Enkephalin hydrolysis by mouse plasma in vitro

Life Sciences, Vol. 50, pp. 667-675 Printed in the USA

ENKEPHALIN Shinji

Pergamon Press

HYDROLYSIS

BY MOUSE PLASMA IN VITRO

Shibanoki 1, Susan B. Weinberger 2, Gery Schulteis 2, Koichi Ishikawa I, and Joe L. Martinez, Jr. 2

iDepartment of Pharmacology, Nihon University School of Medicine, Tokyo, Japan; 2Department of Psychology, university of California, Berkeley, CA (Received in final form January 3, 1992) Summary Hydrolysis of [Leu]- and [Met]enkephalin was determined in samples of pooled whole mouse plasma in vitro by using HPLC-ECD to measure accumulation of Tyrcontaining metabolites. More Tyr-Gly-Gly accumulated from [Met]enkephalin than from [Leu]enkephalin hydrolysis, and [Met]enkephalin's half-life in mouse plasma was approximately half that of [Leu]enkephalin. Comparisons of metabolite formation in the presence versus the absence of inhibitors with high selectivity for various peptidases demonstrated that a bestatinsensitive aminopeptidase, presumably aminopeptidase M, as well as enkephalinase and angiotensin converting enzyme, participate in the hydrolysis of enkephalin in mouse plasma. Peripheral administration of enkephalin and other opioid peptides leads to powerful effects on learning and memory in several mammalian species (see 1 and 2 for recent reviews). Recent studies in our laboratory suggest that understanding the enzymatic hydrolysis of enkephalin in plasma may be important to understanding some of its effects on conditioned behavior. For example, the overall rate of [Leu]enkephalin hydrolysis by rat plasma in vitro changes in association with training on a one-way active avoidance task. Both increases and decreases in hydrolysis rate were noted when blood samples collected from individual rats before and after the first avoidance training trial were compared (3). Studies in mice indicate that administration of [Leu]enkephalin metabolites containing at least an N-terminal Tyr-Gly sequence results in an impairment of one-way active avoidance conditioning similar to that produced by [Leu]enkephalin itself (4-6). In order to interpret the conditioning effects produced in mice by enkephalin and its Tyr-containing metabolites, it is necessary to determine which enyzmes normally participate in enkephalin hydrolysis in mouse plasma. Previous studies indicate

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Enkephalin Hydrolysis by Mouse Plasma

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that [Leu]enkephalin has a short (2-2.5 min) half-life in rat plasma in vitro (7,8) and an even shorter (less than 1 min) halflife in vivo when administered i.p. to rats (9). A m i n o p e p t i d a s e M and a n g i o t e n s i n converting enzyme appear to be the major peptidases that hydrolyze enkephalins in rat plasma (7,8). By contrast, [Leu]enkephalin is hydrolyzed more rapidly by chick plasma than by rat plasma in vitro (half-life 0.7-1 min in chick plasma), and a b e s t a t i n - s e n s i t i v e aminopeptidase, p r e s u m a b l y a m i n o p e p t i d a s e M, accounts for over 99% of the in vitro enkephalin hydrolysis by plasma collected from the chick (i0). In the present study we used high performance liquid c h r o m a t o g r a p h y with electrochemical detection (HPLC-ECD) to characterize the time course of, and the enzymes involved in, the hydrolysis of [Leu]- and [Met]enkephalin by mouse plasma i_nn vitro. Previous studies in our laboratory indicated that [Leu]enkephalin is rapidly taken up and hydrolyzed in vivo following its i.p. or s.c. administration to mice (Janak, Schulteis, and Martinez, submitted for publication). As detailed below, the results of the present study indicate that, as in rats and chicks, a b e s t a t i n - s e n s i t i v e aminopeptidase, p r e s u m a b l y aminopeptidase M, is the primary enzyme that hydrolyzes enkephalin in mouse plasma in vitro. However, hydrolysis is considerably slower in the mouse than it is in the rat or the chick, and dipeptidyl carboxypeptidases play a larger role in enkephalin hydrolysis by mouse plasma than they do in the hydrolysis of enkephalin by rat or chick plasma. Methods Animals. The subjects were male Swiss-Webster mice (Harlan Sprague-Dawley, Inc., Indianapolis, IN) aged 56-60 days on arrival. The animals were housed in groups of three under standard laboratory conditions, in accordance with NIH guidelines. All experimental procedures were approved in advance by the Institutional Animal Care and Use Committee at the U n i v e r s i t y of California at Berkeley. Experiments were completed within one week of the animal's arrival at our colony. Blood Collection. Animals were brought to the testing room at least one hour prior to sample collection. They were sacrificed by decapitation and trunk blood was collected into h e p a r i n i z e d m i c r o c e n t r i f u g e tubes. Plasma was separated by m i c r o c e n t r i f u g a t i o n and incubated at 37°C for at least I0 mins to permit hydrolysis of endogenous enkephalins. Aliquots of plasma from 5-6 mice were pooled for each assay. Plasma Hydrolysis of [Leu]- and rMet]enkephalin. Aliquots (115 ~i) of pooled plasma were combined with saline (i0 ~i) containing one or more of the following peptidase inhibitors: bestatin (prepared in saline to achieve a final concentration of 125-500 BM), puromycin (prepared in saline to achieve a final c o n c e n t r a t i o n of 500 or 5000 ~M), thiorphan (prepared in 5% N,Ndimethyl formamide in saline in achieve a final concentration of

