The degradation of adrenocorticotrophic hormone-(1–4) by mouse brain cytosol

ARCHIVES

OF BIOCHEMISTRY

AND BIOPHYSICS

Vol. 203, No. 1, August, pp. 233-295, 1930

The Degradation

of Adrenocorticotrophic Mouse Brain Cytosol

A. NEIDLE

AND

Hormone-(1

-4) by

M. E. A. REITH

Center for Neurochemistry, Rockland Research Institute,

Ward’s Island,

New York 100.95

Received December 6, 1979 Ser-Tyr-Ser-Met, the N-terminal tetrapeptide of adrenocorticotrophic hormone, is rap idly converted to free amino acids by mouse brain cytosol. Amino acid release was greatly reduced by puromycin, bestatin, EDTA, or o-phenanthroline. In the presence of dithiothreitol, only Se? and Met4 were affected. This, and other data, indicated that the mechanism of breakdown consisted of sequential aminopeptidase action followed by the cleavage of the resulting Ser-Met. Two enzymes were partially purified and shown to account for adrenocorticotrophic hormone-(l-4) degradation. The first of these is an aminopeptidase, the activities of which toward the cleavage of Leu-p-naphthylamide and the release of Ser’ and TyrZ were purified to the extent of 453-, 429, and 426fold, respectively. It has properties and specificity towards aminoacyl p-naphthylamides indicating that it is identical to a neutral arylamidaae purified from the brains of other species. The second enzyme was purified llO-fold and is a sulfhydryl-inhibited dipeptidase of broad specificity (EC 3.4.13.2). Some of the properties of these enzymes and their action on adrenocorticotrophic hormone fragments and other peptides are reported.

Since adrenocorticotrophic hormone (ACTH)’ and some of its fragments can evoke characteristic behavioral responses in animals (l-3), the metabolism of these peptides by the brain is of interest. In a recent study of ACTH-(1-24) breakdown by mouse brain preparations (4) we observed that enzymes present in the cytosol can rapidly convert the hormone to its constituent amino acids. Other oligopeptides are similarly broken down by this preparation including angiotensin, p-endorphin, substance P, glucagon, and the A and B chains of insulin (5). It seems likely therefore that the enzymes involved in ACTH degradation are part of a population of soluble peptide hydrolases capable of acting on a wide variety of substrates. In order to identify these enzymes and define their role in the degradation of ACTH and other peptides, we have been studying the break1 Abbreviations used: ACTH, adrenocorticotrophic hormone; /3NA, p-naphthylamide; DTT, diothiothreitol; SDS, sodium dodecyl sulfate; HTP, hydroxylapatite; CDP I, cytoplasmic dipeptidaae I. 0003-9361/30/09023303$02.00/0 Copyright 0 1980 by Academic Press, All rights of reproductionin any form

Inc. reserved.

233

down of a series of partial ACTH sequences. We have been particularly interested in peptides originating, in the N-terminal region of the hormone, which appears to be especially susceptible to enzymatic attack (4). In this report we describe the breakdown of Ser-Tyr-Ser-Met, the N-terminal tetrapeptide of ACTH. Two enzymes were identified as being primarily responsible for amino acid release. These were purified, and their properties investigated in relation to the degradation of ACTH-(1-4) and other N-terminal ACTH sequences. METHODS AND MATERIALS Mouse brain cytosol. All steps were carried out at 0-4°C. The brains from two adult Swiss mice were homogenized in 10.0 ml of 1% NaCl using a Dounce homogenizer (Kontes Glass Co.). The homogenate was then centrifuged for 30 min at 35,000g and the supernatant solution concentrated to 0.30.5 ml in an ultraiilter (Amicon Corp., PM-10 membrane). In addition to reducing the volume of the enzyme solution, this step removes most of the endogenous amino acids. Appropriate dilutions were

