Eur. J. Biochem. 144, 271-278 (1984) ( FERS 1984

Localization of a narrow-specificity thyroliberin hydrolyzing pyroglutamate aminopeptidase in synaptosomal membranes of guinea-pig brain

Brendan O’CONNOR and Gerard O’CUINN Department of Biochemistry, University College, Galway; and Department of Life Sciences, Regional Technical College, Galway

(Received April 6/June 28, 1984) - EJB 84 0366

In this paper we report the presence of a particulate pyroglutamate aminopeptidase in guinea-pig brain tissue. This activity appears to reside in the synaptosomal membrane and could be released from the membrane by treatment with papain or Triton X-100. By contrast with a previously described broad-specificity soluble pyroglutamate aminopeptidase from guinea-pig brain tissue, the enzyme released from the synaptosomal membrane preparation removed pyroglutamic acid from thyroliberin, acid thyroliberin and < Glu-His-Gly alone of the peptides tested. Unlike the soluble tissue enzyme the present enzyme was inhibited by the presence of EDTA and the activity released from synaptosomal membranes by papain was found to have a relative molecular mass of 230000, almost one order of magnitude greater than that reported for the soluble enzyme.

Research into thyroliberin (< Glu-His-ProNH,) break- down in various tissues has shown that the breakdown of this molecule could be initiated by hydrolysis of either of two bonds. Thyroliberin may be deaminated to acid thyroliberin by a soluble post-proline-cleaving endopeptidase [I - 61 or alter- natively may be converted to <Glu and His-ProNH, by a soluble pyroglutamate aminopeptidase [7]. Previous studies in vitro with rat adenohypophysis [8] and with guinea-pig brain [Y] have shown that these two enzymes acting in concert with a soluble post-proline dipeptidyl aminopeptidase and a soluble proline dipeptidase could effect the complete hydrolysis of thyroliberin to its constituent amino acids. The soluble pyro- glutamate aminopeptidase was found to have a broad substrate specificity and to exhibit activity against a number of biologi- cally active and synthetic peptides which contain a < Glu at the N terminus [8,Y]. By contrast a pyroglutamate aminopeptidase has been purified from rat serum [lo] and from porcine serum [ l l , 121, which displayed a much narrower specificity, hydrolys- ing only thyroliberin or closely related molecules. Preliminary reports have indicated the presence of a second tissue pyroglu- tamate aminopeptidase activity in particulate fractions of brain from guinea-pig [I31 and from rabbit [14]. A subsequent study indicated that the particulate pyroglutamate aminopeptidase may reside in a synaptosomal membrane fraction [15]. In view of the putative neurotransmitter or neuromodulator role for

Abbreviations. < Glu, pyroglutamic acid 5-pyrrolidone-2-carboxy- lic acid; < Glu-MCA, pyroglutamate-7-amido-4-methylcoumarin ; Cbz-Gly-Pro-MCA, N-benzyloxycarbonylglycylproiine-7-amido-4- methylcoumarin; < Glu-His-Pro, acid thyroliberin or deaminated thyroliberin; i Glu-Ah, pyroglutamylalanine; < Glu-Val, pyroglu- tamylvaline; < Glu-Pro, pyroglutamylproline; His: Pro, histidyl- proline diketopiperazine; AMC, 7-amino-4-methylcoumarin; iodoni- trotetrazolium, 2-(p-iodopheny1)-3-~-nitrophenyltetrazo1ium) chlo- ride.

Enzymes. Lactate dehydrogenase (EC 2.1.1.27); succinate dehy- drogenase (EC 1.3.99.1); 5’-nucleotidase (EC 3.1.3.5); pyroglutamate aminopeptidase (EC 3.4.1 1.8); post-proline cleaving endopeptidase (EC 3.4.21.26); post-proline dipeptidyl aminopeptidase (EC 3.4.14. - ); proline dipeptidase (EC 3.4.1 3.9).

thyroliberin [I61 the presence of an enzyme in the synapto- somes, which is capable of degrading this peptide, is of considerable interest. In this paper we confirm the earlier preliminary report of a synaptosomal pyroglutamyl aminopep- tidase and compare its properties with those of the soluble tissue enzyme and with those of the serum enzyme.

MATERIALS AND METHODS

Materials

DL-Dithiothreitol, 2-iodoacetamide, p-chloromercuri- benzoate, N-ethylmaleimide, 1,lO-phenanthroline, 8-hy- droxyquinoline-5-sulphonic acid, benzamidine, bacitracin (B0125), puromycin, bestatin, phenylmethylsulphonyl fluo- ride, ninhydrin, the anorexogenic peptide (< Glu-His-Gly), pyroglutamate aminopeptidase (bovine liver), Triton X-100, thyroliberin, 7-amino-4-methylcoumarin (AMC), pyroglu- tamic acid adenosine triphosphate (disodium salt), 2-(p- iodophenol)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride and papain (papaya latex type 111) were obtained from Sigma Chemical Company (Poole, Dorset, England). Sephadex G-75 Sephadex G-200 and blue dextran were from Pharmacia Fine Chemicals AB (Uppsala, Sweden) cellulose phosphate P81 chromatography paper, Whatman no. 1 and no. 3 chromatog- raphy paper were from Whatman (Maidstone, Kent, England), <Glu-Pro and <Glu-Ala were from Cyclo Chemical (Los Angeles, CA, USA). Acid thyroliberin, luliberin, neurotensin, bombesin, eledoisin, bradykinin potentiating peptide B, pyroglutamate-7-amido-4-methylcoumarin ( < Glu-MCA) and < Glu-Val were from Bachem Feinchemikalien (Bubendorf, Switzerland) [Pro-2,3,4,5-3H]thyroliberin (spec. act. 102 Ci/mmol) was purchased from New England Nuclear (Dreieichenhain, FRG).