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Enkephalin Hydrolysis by Mouse Plasma

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i00 ~M), and captopril (prepared in saline to achieve a final concentration of i00 ~M). Following addition of the appropriate inhibitors, the samples were incubated for i0 min at 37°C. Either [Leu]- or [Met]enkephalin (prepared in saline to achieve a final concentration of 64.8 ~M) then was added to the plasma samples, followed by an additional 2-20 min incubation period. The reaction was stopped by the addition of two volumes of trichloroacetic acid (5% v/v) containing an appropriate internal standard ([Leu]- or [Met]enkephalin, or Tyr-Ala, for substrate and metabolite assays, respectively). Following microcentrifugation, 50 ~i of the supernatant were injected into the HPLC system for analysis. Chromatoqraphy Conditions. Procedures for HPLC-ECD m e a s u r e m e n t of enkephalins and metabolites are detailed in Shibanoki et al. (ii). Briefly, the system is comprised of an ISCO (Lincoln, NE) Model 2350 ternary pump, an ISCO Spherisorb C18 column (4.6 x 250 mm), and an Eicom (Kyoto, Japan) carbon graphite electrochemical detector. The column was maintained at 25°C throughout the separation. For assay of [Leu]- and [Met]enkephalin, the mobile phase was comprised of 0.05 M sodium citrate~citric acid buffer (pH 6.3) containing 19% acetonitrile, the flow rate was 1.2 ml/min, and the applied voltage was set at 1050 mV versus the Ag/AgCl reference electrode. For assay of Tyr, Tyr-Gly, and Tyr-Gly-Gly, a 0.05 M sodium citrate/citric acid buffer (pH 6.3) mobile phase was used, the flow rate was 1.4 ml/min, and the applied voltage was set at 950 mY. Chemicals. Bestatin, puromycin, Tyr, Tyr-Gly, Tyr-Gly-Gly, and Tyr-Ala were purchased from Sigma (St. Louis, MO); thiorphan was purchased from Peninsula Labs (Belmont, CA); [Leu]- and [Met]enkephalin were purchased from Bachem (Torrance, CA); and captopril was donated by the Squibb Institute for Medical Research (Princeton, NJ). Data Analysis. Endogenous [Leu]- and [Met]enkephalin, Tyr-Gly, and Tyr-Gly-Gly levels were not detectable with this assay system (detection limit 0.03 nmol/ml plasma), while endogenous Tyr concentrations were found to be 75-100 nmol/ml of mouse plasma. The amount of Tyr formed as a result of hydrolysis of exogenously added enkephalins was estimated by calculating the difference in the Tyr concentration in the presence versus the absence of added enkephalin. Preplanned comparisons involving dependent t-tests were used to evaluate the significance of the differences in substrate disappearance and in metabolite accumulation in plasma samples incubated with various combinations of substrates and inhibitors. As discussed in Keppel (12), when the number of comparisons is less than the degrees of freedom, Type I error is not inordinately increased.

