Journal
OJ
N w o c h u m i s r r y . 1976 Vol. 26, pp 1159-1165. Pergamon Press. Printed In Great Britam.
REGIONAL AND SUBCELLULAR DISTRIBUTION OF PROTEIN CARBOXYMETHYLASE IN BRAIN AND OTHER TISSUES E. J. DILIBERTO, JR.' and J. AXELROD Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, M D 20014, U.S.A (Receiued 8 August 1975. Accepted 20 October 1975)
Abstract-Protein carboxymethylase, an enzyme that transfcrs the methyl group of S-adenosyl-L-methionine to carboxyl groups of proteins and endogenous acceptor proteins were examined in nerve and endocrine tissues. The highest protein carboxymethylase activity was found in the brain, followed by the testis, pituitary and heart. On the other hand, the tissue with the highest level of endogenous substrate(s) was the pituitary. The nearly identical specific activity ratio for two different protein substrates in all tissues examined, suggests that one enzyme is responsible for carboxymethylase activity in different tissues. The subcellular distribution of the enzyme in brain showed a high concentration in the soluble fraction, presumably representative of the enzyme in the cytosol of cell bodies. Considerable enzyme activity was also found in brain synaptosomes which was increased by osmotic lysis. Protein carboxymethylase was shown to accumulate proximally to a ligation of the rat sciatic nerve. A possible physiological role for protein carboxymethylase in neuronal function is discussed.
THEENZYME, protein carboxymethylase (S-adenosylmethionine: protein-carboxyl methyltransferase, EC 2.1.1.24; protein methylase 11) methylates (esterifies) free carboxyl groups of proteins (KIM& PAIK, 1971; Liss et a!., 1969; MORIN& LISS; 1973). It was initially described as the 'methanol-forming' enzyme (AXELROD & DALY,1965), but the identity of protein carboxymethylase and the 'methanol-forming' enzyme as JR. the same enzyme has been established (DILIBERTO, & AXELROD,1974; MORIN& Lrss, 1973). A study of the substrate specificity of protein carboxymethylase showed that all anterior pituitary hormones were effective methyl acceptors while the posterior pituitary hormones, oxytocin and vasopressin did not serve as substrates (DILIBERTO, JR. & AXELROD, 1974). Recently, investigation of other biologically active proteins showed that the posterior pituitary binding proteins, neurophysins, are also active substrates for protein carboxymethylase (AXELROD & DILIRERTO, JR., 1975; EDGAR& HOPE. 1974). The enzyme was shown to have a limiting K , with respect to S-adenosyl-L-methionine one order of magnitude lower than the apparent K , values of methyltransferases of catecholamine and indolamine metabolism (DILIBERTO, JR. & AXELROD, 1974). As a result of these findings, it was suggested that the enzyme may function in the pituitary by inactivation of protein hormones. In an effort to investigate the physiological significance of protein carboxymethylase, the enzyme activity as well as the presence of endogenous substrates 'Research associate in the Pharmacology-Toxicology Program, National Institute of General Medical Sciences. Abbreviation used: LH, luteinizing hormone (ovine).
was measured in neural and other tissues. Brain regional and subcellular distribution studies were performed to assess the possible role that protein carboxymethylase may have in neuronal function. A preliminary cqmmunication of this work has been published (DILIBERTO, JR. & AXELROD,1975). MATERIALS AND METHODS Matcrials. S-adenosyl-~-~methyl-~H]methionine, 8.54 Ci/mmol and S-adenosyl-~-[methyl-'~C]methionine, 52.3 niCiimmol, were purchascd from New England Nuclear Corp. Luteinizing hormone (LH) (ovine) and thyroidstimulating hormone (bovine) were supplied by NIAMD. Adrenocorticotropic hormone (porcine) and ovalbumin (5 x crystallized) were obtained from Sigma Chemical CO. Sephadex G-100 and QAE-sephadex A-50 were from Pharmacia Fine Chemicals, Inc. Enzyme assay. Protein carboxymethylase activity was assayed by a modification of the method previously described (DILIBERTO, JR. & AXELROD,1974). In this procedure the methyl group from S-adenosyl-~-methionine, which is enzymically transferred to carboxyl groups of proteins, is hydrolysed in alkaline solution to methanol and extracted into an organic solvent. The incubation mixture contained 10 111 of 0.5 M-phosphate buffer (pH 6.0) with or without protein substrate, 10 mg/ml, 50.67 pmol of S-adenosyl-~-[methyl-~H]methionine, 8.54 Ci/mmol, 1 p1 of 0.01 N-NaOH to neutralize the H,S04 in the S-adenosyl-L-methionine solution, and 10 p1 of tissue homogenate in a final volume of 30 pl. The reaction was carried out at 37°C for 15 min and ended by the addition of I50 p1 of 0.5 M-borate buffer (pH 10.0) containing methanol, 0.7% (VjV) and 3 ml of 2:3 mixture of 3-methyl-I-butanol and toluene. After the sample was allowed to stand at ambient temperature for 15 min it was agitated with a vortex (Scientific Industries, Inc.) and centrifuged. Two I-ml aliquots were transferred to two vials; to one, 10 ml of BRAY'S