Determination of enzyme activities

Pyroglutamate aminopeptidase. Pyroglutamate aminopep- tidase was assayed using < Glu-MCA as substrate according to

272

the method of Fugiwara and Tsuru [I71 as modified by Browne and O'Cuinn [9]. Alternatively pyroglutamate aminopeptidase could be measured by using [Pr~-~H]thyroliberin as substrate by a modification of the method of Bauer and Kleinkauf [8]. [ Pro-3H]thyroliberin was diluted with unlabelled thyroliberin to give a specific activity of 0.125 Ci/mmol. 10-p1 aliquots of each sample to be assayed were added to lop1 diluted [Pro- 3H]thyroliberin (0.5 pCi/4nmol) and 10 pl 100 mM imidazole buffer pH 7.4 at 37 "C. The reaction was terminated by addition of 20p1 methanol and 10-p1 aliquots were spotted onto prewashed cellulose phosphate paper, which was then de- veloped using 1.0 M acetic acid. The start segment, containing the highly basic His-ProNH,, was cut out and placed in scintillation vials to which were added 1 ml 2 M ammonia and, 10 min later, 10 ml toluene-based scintillation cocktail. The conversion of thyroliberin to His-ProNH, could be measured by counting the vials in a scintillation spectrometer.

Post-proline cleaving enzyme. Post-proline cleaving enzyme was assayed using Cbz-Gly-Pro-MCA as substrate according to the method of Yoshimoto et al. [I81 as modified by Browne and O'Cuinn [9].

Ouabain-senritive Na' /K' A TPase. Ouabain-sensitive N a + / K f ATPase was estimated by the method of Severson et al. [19] and liberated phosphate was determined by the method of King [20].

Succinate dehydrogenase. Succinate dehydrogenase was assayed according to the method of Pennington [21] as modified by Porteous and Clark [22], in which iodonitrotetra- zolium was used as an artificial electron acceptor.

5'- Ribonucleotir~ase. 5'-Ribonucleotidase was measured by the method of Michell and Hawthorne [23] and released phosphate was determined by the method of King [20].

Lactate dehydrogenase. Lactate dehydrogenase was as- sayed according to the method of Kornberg [24] while occluded lactate dehydrogenase wa5 measured according to the method of Marchbanks [25].

Protein determination

Protein was determined by the Hartree modification [26] of the method of Lowry et al. [27] using bovine serum albumin as standard.

Denzonstration o j two thyroliberin-hydrolyzing pyroglutamate aminopeptidases in different subcellular locations qf guinea-p ig brain

Freshly isolated guinea-pig brains were homogenized in four volumes of ice-cold 0.32 M sucrose containing 100 mM imidazole buffer pH 7.4 using a Potter-Elvehjem homogenizer of clearance 0.075 - 0.125 mm. The tissue was disrupted by twelve up-and-down strokes of the pestle and the crude homogenate was centrifuged at 30000 x g for 30min. The resulting 30000 x g supernatant was stored at 4 ° C while the 30000 x gpellet was washed with two 10-ml aliquots ofwater to release occluded soluble enzyme activity and with two 10-ml aliquots of 0.15 M KCl to minimise adsorption of soluble enzyme activities to particulate structures. The 30000 x g pellet was finally resuspended in 10ml 0.32 M sucrose containing 100 mM imidazole pH 7.4 and assayed along with the 30000 x g supernatant for pyroglutamate aminopeptidase activity using [Pro-3H]thyroliberin and < Glu-MCA as substrates as de- scribed previously. These assays were conducted in buffers containing or omitting dithiothreitol and EDTA, each at a final concentration of 2 mM in the assay.

Subcellular fractionation procedures

Freshly isolated guinea-pig brains were homogenized in four volumes of ice-cold 0.32 M sucrose containing 100 mM imidazole buffer pH 7.4 using a Potter-Elvehjem homogenizer of clearance 0.075 - 0.125 mm. The tissue was disrupted by twelve up-and-down strokes of the pestle. The subsequent subcellular fractionation were based on the methods of Marchbanks [28j and of Whittaker and Barker [29]. Aliquots of each fraction generated were removed and stored at -20 "C until use for further assay.