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Results H y d r o l y s i s of [Leu]- and [Met]enkephalin. As m e a s u r e d by the amount of intact substrate r e m a i n i n g between 2 and 20 min after e n k e p h a l i n addition, the h a l f - l i v e s of [Leu]- and [Met]enkephalin in mouse p l a s m a in vitro were estimated to be 9.1 and 4.6 min, respectively. A s s a y of the amounts of each T y r - c o n t a i n i n g m e t a b o l i t e a c c u m u l a t e d at these same time points indicated that similar enzymes h y d r o l y z e [Leu]- and [Met]enkephalin in mouse plasma, but that there are d i f f e r e n c e s in the relative c o n t r i b u t i o n s of these enzymes to the overall h y d r o l y s i s of the two e n k e p h a l i n s (see FIG. i). Free Tyr was the p r e d o m i n a n t m e t a b o l i t e d e t e c t e d following h y d r o l y s i s of both enkephalins; after a p p r o x i m a t e l y one h a l f - l i f e it accounted for 78% of the T y r - c o n t a i n i n g [Leu]enkephalin m e t a b o l i t e s and 67% of the T y r - c o n t a i n i n g [Met]enkephalin metabolites. Only very small amounts (1-1.2% of the total T y r - c o n t a i n i n g metabolites) of T y r - G l y a c c u m u l a t e d from the h y d r o l y s i s of both enkephalins. By contrast, 20% of the T y r - c o n t a i n i n g [Leu]enkephalin m e t a b o l i t e s and 32% of the T y r - c o n t a i n i n g [Met]enkephalin m e t a b o l i t e s were in the form of T y r - G l y - G l y by the end of a p p r o x i m a t e l y one halflife. These results indicate a larger role for a m i n o p e p t i d a s e a c t i v i t y in [Leu]enkephalin than in [Met]enkephalin hydrolysis,

LE 60

~o

0

10

20

ME

o

_E so

~"

2040 i~ ~o" 0 m

•

Incubation

10

20

/

time

I

(min)

FIG. 1 Time course of the d i s a p p e a r a n c e of substrate ([Leu]enkephalin [LE] or [Met]enkephalin [ME], 64.8 ~M initial concentration, O O) and a c c u m u l a t i o n of p r o d u c t (Tyr, o - - o ; Tyr-Gly, B - - B ; and Tyr-Gly-Gly, A--A) in mouse plasma in vitro. Values are means for four p o o l e d plasma samples.

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671

a n d a l a r g e r r o l e for d i p e p t i d y l c a r b o x y p e p t i d a s e a c t i v i t y in [ M e t ] e n k e p h a l i n t h a n in [ L e u ] e n k e p h a l i n h y d r o l y s i s , by m o u s e p l a s m a in vitro. S t u d i e s w i t h p e p t i d a s e - s e l e c t i v e inhibitors were u n d e r t a k e n t o a t t e m p t to i d e n t i f y t h e s p e c i f i c e n z y m e s i n v o l v e d in t h i s h y d r o l y s i s . R o l e of A m i n o p e p t i d a s e A c t i v i t y in t h e H y d r o l y s i s of [Leu]and [Met]enkephalin. In the p r e s e n c e of t h e a m i n o p e p t i d a s e i n h i b i t o r , b e s t a t i n (13,14), a s i g n i f i c a n t c o n c e n t r a t i o n d e p e n d e n t d e c r e a s e in T y r a c c u m u l a t i o n f r o m [Leu]- a n d [ M e t ] e n k e p h a l i n w a s o b s e r v e d (see T a b l e i). A 9 8 - 9 9 % r e d u c t i o n in T y r a c c u m u l a t i o n f r o m b o t h e n k e p h a l i n s w a s p r o d u c e d by t h e 500 ~ M c o n c e n t r a t i o n of b e s t a t i n . T h i s r e d u c t i o n in T y r f o r m a t i o n w a s a c c o m p a n i e d by a s i g n i f i c a n t c o n c e n t r a t i o n - d e p e n d e n t i n c r e a s e in b o t h T y r - G l y a n d T y r - G l y - G l y a c c u m u l a t i o n . A g r e a t e r i n c r e a s e in T y r - G l y - G l y a c c u m u l a t i o n w a s o b s e r v e d in t h e p r e s e n c e of b e s t a t i n f o l l o w i n g a d d i t i o n of [ M e t ] e n k e p h a l i n , t h a n f o l l o w i n g a d d i t i o n of [Leu]enkephalin, to m o u s e plasma. P u r o m y c i n , a s e l e c t i v e i n h i b i t o r of a m i n o p e p t i d a s e MII (13,14), p r o d u c e d a s i g n i f i c a n t r e d u c t i o n in T y r a c c u m u l a t i o n f r o m [Leu]- a n d [ M e t ] e n k e p h a l i n at a final c o n c e n t r a t i o n of 5000, b u t n o t of 500, ~M. T h e h i g h e r p u r o m y c i n c o n c e n t r a t i o n p r o d u c e d a 4 8 - 6 3 % d e c r e a s e in f o r m a t i o n of free Tyr. In c o n t r a s t to t h e T A B L E i. E f f e c t of B e s t a t i n Tyr-Gly, and Tyr-Gly-Gly Substrate

