0022-3022/78/0601- I653102.00/0
Journal of Neurochemisrry Vol. 30, pp. 1653-1655 Pergamon Press Ltd. 1978. Printed in Great Britain 8 International Sonety lor Neurochemistry Lld.
SHORT COMMUNICATION
Inhibition of brain angiotensin-1 converting enzyme by Bothrops jararaca nonapeptide (SQ 20881) and a prolyl analog (SQ 14225) (Received 21 October 1977. Accepted 3 January 1978)
The activity of the angiotensin converting enzyme was THENONAPEPTIDE inhibitor SQ 20881 obtained originally from Bothrops jararaca venom has been used for elucidat- assayed by fluorometric determination of the rate of proing the role of the renin-angiotensin system in tissues duction of His-Leu from hippuryl-His-Leu. (PIQUILLOUD (ERDOS,1976). Based on the interaction of its C-terminal et al., 1970; YANC& NEFF,1972). Incubations were carried with the hypothetical active center of the angiotensin-1 out at 37°C in a total volume of 0.8 ml, containing 40pmol converting enzyme (EC 3.4.15.1), a new class of inhibitors Hepes buffer, pH 8.0, 240pmol NaC1, and 12-24pg of recently has been developed of which 2-mmethyl-3-mer- brain enzyme protein or 200 pg of plasma enzyme protein. captopropanoyl-L-proline (SQ 14225) is among the most Inhibitor at 3-4 different concentrations was added to the et al., 1977). This analog is not only com- medium 5-10min before addition of the substrate. Alipotent (ONDETTI parable in potency to SQ 20881 but has the potential for quots of 0.2 ml were withdrawn from the incubation mixture at 0, 5 and 15min to insure measurement of initial use as an oral agent in the treatment of hypertension. Extensive work exists on the renin-angiotensin system velocities and the reaction stopped by addition of 0.4ml in peripheral tissues, however, very little is known about of 2 N-NaOH. For determination of I,, values, the final its role in the brain. Among components reported present concentration of the substrate hippuryl-His-Leu was 5 mM, in brain are renin-like enzymes, renin substrates, angioten- for determination of K i values, five to six final concensin-1 converting enzyme, as well as angiotensin-I and I1 trations of substrate were used, ranging from 0 S m ~to (GANTENet af., 1971; YANG& NEW, 1972; and BUCKLEY, 2.5 mM. In cases of linear competitive inhibition experimen1977). Levels of angiotensin-I are reported to be present tal I,, values were compared with theoretical calculated in concentrations higher than that of angiotensin-11, imply- from the formula: I,, = K i (I + S / K , ) (WEBB,1963). ing that the converting enzyme plays a crucial role in regulating the behavioral and biological actions of angiotenRESULTS AND DISCUSSION sin-11. We have recently purified partially from brain an Enzyme used for this study was purified from rat brain angiotensin converting enzyme and compared its properties with that of other tissues (BENUCK& MARKS,1977, by extraction with Triton X-100 followed by column 1978). The availability of purified preparations of brain chromatography on DEAE-cellulose and hydroxylapatite & MARKS1978). enzyme presents an opportunity to test the potency of this as described in detail elsewhere (BENUCK new agent. Data on the properties of this inhibitor should When stored at -20°C it is stable for periods of several facilitate studies on the role of the renin-angiotensin sys- months; its specific activity with the synthetic substrate hippuryl-His-Leu is 75 nmol His-Leu released per min per tem in the CNS. mg protein. The enzyme from porcine plasma used for MATERIALS AND METHODS comparison had a specific activity of 1.2 nmol His-Leu Rats were young males of the Sprague-Dawley strain. released per min per mg protein. Purified preparations Hippuryl-histidyl-leucine (HHL), angiotensin I1 and (Sar were devoid of contaminating enzymes which would Ala*)-angiotensin I1 were purchased from Bachem Fine further degrade the His-Leu released. Under standard Chemicals (Marina del Ray, CA.). Bacitracin was pur- assay conditions the reaction velocity was linear with rechased from Sigma Biochemicals (St. Louis, MO). Plasma spect to enzyme concentration and time with an apparent procine angiotensin converting enzyme was purchased K, of 2.2rnt.4. from Cal Biochem (La Jolla, CA). The inhibitors SQ 20881 Mechanisms of inhibition were compared by two pro(pyroGlu-Trp-Pro-Arg-Pro-Gin-Ile-Pro-Pro) and SQ cedures (1) Isa; the concentration of inhibitor required, 14225 (2-~-methyl-3-mercaptopropanoyl-~-proline) were under optimal assay conditions, to give 50% inhibition provided as gifts from Dr. D. W. CUSHMAN of the Squibb when compared to a reaction run without inhibitor, and Institute for Medical