The crude homogenate was centrifuged at 1000 x g for 10 min. The pellet obtained was washed with 0.32 M sucrose containing 100 mM imidazole pH 7.4. Following recentri- fugation the pellet was resuspended in 0.32 M sucrose contain- ing l00mM imidazole pH 7.4 (fraction PI ) while the super- natants from both centrifugations were combined (fraction s,) and centrifuged at 30000 x g for 30 min. As before the pellet was washed and resuspended in 0.32M sucrose containing 100 mM imidazole p H 7.4 (fraction P,) while the supernatants were combined (fraction S,). Aliquots of fraction P, were placed on discontinuous sucrose gradients consisting of 9 ml each or 1.6M, 1.2M and 0.8 M sucrose. Following centrif- ugation at 135000 x g for 75 min three bands of material were observed in the gradient, one on top of 0.8 M sucrose, one in the 1.2 M sucrose band and one in the 1.6 M sucrose band. Each fraction was diluted with equal volumes of water, centrifuged at I00000 x g for 60min and resuspended in 0.32M sucrose containing 100 mM imidazole pH 7.4 to give fractions A, B and C respectively.

Preparation of synuptosomul membrane fractions

Fraction P, was resolved by centrifugation into fractions A, B and C on discontinuous sucrose gradient as above. Fraction B was pelleted by centrifugation and resuspended in water. The preparation (3 ml) was subjected to gentle hand homogeniza- tion and placed over a second discontinuous sucrose consisting of 8ml 1.6M sucrose, 8ml 1.2M sucrose 6ml 0.6M sucrose and 5 m10.5 M sucrose. Following centrifugation at 135000 x g for 75min four fractions were drawn off from the top of the gradient K (2.5ml), L (9ml), M (8ml) and N (8ml). The particulate matter in fractions L, M and N was pelleted by centrifugation at I00000 x g for 60min and resuspended in 0.32M sucrose (for enzyme marker studies) or in 100M imidazole pH 7.4.

Release of particulate pyroglutamate aminopeptidase from synaptosomal membranes

Synaptosomal membranes were prepared as described above except that fraction M was resuspended in 12 ml100 mM imidazole pH 7.4 (3mg protein ml-I). 2ml synaptosomal membrane suspension were incubated at 30 "C for 60 min with 2ml either 2 M NaC1, papain (0.616 unit), 0.5% v/v Triton X-100, or 0.066% sodium deoxycholate, each in l00mM imidazole buffer pH 7.4. In addition 2-ml aliquots of 100 mM imidazole buffer were added to 2-ml aliquots of synaptosomal membrane suspension and incubated untreated at 30 C for 60min to act as a control. After treatment samples were centrifuged at I00000 x g for 60 min. Supernatants and re- suspended pellets were assayed for pyroglutamate aminopep- tidase using [Pro-3H]thyroliberin as described above.

273

Partial purification by gel filtration of pyroglutamate aminopeptidase activity solubilized ,from synaptosomal membranes by treatment with either papain or Triton X-I00

Synaptosomal membranes were treated with papain as described above. The solubilised pyroglutamate aminopep- tidase was separated from particulate matter by centrifugation at 100000 x g for 60 min. The supernatant was dialyzed against distilled water for 4h , lyophilised and reconstituted in 1 ml l00mM potassium phosphate pH 7.4 containing 0.1 M NaCI. This sample was applied to a Sephadex G-200 column (38 x 1.9 cm) and eluted with 100 mM potassium phosphate buffer

containing 0.1 M NaCl at a flow rate of 4ml/h. I-ml fractions were collected and assayed for pyroglutamate aminopeptidase using [Pr~-~H]thyroliberin as described above. The active fractions were pooled, and dialyzed against distilled water. Following lyophilization the sample was taken up in 100 mM potassium phosphate buffer pH 7.4 and stored in small aliquots at - 20 ’C.

Alternatively synaptosomal membrane preparations were treated with Triton X-100 as described above and the solubi- lized enzyme was separated from particulate matter by centrif- ugation at I00000 x g for 60 min. The supernatant was applied to a Sephadex (3-200 column (38 x 1.9cm) and eluted with 100 mM potassium phosphate pH 7.4 containing 0.25 % v/v Triton X-100 and 0.1 M NaCl at a flow rate of 4ml/h. I-ml fractions were collected and assayed for pyroglutamate amino- peptidase using [Pro-3H]thyroliberin as described above. The active fractions were pooled and stored in small aliquots at - 20 “C.

High-voltage electrophoresis

High-voltage electrophoresis was used to resolve the clea- vage products resulting from the hydrolysis of pyroglutamyl peptides by the synaptosomal-membrane-bound pyroglu- tamate aminopeptidase and by the pyroglutamate aminopep- tidase preparations solubilized from the membrane and par- tially purified by chromatography on Sephadex G-200 gels. The samples were applied to Whatman no. 3 paper and subjected to electrophoresis at a constant voltage (35 Vjcm) for 60 min using the buffer system pyridine/acetic acid/water (1 : 10:89) pH 3.5. After electrophoresis the cleavage products and standards were visualised with Pauly’s reagent or by chlorination.