Inhibitor Tyr

LE a

None

Product (nmoles/ml Tyr-Gly + 1.6

0.79

+ 0.14

plasma) Tyr-Gly-Gly 4.39

+ 0.32

125 ~ M b B 250 ~M B 500 ~M

4.0 + 1 . 9 . 1.4 _+ 0.9* 0.2 _+ 0.2*

1.24 _+ 0.18, 1.37 _+ 0.35* 1.48 + 0.25*

8.83 _+ 1.099.59 _+ 1.19, 9.89 + 0.97*

P 500 ~M c P 5000 ~ M

29.1 _+ 3.2 15.6 _+ 1.8"

0.87 + 0.05 0.44 _+ 0.17,

5.71 + 0.88 11.51 _+ 0.90*

None

45.3

0.79

B

ME d

32.7

a n d P u r o m y c i n on F o r m a t i o n of Tyr, f r o m [Leu]- a n d [ M e t ] e n k e p h a l i n

B 125 ~ M B 250 ~ M B 500 ~ M P 500 ~ M P 5000 ~ M

+ 1.5

+ 0.16

8.61

+ 1.36

5.1 + 1.3" 1.4 _+ 1.6" 1.0 + 0.9*

1.30 _+ 0.30* 1.53 + 0.27* 1.55 + 0.16,

19.81 20.18 21.63

_+ 2.07* _+ 1.23, _+ 1.70-

41.9 _+ 2.8 28.4 _+ 3.3*

0.87 _+ 0.15 0.48 _+ 0.15,

10.90 18.30

_+ 2.71 + 1.04,

a[Leu]enkephalin; b B e s t a t i n ; Cpuromycin; d [ M e t ] e n k e p h a i i n Substrate concentration: 64.8 ~M; I n c u b a t i o n time: I0 min. V a l u e s a r e m e a n s ± S.D. for f o u r p o o l e d p l a s m a samples. * p < 0 . 0 5 c o m p a r e d to "none" group.

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pattern of metabolite accumulation observed in the presence of bestatin, a reduction of Tyr accumulation from hydrolysis of both enkephalins in the presence of puromycin was accompanied by a significant increase in Tyr-Gly-Gly accumulation and a significant reduction in Tyr-Gly accumulation. A combination of bestatin (500 BM) and puromycin (5000 ~M) resulted in a reduction in Tyr accumulation from both enkephalins similar to that produced by bestatin alone, but significantly greater than that produced by puromycin alone. Role of Dipeptidyl Carboxypeptidase Activity in the Hydrolysis of [Leu]- and [Met]enkephalin. The contribution of dipeptidyl carboxypeptidase activity to the hydrolysis of [Leu]and [Met]enkephalin was studied in the presence of the aminopeptidase inhibitor, bestatin (500 ~M), in order to minimize aminopeptidase activity against the Tyr-Gly and Tyr-Gly-Gly metabolites. Thiorphan (i00 ~M), an inhibitor of "enkephalinase" (E.C. 3.4.24.11) (15,16), significantly and markedly reduced Tyr-Gly-Gly formation from both enkephalins in the presence of

~

LE

ME

1.0

m Q.

0

TGG

c

".:

.:

0

o ; o +

o ; o

+

+

+ +

Inhibitor

FIG.

2

Effects of captopril (C; i00 #M) and thiorphan (T; i00 ~M) on the accumulation of Tyr-Gly (TG) and Tyr-Gly-Gly (TGG) in mouse plasma, measured i0 min after addition of [Leu]- or [Met]enkephalin (LE or ME, 64.8 ~M) in the presence of bestatin (500 ~M). Values are means ± S.D. for six pooled plasma samples. ~ p<0.05 compared to bestatin alone; ~ p<0.05 compared to bestatin/thiorphan combination; ~ p<0.05 compared to bestatin/captopril combination.