ADRENOCORTICOTROPHIC

HORMONE-BREAKDOWN

IN MOUSE

BRAIN

CYTOSOL

289

made with 1% NaCl and proteins were measured and 20 mM Tris buffer pH 7.0. Twenty mouse using a modification (6) of the method of Lowry brains (about 10 g) were homogenized in 0.25~ et al. (7). The preparation contained some micro- sucrose and the homogenate was centrifuged for somes along with the soluble fraction of brain. 30 min at 35,000g. The supernatant solution was However, since after centrifugation for 1 h at 100,OOOg then fractionated with ammonium sulfate and the the pellet had little activity against the substrates material precipitating between 50 and 75% of of the present study higher-speed centrifugation saturation was disolved in 2.0 ml of water. The was not routinely done. solution was placed on a column of Sephadex G-50 Incubations. For studies on amino acid formation (Pharmacia Inc.), 1.7 x 9.0 cm, and eluted with from peptides and on the effects of inhibitors, water. Fifteen 1.0 ml fractions were collected. Fracincubations were carried out in 0.04 M phosphate tions 8-10 containing the major portion of the buffer, pH 7.0, at 37”C, for 30 min. Additions arylamidase activity were combined, and applied to a were made as noted in the legends to the tables. column of diethyl aminoethyl cellulose (cellex D, Incubation volume was 0.1 ml. The reactions were Bio-Rad Laboratories 0.8 x 4.0 cm). The column was stopped by the addition of 3% sulfosalicylic acid eluted with three 1.5-ml portions of 0.1 M NaCl and after centrifugation to remove protein, portions followed by ten 1.5 ml portions of buffer increasing were applied to the amino acid analyzer or total in NaCl molar&y at the rate of 0.015 M per fraction amino acid and peptide content were measured (0.25 M at fraction 13). Fractions 5-8 were combined using the ninhydrin reaction. and applied to an hydroxylapatite column, 0.8 x 8.0 cm (HTP, Bio-Rad Laboratories), and eluted stepAmino acid analyses. An autoanalyzer (Technicon Instrument Co.) with a column of Aminex A-6 wise with three 1.5 ml portions of phosphate buffer, pH 6.8, at each of the following phosphate concentra(Bio-Rad Laboratories) (0.6 x 70 cm) and a lithium citrate gradient (8) was employed. o-Phthalaldehyde tions: 0.10, 0.15, 0.20, and 0.25 M. Fractions 6-9 were combined, concentrated to a small volume by ultrawas used as a fluorogenic reagent (9). filtration (Amicon PM-lo), and diluted to 4.0 ml with Enzyme assays. Arylamidase was measured fluoriwater. The DEAE and hydroxylapatite steps were metrically using a Turner Model 111 fluorometer. then repeated. Fractions 7-10 from the final column Filters used were 7-60 (maximum transmission at contained the major portion of the activity. This 360 nm) and 2-A (sharp cutoff at 450 nm). Test material was concentrated to 0.2 ml by ultrafiltration, tubes (12 x ‘72 mm) served as cuvettes. Each tube diluted to 1.1 ml with 0.25 M sucrose, and frozen contained 0.1 mM aminoacyl j3-naphthylamide (BNA), at -70°C in 0.l-ml portions. 1.0 mM dithiothreitol (DTT), 0.05M phosphate bufPurification of the SH-inhibited dipeptidase. All fer pH 6.5, and enzyme in a final volume of 2.0 ml. The sample compartment was maintained at solutions contained 0.1 mM DTT. Mouse brain (2.5 g) 37°C. Tubes of reaction mixture at 37°C were placed was homogenized in 12.5 ml of 1% NaCl and in the sample compartment and enzyme was added. centrifuged at 35,000g for 30 min. Ammonium sulfate After 2-3 min the instrument was adjusted to zero was then added in portions and the fraction preand readings were taken for 5 min at 1-min cipitating between 40 and 60% of saturation disintervals. Activity was calculated from the rate of solved in 10 ml of 0.02 M Tris buffer pH 7.0 increase in fluorescence during a linear portion of (Tris). This solution was concentrated to 0.5 ml by ultrafiltration, brought to 5.0 ml with Tris, and the reaction. Standard curves of P-naphthylamine applied to a column of DEAE-cellulose, 5.0 x 1.2 cm. fluorescence were used for calibration. Peptidase activity was measured using an The column was eluted with thirty 1.5 ml portions autoanalyzer connected to a batch sampler (Technicon of Tris containing a linear gradient of NaCl ranging from 0 to 0.4~. The tubes containing the major Instrument Corp.). Ninhydrin reduced with hydrazine was the calorimetric reagent. After incubations of portion of the dipeptidase activity were combined and applied to a column of hydroxylapatite, 3.0 substrate (1 mM), enzyme, and phosphate buffer x 1.2 cm. The column was eluted with twenty l.O0.05 M, pH 7.0 in 0.1 ml, 1.15 ml of 3% sulfosalicylic ml portions of buffer containing a linear phosphate acid was added and the tubes were centrifuged. Portions of the supernatant were diluted with 2 vol gradient, pH 7.0, from O-O.2 M. Individual tubes containing appreciable activity were brought to 0.25 M of 0.4~ sodium acetate buffer, pH 4.5, and transsucrose and stored at -70°C. ferred to sample cups for ninhydrin determinations. Small amounts of protein could be tolerated in these assays if appropriate blanks were run so that RESULTS when partially purified enzymes were assayed, centrifugation after the addition of sulfosalicylic acid Time Course of Amino Acid Release could be omitted. Arylamidase purification. All steps were carried ACTH-(l-4) was incubated for various out at 0-4°C in solutions containing 1.0 mM DTT times with the soluble fraction of mouse