1159
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E. J. DILIBEKTO, JR. and J. AXELKOD
solution (1960) was added and the sample counted by liquid scintillation spectrometry (Beckman Ls-250). The second aliquot was evaporated for 1 h at 90°C in a chromatography Oven and radioactivity was determined. The difference in radioactivity was taken as a measure of methanol formation. The blank consisted of the tissue homogenate heated to 100°C for 5 min prior to incubation. Since luteinizing hormone (LH) was found to be the best substrate (DILIBERTO, JR. & AXELROD, 1974) a near saturating concentration (approx 90%) of LH was used as the substrate for most experiments. Endogenous acceptor protein activity was estimated by incubating in the absence of added protein. A unit of activity equals I pmol of [3H]methyl transferred per 15 min. Preparation of tissuefor assay. Various tissues from male Sprague-Dawley (Zivic-Miller Laboratories, Allison Park, Pa.) rats were homogenized in 10 vol of cold water with an all glass homogenizer. Enzyme purification. Protein carboxymethylase was purified by a modification of the procedure previously de1974). The most active scribed (DILIBERTO, JR. & AXELROD, fractions from the Sephadex G-100 chromatography (92 ml) were pooled, Iyophilized and dissolved in 15 ml of 5 mM-ethanolamine buffer containing 5 mM-EDTA and 2.4 m~-2-mercaptoethanolat pH 9.7 and dialyzed against the same buffer overnight with two buffer changes. The dialyzed preparation was applied to a column (2.5 x 33 cm) of QAE-Sephadex A-50 that had been equilibrated with the 5 KIM-ethanolamine buffer, pH 9.7. The column was eluted with 180 ml of the ethanolamine buffer followed by a linear concentration gradient to 0.325 N-NaCI in the ethanolamine buffer. The most active fractions were pooled, concentrated, dialyzed and stored at -20°C. Under these conditions there was no loss in activity for several months. The enzyme was purified about 2000-fold with a specific activity of 8.69 nmol of [14C]methyl transferred/min per mg protein. Stability of protein-methyl esters. T o test for the stability of enzymically produced protein-methyl esters as a function of pH, the following procedure was used: An incubation mixture containing 300 pl 0.5 wphosphate buffer, pH 6.0, 12 mg ovalbumin (as substrate), 23.1 nmol of S-adenosyl-~-[methyl-'~C]methionine,52.3 mCi/mmol and purified protein carboxymethylase in a final volume of 1.60 ml was incubated for 30 min at 37°C. The reaction was ended by the addition of 20 pl of 4 N-HCI and allowed to stand for 5 min at room temperature to inactivate the enzyme. The mixture containing [14C]methyl-ovalbumin was neutralized with 20 ,d of 4 N-NaOH; 100 pl of the mixture was added to 1.0 ml of phosphate buffer at various pH's and incubated for 1 h at 37°C. The reaction was stopped by the addition of 1.0 ml 30% trichloroacetic acid (TCA). The TCA precipitates were collected on membrane filters (Millipore Corp.), washed with 10% TCA and counted, using 10 ml Bra)'s solution. A portion of the sample before the second incubation was added to buffer at pH 6.0 and precipitated immediately with TCA. Subcellular fractionation. Whole rat brain, excluding the cerebellum, was homogenized in 10 vol (w/v) of 0.3 M-SUCrose and subjected to subcellular fraction by the method (1965). P1 was obtained by centrifugation of WHITTAKEK at 1000 y for 10 min; the pellet was washed twice by resuspending in 0.3 M-sucrose and centrifuging again at 1000 g for 10 min to obtain P,"(Fig. 3). The supernatant fractions (S, + S1' + S,") were combined and centrifuged a1 17.500 g for 30 rnin to obtain the crude mitochondrial
pellet (P2).The supernatant fluid from this fraction (SZ) was decanted and centrifuged at 100,000 g for 60 min (Beckman, Type 30 rotor) to obtain the rnicrosomal pellet (P3)and soluble supernatant (&). The crude mitochondrial pellet was either gently resuspended in 0.3 M-sucrose and layered on a discontinuous sucrose gradient of equal volumes of 0.8 and 1.2 M-sucrose, which was centrifuged at 53,000 g for 2 h (Beckman, SW-27 rotor), or resuspended in water to obtain a lyzed Pz. The 0.8-1.2 M-sucrose interface containing the synaptosomes was diluted by adding 0.3 M-sucrose dropwise and centrifuged at 100,000 g for 60 min. The synaptosomal pellet was resuspended in either 0.3 M-sucrose or water to obtain unlyzed and lyzed synaptosomes, respectively. Axonal transport. Under light ether anaesthesia ligations of sciatic nerve (male Sprague-Dawley rats) were performed with 4-0 surgical silk. Rats were killed by decapitation at 80 h after ligation and a 1 cm segment proximal to the ligation and a 1 crn segment from the corresponding portion of the unligated contralateral sciatic nerve were removed for enzyme assay.