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bestatin (see FIG. 2). Thiorphan reduced Tyr-Gly-Gly accumulation from [Leu]enkephalin hydrolysis by 82-94% and Tyr-Gly-Gly accumulation from [Met]enkephalin hydrolysis by 91-94%. By contrast, in the presence of bestatin, the angiotensin converting enzyme (ACE)-selective inhibitor, captopril (i00 ~M) (14), had a variable effect on Tyr-Gly-Gly formation. Captopril reduced Tyr-Gly-Gly accumulation from [Leu]enkephalin hydrolysis by 20-80% and Tyr-Gly-Gly accumulation from [Met]enkephalin hydrolsis by 5-50% in various samples of pooled mouse plasma. A combination of captopril, thiorphan, and bestatin produced significantly greater inhibition (>99%) of Tyr-Gly-Gly accumulation from both enkephalins than did either a captoprilbestatin or a thiorphan-bestatin combination. In the presence of bestatin, neither captopril nor thiorphan altered Tyr-Gly formation from either enkephalin. Discussion The results of the present study indicate that mouse plasma has its own unique pattern of enkephalin hydrolysis. The in vitro hydrolysis of enkephalin by mouse plasma is considerably slower than that in rat (7,8) or chick (i0) plasma, and somewhat different enzymes are involved in these three species. Free Tyr is the predominant Tyr-containing metabolite that accumulates following [Leu]- and [Met]enkephalin hydrolysis by mouse, rat (8), and chick (i0) plasma in vitro, and in all three species there is very little accumulation of Tyr-Gly. The major source of inter-species variability in enkephalin hydrolysis is in the amount of Tyr-Gly-Gly that accumulates and in the enzymes that contribute to its production. These differences are considered below. That free Tyr was the predominant metabolite to accumulate following enkephalin hydrolysis in mouse plasma indicates that a major role is played by aminopeptidase(s) in this species. Bestatin, an inhibitor with relatively high selectivity for aminopeptidase M (E.C. 3.4.11.2) (13,14), blocked 98-99% of the Tyr production. It also produced an increase in both Tyr-Gly and Tyr-Gly-Gly accumulation, indicating that its effects were limited to inhibition of aminopeptidase activity. By contrast, concentrations of the aminopeptidase MII inhibitor, puromycin (13,14), that significantly reduced accumulation of free Tyr also reduced Tyr-Gly accumulation. Evidently puromycin only inhibited aminopeptidase activity in mouse plasma at concentrations that inhibited other enzymes as well. These data, together with the finding that a bestatin-puromycin combination produced a similar reduction in Tyr accumulation to that produced by bestatin, but not that produced by puromycin, alone, indicate that enkephalin is hydrolyzed in mouse plasma by a bestatin-sensitive aminopeptidase that most likely is aminopeptidase M. Bestatin resulted in a larger increase in Tyr-Gly-Gly accumulation from [Met]enkephalin than it did from [Leu]enkephalin hydrolysis by mouse plasma, indicating that dipeptidyl carboxypeptidase activity makes a larger contribution to [Met]enkephalin than it

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Enkephalin Hydrolysis by Mouse Plasma

does to [Leu]enkephalin hydrolysis

Vol. 50, No. 10, 1992

in this species.

