Evidence for conversion of N-Tyr-MIF-1 into MIF-1 by a specific brain aminopeptidase

Neurochem. Int. Vol. 6, No. 3, pp. 347 353, 1984 Printed in Great Britain. All rights reserved

0197-0186/84 $3.00 + 0.00 Copyright (~3 1984 Pergamon Press Ltd

EVIDENCE FOR CONVERSION OF N-TYR-MIF-1 INTO MIF-1 BY A SPECIFIC BRAIN AMINOPEPTIDASE NEVILLE MARKS, MARTIN J. BERG, ABBA J. KASTIN*t and DAVID H. CoY t Center for Neurochemistry, Nathan S. Kline Institute for Psychiatric Research, Ward's Island, NY 10035, *Endocrinology Section, VA Medical Center and tDepartment of Medicine, Tulane University School of Medicine, New Orleans, LA 70146, U.S.A. (Received 21 September 1983; accepted 24 October 1983) Abstract--N-Tyr-MIF-1 (Tyr-Pro-Leu-Gly.NH2), an immunoreactive neuropeptide exhibiting saturable high affinity binding in rat brain was found to be converted into MIF-1 (Pro-Leu-Gly.NH 0 by a specific brain aminopeptidase present in rat brain homogenates or cytosol, but with low activity associated with synaptosomal plasma membranes and microsomes. Conversion occurred at a rate of 16 #mol per g w/wt per h and was unaffected by puromycin but inhibited by bestatin (I50, 5 × 10-5 M). Aminopeptidases purified from cytosolic fractions of rat brain (arylamidase), mouse brain (Mn2+-activated aminopeptidase) or porcine kidney (leucine aminopeptidase) were inactive towards N-Tyr-MIF-I but degraded MIF-I with release of Leu-GIy-NH 2 as detected by RP-HPLC procedures. Morphiceptin (Tyr-Pro-Phe-Pro.NH2) , a /~ opioid agonist, also acted as a substrate for the N-Tyr-MIF-1 converting enzyme with cleavage of the Tyr-Pro bond. These tetrapeptides, but not MIF-1 or its N-blocked analogs, were degraded in vitro by a metalloendopeptidase purified from kidney membranes. Since dipeptide products were not detected for crude extracts, a significant role for brain metalloendopeptidase on turnover can be excluded. Thus the results point to the presence of a specific (X-Pro-degrading) aminopeptidase in brain cytosol as an enzyme responsible for converting N-Tyr-MIF-I and inactivating morphiceptin.

N-Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) is a neuropeptide discovered by use of specific antisera directed against the tetrapeptide, but having low crossreactivity towards MIF-1 (Pro-Leu-Gly.NH2) and oxytocin (Kastin et al., 1981a). Immunoreactive tetrapeptide, present in relatively high concentrations in rat pineal gland, striatum, thalamus, and hypothalamus, was shown to change in concentration as a result of some neuroendocrine manipulations (e.g. pinealectomy) leading to interest into its formation and metabolism (Kastin et al., 1981b). The biological profile of N - T y r - M I F - I together with evidence for the existence of saturable high affinity binding sites in discrete rat brain areas (cortex, striatum, amygdala) (Zadina et al., 1982) supports the existence of this tetrapeptide as a novel neuropeptide targeted towards a specific receptor. Based on these observations, the present studies were directed to examination of its mode of inactivation and whether it could serve as an alter-

nate source for MIF-1. The remarkable spectrum of biological activities o f M I F - 1 (Kastin et al., 1980) has led to detailed studies on its metabolism by brain extracts (Hui et al., 1980; Marks and Walter, 1972; Neidle et al., 1980; Simmons and Brecher, 1973; deWildt et al., 1982), by sera of different species (Walter et al., 1975; Witter et al., 1980), or by purified enzymes such as arylamidases (Marks and Walter, 1972; Neidle et al., 1980) or divalent activated aminopeptidase similar in properties to leucine aminopeptidase (Neidle, 1981; Neidle et al., 1980; Simmons and Brecher, 1973). Most of these studies confirm observations that MIF-1 serves as a substrate for brain aminopeptidases with release of L e u - G I y ' N H 2 as an inactive metabolite (Marks and Walter, 1972). In the present study, we report the presence of an enzyme converting N-Tyr-MIF-1 into M I F - I but distinct from that of known brain aminopeptidases, or leucine aminopeptidase of other tissues. EXPERIMENTAL PROCEDURES