RESULTS
Stability of protein methyl esters. To aid in our investigation of the role of 'protein carboxymethylase, the stability of enzymically produced protein-methyl ester of ovalbumin was examined as a function of p H (Fig. 1). The product was more stable at a n acid p H and the rate of spontaneous hydrolysis increases with increasing pH. After incubation a t physiological p H a n d temperature for 1 h, less than 25% of the methyl ester remained. indicating that the methylated proteins are labile a n d probably undergo rapid hydrolysis in viuo. Distribution of protein carboxymethylase in the central and peripheral nervous system. The distribution of protein carboxymethylase in various brain regions a n d peripheral nerve a n d ganglion was determined (Table 1). Tissues were assayed in the presence of added LH (exogenous) as a measure of total enzyme activity o r in the absence of LH (endogenous) which is a measure of the endogenous acceptor proteins to undergo enzymic carboxymethylation. T h e enzyme activity was similar in all brain regions and was slightly lower in the cerebellum. T h e sciatic nerve and the superior cervical ganglion has a lower protein carboxymethylase activity (Table I). In the absence of exogenous substrate only 10% of the activity was observed, indicating that, unlike thc pituitary (vide infra), the brain a n d peripheral nerves have less endogenous substrate(s) for enzymic methylation. Thus, in the presence of LH most brain regions have twice the specific activity of the pituitary, but far less endogenous substrate(s) (Tables 1 and 2). Protein carboxymethylase in carious rat tissucs. Protein carboxymethylase activity was determined for a variety of rat tissues in the presence of several protein substrates or the absence of added substrate (Table 2). T w o specific activity ratios were calculated: (1) the ratio of protein carboxymethylase activity with LH
Protein carboxymethylase in brain and other tissues
I
I
6.0
7.0
I
8.0
PH
FIG.1. Effect of pH on the stability of a methylated pro-
tein. Methylated protein was formed by incubating ovalbumin with purified protein carboxymethylase and S-adenosyl-L-[methyl-'4C]methionine in phosphate buffer, pH 6.0, for 30 min at 37°C. After inactivation of protein carboxymethylase by acid treatment, the enzymically produced [14C]methyl-protein was incubated a second time in phosphate buffer at various pH's for I h at 37°C and stopped by the addition of trichloroacetic acid. The precipitates were collected on membrane filters, washed with 10% trichloracetic acid and counted. The amount of ['4C)methylprotein formed in the first reaction was obtained from samples not incubated in the second reaction. The fraction of [14C]methyl-proteinremaining is the ratio of the counts after incubation at the various pHs to the counts of ['4C]methyl-protein formed in the first reaction. TABLE 1.
1161
as the protein substrate to the activity with ovalbumin as substrate and (2) the ratio of activity in the presence and absence of added LH. The former ratio was used to determine whether protein carboxymethylase in various tissues was a single enzyme; the later ratio was used to estimate the level of endogenous substrate(s) present in a given tissue; the lower the ratio the greater the amount of endogenous substrate(s). The highest enzyme activity of any tissue examined was found in the brain (Tables 1-3); the mammary glands had the lowest enzyme activity. The specific activity ratio of LH to ovalbumin was nearly identical for all tissue, suggesting that protein carboxymethylase in different tissues is the same enzyme. The pituitary had a lower ratio due to the large amount of endogenous substrates which are more active than ovalbumin. The results (Tables 2 and 3) show that the pituitary has the greatest amount of endogenous protein substrate@) and the pancreas, mammary gland and testis have the least. Some tissues, such as the thyroid and seminal vesicle, have a substantial amount or endogenous substrate(s). A comparison of protein carboxymethylase activity in various endocrine tissues is shown in Table 3. The activity of the posterior lobe of the pituitary was found to be twice that of the anterior lobe while both lobes appeared to have a large amount of endogenous substrate(s). Of the endocrine tissues, the testis had the highest specific activity and the pineal the lowest, with the other tissues intermediate. Sex differences of protein carboxymethylase actioity. Protein carboxymethylase activity in male and female pituitaries and hypothalami was studied in the presence or absence of added LH (Fig. 2). In general, the specific activity of male tissues was significantly
PROTEIN CARBOXYMETHYLASE ACTIVITY IN THE CENTKAL AND PERIPHERAL NERVOUS SYSTEM
Specific Activity (units/mg t i s s u e )
Exogenous
Endogenous
Hypothalamus ( 5 )
15.33 f 0.31
1 . 6 0 i 0.06
Striatum (5)
16.87 ? 0 . 2 0
1.66 f 0 . 0 2
Midbrain ( 5 )
14.38
0.22
1.43 i 0.05
Hippocampus ( 5 )
15.44 f 0.23
1.83 i 0.05
Cerebellum (5)
1 1 . 5 8 f. 0 . 2 8
1.224
Brain s t e m ( 5 )
13.81 i 0.20
1 . 3 1 k 0.08
C o r t e x (5)
15.03 f 0.51
2.25*
?
S c i a t i c nerve (4)
2 . 6 2 f. 0 . 0 8
S i p e r i o r c e r v i c a l g a n g l i o n (9)
5.09
*
0.23 t 0.01
0.24
P r o t e i n c a r b o x y m e t h y l a s e a c t i v i t y was d e t e r m i n e d i n t h e p r e s e n c e ( e x o g e n o u s ) a n d a b s e n c e ( e n d o g e n o u s ) o f LH, 3 . 3 mg/ml o f i n c u b a t i o n m i x t u r e . t h e number o f a n i m a l s from w h i c h t i s s u e s were t a k e n for a s s a y .
* T i s s u e from o n l y one a n i m a l was a s s a y e d .
Numbers i n p a r e n t h e s e s a r e V a l u e s a r e means
*
S.E.M.
E. J. DILIBERTO, JR. and J. AXELROD
1162
TABLE 2.