290

NEIDLE

brain homogenates and the amino acid composition of the mixture was determined by column chromatography (Table I). From these data it is possible to make an approximate assignment of the distribution of serine between positions 1 and 3, since Ser3 must be less than Met4. At all time points Se9 is higher than Ty? indicating an initial N-terminal attack on the tetrapeptide. This effect is not very pronounced, however, except at the earliest time point, and other bond cleavages must follow in rapid succession. If Tyr2 release is taken as a measure of ACTH-(1-4) degradation, its rate of disappearance was 1.7 pmollmg protein/h. This is approximately 30-fold the initial rate of ACTH-(l-24) breakdown under similar conditions (4) and is in agreement with the generality that smaller peptides are usually degraded more rapidIy than larger ones. The Effects of Inhibitors

In addition to SH and metal chelating reagents, we were interested in puromycin which can act as a competitive inhibitor of arylamidase (lo), and bestatin which inhibits various exopeptidases including leucine aminopeptidase (11). Puromycin, bestatin, EDTA, and o-phenanthroline greatly reduced the liberation of all amino acids from ACTH-(l-4) (Table II). DTT TABLE I AMWO ACID RELEASE FROMACTH-(1-4) AS A FUNCTION OF INCUBATION TIME Incubation time (min)

nmol released/nmol substrate Ser 1, 3”

Tyr 2

Met 4

6 15 25 50 75

0.08 0.22 0.39 0.77 1.21

0.03 0.11 0.20 0.41 0.64

0.02 0.10 0.16 0.35 0.55

Note. ACTH-(l-Q, 40 nmol, was incubated at 37°C with 20 ~1 of an ultrafiltrate of mouse brain cytosol (10 pg protein) in 0.04 M phosphate buffer, pH 6.5. The final volume was 0.1 ml. (LPositions of the residue in ACTH-(1-I).

AND REITH TABLE II EFFECT OF INHIBITORS ON AMINO ACID LIBERATION FROMACTH-(l-4) BY BRAIN EXTRACT nmol released/ nmol substrate Additions

Ser 1, 3”

Tyr2

Met4

None hromycin, 0.5 mM EDTA, 2 mM o-Phenanthroline, 0.5 mM Bestatin, 50 pg/ml DTT, 1 mM

1.44 0.20 0.12 0 0.03 0.89

0.83 0.09 0.15 0 0.02 0.86

0.70 0.08 0 0 0 0.12

Note. Incubation mixtures contained 40 nmol ACTH-(l-4), 20 ~1 0.2 M phosphate buffer, pH 6.5, and 29 pg of brain extract protein in a final volume of 100 ~1; the duration of the incubation was 30 min. Brain extract and inhibitors were preincubated for 30 min before addition of the substrate. The amino acid released in the absence of ACTH-(1-4) was negligible. The average concentrations found in two experiments are presented. Individual measurements fell within a range of *5% of the mean values. a Positions of the residue in ACTH-(1-I).

selectively inhibited serine and methionine release. Since Se9 liberation must be greater than that of Tyr2, the effect on serine was limited to the residue in position three. These results indicate that SerMet is an intermediate in ACTH-(1-4) degradation and that it is cleaved by an SH-inhibited dipeptidase. The presence of such an enzyme in brain cytosol was confirmed by the strong inhibition of Ser-Met hydrolysis by 1 mM DTT. Ser-Tyr hydrolysis was also prevented by the presence of DTT, providing additional evidence that the N-terminal residues are not cleaved by way of a dipeptide intermediate. The Mechanism of ACTH-(l-4)

Degradation

From the results presented above, it seemed likely that ACTH-(l-4) was degraded in three steps as follows: (i) Ser-Tyr-Ser-Met --, Ser + Tyr-Ser-Met (ii)

Tyr-Ser-Met

+- Tyr + Ser-Met

(iii)

Ser-Met + Ser + Met.