Research, Princeton, NJ. (2) kinetics in terms of competitive vs noncompetitive inhiThe enzyme from rat brain was extracted with Triton bition by measurement of K ivalues. To insure initial veloX-100 and purified on DEAE cellulose and hydroxylapatite cities, the measurements were made at short time periods as described in detail elsewhere (BENUCK & MARKS,1978). and K ivalues computed by two alternative Dixon plots For comparison, enzyme from procine plasma was used (I/V vs I/S; and IjV vs i) (DIXON& WEBB,1964). in some experiments. Our results indicate, in terms of I,,, values, that the relative potency of the prolyl analog SQ 14225 was similar Ahhreuiations used: HHL, Hippuryl-histidyl-leucine; to that of SQ 20881 and was some 250-500 fold higher Hepes, N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic than angiotensin-I1 and its Sar'Ala"-analog (saralasin) acid. when incubated with the brain enzyme (Table I). It was
'-
1653
Short communication
1654 TABLE1. INHIBITION OF
ANGIOTENSIN-CONVERTING ENZYME BY PEPTIDES
SQ 14225 SQ 20881 (Sar'-Ala8)Angiotensin I1 Angiotensin I1 Bacitracin
Brain
Lung
0.04 0.08
0.01. 0.56t
601
20 10 100
-
A N D OTHER
Ki (PM) Plasma
Is0 ( P M )
Peptide
SQ 14225
Brain
0.01 0.012 4 4 26
Lung
0.002* 0.lt
0.01 0.008
-
3 10
lorj -
~
* CUSHMAN et al., (1977).
t CHEUNG& CHUSHMAN (1973). $ LANZILLO & FANBURG (1974).
5 SOFFER(1976). Values were measured using a purified preparation of converting enzyme from rat brain and porcine plasma and compared to those published for the lung. In all cases the values were measured using hippuryl-His-Leu as the substrate. For brain and plasma enzyme the rate of hydrolysis in presence and absence of inhibitor was performed by the fluorometric measurement of His-Leu as described in the Methods. The reaction mixture contained 1Opmol of Hepes buffer, pH 8.0, 60pmol of NaCI, and 3-6pg of brain enzyme protein or 50pg of plasma enzyme protein in 0.2ml. For measurements of K isubstrate concentrations ranging from 0.5 mM to 2.5 mM were used; for I,, the optimal substrate concentration of 5 mM was employed. All inhibitors were shown to be competitive using the graphical methods noted in the Methods section and illustrated in the cases of SQ 14225 and SQ 20881 in Fig. 1.
previously shown that the nonapeptide can inhibit converting enzyme purified from serum (DASer ai., 1977) lung & CUSHMAN, 1973) and brain (BENUCK & MARKS, (CHEUNG 1978). In an earlier report, YANG & NEFF (1972) also observed a 53% inhibition of crude brain enzyme at an inhibitor concentration of 0.1 PM. The present study is the first with SQ 14225 and the purified brain enzyme; the I,, obtained when compared to that of SQ 20881 differs from the ratio obtained with the lung enzyme, where the et nonapeptide is approx 50 fold less potent (CUSHMAN al., 1977). The reasons for this difference are unclear, but could be related to the purity of the enzyme used, the conditions of assay, or some intrinsic property of the enzyme in the different tissues. In studies on kinetics of inhibition, SQ 14225, SQ 20881, angiotensin I1 and its analog acted as competitive inhibitors. The plots of the effects of SQ 14225 and SQ 20881 on brain enzymatic activity at different substrate concen-
trations are included in Fig. 1 from which a K i value was computed (Table 1). The values obtained are 400 fold smaller than the K i values for the two angiotensin peptides. Comparison with plasma enzyme indicated a similar range of values for all four peptides (Table 1). In lung, however, the values obtained by CUSHMAN and coworkers differed with respect to SQ 14225, which was 5 fold smaller than that obtained with brain enzyme, and SQ 20881 with a value 10 fold larger than with brain enzyme (CHEUNG& CUSHMAN, 1973; CUSHMAN et a[., 1977). To contrast the effects of SQ 14225 with that of a nonspecific inhibitor, the Is, and K i of the peptide bacitracin was examined. Bacitracin had been reported to inhibit amino- and carboxypeptidases, but its mechanism of action has not been et al., 1977). Present studies gave an elucidated (PATTHY, Is, value which was about lo3 fold less potent than the prolyl derivative, the K i plot indicated competitive inhibition. The experimental Is, values obtained correspond SQ 2088 I
SO 14225
30r
30
r
/
30 nw 20nu
10 nu 5nu Onr
FIG. 1. Double reciprocal plots of the activity of purified angiotensin I-converting enzyme of rat brain in the presence of the inhibitors SQ 14225 and SQ 20881. Concentrations of inhibitor are shown in the figure. The velocity of reaction was measured by the fluorimetric assay of His-Leu using hippurylHis-Leu (HHL) as substrate as described in the Methods section and Table 1.