Substrate specificity of the particulate pyroglutamate arninopeptidase

The specificities of the membrane-bound, Triton-X-I 00- solubilized, and papain-solubilized enzymes were determined using a number of naturally occurring and synthetic pyroglu- tamyl peptides. loop1 potential substrates (1 mM in l00mM potassium phosphate buffer pH 7.4) were incubated for 16 h at 37 ‘C with 100 p1 either fraction M (synaptosomal membrane fraction), post-G-200 Triton-X-I 00-solubilized enzyme prep- aration or post-G-200 papain-solubilized enzyme preparation. The products were resolved by high-voltage electrophoresis and visualized by chlorination. The products were compared with those obtained when the peptides were incubated with a commercially available partially purified preparation of the soluble pyroglutamate aminopeptidase (from bovine liver) in the presence of 2 m M EDTA and 2 m M dithiothreitol. This enzyme was separated from a contaminating post-proline

endopeptidase activity by gel filtration on a Sephadex G-75 column (32 x 1.5cm) prior to use.

Inhibitor studies on the particulate pyroglutamute aminopeptidase

The effects of various inhibitors on the membrane-bound, Triton-X-I 00-solubilized and papain-solubilized enzyme activ- ities were assessed as follows. lop1 sample was first prein- cubated with lop1 potential inhibitor (made up in l00mM potassium phosphate buffer pH 7.4) for 5 min at 37 ‘C. Then 10 pl radiolabelled thyroliberin substrate (0.5 pCi/4 nmol in lOpl) was added to each test and all tests were incubated at 37 C for 30 min. Appropriate controls and blanks were set up simultaneously. The residual pyroglutamate aminopeptidase was assayed using [Pro-3H]thyroliberin as substrate as de- scribed previously.

RESULTS

Demonstration of’ two thyroliberin-hydrol-vzing pyroglutamate aminopeptiduses in different subcellular locations of’ guinea-pig brain

When guinea-pig brain homogenates were resolved by centrifugation into a 30000 x g pellet and a 30000 x g super- natant both fractions were tested for pyroglutamate aminopep- tidase activity using [Pr~-~H]thyroliberin and < Glu-MCA as substrates. The results obtained are presented in Table 1. When 2 mM dithiothreitol and 2 mM EDTA were present in the assay buffer the 30000 x g supernatant fraction showed considerable pyroglutamate aminopeptidase activity against both substrates whereas negligible activity against both substrates was re- corded in the absence of 2 mM dithiothreitol and 2 mM EDTA. This result indicates the presence in the 30000 x g supernatant of a pyroglutamate aminopeptidase, which can hydrolyze both thyroliberin and <Glu-MCA, and the dependence of this activity on the presence of dithiothreitol and EDTA. On the other hand when the assay buffer contained 2mM dithio- threitol and 2 mM EDTA the 30000 x g pellet showed negli- gible activity witheither thyroliberin or < Glu-MCA. However, when dithiothreitol and EDTA were absent from the assay buffers the 30000 x g pellet displayed considerable pyroglu- tamate aminopeptidase activity against thyroliberin alone. This result indicates the presence of a particulate pyroglu- tamate aminopeptidase, which is inhibited by dithiothreitol or EDTA and which can remove pyroglutamic acid from thyroli- berin but not from <Glu-MCA.

Identification (if the subcellular component in the 30000 x gpellet which contains the pyroglutamyl aminopeptidase

Initially crude homogenate of guinea-pig brain was frac- tionated by differential centrifugation into a nuclear pellet (PI) a mitochondrial or 30000 x g pellet (PJ and a 30000 x g supernatant (S2). Centrifugation of the 30000 x g pellet on a discontinuous sucrose gradient produced a myelin fraction (A), a synaptosomal fraction (B) and a mitochondrial fraction (C). As shown in Table 2 the specific activities of ouabain-sensitive Na+/K’ ATPase and 5’-ribonucleotidase, enzyme markers for plasma membranes, rose in the synaptosomal fraction relative to the fraction applied to the gradient, as did the specific activity of occluded lactate dehydrogenase, a marker for intact synaptosomes. The specific activity of pyroglutamate amino- peptidase also rose in the synaptosomal fraction but fell in the

274

myelin and mitochondria1 fractions relative to the 30000 x g pellet applied to the gradient, as was the case for each of the marker enzymes mentioned above. 75 % of the original pyro- gylutamate aminopeptidase from the crude homogenate was recovered in the synaptosomal fraction. The synaptosomes, recovered from fraction B by centrifugation and lysed by suspension in distilled water, were resolved by centrifugation on a second discontinuous sucrose density gradient in to an occluded cytoplasm fraction (K) a synaptosomal vesicle frac- tion (L) a synaptosomal membrane fraction (M) and an intraterminal mitochondria fraction (N). The synaptosomal membrane fraction (M) was found to contain the majority (67 %) of the pyroglutamate aminopeptidase activity applied to the second gradient. The specific activities of pyroglutamate aminopeptidase, ouabain-sensitive N a f / K + ATPase and 5'- ribonucleotidase rose in this band alone relative to the fraction applied to the gradient.

Release of pyroglutamate aminopeptidase fiom sjnaptosomal nienibranes

Table 3 shows the effect of various treatments on the release of pyroglutamate aminopeptidase from the synaptosomal membranes preparations. It can be seen that treatment of the

membranes with 1 M NaCl effected little release of the enzyme and the majority of the activity was retained on the membrane. Use of papain was most effective, releasing the vast majority of the enzyme and achieving an excellent recovery. When Triton X-100 was employed the efficiency in releasing the enzyme was reduced and the recovery of activity was also reduced. Sodium deoxycholate was a less efficient releasing agent than Triton X-I 00 although the recoveries of activity were comparable to those obtained with Triton X-100. The concentrations of papain and Triton X-100 employed were determined in a preliminary study (results not shown) to be most effective concentrations for each reagent in releasing the membrane bound activity.