ADRENOCORTICOTROPHIC

HORMONE-BREAKDOWN

IN MOUSE BRAIN CYTOSOL

291

TABLE III

PURIFICATIONOFMOUSE BRAINNEUTRALARYLAMIDASE Total units” 35,000g supernatant (NH&S04, 50-75% Sephadex G-56 DEAE-cellulose Hydroxylapatite DEAE cellulose 2 Hydroxylapatite 2

Specific activity (units/mg)

8.7 6.1 6.1 4.6 3.0 2.0 1.2

0.03 0.14 0.15 0.60 3.00 4.47 13.70

Purification (-fold)

Overall recovery (%)

1 4.3 4.6 19 95 141 433

70 70 53 35 23 13

n A unit is defined as 1 pmol Leu-pNA hydrolyzed/min.

The questions that remained concerned the nature of the enzymes responsible for these reactions. Pur&cation peptidase

and Properties

of the Amino-

Some of the properties of this enzyme were already known from the inhibition experiments of Table II; they are similar to those of a group of aminopeptidases isolated from bovine brain (12) and pituitary (13) and the brains of pig (14), monkey (lo), and rat (15). Although the characteristics of these enzymes (and the names given to them) differ somewhat from author to author, they have many features in common. In all cases they represent the major activity of the soluble portions of brain homogenates toward the p-naphthylamides of leucine, alanine, and lysine. To establish whether such an arylamidase takes part in ACTH-(1-4) degradation, we purified the leucyl p-naphthylamine-cleaving enzyme of mouse brain cytosol and measured its ability to hydrolyze the N-terminal residues of the peptide. The method chosen for purification was a small-scale modification of one previously used for the preparation of pig brain neutral arylamidase (14). The last ammonium sulfate precipitation was eliminated in order to have a final enzyme free of ammonia. Instead, ultrafiltration to remove salt and additional chromatographic steps using DEAE-cellulose and hydroxylapatite were employed (see Methods and Materials and Table III). The

final preparation, obtained in 13% yield from brain supernatant, was 433-fold purified. Upon electrophoresis in the presence of SDS (16), a single major and several minor bands were observed (Fig. 1A). In the absence of SDS, the major band coincided with the position of leucine+NA hydrolyzing activity (Fig. 1B). From these data and the position of elution of activity from a column of Sephadex G-150, it appears that the enzyme consists of a single polypeptide chain with a molecular weight of about 100,000. The order of reactivity shown by the enzyme toward a series of aminoacyl pnaphthylamides was similar to that of the enzyme from most other species (lo-13), but differed somewhat from that reported for the rat preparation (15). The close agreement between the order of reactivity of these substrates when crude supernatant is compared to neutral arylamidase suggests that a single enzyme of broad specificity is responsible for the major portion of many of these activities. In addition to its action on p-naphthylamides, arylamidase can act as an aminopeptidase cleaving both peptidyl p-naphthylamides (14, 17) and a variety of small peptides (10, 14). The purified enzyme released Ser and Tyr from the N-terminal positions of ACTH(l-4) at rates of 5.4 and 4.7 pmol/mg protein/min, respectively. The increases in specific activity, over that of the cytosol, for leucyl p-naphthylamide, Ser’ and Tyr2 hydrolysis were 453-, 429-, and 426-fold,