CoMPARIWN OF THE CARBOXYMETHYLASE OF DIFFERENT PROTEIN SUBSTRATES BY VARIOUS RAT TISSUES Specific A c t i v i t y (units/ng t i s s u e ) Protein a h s t r a t e s
Specific Activity Ratios
LH
Ovalbumin
Endogenous
Others+
P i t u i t a r y (4) Spleen(4) Adrenal(4)
8 . 0 5 f 0.07
5.35 f 0 . 0 2 2.99 f 0 . 1 0 1.78 f 0.05
4.04 t 0.11 0.38 f 0 . 0 4 0.32 f 0.01
7.69
Lung(4) Thyroid(4)
4.88 3.66 2.53 2.62 3.68 4.96 3i43 7’.41
2.16 1.57 0.95 1.22 1.66 2.14 1;45 3.20 1.17
0.60 f 0.11 0.89 f 0 . 1 1 0.29 2 0.10 0 . 2 3 t 0.01 0.28 f 0 . 0 1 0 . 5 0 t 0.01 o i i 4 t O;o4 0.40 f 0 . 0 2
Kidney (4) Vas d e f e r e n n ( 5 ) Seminal v e s i c l e ( 5) P r o s t a t e (5) Uterus(5) Mammary g l a n d s ( 5 )
5.62 t 0 . 1 4 4.43 f 0.06 t 0.18 t 0.31
t 0.08
t 0.08 t 0.19 0.21
*
oi04
t i 0.21 3.01 t 0 . 0 7
0.14 0.11 t 0.10
f f
f 0.06
t 0.07 0.15
*
f
0;oi
i 0.14 t 0.04
7.72 f 0 . 2 4 2.75 f 0.35 2 . 6 3 t 0.09 3.06 f 0.11 0.86 t 0 . 1 2
f
LWOvalbumin
O.Ol(TSH)
3.89 t O.O3(ACTH) 3.95 f 0.03(TSH) 2.64 f 0.08(TSH)
0.26 t 0.03 0.81 f 0 . 1 4 0 . N t 0.03
LWEndogenous 2.0 14.8 13.8
1.5
1.9 2.5 2.3 2.3 2.7 2.2 2.2 2.3 2.4 2.3 2.6
8.1 4.1 8.7 11.4 14.2 9.9
24.5 18.5 11.6
9.5 5.5 10.1 11.3 21.5
0.26 t 0.03
0.27 f 0 . 0 2 0.04 f 0.01
S p e c i f i c a c t i v i t y of p r o t e i n c e r h o x y n e t h y l a s e was e x a n i n e d i n t h e p r e s e n c e o r a b s e n c e (endogenous) of p r o t e i n s u b s t r a t e s , 3.3 ng/ml of i n c u b a t i o n m i x t u r e . Numbers i n p a r e n t h e s e s a r e t h e number of a n i m a l s from which t i s s u e s were taken f o r assay. Values a r e means f S.E.Y. * P S I , t h y r o i d - s t i m u l a t i n g hornone; ACTH, a d r e n o c o r t i c o t r o p i c hornone.
greater than of female. There was a wider standard error for the female pituitaries than the male when assayed in the absence of added LH, possibly reflecting a variation in the levels of endogenous substrate(s) during the estrus cycle. The range in activity for females was 1 . U . 2 0 units/mg tissue, while for the male it was 4.60-4.94 units/mg tissue. Subcellular distribution of protein carboxymethylase in rat brain. A subcellular localization of the enzyme was performed in rat brain (Fig. 3). The greatest proportion of the enzyme appears in the 1,OOO g supernatant fraction, i.e. 95.1% present in the combined S, fractions; similarly, S2 and S3 contain the major portion of the enzyme. The specific activity progressively increased from the S1 to S3 fraction. The large con-
tent of the enzyme in the supernatant fraction reflects its high localization in the cytosol of neurons or glial cells. Of special interest was the finding that a large amount of enzyme activity is present in the P2 fraction which was increased 3-fold by osmotic lysis (Fig. 3). Fractionation of the P2 fraction on a discontinuous sucrose gradient produced a fraction of synaptosomes which, after dilution and centrifugation at 100,OOO g for 60 min, was resuspended in either 0.3 M-sucrose or HzO. Osmotic lysis of the synaptosomes caused an increase in total and specific activity of greater than 5-fold. Since the supernatant fraction after centrifugation of the synaptosomes had no enzyme activity, no lysis was obtained by dilution of the synaptosomes from the sucrose gradient. The PI”
TABLE 3. PROTEIN CARLKIXYMETHYLASE
ACTIVITY IN ENOOCRINE TISSUES
S p e c i f i c a c t i v i t y (units/mg t i s s u e )
Specific a c t i v i t y r a t i o Exogenous/Endogenous
Exogenous
Endogenous
P i t u i t a r y g l a n d (14)
8.85 f 0 . 0 7
4.73 f 0.04
1.9
P o s t e r i o r l o b e (5)
1 1 . 4 4 i 0.46
*
0.22
2.1
3.02 ? 0 . 1 5
2.1
A n t e r i o r l o b e (5)
6.37
+_
0.18
5.39
P i n e a l (4)
1.82 f 0.11
Adrenal (4)
4.43
f
0.06
0.32 f 0.01
13.8
Thyroid (4)
3.66
?
0.31
0.89
0.11
4.1
P a n c r e a s (4)
3.43
f
0.04
0.14 f 0.04
24.5
1 2 . 3 2 f 0.25
0 . 3 5 ?r 0 . 0 1
35.2
3 . 2 5 ? 0.07
0.22 2 0.02
14.8
Testis (5) O v a r i e s (5)
f
P r o t e i n c a r b o x y m e t h y l a s e a c t i v i t y was d e t e r m i n e d i n t h e p r e s e n c e (exogenous) and a b s e n c e ( e n d o g e n o u s ) of LH, 3.3 mg/ml of i n c u b a t i o n m i x t u r e .
Numbers i n p a r e n t h e s e s
a r e t h e number o f a n i m a l s from which t i s s u e s w e r e t a k e n f o r a s s a y .
*
S.E.M.
V a l u e s a r e means
Protein carboxymethylase in brain and othcr tissues PROTEIN CARBOWMETHYLASEACTIVITY IN MALE AND FEMALE RATS Hypothalamus
(5)
0
Male Female
8 Pituitary
Is
6 .$
114)
E E“ 8.0 -
jg 8’
C ._
d
a 2
4.0
-
I
L
LH Present13.3 mg/ml)
- _
+ +
_ _
+ +
P
OJ
N w o c h u m i s r r y . 1976 Vol. 26, pp 1159-1165. Pergamon Press. Printed In Great Britam.