Substrate specijkities of the synaptosomal-membrane-bound pyroglutamate aminopeptidase and o j the pyroglutamate aminopeptidase preparations released ,fiorn the membrane by treatment with papain or with Triton X-100

When the papain released enzyme was fractionated on a G-200 column and Triton-X-100-released enzyme was frac- tionated on a G-200 column equilibrated with buffer contain- ing 0.25 % v/v Triton X-100 the pyroglutamate aminopeptidase activities, as detected using [Pro-3H]thyroliberin as substrate,

Table 1. Demonstration of two thyroliberin-hydrolyzing pyroglutamate aniinopeptidases in dtfierent .subcellular locations of guinea-pig brain Activities were measured as described in Materials and Methods and expressed as nmol pyroglutamic acid released/min for the total fraction ~

Fraction Substrate : [Pr~-~H]thyroIiberin Substrate: < Glu-MCA

+2mM dithiothreitol ~ 2mM dithiothreitol +2mM dithiothreitol -2mM dithiothreitol + 2 mM EDTA - 2 mM EDTA + 2 mM EDI'A - 2 mM EDTA

30000 x g supernatant 44.08 30000 x g pellet 4.86

2.18 70.30

80.00 3.60

2.00 0.90

Table 2. Localisation oj' particulate thyroliberin-metabolizing pyroglutamate aminopeptidase and oilier enzyme markers in various subcellular ,fractions derived from guinea-pig brain Specific activities of each enzyme are expressed as nmol product min-' mg-'. n.d. indicates not done, - indicates no activity detected

Subcellular fraction Pyrogluta- Specific activities of mate amino- peptidase in pyro- ouabain- 5'-nucleo- succinate lactate occluded each fraction glutamate sensitive tidase dehydro- dehydro- lactate as a per- amino- Na+/K+ genase genase dehydro- centage of peptidase ATPase genase that in crude extract

% nmoI min-' mg-'

Crude extract Nuclear pellet (PI) 1000 x g supernatant (S,) 30000 x g supernatant (S,) Mitochondrial pellet (P') Myelin fraction (A) Synaptosomal fraction (B) Mitochondrial fraction (C) Occluded cytoplasm (K) Synaptic vesicle fraction (L) Synaptic membrane fraction (M) Intraterminal mitochondria

fraction (N)

100 10 99

6 98

9 75

3.5 0.16 2.5

50.6

12.8

0.115 0.037 0.120 0.014 0.238 0.114 0.428 0.107 0.031 0.226 0.706

0.257

46.8 21.3 48.1

71.6 18.3 73.5 10.1

0.46

-

-

89.4

19.3

1.59 1.12 1.62 0.17 3.08 1.42 7.41 2.10 1.19 3.86 8.45

1.77

2.02 0.78 1.72 0.45 2.48 0.28 0.05 3.98 -

-

0.56

1.18

245 x lo3 164 x lo3

5 1 0 ~ 1 0 ~ -

446 x 10' 414 x 10'

269 103 602 102

7 103 152 x 102 52 x lo3 922 x 10'

170 x lo3 2540 x 10' 29 x lo3 284 x 10'

n.d. n.d. n.d. n.d. n.d. n.d.

n.d. n.d

were collected from each column, pooled and each preparation was separately tested along with the synaptosomal-membrane- bound preparation for substrate specificity. A number of peptides containing pyroglutamic acid in the N-terminal position were used and the results are presented in Table4. Each preparation could bring about release of pyroglutamic acid from tripeptides commencing with pyroglutamic acid and histidine in the N-terminal and central positions respectively, namely thyroliberin, acid thyroliberin and the putative anor- exogenic peptide. No pyroglutamic acid released could be detected when each preparation was tested with neurotensin, bombesin, eledoisin or bradykinin potentiating factor B nor with the dipeptides < Glu-Ala, < Glu-Val or < Glu-Pro. Fig. 1 shows the high-voltage electrophoretogram profiles of prod- ucts obtained when each preparation was incubated with luliberin and with thyroliberin. I t can be seen that synapto- soma1 membrane preparations can effect the rclease of pyroglu- tamic acid from luliberin whereas this capacity is not retained by either of the solubilized pyroglutamate aminopeptidases.

