight cutoff of IO,ONO were employed (centricon 10, Amicon Corp.). One ml of 0.05 M Tris buffer, pH 9.2, 8.6, 7.4, or 6.0 was combined with a 250 ul aliquot of Pre-PNMT that had been mixed with 3H-NE. These mixtures were then separated by f i l t r a t i o n under centrifugal force. Retained protein was rinsed by several steps of dilution with 1 ml of buffer at the appropriate pH and re-centrifugation. At the end of each centrifugation, aliquots of the f i l t r a t e from each of the four concentrators were assayed for 3H-NE activity by liquid s c i n t i l l a tion counting. The results indicated that during the f i r s t three cycles of dilution and u l t r a f i l t r a t i o n over 200% more ~H-NE was dissociated from protein at pH 6.0 than at the other hydrogen ion concentrations tested. Three cycles of dilution and u l t r a f i l t r a t i o n of PNMT with a pH 6.0 buffer increased the signal-to-noise ratio for 30 nM NE in the NE assay from 3.1 (11855/3879) to 14.8 (8176/553). DISCUSSION These experiments leave l i t t l e doubt that NE endogenous to the adrenal medulla can bind avidly and reversibly to protein that copurifies with PNMT and can thereby interfere with assays based upon this enzyme. The characteristics of the NE binding are intriguing. Because NE is a phenolic amine with two overlapping deprotonations (pK1 = 8.73, pK2 = 9.84), i t exists in equilibrium between cationic, anionic, neutral, and zwitterionic forms. According to Martin (4), the ratio of zwitterionic to neutral forms of the two groups is about 3 for NE and 4 for E. While the zwitterionic form would not predominate at neutral pH, i f this form favored binding to protein, return towards equilibrium would readily generate more NE that was suitable for binding. Such effects might explain why protein binding of NE becomes prevalent at pH 7. Our results support the conclusion that at an intravesicular pH of 5.6 (5), NE is free (6). But they imply that intracellular NE not sequestered in aminergic vesicles is bound to protein. It is possible that protein binding of NE has physiologically important consequences. For example, i t seems unlikely that the methylation of NE in adrenal chromaffin cells is a solution-phase reaction. This line of reasoning suggests that further study of the subcellular localization of catecholamine synthesis would prove rewarding. As a practical matter, the binding of NE to protein at neutral pH can be a nuisance to the investigator. For example, a biochemist interested in the catalytic properties of PNMT, per se, must be aware of potential artifacts from endogenous substrate in supposedly pure preparations of the enzyme. In a study e x p l i c i t l y focussed on the binding of catecholamine to dialyzed chromaffin granule material, Uvn~s and Eborg (7) were forced to work at acid pH because NE binding at neutrality was non-saturable. In our experiments, NE binding
Vol. 36, No. 9, 1985
Catecholamine Binding by PNMT
887
rendered standard preparations of PNMT useless for the purpose of enzymatic NE assays. We have no ready explanation for the unexpected exacerbation of the l a t t e r problem in our recent work. Perhaps i t had been avoided previously by chance, owing to unintentional drops in pH during the ammonium s u l f a t e f r a c t i o n a t i o n of PNMT. Of course, a moderate degree of NE contamination may be t o l e r a b l e since i t merely reduces assay s e n s i t i v i t y without introducing systematic errors of measurement, so long as approximate blanks are used. In any event, since the PNMT p u r i f i c a t i o n e n t a i l s both d i a l y s i s and gel f i l t r a t i o n , performing these steps at pH 6.0 o f f e r s an easy way to avoid such contamination and enhance assay s e n s i t i v i t y . REFERENCES 1. 2. 3. 4. 5. 6. 7.
D.P. HENRY, B.J. STARMAN, D.G. JOHNSON AND R.H. WILLIAMS, L i f e . Sci. 16 375-384 (1975). J~AXELROD, J. Biol. Chem. 237 1657-1660 (1962). R.W. FULLER, S. HEMRICK-LUECKE, R.E. LOOMEY, J. HORNG, R.R. RUFFALO and B.B. MOLLOY, Biochem. Pharmacol. 30 1345-1352 (1981). R.B. MARTIN, J. Phys. Chem. 75 2657-2661 (1971). R.G. JOHNSON AND A. SCARPA, ~ Biol. Chem. 251 2189-2191 (1976). P.R. SHARP AND E.P. RICHARDS, Biochim. Biophys. Acta 497 14-28 (1977). B. UVN~S and C.-H. ~BORG, Acta Physiol. S c a n t 109 345-354 (1980).