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Inactivation of thyrotropin-releasing hormone (TRH) and (3Me-HIS) TRH by brain peptidases studied by high-performance liquid chromatography
Neuroscience Lette~, 2~ ( ~982) 6 ~-65
6~
=.Isev~er/NorL~-Ho~iand Scientific Publishers L~d.
INACT~rATION OF TNYNOTNOPIN=IIELEAIII[NG NONMON.E (TNII) AND (3Me~Nig) TNH BY, BNAgN PEPTiDASES STUDIED ~'~ HgGN,PERFO~MANCE L~QU~D CHROMATOGNAP~Y
E.C. GRIFI"ITHS, J.R. McDERMOTT* and A.t. SMITH*
Department of Physiology, Stopford Building, Um;verMty of Ma~chester, /vIanchester M ~ 9J~Tand *MRC Neuroendocrdnology Unit, Newcastle General Hospital, Newc~stle.upon-Tyne NE4 :LeE (UoK.) (Received July 13th, 1981; Accepted OcV.~ber 22nd, 1981)
High-performance liquid chromatography (HPLC) has been used to separa~:e and identify the metabolites formed from thyrotrophin-releasing-hormone (TRH) and its hyperactive analogue, (3McHis)TRH, by subceUular fractions from rat co~ex, hypothalamus and thalamus. Deamidatien by -~he proline endopeptidase and formation of Nstidy~proline diketopiperazines by ff,e pyrogtmamy ~. aminopeptidase were found to be the major mechamsms of brain ii~acfivatio~ of both pop,ides; (3Moo His)TRH was slightly more stable than TRH in the presence of the brain peptidase~;, and w~th e~)banced receptor binding affinity, this could explain its increased biological activity. The HPLC system t!sed may be applicable to determining the mechanisms of brain inactivation of other TRH analogues and couM also be used to define the pathways for inactivation of larger neuropepddes as well.
In addition to its action in stimulating pituitary release of tbyro~rophia a~d prolactin, thyrotrophin-releasing hormone (TRH; pGtu-His-ProNHa) aiso has independent actions within the central nervous system and there is now sufficient evidence to suggest that TRH functions :~s a neurotransmitter [9]. Among the expected criteria for a peptide neurotransrat~ter is the presence of a rapid inactio ration system. Three brain peptidases have been identified which can cleave the ~ripeptide: a pyroglutamyl aminopeptidase, a histidyl-proline irnidopeptidase and a proline endopeptidase [7, 12, 13]. Two TRH brain metabo~ites, deamido-TRH (TRH-OH) and histidyl-proline diketoNperazine (cydo-His-Pro), are produced by the proline endopeptidase and pyroglu,:amyl amino-peptidase, respectively, and are believed to have their own biological activities [1, 1i]. High-performance liquid chromatography (HPLC) has been used v) separage these me~abolite~ ~om TRH and m follow ~he inactivation of TRH in vitro or~ rat b~ain. The mac~iva.~iol~ pathway of pGiu-NaimmethyloHis-ProNH2, (3MeoHis)~iRH~. a hyperactive TRH analogue, has also been_ studied by HPLC. A supernatant (so.~uble/cymplasmic) fraction and a pardc~!a~e fraedo~ containing microsomes and mitoehondr{a weP prepared from cerebra! cortex, 03PA-39.40/82/~,~-00~?/$ 02.75 © Etsevier/Norl:hoHoltaud Scientific PuNishers Ltd.