Abbreviations: MIF-1, melanotropin inhibiting factor; LAP, leucine aminopeptidase; PLG, prolyl-leucylglycinamide

Preparation of subcellular fractions Male Sprague-Dawley rats, 4-6 weeks old, were decapitated and whole brains were removed. All of the following 347

348

N. MARKS el al.

procedures were performed at 4 C. Brain was homogenized in 10 vol of 0.32 M sucrose and centrifuged at 1000 g for 10 rain to remove debris: the resulting supernatant was centrifuged at 17,500 g for 20 rain: to yield the postmilochondrial supernatant and pellet. The postmitochondrial pellet was homogenized and applied to a sucrose density gradient of 0.32 0.8 1.2 M with eentrifugation at 50,000 g for 2 h. Synaptosomes were located at the 0.8 1.2 M boundary. These were collected and osmotically shocked for 1 h in I 1 volumes of ice-cold 20 mM Tris HCI, pH 7.6; synaptosomal plasma membranes (SPM) were prepared by centrifugation at 16,500 g for 20 rnin and homogenized in 20 mM Tris HCI pH 7.6 plus 0.2". (v:v) Triton X-100. The post-mitochondrial supernatant was centrifuged at 100,000 g tot 1 h to yield post-microsomal supernatant and microsomes. Microsomes were homogenized in 20 mM Tris HC1 pH 7.6 plus 0.2°11 v/v Triton X-100.

Preparation ~/ arylamidase Posl-mitochondrial supernatant containing 1 mM dithiothreitol (Cleland's reagent) was applied to an anion exchange column of Cellex D (Bio-Rad, Richmond, CA) equilibrated with 20 mM Tris-HC1, pH 7.6, I mM Cleland's reagent, and 0.01 mM ZnC1,. The column was eluted with two bed volumes of buffer followed by a linear gradient of 0.05-0.25 M NaCI. Active fractions were pooled and applied to another anion exchange column of DE53 cellulose (Whatman, Piscataway, N J) and treated as above. Active fractions were pooled and dialyzed overnight against 50 mM sodium phosphate, pH 7.0 plus I mM Cleland's reagent and 0.01 mM ZnC1,. The sample was applied to a column of hydroxylapatite (Calbiochem. San Diego, CA) equilibrated in the same buffer. The column was eluted with three bed volumes of buffer followed by a linear phosphate buffer gradient of 50-250 raM. Active fractions were dialyzed overnight against 20 mM Tris HC1 pH 7.6 containing 1 mM Cleland's reagent. The enzyme was homogeneous when analyzed by the gel-electrophoretic method of Weber and Osborn (1969) and gave a single band of M~ 103,000. Enzyme was stored in small aliquots in 50". v/v glycerol with 1 mM Cleland's reagent at - 2 0 C. Purfhcation O/ metalloendopeptidase Enzyme was purified from rat kidney membranes as described previously (Benuck et al., 1982). Pur(licalion ~/ leucine aminopeptidase Mn 2~ activated aminopeptidasc of mouse brain cytosol was furnished by Amos Neidle (Nathan S. Klinc Institute, Institute for Psychiatric Research, Ward's Island, NY). The enzyme was purified by the method described by Neidle (1981). EnQvme assays Arylamidase was assayed by use of 0.04 /Lg of enzyme plus 75 nmol leucyl-2-naphthylamide, 0.3 /tmol Cleland's reagent, and 120/~mol Tris-HCl, pH 7.6 in a final volume of 3 ml. The reaction was carried out at 3 7 C with continuous monitoring of released 2-naphthylamide by a Turner Model 110 Fluoromonitor with excitation at 365 nm and emission at 415 nm. Purified endopeptidase was tested using 0.02 ,ug of enzyme plus 10 nmol leucine-enkephalin (Vega Biochemicals. Tucson. AZ) plus 4 /tmol of Tris-HCI. pH 7.6 in a final