Short communication
1655
closely with theoretical, based on the formula given in the REFERENCES Methods. BENUCK M. & MARKSN. (1977) Fedn Proc. Fedn Am. Socs It is apparent from comparison of I,, and K i values, exp. Biol. 36, 803. that the Bothrops jararaca peptide and its prolyl analog BENUCKM. & MARKSN. (1978) J. Neurochem. 30, SQ 14225 are considerably more potent inhibitors of the 729-734. angiotensin converting enzyme than either angiotensin I1 BUCKLEY J. P. (1977) Biochem. Pharmac. 26, 1-3. or saralasin. Similar mechanisms of action for SQ 20881 CHEUNG H. S. & CUSHMAN D. W. (1973) Biochim. biophys. and SQ 14225 can be predicated on the basis of their strucActa 293, 451463. ture, especially with respect to the C-terminus. Comparison CUSHMAN D. W., CHEUNGH. S., SABOA. F. & ONDETTI of the structure of various B . jararaca peptides which inM. A. (1977) Biochemistry 16, 54845491. hibit the angiotensin converting enzyme (CHEUNG & CUSH- DAS M., HARTLEY J. L. & SOFFERR. L. (1977) J. biol. MAN, 1973) indicate that C-terminal proline groups are Chem. 252, 1316-1319. essential. Further, SQ 20881 contains an N-terminal pyro- DIXONM. & WEBBE. C. (1964) Enzymes, 2nd ed. Academic Glu as well as a C-terminal Pro-Pro, both of which protect Press, New York. the peptide from hydrolysis by exopeptidases. SQ 14225 ERDOSE. G. (1976) Am. J. M e d . 60, 749-759. is a potent inhibitor of the brain enzyme, and since it is GANTEND., MARQUEZ-JULIO A.. GRANGER P., HAYDUK K., smaller in molecular dimension than other B. jararaca pepKARSUNKY K. P., BOUCHERR. & GENESTJ. (1971) Am. tides, it might facilitate studies on the role of the reninJ. Physiol. 221, 1733-1737. angiotensin system in the CNS. In this respect, VOLLMER LANZILLO J. J. & FANBURC B. L. (1974) J. biol. Chem. & BOCCAGNO (1977) showed that SQ 14225, following in249, 2312-2318. tracerebroventricular injection, is a potent inhibitor of ONDETTI M. A,, RUBINB. & CUSHMAN D. W. (1977) Science angiotensin-I conversion in the CNS. 196,441443. PATTHY A., GRAFL., KENESSEY A,, SZBKELY J. I. & BAJUSZ S. (1977) Biochem. biophys. Res. Commun. 19, 254259. Acknowledgements-This work was supported in part by Y. A,, REINHARZ A. & ROTH M. (1970) Bioa grant from USPHS-NS-12578. We are indebted also to PIQUILLOUD chim. biophys. Acta 206, 136142. Dr. D. W. CUSHMAN for a supply of the inhibitors SQ SOFFERR. L. (1976) Ann. Reo. Biochem. 45, 73-94. 20881 and SQ 14225. VOLLMER R. R. & BOCCAGNO J. A. (1977) Eur. J. Pharmac. 45, 117-125. Research Institute for Neurochemistry, M. BENUCK WEBBJ. L. (1963) Enzyme and Metabolic Inhibitors, Vol. Rockland Research Institute, N. MARKS 1, p. 106. Academic Press, New York. Ward’s Island. YANGH.-Y. T. & NEFF N. H. (1972) J. Neurochem. 19, NY 10035, U.S.A. 2443-2450.