hypotha|amus and thalaraus of adult male Sprague-Daw|e~' rats [6]. A]iquots of each fraction (equiva!ent to 2 hypotha|arai) were incubated with 190 ~g TRH or (3Me-His)TRH at pH 7.38 and 37°C for varying time intervals. Samples were boiled to terrainate enzyme activity, freeze-dried and the residue extracted with2 ral 80% methanol containing OA % ~rifluoroacetic ~aCid:{TFA)o;:A:!eontr01!~cu~fi 0n containing fraction pre-bofled for: I o m i n ~ d :hheninc:Uba~ with jth¢i~pfid~:W~ prepared. After centrifugation, the e×tract was dried down and redissoIved in 200 #l 0.08% TFA for HPLC on a Bonapak C-18 column, as previously described [8]° Of e~zh sample 40 #| was injected from a Wispautosampler (WatersAssociates) and the column eluded at I ral/rain flow-ra~ with the following linear gradients: for TRH, 5-199% B over I0 rnin where A = (?:,08%TFA and B = 7 % acetonitrile; for (3Me-His)TRH, 3-24°70 B over 10 rain where A =0.08% TFAand B = 70% acetonitrile. The acetonitrile in both cases contained 0.08°70 TFA. The eluent was monitored at 206 nra and products forraed frora both peptides were identified by their retention times on the column (see below). TRH was obtained from Urfiscience, Cambridge, U.K. and (3Me-His)TRH from Calbiochera, Bishops Stortford, U.K. Retentio~ times for TRH- and (3MeoHis)TRH-related pepfides were found to be as follows in their appropriate gradient conditions: TRH, 12.2 rain; cyclo-His-Pro, 10.7 rain; TRH-OH, 14.0 rain; (3Me-His)TRH, 12.9 rain; cy¢lo-(3Me-His)Pro, 12.5 rain; (3Me-His)TRH-OH, 14.0 rain. Only TRH-OH was formed from TRH by the soluble fraction of cortex, th~laraus and hypo~:ha|araus, indicating the presence of the proline er~dopeptidase in this fraction. In the particulate fraction, cycIo-His-Pro and a small amount of TRH-OH were formed by the pyroglutarayl arainopepfidase and pro|ine endopepfidase. The tirae-course of TRH inactivation by the cortical solub|e fraction (see Fig. 1) clearly shows the appearance of TRH-OH (peak 2) with cono~raitan~ disappearance of TRH (peak i) over the 15-18@ rain incubation period. TRH incubation with the particulale fraction (a|so in Fig. l) produced cycio-His-Pro (peak 3) as a major degradation product with a very small amount of TRH-OH. Once formed, the raetabolites seemed stable to further degradation. Fig. 2 show~ that inc~abation of (3Me.His)TRH (peak 1) with the two fractions resuited in substra~e ~iisappearance over the 30-1.80 rain incubation period, with (3Me.His)TRH-OH (peak 2) formation in the supernatant fraction and (3Me)histidyl-proline diketopiperazir~e (cycIo-~3Me-His)TRH-OH)in t h e particulate fraction. C~mparing the inactivation of the two peptides, (3Me-His)TRH would appear to b~~only slightly more resistant to CNS degradation than TRH. From the,~e results, the technique of HPLC can clearly be used to separate and identi ~y the principal brain raetabolites formed from TRH and one of its hyperactive analog~es~ (3Me-His)TRH. The presence of proline endopeptidase and pyrogluta~yl peptida~ in .supernatan¢ and partlc,~late,-fractmns, ~respect~ ely, cant,be defii,,ed by their prod~action of appropriatedearaidated:peptides and diketQpiperazine~ frem the parent tl:ipepfides~ thus showing ~that TRH and)(3h4e-His)TgH inactivated by s~flar pathways in rat braim tn the case of the d~ei:op~perazme
63
1
_j! ~n
3g~n
CT P
w
Fig. 1. HPLC profiles of products formed from TRH after incubation ~ith sepematant (S) and particulate (P) fractioes of rat cortex (CT] far ~5-~g80 rain. Pe:~?; ] ~ TRI~]; peak 2 = TRH-OH; peak 3 = cyclo-His-Pro. Vertical arrow shews the injection point.