292

NEIDLE

AND REITH

respectively. The copurification of all three activities suggest that the formation of Tyr’ from Tyr- Ser-Met is also catalyzed by neutral arylamidase. Thus, with respect to ACTH-(l-4), the neutral arylamidase acts as an aminopeptidase, sequentially removing the two N-terminal residues. The mouse brain enzyme also shows some activity toward ACTH-(l-24), -(l-10), and -(4-10). Within this series of substrates there is a marked decrease in activity with increasing size, even when the same N-terminal sequence is present. Thus the rate of release of Ser’ from ACTH(l-10) and ACTH-(1-24) took place at 23 and 1.7% of that from ACTH-(l-4), respectively. The rate of Met4 release from ACTH(4- 10) was 27% of that of Ser’ from the l-4 fragment and small quantities of the following three amino acids were released in decreasing amounts. The requirement for sulfhydryl groups by neutral arylamidase and its inhibition by EDTA, o-phenanthroline, puromycin, and bestatin are similar to the effects of these substances on ACTH-(1-4) degradation by mouse brain cytosol (Table II). No activity was observed in the absence of DTT or in the presence of puromycin (0.5 mM> or bestatin (50 pg/ml). Inhibitions of 40 and 93% were caused by EDTA (1.0 InM) and o-phenanthroline (0.5 mM), respectively. The increased requirement for SH of the purified enzyme compared to the cytosol indicates that reversible oxidation of an essential thiol group had taken place during isolation procedure. Seryl Methionine Cleavage The accumulation of seryl methionine in the presence of DTT indicated that a dipeptidase similar to that found in ascites cells (18), pig brain (19), and monkey (20) and pig intestine (21) might be responsible for its degradation. To define the role of the enzyme in peptide degradation in mouse brain extracts, we undertook its isolation. Upon purification, as has been reported for these dipeptidases, the SerMet-splitting enzyme appeared to be highly unstable. However, by carrying out the entire purification procedure in 1 day and

A.

1

2

a.

12

FIG. 1. Electrophoresis of the purified arylamidaae. (A) Slab gel electrophoresis in the presence of SDS and 2-mercaptoethanol according to Laemmli (16). (1) Forty microliters of purified enzyme. (2) Molecular weight standards (Bio-Rad). Top to bottom; phosphorylase B (M, 94,000), bovine serum albumin R%OOO), ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (21,000), and lysozyme (14,300). (B) Disc gels using the same system as in A, but omitting mercaptoethanol and SDS. Forty microliters of enzyme was applied to each tube. (1) Stained with Coomassie blue. (2) Incubated for 15 min at 3’7°C in 0.05 M phosphate buffer, pH 6.5, containing 1 mM leucyl+NA, followed by 30 min in a 0.4% solution of diazo blue B in 0.3 M sodium acetate, pH 4.5, containing 2% Brij (30% aqueous solution).

keeping a small amount of DTT (10m4M) in all solutions, a purification of llO-fold with a recovery of 17% was obtained (Table IV). The final preparation, stored at -70°C in the presence of 0.25 M sucrose, retained activity for several weeks. At this purification, a complex pattern of protein bands was found after electrophoresis in the presence of SDS and further characterization of the molecular properties of the dipeptidase was not attempted. The peptidase was virtually unaffected by puromycin but was inhibited by bestatin (100% at 50 kg/ml), DTT (100% at 1.0 mM), o-phenanthroline (82% at 0.5 mM>, and EDTA (50% at 2 mM>. The action of the latter two inhibitors indicated that the enzyme has a metal requirement. The SH inhibition has also been linked to a metal requirement, since the order of effective-

ADRENOCORTICOTROPHIC

HORMONE-BREAKDOWN

IN MOUSE BRAIN CYTOSOL

293

TABLE IV PURIFICATION OF A DIPE~TIDASE FROM MOUSE BRAIN

Step

Total units”

Specific activity (units/mg)

Purification (-fold)

Overall recovery (%I

35,OOOgsupernatant (NH&SO1, 40-60% DEAE Hydroxylapatite

70.50 51.35 18.67 11.99

0.6 1.7 6.5 65.8

2.8 11 110

73 26 17

’ A unit is defined as 1 pmol Ser-Met hydrolyzetimin.

ness of a group of sulfhydryl reagents paralleled their metal-chelating ability (18). Zinc has been suggested as the cofactor for both the ascites and intestinal dipeptidases. The Ser-Met-cleaving enzyme appeared to be a true dipeptidase with little or no activity directed toward substrates with three or more residues (Table V). With respect to its specificity, instability, and the effects of inhibitors, it greatly resembles TABLE V RELATIVE RATES OF HYDROLYSIS FOR DIFFERENT SUBSTRATES OF THE MOUSE BRAIN EXTRACT AND THE PURIFIED DIPEPTIDASE qnol

Brain Substrate &r-Met Ser-Tyr Ah-Ah Val-Vs.1 Gly-Leu Tyr-Tyr Pro-Ala Ala-Pro LyS-Ah Ala-Asp Asp-Ala Ala-His His-Ala Trp-Cly Ala-Ala-Ala< Leu-Gly-Gly ACTH-(l-4) ACTH-(I-10)