When combined with bestatin, the enkephalinase inhibitor, thiorphan (15,16), produced in a stable decrease in Tyr-Gly-Gly accumulation from both enkephalins, indicating that enkephalinase plays a role in enkephalin hydrolysis in mouse plasma. The Tyr-Gly-Gly decrease was slightly more pronounced for [Met]- than it was for [Leu]enkephalin. These data are consistent with the somewhat greater reactivity of [Met]- than of [Leu]enkephalin with enkephalinase, as reported in other species (17). By contrast, the effects of the ACE inhibitor, captopril (15), on Tyr-Gly-Gly production were highly variable in individual samples of pooled mouse plasma, and captopril had a greater effect on Tyr-Gly-Gly production from [Leu]enkephalin than it did on Tyr-Gly-Gly production from [Met]enkephalin. These findings clearly indicate a role for ACE in enkephalin hydrolysis in mouse plasma, although the magnitude of its contribution could not be determined with certainty in the present study based on samples of pooled plasma. In the presence of bestatin, a combination of captopril and thiorphan produced almost complete inhibition of Tyr-Gly-Gly production from both enkephalins. This inhibition was significantly greater than that produced by either inhibitor alone. These data suggest that two or more dipeptidyl carboxypeptidases contribute to enkephalin hydrolysis in mouse plasma. These data also suggest that, although relatively high concentrations of inhibitors were employed, selectivity of these inhibitors for their respective peptidases was not reduced significantly. Only one dipeptidyl carboxypeptidase, ACE, participates in enkephalin hydrolysis in rat plasma (7,8). Our investigation of the source of the variability in effectiveness of captopril, and of the specific dipeptidyl carboxypeptidase(s) that hydrolyze enkephalin in this species, is detailed in separate studies of plasma samples from individual mice, reported elsewhere (Schulteis, Rodriguez, Weinberger, and Martinez, in preparation). The present study indicates that aminopeptidase M, enkephalinase, and ACE all participate in the hydrolysis of enkephalin by mouse plasma in vitro. These results contrast with our previous findings that aminopeptidase M and ACE together account for virtually all of the hydrolysis of enkephalins by rat plasma (7,8), and that aminopeptidase M alone accounts for virtually all of the hydrolysis of enkephalins by chick plasma (i0). In addition, enkephalin hydrolysis is considerably slower in mouse, than it is in rat or chick, plasma. Whether these interspecies differences in rate of enkephalin hydrolysis are attributable to interspecies differences in amounts of peptidases in plasma, or to other factors which might affect the activity of a given peptidase, awaits further study. Acknowledqements These studies were supported by NIDA #DA04195 and NIDA #DA04795. Results of these studies are reported in part in S.

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Enkephalin Hydrolysis by M o u s e P l a s m a

SHIBANOKI, S.B. WEINBERGER, G. SCHULTEIS, K. ISHIKAWA, MARTINEZ, JR., Soc. Neurosci. Abstr. 15 368 (1989).

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and J.L.

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3. J.L. MARTINEZ, JR. and S.B. WEINBERGER, Behav. Neurosci. 102 404-408 (1988). 4. S.B. W E I N B E R G E R and J.L. MARTINEZ, JR., Life sci. 4_/3769-776 (1988) .

5. J.L. MARTINEZ, JR., S.B. WEINBERGER, P.H. JANAK, and G. SCHULTEIS, Psychol. Sci. ! 205-208 (1990). 6. P.H. JANAK and J.L. MARTINEZ, JR., Pharmacol. Biochem. Behav. 3_2_7655-659 (1990). 7. S.B. W E I N B E R G E R and J.L. MARTINEZ, JR., J. Pharmacol. Exp. Therap. 247 129-135 (1988). 8. S. SHIBANOKI, S.B. WEINBERGER, K. ISHIKAWA, and J.L. MARTINEZ, JR., Reg. Pep. 3_22 267-278 (1991). 9. G. SCHULTEIS, S.B. WEINBERGER, and J.L. MARTINEZ, JR. Peptides i__O0913-919 (1989). i0. S. SHIBANOKI, S.B. WEINBERGER, D. BENISTON, K.A. NUDELMAN, G. SCHULTEIS, E.L. BENNETT, M.R. ROSENZWEIG, K. ISHIKAWA, and J.L. MARTINEZ, JR. J. Pharmacol. Exp. Therap. 256 650-655 (1991).

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12. G. KEPPEL, Design and Analysis: A R e s e a r c h e r ' s Handbook, 3rd Ed., Prentice Hall, Englewood Cliffs (1991). 13. B. GIROS, C. GROS, B. SOLHONNE, and J.C. SCHWARTZ, Molec. Pharmacol. 2-9 281-287 (1986). 14. L.B. HERSH, N. ABOUKHAIR, and S. WATSON, Peptides 8 523-532 (1987).

15. J.C. SCHWARTZ, Trends Neurosci. Feb. 45-48 (1983). 16. B.P. ROQUES, M.C. FOURNIE-ZALUSKI, E. SOROCA, J.M. LECOMTE, B. MALFROY, C. LLORENS, and J.C. SCHWARTZ, Nature (Lond.) 288 286-288 (1980). 17. L.B. HERSH, J. Neurochem. 4_/3 487-493 (1984).