Journal of Neurochemisrry Vol. 30, pp. 1653-1655 Pergamon Press Ltd. 1978. Printed in Great Britain 8 International Sonety lor Neurochemistry Lld.
SHORT COMMUNICATION
Inhibition of brain angiotensin-1 converting enzyme by Bothrops jararaca nonapeptide (SQ 20881) and a prolyl analog (SQ 14225) (Received 21 October 1977. Accepted 3 January 1978)
The activity of the angiotensin converting enzyme was THENONAPEPTIDE inhibitor SQ 20881 obtained originally from Bothrops jararaca venom has been used for elucidat- assayed by fluorometric determination of the rate of proing the role of the renin-angiotensin system in tissues duction of His-Leu from hippuryl-His-Leu. (PIQUILLOUD (ERDOS,1976). Based on the interaction of its C-terminal et al., 1970; YANC& NEFF,1972). Incubations were carried with the hypothetical active center of the angiotensin-1 out at 37°C in a total volume of 0.8 ml, containing 40pmol converting enzyme (EC 3.4.15.1), a new class of inhibitors Hepes buffer, pH 8.0, 240pmol NaC1, and 12-24pg of recently has been developed of which 2-mmethyl-3-mer- brain enzyme protein or 200 pg of plasma enzyme protein. captopropanoyl-L-proline (SQ 14225) is among the most Inhibitor at 3-4 different concentrations was added to the et al., 1977). This analog is not only com- medium 5-10min before addition of the substrate. Alipotent (ONDETTI parable in potency to SQ 20881 but has the potential for quots of 0.2 ml were withdrawn from the incubation mixture at 0, 5 and 15min to insure measurement of initial use as an oral agent in the treatment of hypertension. Extensive work exists on the renin-angiotensin system velocities and the reaction stopped by addition of 0.4ml in peripheral tissues, however, very little is known about of 2 N-NaOH. For determination of I,, values, the final its role in the brain. Among components reported present concentration of the substrate hippuryl-His-Leu was 5 mM, in brain are renin-like enzymes, renin substrates, angioten- for determination of K i values, five to six final concensin-1 converting enzyme, as well as angiotensin-I and I1 trations of substrate were used, ranging from 0 S m ~to (GANTENet af., 1971; YANG& NEW, 1972; and BUCKLEY, 2.5 mM. In cases of linear competitive inhibition experimen1977). Levels of angiotensin-I are reported to be present tal I,, values were compared with theoretical calculated in concentrations higher than that of angiotensin-11, imply- from the formula: I,, = K i (I + S / K , ) (WEBB,1963). ing that the converting enzyme plays a crucial role in regulating the behavioral and biological actions of angiotenRESULTS AND DISCUSSION sin-11. We have recently purified partially from brain an Enzyme used for this study was purified from rat brain angiotensin converting enzyme and compared its properties with that of other tissues (BENUCK& MARKS,1977, by extraction with Triton X-100 followed by column 1978). The availability of purified preparations of brain chromatography on DEAE-cellulose and hydroxylapatite & MARKS1978). enzyme presents an opportunity to test the potency of this as described in detail elsewhere (BENUCK new agent. Data on the properties of this inhibitor should When stored at -20°C it is stable for periods of several facilitate studies on the role of the renin-angiotensin sys- months; its specific activity with the synthetic substrate hippuryl-His-Leu is 75 nmol His-Leu released per min per tem in the CNS. mg protein. The enzyme from porcine plasma used for MATERIALS AND METHODS comparison had a specific activity of 1.2 nmol His-Leu Rats were young males of the Sprague-Dawley strain. released per min per mg protein. Purified preparations Hippuryl-histidyl-leucine (HHL), angiotensin I1 and (Sar were devoid of contaminating enzymes which would Ala*)-angiotensin I1 were purchased from Bachem Fine further degrade the His-Leu released. Under standard Chemicals (Marina del Ray, CA.). Bacitracin was pur- assay conditions the reaction velocity was linear with rechased from Sigma Biochemicals (St. Louis, MO). Plasma spect to enzyme concentration and time with an apparent procine angiotensin converting enzyme was purchased K, of 2.2rnt.4. from Cal Biochem (La Jolla, CA). The inhibitors SQ 20881 Mechanisms of inhibition were compared by two pro(pyroGlu-Trp-Pro-Arg-Pro-Gin-Ile-Pro-Pro) and SQ cedures (1) Isa; the concentration of inhibitor required, 14225 (2-~-methyl-3-mercaptopropanoyl-~-proline) were under optimal assay conditions, to give 50% inhibition provided as gifts from Dr. D. W. CUSHMAN of the Squibb when compared to a reaction run without inhibitor, and Institute for Medical Research, Princeton, NJ. (2) kinetics