SOUIW”

1 1 2 2 3 2 4 4 1 1 1 1 1 5 6 4 7 8

eleavedlminlmg

extract

protein Purieled dipeptidase

-DlT

+DTTb

-DTT

+Dl-T

0.60 0.12 0.48 0.44 0.22 0.01 0.98 0.05 0.03 0.01 0.02 0.03 0.002 0.14 0.28 0.08 0.01

0.06 0.01 0.07 0.08 0.03 0.01 0.03 0.00 0.03 0.90 0.002 0.01 0.007 0.15 0.19 0.05 -

65.8 5.3 50.0 57.9 27.6 2.4 3.4 0.0 0.0 0.4 0.0 2.3 0.3 0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0

the corresponding enzyme of other tissues. It is active against a wide range of neutral dipeptides, some exceptions being TrpGly and Ala-Pro. The presence of more than one enzyme in the preparation cannot be ruled out at the present state of purification. The activity of the dipeptidase toward substrates with acidic or basic residues appeared limited and depended on amino acid sequence. Thus Lys-Ala was not cleaved at all and the activity toward Asp-Ala and His-Ala was much lower than that toward Ala-Asp and Ala-His, respectively. A comparison between the activity of the supernatant preparation and that of the purified dipeptidase is also included in Table V. The Ser-Met-splitting enzyme appears to represent the total hydrolytic activity of the supernatant with regard to many of these substrates. It should be kept in mind, however, that the specific activity of the dipeptidase is very high in relation to other enzymes with overlapping specificity, and so enzymes that make only a minor contribution to the splitting of a particular dipeptide under the conditions of Table V can still be of physiological importance. As regards ACTH fragments, the purified enzyme contains all or almost all of the Ser-Tyr- and Ser-Met-cleaving activity present in the cytosol. It is inactive toward ACTH-(l-4) and other ACTH sequences with three or more residues. DISCUSSION

’ 1, Vega; 2, Mann; 3, Cycle; 4, Sigma; 5, Chemalag; 6, Miles; 7, Baehem; 8, Organon. ’ The concentration of DTT was 1 mu. F For this and other peptides with more than two residues, the values represent pmol of amino acid released/min/mg.

The action of mouse brain cytosol in hydrolyzing ACTH-(l-4) to its constituent amino acids can be accounted for almost

294

NEIDLE

AND REITH

entirely, by the action of two enzymes, an aminopeptidase and a dipeptidase. Based on its spectrum of activity toward amino acyl-/3-naphthylamides, the aminopeptidase is similar to arylamidases isolated from brain and other mammalian tissues. Although peptides are the presumed endogenous substrates for the arylamidases, little is known about their specificity toward these substrates. From our experiments with a small number of ACTH fragments and other peptides, the mouse brain enzyme appears to be an aminooligopeptidase of broad specificity. Because of its high specific activity toward substrates such as Leu-Gly-Gly and LeuP-naphthylamide, care must be taken to differentiate it from other enzymes such as aminotripeptidase (EC 3.4.11.4) and leucine aminopeptidase (EC 3.4.11.1). Inhibition by puromycin and activation by sulfhydryl-containing compounds may be useful for this purpose. Although arylamidase does not seem to be an appropriate name for this enzyme, a more descriptive name based on its activity toward naturally occurring substrates would require more information on peptide specificity than is currently available. The dipeptidase cleaving seryl methionine has a well-defined specificity, very similar to that of the “ascites dipeptidase” of Patterson et al. (22), the “intestinal dipeptidase” of Noren et al. (21), and the “master dipeptidase” of Das and Radakrishnan (23). These enzymes, in turn, resemble the glycylleucine dipeptidase of Smith (24), which has been given the IUPAC designation EC 3.4.13.2. Again, none of these names seem satisfactory. Since this enzyme is the best characterized of the dipeptidases and its localization in the cytoplasm has been established, we propose that it be called cytoplasmic dipeptidase I (CDP I), in accordance with the nomenclature of McDonald et al. (25). Although the two enzymes that we have characterized appear to be the major activities which degrade ACTH-(l-4), it is likely that minor pathways involving other degradative enzymes are also present and acting simultaneously. These secondary pathways might play a disproportionately