REGIONAL AND SUBCELLULAR DISTRIBUTION OF PROTEIN CARBOXYMETHYLASE IN BRAIN AND OTHER TISSUES E. J. DILIBERTO, JR.' and J. AXELROD Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, M D 20014, U.S.A (Receiued 8 August 1975. Accepted 20 October 1975)
Abstract-Protein carboxymethylase, an enzyme that transfcrs the methyl group of S-adenosyl-L-methionine to carboxyl groups of proteins and endogenous acceptor proteins were examined in nerve and endocrine tissues. The highest protein carboxymethylase activity was found in the brain, followed by the testis, pituitary and heart. On the other hand, the tissue with the highest level of endogenous substrate(s) was the pituitary. The nearly identical specific activity ratio for two different protein substrates in all tissues examined, suggests that one enzyme is responsible for carboxymethylase activity in different tissues. The subcellular distribution of the enzyme in brain showed a high concentration in the soluble fraction, presumably representative of the enzyme in the cytosol of cell bodies. Considerable enzyme activity was also found in brain synaptosomes which was increased by osmotic lysis. Protein carboxymethylase was shown to accumulate proximally to a ligation of the rat sciatic nerve. A possible physiological role for protein carboxymethylase in neuronal function is discussed.
THEENZYME, protein carboxymethylase (S-adenosylmethionine: protein-carboxyl methyltransferase, EC 2.1.1.24; protein methylase 11) methylates (esterifies) free carboxyl groups of proteins (KIM& PAIK, 1971; Liss et a!., 1969; MORIN& LISS; 1973). It was initially described as the 'methanol-forming' enzyme (AXELROD & DALY,1965), but the identity of protein carboxymethylase and the 'methanol-forming' enzyme as JR. the same enzyme has been established (DILIBERTO, & AXELROD,1974; MORIN& Lrss, 1973). A study of the substrate specificity of protein carboxymethylase showed that all anterior pituitary hormones were effective methyl acceptors while the posterior pituitary hormones, oxytocin and vasopressin did not serve as substrates (DILIBERTO, JR. & AXELROD, 1974). Recently, investigation of other biologically active proteins showed that the posterior pituitary binding proteins, neurophysins, are also active substrates for protein carboxymethylase (AXELROD & DILIRERTO, JR., 1975; EDGAR& HOPE. 1974). The enzyme was shown to have a limiting K , with respect to S-adenosyl-L-methionine one order of magnitude lower than the apparent K , values of methyltransferases of catecholamine and indolamine metabolism (DILIBERTO, JR. & AXELROD, 1974). As a result of these findings, it was suggested that the enzyme may function in the pituitary by inactivation of protein hormones. In an effort to investigate the physiological significance of protein carboxymethylase, the enzyme activity as well as the presence of endogenous substrates 'Research associate in the Pharmacology-Toxicology Program, National Institute of General Medical Sciences. Abbreviation used: LH, luteinizing hormone (ovine).
was measured in neural and other tissues. Brain regional and subcellular distribution studies were performed to assess the possible role that protein carboxymethylase may have in neuronal function. A preliminary cqmmunication of this work has been published (DILIBERTO, JR. & AXELROD,1975). MATERIALS AND METHODS Matcrials. S-adenosyl-~-~methyl-~H]methionine, 8.54 Ci/mmol and S-adenosyl-~-[methyl-'~C]methionine, 52.3 niCiimmol, were purchascd from New England Nuclear Corp. Luteinizing hormone (LH) (ovine) and thyroidstimulating hormone (bovine) were supplied by NIAMD. Adrenocorticotropic hormone (porcine) and ovalbumin (5 x crystallized) were obtained from Sigma Chemical CO. Sephadex G-100 and QAE-sephadex A-50 were from Pharmacia Fine Chemicals, Inc. Enzyme assay. Protein carboxymethylase activity was assayed by a modification of the method previously described (DILIBERTO, JR. & AXELROD,1974). In this procedure the methyl group from S-adenosyl-~-methionine, which is enzymically transferred to carboxyl groups of proteins, is hydrolysed in alkaline solution to methanol and extracted into an organic solvent. The incubation mixture contained 10 111 of 0.5 M-phosphate buffer (pH 6.0) with or without protein substrate, 10 mg/ml, 50.67 pmol of S-adenosyl-~-[methyl-~H]methionine, 8.54 Ci/mmol, 1 p1 of 0.01 N-NaOH to neutralize the H,S04 in the S-adenosyl-L-methionine solution, and 10 p1 of tissue homogenate in a final volume of 30 pl. The reaction was carried out at 37°C for 15 min and ended by the addition of I50 p1 of 0.5 M-borate buffer (pH 10.0) containing methanol, 0.7% (VjV) and 3 ml of 2:3 mixture of 3-methyl-I-butanol and toluene. After the sample was allowed to stand at ambient temperature for 15 min it was agitated with a vortex (Scientific Industries, Inc.) and centrifuged. Two I-ml aliquots were transferred to two vials; to one, 10 ml of BRAY'S