Table 3. Relijase oJ the particulate pyroglutamate aminopeptidase from the synuptosomal menibrune

Treatmcn t Percentage Percentage of original of original pyroglutamate pyroglutamate aminopeptidase aminopeptidase remaining in released to 100000 x g 100000 x g pellet supernatant

%

Control (no treatment) 91 6

Salt ( 1 M NaCI) 70 10

of activity) 10 87

(0.25 %) 26 44

Papain (0.616 unit

Triton X-100

Sodium deoxy- cholate (0.033 %) 35 25

275

0

8 0 0 0 0 0

0

. 0 @ 0 0

e 0 0 0 0

0

e 0

1 2 3 L 5 6 7 8 9 1 0 1 1 1 2

0 0 0 0 0 0

Fig. 1. Separation by high-voltage elcctriplioresis of degradation p o d - ucts produced by the action of a synaptosomal membrane preparation and ofpyroglutamate aminopeptidase activities solubilized f rom synap- tosomal membranes on thyroliberin and luliberin respectively. 100 p1 each enzyme preparation was incubated with 100 p1 1 mM peptide and 100 p1 100mM potassium phosphate buffer pH 7.4 at 37 <‘C for 16 h. 30 p1 each reaction mixture was spotted into Whatman no. 3 paper and products were separated by high-voltage electrophoresis (35 V/cm) in a pyridine/acetic acid/water system (I : 10: 89, v/v/v) pH 3.5 for 1 h. Appropriate available standards were also applied to Whatman no. 3 paper. After electrophoresis the breakdown products and standards were visualized by either Pauly’s reagent or by chlorination. The sequence of application from left to right on the Whatman no. 3 paper, of standards and reaction mixtures which contain the following peptides and enzyme preparations are indicated by incrcasing number. (1) <Glu, (2) His, (3) His:Pro, (4) thyroliberin, (5) thyroliberin and commercial bovine liver pyroglutamate aminopeptidase, (6) thyroli- berin and synaptosomal membrane preparation, (7) thyroliberin and pyroglutamate aminopeptidase activity released from synaptosomal membranes by papain and partially purified by Sephadex (3-200 chromatography, (8) luliberin, (9) luliberin and commercial bovine liver pyroglutamate aminopeptidase from bovine liver, (10) luliberin and synaptosomal membrane preparation, (1 1) luiberin and pyroglu- tamate aminopeptidase activity released from synaptosomal mem- branes by papain and partially purified by Sephadex G-200 chroma- tography and (12) luliberin and pyroglutamate aminopeptidase ac- tivity released from synaptosomal membranes by Triton X-100 and partially purified by Sephadex G-200 chromatography

Table 4. The ability of synaptosomal-membrane-bound pyroglutamate aminopeptidase and pyroglutamate aminopeptidase activitirs releasedj-om synaptosomal membranes to release pyroglutamic acid,from various peptides Each peptide was incubated with the enzyme preparation for 16 h and the products separated by high-voltage electrophoresis and visualized as described in Materials and Methods. + indicated that pyroglutamate acid was visualized as a product; - indicated that pyroglutamic acid was not produced as a product

Substrate Synaptosomal-membrane- Papain-solubilized Triton-solubilized bound pyroglutamate pyroglutamate pyroglutamate aminopeptidase aminopeptidase aminopeptidase

Thyroliberin + + + Acid thyroliberin + + + Luliberin + < Clu-His-Gly + + + Neurotensin - - -

Bombesin Eledoisin

< Glu-Ala < Glu-Val < Glu-Pro - -

- -

- - -

- - -

Bradykinin potentiating factor B - - -

- - -

- - -

-

276

Table 5. The <lpc.ts of inhibitors OIZ the activity of a sqinaptosomal-membrane-hound ‘pyroglutamate aminopeptiduse’, and of’ a pjroglutarnute urniiiopeptiduse preparation solubilized from synaptosomal membranes by treatment with either papain or Triton X-100 Each enzyme activity was determined as described in Materials and Methods. The enzyme was pre-incubated with the inhibitor to be tested for 5min at 37 C and the reaction was initiated by the addition of substrate

Inhibitor Enzyme activity in the presence of inhibitor as a percentage of activity in an untreated sample

synap tosomal-membrane- papain-solubilized Triton-X-1 00-solu- bound pyroglutamate pyroglutamate bilized pyroglutamate aminopep tidase aminopeptidase aminopep tidase

mM %

2-Iodace tamide

N-Ethylmaleimide

p-C hloromerihenzoate

Dithiothreitol

EDTA

8-Hydroxyquiniline

1 ,lo-Phenanthroline

Phenylmethylsulfonyl fluoride”

Benzamidine

Baci tracin

Puromycin

1 .o 100 10.0 90

1.0 98 10.0 82 0.5 100 5.0 71 0.005 98 0.05 102 0.5 118 5.0 83 2.0 7

10.0 0

1.0 25 10.0 10 0.1 95 1 .o 20 1 .o 100

10.0 78 1 .o 100

10.0 99 1 .O 97

10.0 78 1 .o 97 5.0 40

99 80 91 75 91 71

98 96 89 33

1 0

20 0

90 14

100 85

100 93 94 52

77 29

98 75

n.d. n.d.

83 69

n.d. n.d. n.d. n.d.

2 0

18 0

88 10

n.d. n.d. 100 90

n.d. n.d. 80 33

* Assayed in the presence of 5 % acetone.

Moreover while the membrane-bound preparation caused the degradation of luliberin to at least six breakdown products the solubilized pyroglutamate aminopeptidase activities have no effect on luliberin. Fig. 1 also shows that while the synapto- soma1 membrane preparation could degrade thyroliberin to pyroglutamic acid, histidine and histidylproline diketopipe- razine (His : Pro), which formed spontaneously and non- enzymatically by cyclisation of His-ProNH,, the solubilized preparations degraded thyroliberin to pyroglutamic acid and hsitidylproline diketopiperazine alone; the same products obtained when thyroliberin was incubated with a commercial preparation of bovine liver pyroglutamate aminopeptidase.