in terms of competitive vs noncompetitive inhiThe enzyme from rat brain was extracted with Triton bition by measurement of K ivalues. To insure initial veloX-100 and purified on DEAE cellulose and hydroxylapatite cities, the measurements were made at short time periods as described in detail elsewhere (BENUCK & MARKS,1978). and K ivalues computed by two alternative Dixon plots For comparison, enzyme from procine plasma was used (I/V vs I/S; and IjV vs i) (DIXON& WEBB,1964). in some experiments. Our results indicate, in terms of I,,, values, that the relative potency of the prolyl analog SQ 14225 was similar Ahhreuiations used: HHL, Hippuryl-histidyl-leucine; to that of SQ 20881 and was some 250-500 fold higher Hepes, N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic than angiotensin-I1 and its Sar'Ala"-analog (saralasin) acid. when incubated with the brain enzyme (Table I). It was
'-
1653
Short communication
1654 TABLE1. INHIBITION OF
ANGIOTENSIN-CONVERTING ENZYME BY PEPTIDES
SQ 14225 SQ 20881 (Sar'-Ala8)Angiotensin I1 Angiotensin I1 Bacitracin
Brain
Lung
0.04 0.08
0.01. 0.56t
601
20 10 100
-
A N D OTHER
Ki (PM) Plasma
Is0 ( P M )
Peptide
SQ 14225
Brain
0.01 0.012 4 4 26
Lung
0.002* 0.lt
0.01 0.008
-
3 10
lorj -
~
* CUSHMAN et al., (1977).
t CHEUNG& CHUSHMAN (1973). $ LANZILLO & FANBURG (1974).
5 SOFFER(1976). Values were measured using a purified preparation of converting enzyme from rat brain and porcine plasma and compared to those published for the lung. In all cases the values were measured using hippuryl-His-Leu as the substrate. For brain and plasma enzyme the rate of hydrolysis in presence and absence of inhibitor was performed by the fluorometric measurement of His-Leu as described in the Methods. The reaction mixture contained 1Opmol of Hepes buffer, pH 8.0, 60pmol of NaCI, and 3-6pg of brain enzyme protein or 50pg of plasma enzyme protein in 0.2ml. For measurements of K isubstrate concentrations ranging from 0.5 mM to 2.5 mM were used; for I,, the optimal substrate concentration of 5 mM was employed. All inhibitors were shown to be competitive using the graphical methods noted in the Methods section and illustrated in the cases of SQ 14225 and SQ 20881 in Fig. 1.
previously shown that the nonapeptide can inhibit converting enzyme purified from serum (DASer ai., 1977) lung & CUSHMAN, 1973) and brain (BENUCK & MARKS, (CHEUNG 1978). In an earlier report, YANG & NEFF (1972) also observed a 53% inhibition of crude brain enzyme at an inhibitor concentration of 0.1 PM. The present study is the first with SQ 14225 and the purified brain enzyme; the I,, obtained when compared to that of SQ 20881 differs from the ratio obtained with the lung enzyme, where the et nonapeptide is approx 50 fold less potent (CUSHMAN al., 1977). The reasons for this difference are unclear, but could be related to the purity of the enzyme used, the conditions of assay, or some intrinsic property of the enzyme in the different tissues. In studies on kinetics of inhibition, SQ 14225, SQ 20881, angiotensin I1 and its analog acted as competitive inhibitors. The plots of the effects of SQ 14225 and SQ 20881 on brain enzymatic activity at different substrate concen-
trations are included in Fig. 1 from which a K i value was computed (Table 1). The values obtained are 400 fold smaller than the K i values for the two angiotensin peptides. Comparison with plasma enzyme indicated a similar range of values for all four peptides (Table 1). In lung, however, the values obtained by CUSHMAN and coworkers differed with respect to SQ 14225, which was 5 fold smaller than that obtained with brain enzyme, and SQ 20881 with a value 10 fold larger than with brain enzyme (CHEUNG& CUSHMAN, 1973; CUSHMAN et a[., 1977). To contrast the effects of SQ 14225 with that of a nonspecific inhibitor, the Is, and K i of the peptide bacitracin was examined. Bacitracin had been reported to inhibit amino- and carboxypeptidases, but its mechanism of action has not been et al., 1977). Present studies gave an elucidated (PATTHY, Is, value which was about lo3 fold less potent than the prolyl derivative, the K i plot indicated competitive inhibition. The experimental Is, values obtained correspond SQ 2088 I
SO 14225
30r
30
r
/
30 nw 20nu
10 nu 5nu Onr
FIG. 1. Double reciprocal plots of the activity of purified angiotensin I-converting enzyme of rat brain in the presence of the inhibitors SQ 14225 and SQ 20881. Concentrations of inhibitor are shown in the figure. The velocity of reaction was measured by the fluorimetric assay of His-Leu using hippurylHis-Leu (HHL) as substrate as described in the Methods section and Table 1.