1159
1.160
E. J. DILIBEKTO, JR. and J. AXELKOD
solution (1960) was added and the sample counted by liquid scintillation spectrometry (Beckman Ls-250). The second aliquot was evaporated for 1 h at 90°C in a chromatography Oven and radioactivity was determined. The difference in radioactivity was taken as a measure of methanol formation. The blank consisted of the tissue homogenate heated to 100°C for 5 min prior to incubation. Since luteinizing hormone (LH) was found to be the best substrate (DILIBERTO, JR. & AXELROD, 1974) a near saturating concentration (approx 90%) of LH was used as the substrate for most experiments. Endogenous acceptor protein activity was estimated by incubating in the absence of added protein. A unit of activity equals I pmol of [3H]methyl transferred per 15 min. Preparation of tissuefor assay. Various tissues from male Sprague-Dawley (Zivic-Miller Laboratories, Allison Park, Pa.) rats were homogenized in 10 vol of cold water with an all glass homogenizer. Enzyme purification. Protein carboxymethylase was purified by a modification of the procedure previously de1974). The most active scribed (DILIBERTO, JR. & AXELROD, fractions from the Sephadex G-100 chromatography (92 ml) were pooled, Iyophilized and dissolved in 15 ml of 5 mM-ethanolamine buffer containing 5 mM-EDTA and 2.4 m~-2-mercaptoethanolat pH 9.7 and dialyzed against the same buffer overnight with two buffer changes. The dialyzed preparation was applied to a column (2.5 x 33 cm) of QAE-Sephadex A-50 that had been equilibrated with the 5 KIM-ethanolamine buffer, pH 9.7. The column was eluted with 180 ml of the ethanolamine buffer followed by a linear concentration gradient to 0.325 N-NaCI in the ethanolamine buffer. The most active fractions were pooled, concentrated, dialyzed and stored at -20°C. Under these conditions there was no loss in activity for several months. The enzyme was purified about 2000-fold with a specific activity of 8.69 nmol of [14C]methyl transferred/min per mg protein. Stability of protein-methyl esters. T o test for the stability of enzymically produced protein-methyl esters as a function of pH, the following procedure was used: An incubation mixture containing 300 pl 0.5 wphosphate buffer, pH 6.0, 12 mg ovalbumin (as substrate), 23.1 nmol of S-adenosyl-~-[methyl-'~C]methionine,52.3 mCi/mmol and purified protein carboxymethylase in a final volume of 1.60 ml was incubated for 30 min at 37°C. The reaction was ended by the addition of 20 pl of 4 N-HCI and allowed to stand for 5 min at room temperature to inactivate the enzyme. The mixture containing [14C]methyl-ovalbumin was neutralized with 20 ,d of 4 N-NaOH; 100 pl of the mixture was added to 1.0 ml of phosphate buffer at various pH's and incubated for 1 h at 37°C. The reaction was stopped by the addition of 1.0 ml 30% trichloroacetic acid (TCA). The TCA precipitates were collected on membrane filters (Millipore Corp.), washed with 10% TCA and counted, using 10 ml Bra)'s solution. A portion of the sample before the second incubation was added to buffer at pH 6.0 and precipitated immediately with TCA. Subcellular fractionation. Whole rat brain, excluding the cerebellum, was homogenized in 10 vol (w/v) of 0.3 M-SUCrose and subjected to subcellular fraction by the method (1965). P1 was obtained by centrifugation of WHITTAKEK at 1000 y for 10 min; the pellet was washed twice by resuspending in 0.3 M-sucrose and centrifuging again at 1000 g for 10 min to obtain P,"(Fig. 3). The supernatant fractions (S, + S1' + S,") were combined and centrifuged a1 17.500 g for 30 rnin to obtain the crude mitochondrial
pellet (P2).The supernatant fluid from this fraction (SZ) was decanted and centrifuged at 100,000 g for 60 min (Beckman, Type 30 rotor) to obtain the rnicrosomal pellet (P3)and soluble supernatant (&). The crude mitochondrial pellet was either gently resuspended in 0.3 M-sucrose and layered on a discontinuous sucrose gradient of equal volumes of 0.8 and 1.2 M-sucrose, which was centrifuged at 53,000 g for 2 h (Beckman, SW-27 rotor), or resuspended in water to obtain a lyzed Pz. The 0.8-1.2 M-sucrose interface containing the synaptosomes was diluted by adding 0.3 M-sucrose dropwise and centrifuged at 100,000 g for 60 min. The synaptosomal pellet was resuspended in either 0.3 M-sucrose or water to obtain unlyzed and lyzed synaptosomes, respectively. Axonal transport. Under light ether anaesthesia ligations of sciatic nerve (male Sprague-Dawley rats) were performed with 4-0 surgical silk. Rats were killed by decapitation at 80 h after ligation and a 1 cm segment proximal to the ligation and a 1 crn segment from the corresponding portion of the unligated contralateral sciatic nerve were removed for enzyme assay.