The effects of ,functional reagents on the activity of synaptosomal-bound pyroglutamate aminopeptidase nnd of pyroglutamate aminopeptidase preparations solubilized .from synaptosomal membranes by treatment with papain or Triton X-100

Table 5 shows that 2-iodoacetamide, N-ethylmaleimide, p-chloromercuribenzoate, phenylmethylsulphonyl fluoride and benzamidine had little inhibitory effect on each prepara- tion at low concentrations (0.5 mM or l.OmM) and effected

only modest inhibition at 5.0 mM or 10.0 mM concentrations. Bacitracin and puromycin displayed a greater inhibitory effect with each preparation. However, the chelating agents EDTA (2 mM), 8-hydroxyquinoline (1 mM) and 1,lO-phenanthroline (1 mM) inhibited at least 75 % of the pyroglutamate aminopep- tidase in each preparation. EDTA and 8-hydroxyquinoline had only a slightly inhibitory effect on the activity of guinea-pig serum pyroglutamate aminopeptidase (results not shown). Pretreatment of serum pyroglutamate aminopeptidase with papain (3.08 units/lOmg protein) followed by filtration on Sephadex G-200 did not give rise to any increased sensitivity to EDTA and 8-hydroxyquinoline on the part of the eluted serum pyroglutamate aminopeptidase activity.

The effect of dithiothreitol on the synaptosomal- membrane-bound pyroglutamate aminopeptidase was negli- gible at low concentrations (0.005 mM and 0.05 mM). Raising the concentration of dithiothreitol to 0.5 mM brought about a consistent and reproducible stimulation of activity. Further increase in dithiothreitol concentration to 5.0 mM resulted in a modest reduction in activity. Low concentrations of dithio- threitol had little effect also on the post-G-200 papain-released pyroglutamate aminopeptidase. However, 0.5 mM dithio- threitol had a mildly inhibitory effect in contrast with its effect

277

on the membrane-bound pyroglutamate aminopeptidase and raising of the concentration of this sulphydryl reagent brought about significant reduction in the activity of the papain- solubilized enzyme.

DISCUSSION

The presence of a soluble pyroglutamate aminopeptidase in brain and hypothalamic tissue has been well documented [8,9] and is similar to soluble pyroglutamate aminopeptidase in other animal tissues [30- 321 and in microorganisms [33 - 361 in respect of broad substrate specificity and other physi- cochemical properties. Previous work had indicated the pre- sence of thyroliberin-degrading enzymes, which appeared to be associated with plasma membranes [37, 381 and the possible presence of an additional pyroglutamate aminopeptidase, which was located in a particulate site, was indicated by work on rabbit [I41 guinea-pig [39] and rat [40] brain mitochondria1 pellets. The publication of a method to determine pyroglu- tamate aminopeptidase using [Pr~-~H]thyroliberin as substrate [8] permitted these findings to be investigated more fully and showed that the presence or absence of dithiothreitol and EDTA in assay buffers determined which of the two pyroglu- tamate aminopeptidase activities in brain tissues was being assayed. The results in Table1 also showed that while the broad-specificity soluble activity, which required dithiothreitol and EDTA, could hydrolyse both thyroliberin and <Glu- MCA, the particulate enzyme, which was inhibited by these agents, could only hydrolyse thyroliberin thus hinting at a norrower substrate specificity. Further fractionation of the post-mitochondria1 pellet by centrifugation on two successive discontinuous sucrose density gradients and the use of specific enzyme markers indicated that the particulate pyroglutamate aminopeptidase is located in the synaptosomal membrane fraction. The fact that little pyroglutamate aminopeptidase activity was found in the occluded cytoplasm fraction from the second gradient and that washing the synaptosomal prepara- tion with NaCl effected little release of this enzyme suggested that the enzyme was not occluded in the synaptosomes and does not arise on the synaptosomal membranes as a result of adsorption of either the soluble tissue enzyme [8,9] or a serum enzyme [lo- 121.

The substrate specificity of synaptosomal-bound pyroglu- tamate aminopeptidase and of the two preparations solubilized from the synaptosomal membrane by papain and Triton X- 100, respectively, differentiate these activities sharply from the soluble tissue enzyme, which hydrolyzed all of the substrates which were presented to the particulate enzyme except eledoisin and <Glu-Pro [9], both of which contain <Glu-Pro. In hydrolyzing only thyroliberin, acid thyroliberin and < Glu- His-Gly the present enzyme is closer in its specificity to the porcine serum, which hydrolyzed only thyroliberin and acid thyroliberin [ll]. A relative molecular mass of 230000, ob- tained for pyroglutamate aminopeptidase activity solubilized by papain, is close to a relative molecular mass of 260000, reported for the porcine serum enzyme [I 11, but is almost one order of magnitude larger than the value of 24000 obtained for the soluble tissue enzyme 191. The synaptosomal enzyme and its solubilized preparations, in common with the porcine serum enzyme, are only slightly reduced in activity by the presence of either iodoacetamide, N-ethylmaleimide or p-chloromer- curibenzoate each at 1 mM. By contrast the soluble tissue enzyme is completely inhibited by similar concentrations of the same compounds. Use of EDTA and 8-hydroxyquinoline

offered the greatest discrimination between the activity of the synaptosomal membrane and that of porcine serum in partic- ular. EDTA (5.0mM) had no effect on the soluble tissue enzyme [9] and only a slight inhibitory effect on the porcine serum enzyme [ll]. Likewise 8-hydroxyquinoline (1 mM) re- duced the activity of the porcine serum enzyme by 20 % [I I] but reduced the activities of the synaptosomal membrane prepara- tions by at least 75 %. Papain treatment of the guinea-pig serum enzyme did not effect its low sensitivity to EDTA and 8-hy- droxyquinoline.