formation, the pyroglutamyl peptida,se first yield5 His-ProNH2 from TRH, which then spontaneously ¢yd~es at neu~rat pH to cyclo-lk]is-Pro [t2]; a similar mechanism obviously occurs for (3Me-His)TRH. Since b~th TRH-OH arid cyck> His-Pro have their own biologieai activity [1~ 1 I], the ability to separate these metabolites from their parent peptides cou!d be very important; b~th metabelites have beer~ detected by radioimmunoassay in rat brain [3, 15], so their separatioa and measurement could now be achieved with e~se by ¢ombiaing the rapid reprodudbility of HPLC system described here with ~he respective radioimmunoassayso This would provide information not only on TRH turaover in the brain bu~ also o~ the relative rates of formatm, and Nays~olog~cN significaace of the two metabolites
[5]. From the HPLC rezt~Rs (3Me-His}TRH seems to be .~igb~iy ~cs.s readi!y degrada~ hie than TRH .in-the CNS, which would in part explain this analog~e's e~.har~ced biologiea~ activity and coafkm previous radioimmm~eas~ay data on the anategue's inactivation 1t012 However, m~ altemative explaaatioa would be that its i~creased activity is d~e to a comb ir~ation of i~:reased s~abiiity to m~ct~v,~t~or~ with ~he
r$0mm
•~
t,$n
:ii
Fig. 2. HPLC profiles of products formed from (3Me-HLs)TRH after ~ncubation with supematant (S) and partkulate (P) fractions of rat cortex (C~) for 30-189 rain. Peak 1 = (3Me.His)TRH; peak 2 = C~Me-}-Iis.)'I'RH-OH; peak 3 = cydo-(3Me-His)Pro. Vertical arrow shows/he injection point.
iacreased affinity of (3Me-His)TRH. fo~ TRH binding sites at pituitary and CNS levels shown recently by Taylc~r arm Burr [14], F~om the ap.~rent success with separating (3Me-His)TRH from its metabolites theHFLC method described may be eql~.~ally.applicable to the identification of brain metabolites formed from other TRH an~logues as well and could provide useful information on their relative ~smbilitie~ to ~te proline e_udopepti/dasearid pyro#ytmnyt aminopept~d~e present in the two subcel~utar fracfioas used. The ~p~d.and ease of metabolite separation from TRH ~nd (3Me-His)TRH by H P L C sure, is that this technique, could be widely, used for stud,ring, the mechanis~as of inactivation of other neuropeptides in vitro, The identification of the pahtways involved couM then be applied to investigating the m~hani,sms, of in vivo i~ac~iva.tion t~ deter~fine how ~europeptide turnover.at sites .~)fproduction and release, a~d how biotransformafi~":Of-;neurepeP~des:m,-other::::biolOgically~a~tive : Prel!mtaary resu]~ with lu~em~z!ng/h~tm~e~!eieas~ag hormone confirm that this HPLC method will ~deed fu!ffl ~ueh a;tr~ise~ [2],
65
C " ~ : ~ s"s fo~" technical assis~ace, the Wei~ce~e and We wish to thank Miss ~' " 6" o . ", L~wenste~n Trusts for ~mancm! :~ pport to ~~:.C.O. ~md Miss E. Whittaker for assistance iu preparing this manuscript, r
i ~ ¢ h i , G;, Lauhay, No a~ad Rips, R~ Induction of we~ dog shakes by intracereb~l 'acid' TRH in rats, Neurosci. Left., ~60980) 209-2~2, 21Edward~n. J:A:; ~Griffi~S,!E.C~ McDermotL LR. and Slrfith, A,I.) lnactivaaon of luteinizing < h0rmone'rdeasing h0rm0n¢ byrat br~, peptidases, J, Physiol, 0~ond.), in pres3. 3 Emerson, C.H,)Vogel, w , a n d cutn:ie:~ B.L;, Concentrations of thyro~rophin releasing hormone (TRH).and d ~ d o . T R H , and TRHdeamidase activity ~n brain, Endocrinology, 107 (~980) 4 Gfiffiths' E.C, and KeLly, J.A., Mechmaisms of inactivation of hypoth~lamic regutmory hormones, Mo!ec. cell. Endoer., 14(1980) 3-17. 