volume of 100 t~1. Reaction ~as pcrlormed at 37 C for 15 min and terminated by the addition of 20 i~1 of 201'i; (v/v) perchloric acid. Aliquots of 50 itl were injected into a 5 I~ C~s reverse-phase Spherisorb column (Applied Science, State College, PAl for analysis by high performance liquid chromatography (HPLC) and isocratically eluted with 5",. acetonitrile in 0. I M phosphate pH 3.0 with u.v. detection of released Tyr-Gly-Gly at 214 nm. Leucine aminopeptidase from brain was assayed using 0.14 Itg of enzyme, 50 nmol Tyr-Gly-Gly, 2.5 ltmol Mn('L, and 5.0 t~mol of bicine buffer, pH 8.5 in a final volume 100 /~1. Reaction was terminated after incubation at 37 C for 5 rain with 20 itl of perchloric acid (v:v). Porcine kidney (Sigma lot No. II1F-8016t LAP was incubated under identical conditions but using 10 pg of protein (sp. act. of 140 units per mg using Leu-NH: per rain). Samples were centrifuged, and 50 tzl aliquots were applied to a column under the conditions stated above for HPLC analysis of frec tyrosine and remaining substrate. Brain LAP had a specific activity of 79.5 ,umol with the tripeptide substratc or 170 units per mg protein per min using Leu.NH~ as the substrate. Assay.v ~![ M l f - 1 and analog.~ Aliquots of homogenate, posl-mitochondrial or microsomal supernatants, SPM (30 80/~g protein), microsomcs (640 /tg) or purified enzymes (0.9 1.2 Itg of protein) wcrc added to 2/tmol of Tris- HCI, pH 7.6, 100 nmol of substrate and 0.2/tmol ot cysteine to a final w~lume of 200 itl. Aflcr incubations of up to 21 h a l 37 C, reactions were terminated by addition of 50 t~l of 20", (v/v) perchloric acid. Samples were centrifuged, and 10 ,ttl aliquots applied to a 5 l~ Cis Adsorbosphere column (Applied Sciences) for HPLC analysis by a procedure described previously (Benuck and Marks. 1980). RESU ISI'S T h e m e t a b o l i s m o f T y r - M I F - 1 / M I F - I was investigated with rat brain h o m o g e n a t e , p o s t m i t o c h o n d r i a l or m i c r o s o m a l s u p e r n a t a n t s , purified S P M , a n d m i c r o s o m e s . Rates o f p e p t i d e m e t a b o l i s m were quantified on the basis o f s u b s t r a t e utilization or a p p e a r a n c e o f p r o d u c t s as identified by R P - H P L C procedures. M I F - 1 was d e g r a d e d by rat brain h o m o g e n a t e s at a rate o f 12.2 l~mol per g wet weight p e r h or 128 /~mol per g p r o t e i n per h with a p p e a r a n c e o f LeuGIy. NH2 as a p r o d u c t (Fig. 1). M i n o r peaks observed in the c h r o m a t o g r a m were derived from n o n specific materials associated with h o m o g e n a t e s and are insignificant c o m p a r e d to the peak for LeuG l y - N H 2 which has a r a t h e r low a b s o r p t i o n at 21(~214 n m as c o m p a r e d to M I F - I , Values tbr M I F - I d e g r a d a t i o n by h o m o g e n a t e were 2-fold higher than by s u p e r n a t a n t , a n d 4(~58 higher fold t h a n with purified S P M or m i c r o s o m e s (Table 1). Rates were 1.2-5 fold higher for m e t a b o l i s m o f N - T y r - M I F - I as c o m p a r e d to M I F - 1 in all three fractions. D e g r a d a t i o n o f N - T y r - M 1F- 1 was a c c o m -

Conversion of N-TYR-MIF-I 0.0067

into M I F - I

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(B)

¢4 3~

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Fig. 1. Profile o f p r o d u c t s separated on a reverse phase C~8 c o l u m n after i n c u b a t i o n o f MIF-1 ( P r o - L e u - G l y . NH2, P L G ) with rat brain h o m o g e n a t e s for 1 h (B) or with purified rat brain a r y l a m i d a s e for 21 h (D) at 37°C, as c o m p a r e d to i n c u b a t e d c o n t r o l s (A, C). I n c u b a t i o n s were a c c o m p a n i e d by decrease in P L G with a p p e a r a n c e o f L e u - G I y . N H 2 ( L G ' N H 2) as the product. L e g e n d to Table 2.

panied by release of Tyr and MIF-1 as the only identifiable products as illustrated for the postmitochondrial supernatant (Fig. 2). Breakdown of N-Tyr-MIF-1 by this fraction was not linear with time but appeared to reach a maximum value within

10 min of incubation under the conditions selected (Fig. 2). Morphiceptin (Chang et al., 1982), a tetrapeptide with an N-terminus dipeptide similar to that of N-Tyr-MIF-1, was degraded by homogenate and

Table 1. Degradation of N-Tyr-MIF-1/MIF-I peptides and morphiceptin by rat brain homogenate or subcellular fractions