large role in the metabolism of N-terminal ACTH fragments, if, for example, they have a preferential localization within the brain relative to the distribution of the hormone, or if they have higher affinity for the terminal sequences than the enzymes that we have described. Even if the reaction sequence involving arylamidase and CDP I is the obligatory degradative pathway for ACTH-(l-4), larger fragments may not give rise to this intermediate and so these reactions would not have a direct link to ACTH metabolism. Our interest in these enzymes, at present, is based on the possibility that they are part of a group of highly active peptidases of low specificity that are responsible for the final steps in the metabolism of a wide variety of peptides within the brain, including those arising from other ACTH sequences. We expect that studies, similar to those reported here, involving other ACTH fragments will help to clarify these relationships. The rate-limiting steps in ACTH metabolism (including those which modulate its physiological activities) occur earlier in the degradation sequence (4). It seems possible that when these are determined, an overall pattern will be found in which the earlier steps besides altering the function of the hormone specifically give rise to fragments which can rapidly and efficiently be converted to free amino acids by terminal degradative pathways such as those reported here. ACKNOWLEDGMENTS We are indebted to Dr. M. Banay-Schwartz for help with gel electrophoresis and to Mrs. L. Bazzurro for technical assistance. REFERENCES 1. DE WIED, D., WITTER, A., AND GREVEN, H. M. (1975) Bioc/z~~m.Phurwzacol. 24, 1463-1468. 2. DE WIED, D., AND GISPEN, W. H. (1977) in Peptides in Neurobiology (Gainer, H., ed.), pp. 397-448, Plenum, New York. 3. DE WIED, D. (1977) Life Sci. 20, 195-204. 4. REITH, M. E. A., NEIDLE, A., AND LAJTHA, A. (1979) Arch. Biochem. Biophys. 195, 478-484. 5. REITH, M. E. A., AND NEIDLE, A. (1979) Trans. Amer. Sot. Neurochem. 10. 104.

ADRENOCORTICOTROPHIC

HORMONE-BREAKDOWN

6. PETERSON, G. L. (1977) Anal. Biochem. 83,346356. 7. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 8. PERRY, T. L., STEDIVAN, D., AND HANSEN, S. (1968) J. Chromatogr. 38, 460-466. 9. ROTH, hi., AND HAMPAI, A. (1973) J.‘Chromatogr. 83, 353-356. 10. HAYASHI, M. (1978) J. Biochem. 84, 1363-1372. 11. SUDA, H., TAKAAKE, A., TAKEUCHI, T., AND UMEZAWA, H. (1976) Arch. Biochem. Biophys. 177, 196-200. 12. BRECHER, A. S., AND SUSZKIW, J. B. (1969) Bioch.em. .I. 112, 335-342. 13. EWIS, S.. AND PERRY, M. (1966) J. Biol. Chem. 241, 3679-3686. 14. NEIDLE, A., AXD LAJTHA, A. (1976) Prob. Brain Biochem. 4. 48-.58. 15. MARKS, N., DATTA, R. K., AND LAJTHA, A. (1968) J. Bid. Chem. 243, 2882-2889.

IN MOUSE

BRAIN

CYTOSOL

2%

16. LAEMMLI, U. K. (1970) Nature flondon) 227, 680435. 17. SUSZKIW, J. B., AND BRXHER, A. S. (1970) Biochemistry 9, 4008-4017. 18. HAYMAN, S., AND PATTERSON, E. K. (1971) J. Biol. Chem. 246, 600-669. 19. NEIDLE, A., AND CHEDEKEI., M. (1971) Trans. Amer. Sot. Neurochem. 2, 98. 20. DAS, M., AND RADHAKRISHNAK, A. N. (1972) Biochem. J. 128, 463-465. 21. NOREN, O., Sj&zTRI)M, H., AND JOSEFSSOK, L. (1973) B&him, Biophys. Acta 327,446-456. 22. PA?TERSOX, E. K., GATMAITAN, J. S., AXVII HAYMAN, S. (1973) Biochemistry 12,3701-3709. 23. DAS, M., AND RADHAKRISHXAN, A. N. (1973) Biochem. J. 135,609-615. 24. SMITH, E. L. (1968) J. Bid. Chem. 176, 9-19. 25. MCDONALD, J. K., AND SCHWABE, C. (1977) in Protcinases in Mammalian Cells and Tissues (Barrett, A. J., ed.), pp. 312-391, NorthHolland, Amsterdam.