5 Griffiths, E.C, and Webster, V.A.D., Thyrotrophhl re!~a~ing hormone and i~ me~abolites as modulators of CNS activity, Lancet, 1.(198!) 834-835. 6 Gfiffiths, E.C., Kelly, J.A., Klootwijk, W. and Visser, T.J., Enzyn~Jc formation of TRHoOH from TRH by rat hypothalamus, Molec. ceil. Endocr., 18 (1980) 59--67. 7 Matstfi, T., Prasad, C. and Peterkofsky, A., Metabdisa~ of thyrotropin°releasing hormone: isolafion arid characterization of an imidopeptidase for hisfidyl-prolineamide, J. biol. ,Chem., 254 (1979) 2439-2445. 8 McDermott, J.R., Smith, A.I., Biggins, J.A., AI-NoaemJ, M.C. and Edwardson, J.A., Characterization and determination of neuro~ptides by high-perfo~xnance liquid chromatography and radio~inmaunoassay, J. Chromatogr., 222 098t) 371-379. 9 Morley, J.E., Extra-hylmth~darr~c thyrotropin releasir~g hormone (TRH): its distribution and ffs functions, Life Sei., 25 (1979) 1539-d550. I0 Oliver, C., Gillioz, P., Girand, P. and Coute-Devo~x, B., Degradation of TRH and its analogues by rat serum and brain homogenate, Biochem. Biophys. rcs. Congnun., 84 (1978) 1097-1102. 11 Peterkofsy, A. and Battain~, F., The biological acclivities of the neuropepfide histidybproline diketopiperazine, Neuropeptides, I (1'980) 105-115. t2 Prasad, C. and Peterkofsky, A., Demonstration of pyroglutamyt ~eptidase and amidase activb ties in hamster hypothatamic extracts, Jl. biol. Chem., ~51 (1976) 322~- 3234. 13 Rupnow, 3.H., Taylor, W.L. and D~xon, J.E., Pm~ficafion and ch~:-acterizat:,on of a thyrotrop~n-releasing hormone deamidase from rat brain, Biochemist,'y, 18 (1979) ~2(~6-]2|2. 14 Taylor, R.L. and Bu~rt, D,R., Preparation of [-~H-3-Me-HisZ]TRH as an improved ligand for T~,H receptors, Neuroend~verinology, 32 (1981) 3 t0-316. 15 Yanagisawa, T., Prasad, C. and Peterkofsky, Ao, The subcdlular a~d organ distribution and natural form of t~istidyI-prolitnediketopiperaziine in rat brain de~:ermined by a specific radioimmunoassay, 3. biol. Chem., 255 (19?~)) 10290-10298.
6~
=.Isev~er/NorL~-Ho~iand Scientific Publishers L~d.
INACT~rATION OF TNYNOTNOPIN=IIELEAIII[NG NONMON.E (TNII) AND (3Me~Nig) TNH BY, BNAgN PEPTiDASES STUDIED ~'~ HgGN,PERFO~MANCE L~QU~D CHROMATOGNAP~Y
E.C. GRIFI"ITHS, J.R. McDERMOTT* and A.t. SMITH*
Department of Physiology, Stopford Building, Um;verMty of Ma~chester, /vIanchester M ~ 9J~Tand *MRC Neuroendocrdnology Unit, Newcastle General Hospital, Newc~stle.upon-Tyne NE4 :LeE (UoK.) (Received July 13th, 1981; Accepted OcV.~ber 22nd, 1981)
High-performance liquid chromatography (HPLC) has been used to separa~:e and identify the metabolites formed from thyrotrophin-releasing-hormone (TRH) and its hyperactive analogue, (3McHis)TRH, by subceUular fractions from rat co~ex, hypothalamus and thalamus. Deamidatien by -~he proline endopeptidase and formation of Nstidy~proline diketopiperazines by ff,e pyrogtmamy ~. aminopeptidase were found to be the major mechamsms of brain ii~acfivatio~ of both pop,ides; (3Moo His)TRH was slightly more stable than TRH in the presence of the brain peptidase~;, and w~th e~)banced receptor binding affinity, this could explain its increased biological activity. The HPLC system t!sed may be applicable to determining the mechanisms of brain inactivation of other TRH analogues and couM also be used to define the pathways for inactivation of larger neuropepddes as well.