Sabstrate Pro-Leu-Gly'NH 2 Tyr-Pro-Leu-Gly. NH 2 Tyr-Pro-Phe-Pro- NH 2

Homogenate Supt* SPM Microsomes #mol per g wet wt (Act.) or protein per h (Sp. act.) Act. Sp. act. Act. Sp. act. Act. Sp. act. Act. Sp. act. 12.2 15.8 4.6

128 165 47.5

6.0 10.0 6.9

201 334 230

0.21 1.0 0

42 198

0.32 0.42 1.1

23.5 38.0 102.0

*Post-mitochondrial supernatant; SPM denotes synaptosomal plasma membrane. Incubation mixture of 200 ,ul 10 mM Tri~HCI, pH 7.6 contained 80 #1 of tissue fractions (3~660 #g protein), 100 nmol substrate, 0.2 #mol cysteine was incubated at 37°C for periods up to 21 h. Reaction terminated with 50 #1 perchloric acid (20~ w/v) and aliquots analyzed by reverse phase in a C~8 column as described in the Methods Section. No breakdown was observed for pyroGlu-Leu-Gly'NH 2 or cycloLeu-Gly. Values are the means of 3~4 determinations agreeing within 12~.

350

N. MARKSet al.

002

No metabolism was observed for N-blocked MIFl analogs pyroGlu-Leu-Gly.NH2 or cyclo(Leu-Gly) by brain homogenates when incubated under conditions that led to breakdown of N-Tyr-MIF-1/MIF-1. Purified e n z y m e s >-

L L: 0 e ~

4i

I0

3

Min

20

30

I

l

TYR--PRO--

LEU-GLY- NH2

1

Peptides were incubated with enzyme purified from brain by the procedures of Marks et al. (1968) with modifications as described in the Methods section for arylamidase, and compared with the action of two leucine aminopeptidases, one purified from kidney cytosol and another from mouse brain (Neidle, 198 l). The results summarized in Table 2 showed that MIF-1 served as a substrate for aminopeptidases, but that N-Tyr-MIF-I and morphiceptin or the Nblocked MIF-1 analog were not degraded even upon prolonged incubations of up to 21 h. Degradation of M1F-1 was accompanied by appearance of LeuGly-NH: as an intermediate. The rapid disappearance of the dipeptide upon longer incubations

TYROSINE

/:

~

O

~

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PRO-LEU-GLY-NH2

L

L

I

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2. Conversion

of

N-Tyr-MIF-1

\

(Tyr-Pro-Leu-

Oly. NH2) by rat brain post-mitochondrial supernatant with time of incubation. The insect depicts the HPLC profile at 60 min with identification of products. Leu-Gly.NH 2 coelutes with Tyr but has an insignificant absorption at the sensitivity employed (3-fold lower than Fig. 1).

i

I

10

post-mitochondrial supernatant, but not by SPM, with release of Tyr and one other product tentatively identified as Pro-Phe-Pro.NH2 (Table 1, Fig. 3). Rates of morphiceptin metabolism were one third lower in the homogenate and 0.7 that of N-Tyr-MIF1 in the supernatant fraction. A similar spectrum of products but with slightly lower rates of metabolism were observed for all three peptides when incubated with post-microsomal supernatants. Morphicepton was degraded by microsomes at rates higher than for MIF-1 or N-Tyr-MIF-1, but at a lower rate by homogenates or SPM. Metabolism of morphiceptin, unlike N-Tyr-MIF-I, was proportional with time of incubation up to periods of 1 h (Figs 2, 3). The conversion of N-Tyr-MIF-1, or metabolism of morphiceptin, by the post-microsomal supernatant fraction was unaffected by 10 5M puromycin but was inhibited by bestatin with an 150value of 5.0 x 10 5M (data not shown).

TYROSINE ~

20

i

30

,

-8

/ ~ T ° MORPHICEPTIN YR-PRO-PHE-PRO- NH2)

/ 0

/ I

30

I

60

Time ( m i n )

Fig. 3. Degradation of morphiceptin (Tyr-Pro-PhePro. NH2) by post-mitochondrial supernatant with time of incubation at 37uC. Inset depicts HPLC profile obtained at 60 min with identification of products.