These results indicate that while substrate specificity, molecular size, subcellular location and sensitivity to inhibitors effectively distinguish between the soluble brain pyroglutamate aminopeptidase and the brain synaptosomal enzyme, the latter activity is only different from the serum enzyme by virtue of its topology and its sensitivity to EDTA and 8-hydroxyquinoline. It is possible that these activities represent membrane-bound and secreted forms of the same gene product.

Of some interest was the finding that while the synapto- soma1 membrane preparations could release pyroglutamic acid from luliberin, this ability was not retained by pyroglutamate aminopeptidase activities released from the membrane by either papain or Triton X-100 and partially purified by gel filtration on Sephadex G-200. A number of possible expla- nations may be proposed to explain these results. (a) That a luliberin-specific pyroglutamate aminopeptidase is released from the membrane, which eluted from the Sephadex G-200 column at an elution volume distinct from that of thyroliberin pyroglutamate aminopeptidase. However, when the eluates from the column were scanned for pyroglutamate aminopep- tidase using luliberin as substrate no pyroglutamate-releasing activity could be observed. It is possible, however, that the releasing agents may either destroy such an enzyme or fail to effect its release. (b) That the substrate specificity of the synaptosomal-membrane-bound pyroglutamate aminopep- tidase may be altered on release from the membranes. (c) That luliberin, which is not acted upon by the synaptosomal- membrane-bound pyroglutamate aminopeptidase, is, however, acted upon by endopeptidase activities of the membrane to produce breakdown products, which may then be acted upon by the membrane-bound pyroglutamate aminopeptidase. Fig. I indicated that a number of activities were present in the membrane which effected breakdown of luliberin and a number of reports have been published describing chymotrypsin-like [41] and non-chymotrypsin-like [42] endopeptidase activities in bovine adenohypophysis, which can hydrolyze luliberin. Fig. 1 also indicates that the thyroliberin pyroglutamate aminopep- tidase activities released from the membrane and recovered from Sephadex G-200 columns had no effect when incubated with luliberin indicating that these preparations were free of luliberin-metabolising peptidases. This explanation is under investigation at present. The fact that histidine was a product only when thyroliberin was incubated with the synaptosomal membrane preparation and not with the solubilized pyroglu- tamate aminopeptidase preparations indicated that a further degradation enzyme for thyroliberin or its products was present on the synaptosomal membranes.

A number of workers have reported the presence of thyroliberin in extra hypothalamic brain [43 -451 and Winokur et al. [46] have localized thyroliberin-like immunoreactivity to subcellular fractions of rat brain consisting largely of synapto- somes and synaptic vesicles. In addition high-affinity receptors for thyroliberin have been found in extra hypothalamic brain of rat [47] and monkey [48]. It has been demonstrated that calcium-dependent potassium-stimulated release of radioim-

218

munoassayable thyroliberin takes place from hypothalamic synaptosome-rich fractions [49]. Thyroliberin is also reported to increase noradrenaline and acetylcholine turnover in the central nervous system [50, 511. These results would suggest that thyroliberin has a role to play as a neurotransmitter or neuromodulator. The presence of a specific inactivation mechanism for thyroliberin on the presynaptic or postsynaptic membrane or in the synaptic cleft would be prerequisite for such a suggestion to gain credence.

This study describes the presence of a particulate pyroglu- tamate aminopeptidase which is enriched in the course of preparing synaptosomal membranes from guinea-pig brain. It is clearly distinct from the soluble enzyme from the same source [9] but appears to share many of its properties with a porcine serum activity including a restriction of its substrate specificity to thyroliberin and closely related peptides. Its localization in the synaptosomal membranes and its restricted substrate specificity suggest a specific role for this enzyme in degrading neurotransmitter or neuroregulator thyroliberin in these struc- tures. It remains to be determined whether the present enzyme, with its restricted substrate specificity, can discharge that function.

We gratefully acknowledge helpful discussions with Dr John Donlon, Prof. M. T. Fitzgerald and Prof. Dan O’Donovan. We thank the Medical Research Council of Ireland for awarding a grant-in-aid to Gerard O’Cuinn and the Department of Education (Dublin, Ireland) for awarding a research maintenance grant to Brendan O’Connor. We also thank Patricia McLaughlin for typing this manuscript.

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B. O’Connor, Department of Biochemistry, University College Galway, National University of Ireland, Galway, Ireland G. O’Cuinn, Department of Life Sciences, Regional Technical College, Galway, Ireland