Short communication
1655
closely with theoretical, based on the formula given in the REFERENCES Methods. BENUCK M. & MARKSN. (1977) Fedn Proc. Fedn Am. Socs It is apparent from comparison of I,, and K i values, exp. Biol. 36, 803. that the Bothrops jararaca peptide and its prolyl analog BENUCKM. & MARKSN. (1978) J. Neurochem. 30, SQ 14225 are considerably more potent inhibitors of the 729-734. angiotensin converting enzyme than either angiotensin I1 BUCKLEY J. P. (1977) Biochem. Pharmac. 26, 1-3. or saralasin. Similar mechanisms of action for SQ 20881 CHEUNG H. S. & CUSHMAN D. W. (1973) Biochim. biophys. and SQ 14225 can be predicated on the basis of their strucActa 293, 451463. ture, especially with respect to the C-terminus. Comparison CUSHMAN D. W., CHEUNGH. S., SABOA. F. & ONDETTI of the structure of various B . jararaca peptides which inM. A. (1977) Biochemistry 16, 54845491. hibit the angiotensin converting enzyme (CHEUNG & CUSH- DAS M., HARTLEY J. L. & SOFFERR. L. (1977) J. biol. MAN, 1973) indicate that C-terminal proline groups are Chem. 252, 1316-1319. essential. Further, SQ 20881 contains an N-terminal pyro- DIXONM. & WEBBE. C. (1964) Enzymes, 2nd ed. Academic Glu as well as a C-terminal Pro-Pro, both of which protect Press, New York. the peptide from hydrolysis by exopeptidases. SQ 14225 ERDOSE. G. (1976) Am. J. M e d . 60, 749-759. is a potent inhibitor of the brain enzyme, and since it is GANTEND., MARQUEZ-JULIO A.. GRANGER P., HAYDUK K., smaller in molecular dimension than other B. jararaca pepKARSUNKY K. P., BOUCHERR. & GENESTJ. (1971) Am. tides, it might facilitate studies on the role of the reninJ. Physiol. 221, 1733-1737. angiotensin system in the CNS. In this respect, VOLLMER LANZILLO J. J. & FANBURC B. L. (1974) J. biol. Chem. & BOCCAGNO (1977) showed that SQ 14225, following in249, 2312-2318. tracerebroventricular injection, is a potent inhibitor of ONDETTI M. A,, RUBINB. & CUSHMAN D. W. (1977) Science angiotensin-I conversion in the CNS. 196,441443. PATTHY A., GRAFL., KENESSEY A,, SZBKELY J. I. & BAJUSZ S. (1977) Biochem. biophys. Res. Commun. 19, 254259. Acknowledgements-This work was supported in part by Y. A,, REINHARZ A. & ROTH M. (1970) Bioa grant from USPHS-NS-12578. We are indebted also to PIQUILLOUD chim. biophys. Acta 206, 136142. Dr. D. W. CUSHMAN for a supply of the inhibitors SQ SOFFERR. L. (1976) Ann. Reo. Biochem. 45, 73-94. 20881 and SQ 14225. VOLLMER R. R. & BOCCAGNO J. A. (1977) Eur. J. Pharmac. 45, 117-125. Research Institute for Neurochemistry, M. BENUCK WEBBJ. L. (1963) Enzyme and Metabolic Inhibitors, Vol. Rockland Research Institute, N. MARKS 1, p. 106. Academic Press, New York. Ward’s Island. YANGH.-Y. T. & NEFF N. H. (1972) J. Neurochem. 19, NY 10035, U.S.A. 2443-2450.