Conversion of N-TYR-MIF-I into MIF-I

351

Table 2. Degradation of N-Tyr-MIF-1/MIF-1 peptides, morphiceptin and enkephalin by purified aminopeptidases or metalloendopeptidase Substrate Products detected MIF-I (Pro)Leu-Gly"NH2 Aminopeptidase* N-Tyr-MIF-I None Arylamidase pyroGlu-Leu-Gly. NH 2 None Brain LAP Kidney LAP Morphiceptin None Leu-enkephalin Tyr; Gly-Gly-Phe-Leu MIF-I None Metalloendopeptidaset N-Tyr-MIF-I Tyr-Pro; Leu-Gly. NH 2 pyroGlu-Leu-Gly. NH 2 None Morphiceptin Tyr-Pro, (Phe-Pro .NH2) Leu-enkephalin Tyr-Gly-Gly; Phe-Leu *Aminopeptidases used and specific activities (/~mol per mg protein/min) were for rat brain arylamidase (MIF-I, 3,8; Leu-enkephalin, 94.8) mouse brain Mn2÷ activated LAP (MIF-I, 18.2; Leu-enkephalin, 12.2), and porcine kidney LAP. tMetalloendopeptidase was purified from rat kidney particulates (N-Tyr-MIF-I, 0.48; Leuenkephalin, 6.0; morphiceptin, 0.24). Cyclo-Leu-Gly did not serve as a substrate for purified enzyme. Products not separated or identified by HPLC are in parentheses.

indicate t h a t it served as a substrate for the a m i n o peptidases. F o r purposes o f c o m p a r i s o n to other N-terminal tyrosyl peptides, the enzymes were i n c u b a t e d with Leu-enkephalin a n d products identified by H P L C . The results showed that e n k e p h a l i n served as a substrate with release o f Tyr a n d the des-Tyr peptide as the only p r o d u c t s detected. The tetrapeptides but not M I F - I or N-blocked M I F - 1 analogs served as substrates for a kidney m e t a l l o e n d o p e p t i d a s e purified from particulate fractions. B r e a k d o w n o f N - T y r - M I F - 1 was a c c o m p a n i e d by release o f Tyr-Pro a n d L e u - G I y . N H 2 as the two p r o d u c t s (Fig. 4); b r e a k d o w n of m o r p h i c e p t i n was a c c o m p a n i e d by a decrease in substrate with the a p p e a r a n c e of a new peak in the H P L C chro002

m a t o g r a m with a retention time o f 20 rain for TyrPro. I n c u b a t i o n of Leu-enkephalin with metalloendopeptidase led to release of Tyr-Gly-Gly a n d Phe-Leu in agreement with o u r previous observations (Benuck a n d M a r k s , 1980; Benuck et al., 1982). D~CUSSION

Evidence is presented that rat brain extracts contain a n enzyme converting N - T y r - M I F - I into MIF-1 that is distinct from that of k n o w n brain a m i n o peptidases (arylamidase, LAP). The present observations for two rodent b r a i n aminopeptidases or porcine kidney L A P agree with those o f Hayashi (1978) for m o n k e y b r a i n arylamidase which did n o t cleave X - P r o - b o n d s such as the A r g L P r o 2 of bra-

002

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(A)

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0¢~

I

I

>~-

I1:

0

z I )-

K I

I0

20

Min

30

10

20

J 3O

Min

Fig. 4. HPLC profiles on a RP-C,s column for N-Tyr-MIF-I (A) incubated for 60 min, and for morphiceptin (B) incubated for 30 rain following incubation with purified rat kidney metalloendopeptidase. Incubations were accompanied by decrease of substrate compared to controls with appearance of two products for N-Tyr-MIF-1 identified as Tyr-Pro and Leu-GIy.NH2, and a new peak for morphiceptin with retention time equivalent to Tyr-Pro.