RESULTS
Stability of protein methyl esters. To aid in our investigation of the role of 'protein carboxymethylase, the stability of enzymically produced protein-methyl ester of ovalbumin was examined as a function of p H (Fig. 1). The product was more stable at a n acid p H and the rate of spontaneous hydrolysis increases with increasing pH. After incubation a t physiological p H a n d temperature for 1 h, less than 25% of the methyl ester remained. indicating that the methylated proteins are labile a n d probably undergo rapid hydrolysis in viuo. Distribution of protein carboxymethylase in the central and peripheral nervous system. The distribution of protein carboxymethylase in various brain regions a n d peripheral nerve a n d ganglion was determined (Table 1). Tissues were assayed in the presence of added LH (exogenous) as a measure of total enzyme activity o r in the absence of LH (endogenous) which is a measure of the endogenous acceptor proteins to undergo enzymic carboxymethylation. T h e enzyme activity was similar in all brain regions and was slightly lower in the cerebellum. T h e sciatic nerve and the superior cervical ganglion has a lower protein carboxymethylase activity (Table I). In the absence of exogenous substrate only 10% of the activity was observed, indicating that, unlike thc pituitary (vide infra), the brain a n d peripheral nerves have less endogenous substrate(s) for enzymic methylation. Thus, in the presence of LH most brain regions have twice the specific activity of the pituitary, but far less endogenous substrate(s) (Tables 1 and 2). Protein carboxymethylase in carious rat tissucs. Protein carboxymethylase activity was determined for a variety of rat tissues in the presence of several protein substrates or the absence of added substrate (Table 2). T w o specific activity ratios were calculated: (1) the ratio of protein carboxymethylase activity with LH
Protein carboxymethylase in brain and other tissues
I
I
6.0
7.0
I
8.0
PH
FIG.1. Effect of pH on the stability of a methylated pro-
tein. Methylated protein was formed by incubating ovalbumin with purified protein carboxymethylase and S-adenosyl-L-[methyl-'4C]methionine in phosphate buffer, pH 6.0, for 30 min at 37°C. After inactivation of protein carboxymethylase by acid treatment, the enzymically produced [14C]methyl-protein was incubated a second time in phosphate buffer at various pH's for I h at 37°C and stopped by the addition of trichloroacetic acid. The precipitates were collected on membrane filters, washed with 10% trichloracetic acid and counted. The amount of ['4C)methylprotein formed in the first reaction was obtained from samples not incubated in the second reaction. The fraction of [14C]methyl-proteinremaining is the ratio of the counts after incubation at the various pHs to the counts of ['4C]methyl-protein formed in the first reaction. TABLE 1.
1161
as the protein substrate to the activity with ovalbumin as substrate and (2) the ratio of activity in the presence and absence of added LH. The former ratio was used to determine whether protein carboxymethylase in various tissues was a single enzyme; the later ratio was used to estimate the level of endogenous substrate(s) present in a given tissue; the lower the ratio the greater the amount of endogenous substrate(s). The highest enzyme activity of any tissue examined was found in the brain (Tables 1-3); the mammary glands had the lowest enzyme activity. The specific activity ratio of LH to ovalbumin was nearly identical for all tissue, suggesting that protein carboxymethylase in different tissues is the same enzyme. The pituitary had a lower ratio due to the large amount of endogenous substrates which are more active than ovalbumin. The results (Tables 2 and 3) show that the pituitary has the greatest amount of endogenous protein substrate@) and the pancreas, mammary gland and testis have the least. Some tissues, such as the thyroid and seminal vesicle, have a substantial amount or endogenous substrate(s). A comparison of protein carboxymethylase activity in various endocrine tissues is shown in Table 3. The activity of the posterior lobe of the pituitary was found to be twice that of the anterior lobe while both lobes appeared to have a large amount of endogenous substrate(s). Of the endocrine tissues, the testis had the highest specific activity and the pineal the lowest, with the other tissues intermediate. Sex differences of protein carboxymethylase actioity. Protein carboxymethylase activity in male and female pituitaries and hypothalami was studied in the presence or absence of added LH (Fig. 2). In general, the specific activity of male tissues was significantly
PROTEIN CARBOXYMETHYLASE ACTIVITY IN THE CENTKAL AND PERIPHERAL NERVOUS SYSTEM
Specific Activity (units/mg t i s s u e )
Exogenous
Endogenous
Hypothalamus ( 5 )
15.33 f 0.31
1 . 6 0 i 0.06
Striatum (5)
16.87 ? 0 . 2 0
1.66 f 0 . 0 2
Midbrain ( 5 )
14.38
0.22
1.43 i 0.05
Hippocampus ( 5 )
15.44 f 0.23
1.83 i 0.05
Cerebellum (5)
1 1 . 5 8 f. 0 . 2 8
1.224
Brain s t e m ( 5 )
13.81 i 0.20
1 . 3 1 k 0.08
C o r t e x (5)
15.03 f 0.51
2.25*
?
S c i a t i c nerve (4)
2 . 6 2 f. 0 . 0 8
S i p e r i o r c e r v i c a l g a n g l i o n (9)
5.09
*
0.23 t 0.01
0.24
P r o t e i n c a r b o x y m e t h y l a s e a c t i v i t y was d e t e r m i n e d i n t h e p r e s e n c e ( e x o g e n o u s ) a n d a b s e n c e ( e n d o g e n o u s ) o f LH, 3 . 3 mg/ml o f i n c u b a t i o n m i x t u r e . t h e number o f a n i m a l s from w h i c h t i s s u e s were t a k e n for a s s a y .
* T i s s u e from o n l y one a n i m a l was a s s a y e d .
Numbers i n p a r e n t h e s e s a r e V a l u e s a r e means
*
S.E.M.
E. J. DILIBERTO, JR. and J. AXELROD
1162
TABLE 2.