352

N. MARKSel a[.

dykinin or substance P, and with older findings on the specificity of purified LAP (Delange and Smith, 1971). Purification of the N-Tyr-MIF-1 converting enzyme is required in order to compare its specificity with other enzymes cleaving X-Pro-bonds (aminopeptidase P), none of which are available in highly purified form from mammalian tissues. Fleminger et al. (1981) and Yaron (1974) purified an enzyme from E. coil acting on the ArgO-Pro 2 bond of kinin-9 and substance P or other X-Pro-substrates that included a synthetic fluorogenic material, Phe(NO2)-Pro-HN-(CH2)-NH-o-aminobenzoyl. The latter substrate has some analogy to N-Tyr-MIF-1 and morphiceptin in having an aromatic (hydrophobic) group adjacent to Pro. With respect to mammalian tissue, Dehm and Nordwig (1970) have partially purified an enzyme from porcine kidney microsomes of higher M~ that degraded a number of tri, tetra- and polypeptides with X-Pro-bonds. This enzyme was also found in soluble fractions and along with the observations of Fleminger et al. (1981) for the presence of enzyme in human serum or calf lung (aqueous) extracts indicate that cytosol is a potential source for aminopeptidase P. In this context, the N-Tyr-MIF-1 converting enzyme was enriched in brain soluble fractions as compared to homogenate. Brain enzyme may, however, differ from that of kidney since very little activity was associated with microsomes or with purified membranes (SPM). For purposes of enzyme characterization, morphiceptin seems more suited than N-Tyr-MIF-1, since degradation was linear with time of incubation. The non-linear conversion of N-Ty~-MIF- 1 with time points to product inhibition, a feature of interest in mechanisms regulating intracellular production of peptides. Brain homogenates are known to contain enzyme(s) capable of recognizing the N-termini of substance P (Benuck and Marks, 1975) and kinin-9 (Marks and Pirottm 1971) but these have not yet been characterized or examined for specificity. The possibility also exists that prolidase (imidodipeptidase) acts on X-Pro polypeptides as reported by Hill and Schmidt (1962) but discounted by Yaron and Mlynar (1968) and Sjostrom et al. ( 1971 ) as being attributable to aminopeptidase P contamination. Prolidase occurs in brain fractions but has not been purified sufficiently for studies on polypeptide specificity (Hui and Lajtha, 1978). The source of M1F-1 in brain and its full biological properties are unresolved questions. The potent central effects of MIF-1, nevertheless, led to studies on its metabolism with some evidence that it may be formed from oxytocin by action of brain endo-

peptidases (Walter et al., 1973). Most studies on MIF-I indicate that cleavage occurs at the Pro-Leu bond and is the primary mode of inactivation, but techniques used to follow metabolism have not demonstrated, unequivocally, release of Leu-Gly. NH 2 as an intermediate, in the present study using HPLC, we showed that Leu-Gly. NH~ is formed as a result of cleavage. The value found for MIF-I degradation of 12.2/tmol per g wet weight per h rat brain homogenate agrees well with those reported by Hui et al. (1980) for rat brain and Neidle et al. (1980) for mouse brain using alternate methods of measurement. The ratio of relative activities for brain arylamidase versus Mn2~-activated aminopeptidase with M1F-I and enkephalin as substrates, suggest that the latter enzyme is more active on MIF-I turnover, while the former more rapidly degrades the pentapeptide (KN~ 64.3 pM). The use of N-Tyr-MIF-I/MIF-I presents opportunities to characterize brain exo- and endopeptidases as illustrated here and for aminopeptidases known to cleave the Pro-Leu bond of MIF-I. N-Tyr-MIF-1 and morphiceptin were shown to act as substrates for a purified metalloendopeptidase, but it is unlikely that the brain enzyme plays a significant role in ti~o since incubation with homogenates did not yield detectable levels of dipeptides. The kidney metalloendopeptidase used has properties identical to that of a brain enzyme (Ben uck et al. 1982), both of which act on Leu-enkephalin at the Gly-Phe site to release the N-terminal tripeptide. As in the case of N-TyrMIF-I, metalloendopeptidase inactivation of enkephalins appears to be less relevant than the action of aminopeptidases. In view of the prevalence of X-Pro-peptides in the brain, there would appear to be need for purification of a specific aminopeptidase acting on the N-terminus. N-Tyr-MIF-I or morphiception appear to be useful substrates tor this purpose. Supported in part by NINCDS (NS12578) (NM) and the Veterans Administration and ONR (AJK). Acknowledgements

REFERENCES

Bhargava H. N., Pandey R. N. and Matwyshyn G. A. (1983) Effects of Pro-Leu-Gly. NH, and cyclo (Leu-Gly) on morphine-induced antinociception and brain 6, /~, and k opiate receptors. Liii' Sci. 32, 2095 2101. Benuck M. and Marks N, (1975) Enzymatic inactivation o1" substance P by a partially purified enzyme from rat brain. Biochem. biophys. Res. Commun. 65, 153-160. Benuck M. and Marks N. (1980) Characterization of a distinct membrane bound dipeptidyl carboxypeptidase

Conversion of N-TYR-MIF-l into MIF-l inactivating enkephalin in brain. Biochem. biophys. Res. Commun. 95, 822-828. Benuck M., Berg M. J. and Marks N. (1982) Rat brain and kidney metalloendopeptidase: enkephalin heptapeptide conversion to form a cardioactive neuropeptide, PheMet-Arg-Phe-NH 2. Biochem. biophys. Res. Commun. 107, 1123-1129.

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