CoMPARIWN OF THE CARBOXYMETHYLASE OF DIFFERENT PROTEIN SUBSTRATES BY VARIOUS RAT TISSUES Specific A c t i v i t y (units/ng t i s s u e ) Protein a h s t r a t e s
Specific Activity Ratios
LH
Ovalbumin
Endogenous
Others+
P i t u i t a r y (4) Spleen(4) Adrenal(4)
8 . 0 5 f 0.07
5.35 f 0 . 0 2 2.99 f 0 . 1 0 1.78 f 0.05
4.04 t 0.11 0.38 f 0 . 0 4 0.32 f 0.01
7.69
Lung(4) Thyroid(4)
4.88 3.66 2.53 2.62 3.68 4.96 3i43 7’.41
2.16 1.57 0.95 1.22 1.66 2.14 1;45 3.20 1.17
0.60 f 0.11 0.89 f 0 . 1 1 0.29 2 0.10 0 . 2 3 t 0.01 0.28 f 0 . 0 1 0 . 5 0 t 0.01 o i i 4 t O;o4 0.40 f 0 . 0 2
Kidney (4) Vas d e f e r e n n ( 5 ) Seminal v e s i c l e ( 5) P r o s t a t e (5) Uterus(5) Mammary g l a n d s ( 5 )
5.62 t 0 . 1 4 4.43 f 0.06 t 0.18 t 0.31
t 0.08
t 0.08 t 0.19 0.21
*
oi04
t i 0.21 3.01 t 0 . 0 7
0.14 0.11 t 0.10
f f
f 0.06
t 0.07 0.15
*
f
0;oi
i 0.14 t 0.04
7.72 f 0 . 2 4 2.75 f 0.35 2 . 6 3 t 0.09 3.06 f 0.11 0.86 t 0 . 1 2
f
LWOvalbumin
O.Ol(TSH)
3.89 t O.O3(ACTH) 3.95 f 0.03(TSH) 2.64 f 0.08(TSH)
0.26 t 0.03 0.81 f 0 . 1 4 0 . N t 0.03
LWEndogenous 2.0 14.8 13.8
1.5
1.9 2.5 2.3 2.3 2.7 2.2 2.2 2.3 2.4 2.3 2.6
8.1 4.1 8.7 11.4 14.2 9.9
24.5 18.5 11.6
9.5 5.5 10.1 11.3 21.5
0.26 t 0.03
0.27 f 0 . 0 2 0.04 f 0.01
S p e c i f i c a c t i v i t y of p r o t e i n c e r h o x y n e t h y l a s e was e x a n i n e d i n t h e p r e s e n c e o r a b s e n c e (endogenous) of p r o t e i n s u b s t r a t e s , 3.3 ng/ml of i n c u b a t i o n m i x t u r e . Numbers i n p a r e n t h e s e s a r e t h e number of a n i m a l s from which t i s s u e s were taken f o r assay. Values a r e means f S.E.Y. * P S I , t h y r o i d - s t i m u l a t i n g hornone; ACTH, a d r e n o c o r t i c o t r o p i c hornone.
greater than of female. There was a wider standard error for the female pituitaries than the male when assayed in the absence of added LH, possibly reflecting a variation in the levels of endogenous substrate(s) during the estrus cycle. The range in activity for females was 1 . U . 2 0 units/mg tissue, while for the male it was 4.60-4.94 units/mg tissue. Subcellular distribution of protein carboxymethylase in rat brain. A subcellular localization of the enzyme was performed in rat brain (Fig. 3). The greatest proportion of the enzyme appears in the 1,OOO g supernatant fraction, i.e. 95.1% present in the combined S, fractions; similarly, S2 and S3 contain the major portion of the enzyme. The specific activity progressively increased from the S1 to S3 fraction. The large con-
tent of the enzyme in the supernatant fraction reflects its high localization in the cytosol of neurons or glial cells. Of special interest was the finding that a large amount of enzyme activity is present in the P2 fraction which was increased 3-fold by osmotic lysis (Fig. 3). Fractionation of the P2 fraction on a discontinuous sucrose gradient produced a fraction of synaptosomes which, after dilution and centrifugation at 100,OOO g for 60 min, was resuspended in either 0.3 M-sucrose or HzO. Osmotic lysis of the synaptosomes caused an increase in total and specific activity of greater than 5-fold. Since the supernatant fraction after centrifugation of the synaptosomes had no enzyme activity, no lysis was obtained by dilution of the synaptosomes from the sucrose gradient. The PI”
TABLE 3. PROTEIN CARLKIXYMETHYLASE
ACTIVITY IN ENOOCRINE TISSUES
S p e c i f i c a c t i v i t y (units/mg t i s s u e )
Specific a c t i v i t y r a t i o Exogenous/Endogenous
Exogenous
Endogenous
P i t u i t a r y g l a n d (14)
8.85 f 0 . 0 7
4.73 f 0.04
1.9
P o s t e r i o r l o b e (5)
1 1 . 4 4 i 0.46
*
0.22
2.1
3.02 ? 0 . 1 5
2.1
A n t e r i o r l o b e (5)
6.37
+_
0.18
5.39
P i n e a l (4)
1.82 f 0.11
Adrenal (4)
4.43
f
0.06
0.32 f 0.01
13.8
Thyroid (4)
3.66
?
0.31
0.89
0.11
4.1
P a n c r e a s (4)
3.43
f
0.04
0.14 f 0.04
24.5
1 2 . 3 2 f 0.25
0 . 3 5 ?r 0 . 0 1
35.2
3 . 2 5 ? 0.07
0.22 2 0.02
14.8
Testis (5) O v a r i e s (5)
f
P r o t e i n c a r b o x y m e t h y l a s e a c t i v i t y was d e t e r m i n e d i n t h e p r e s e n c e (exogenous) and a b s e n c e ( e n d o g e n o u s ) of LH, 3.3 mg/ml of i n c u b a t i o n m i x t u r e .
Numbers i n p a r e n t h e s e s
a r e t h e number o f a n i m a l s from which t i s s u e s w e r e t a k e n f o r a s s a y .
*
S.E.M.
V a l u e s a r e means
Protein carboxymethylase in brain and othcr tissues PROTEIN CARBOWMETHYLASEACTIVITY IN MALE AND FEMALE RATS Hypothalamus
(5)
0
Male Female
8 Pituitary
Is
6 .$
114)
E E“ 8.0 -
jg 8’
C ._
d
a 2
4.0
-
I
L
LH Present13.3 mg/ml)
- _
+ +
_ _
+ +
P