In addition to its action in stimulating pituitary release of tbyro~rophia a~d prolactin, thyrotrophin-releasing hormone (TRH; pGtu-His-ProNHa) aiso has independent actions within the central nervous system and there is now sufficient evidence to suggest that TRH functions :~s a neurotransmitter [9]. Among the expected criteria for a peptide neurotransrat~ter is the presence of a rapid inactio ration system. Three brain peptidases have been identified which can cleave the ~ripeptide: a pyroglutamyl aminopeptidase, a histidyl-proline irnidopeptidase and a proline endopeptidase [7, 12, 13]. Two TRH brain metabo~ites, deamido-TRH (TRH-OH) and histidyl-proline diketoNperazine (cydo-His-Pro), are produced by the proline endopeptidase and pyroglu,:amyl amino-peptidase, respectively, and are believed to have their own biological activities [1, 1i]. High-performance liquid chromatography (HPLC) has been used v) separage these me~abolite~ ~om TRH and m follow ~he inactivation of TRH in vitro or~ rat b~ain. The mac~iva.~iol~ pathway of pGiu-NaimmethyloHis-ProNH2, (3MeoHis)~iRH~. a hyperactive TRH analogue, has also been_ studied by HPLC. A supernatant (so.~uble/cymplasmic) fraction and a pardc~!a~e fraedo~ containing microsomes and mitoehondr{a weP prepared from cerebra! cortex, 03PA-39.40/82/~,~-00~?/$ 02.75 © Etsevier/Norl:hoHoltaud Scientific PuNishers Ltd.
hypotha|amus and thalaraus of adult male Sprague-Daw|e~' rats [6]. A]iquots of each fraction (equiva!ent to 2 hypotha|arai) were incubated with 190 ~g TRH or (3Me-His)TRH at pH 7.38 and 37°C for varying time intervals. Samples were boiled to terrainate enzyme activity, freeze-dried and the residue extracted with2 ral 80% methanol containing OA % ~rifluoroacetic ~aCid:{TFA)o;:A:!eontr01!~cu~fi 0n containing fraction pre-bofled for: I o m i n ~ d :hheninc:Uba~ with jth¢i~pfid~:W~ prepared. After centrifugation, the e×tract was dried down and redissoIved in 200 #l 0.08% TFA for HPLC on a Bonapak C-18 column, as previously described [8]° Of e~zh sample 40 #| was injected from a Wispautosampler (WatersAssociates) and the column eluded at I ral/rain flow-ra~ with the following linear gradients: for TRH, 5-199% B over I0 rnin where A = (?:,08%TFA and B = 7 % acetonitrile; for (3Me-His)TRH, 3-24°70 B over 10 rain where A =0.08% TFAand B = 70% acetonitrile. The acetonitrile in both cases contained 0.08°70 TFA. The eluent was monitored at 206 nra and products forraed frora both peptides were identified by their retention times on the column (see below). TRH was obtained from Urfiscience, Cambridge, U.K. and (3Me-His)TRH from Calbiochera, Bishops Stortford, U.K. Retentio~ times for TRH- and (3MeoHis)TRH-related pepfides were found to be as follows in their appropriate gradient conditions: TRH, 12.2 rain; cyclo-His-Pro, 10.7 rain; TRH-OH, 14.0 rain; (3Me-His)TRH, 12.9 rain; cy¢lo-(3Me-His)Pro, 12.5 rain; (3Me-His)TRH-OH, 14.0 rain. Only TRH-OH was formed from TRH by the soluble fraction of cortex, th~laraus and hypo~:ha|araus, indicating the presence of the proline er~dopeptidase in this fraction. In the particulate fraction, cycIo-His-Pro and a small amount of TRH-OH were formed by the pyroglutarayl arainopepfidase and pro|ine endopepfidase. The tirae-course of TRH inactivation by the cortical solub|e fraction (see Fig. 1) clearly shows the appearance of TRH-OH (peak 2) with cono~raitan~ disappearance of TRH (peak i) over the 15-18@ rain incubation period. TRH incubation with the particulale fraction (a|so in Fig. l) produced cycio-His-Pro (peak 3) as a major degradation product with a very small amount of TRH-OH. Once formed, the raetabolites seemed stable to further degradation. Fig. 2 show~ that inc~abation of (3Me.His)TRH (peak 1) with the two fractions resuited in substra~e ~iisappearance over the 30-1.80 rain incubation period, with (3Me.His)TRH-OH (peak 2) formation in the supernatant fraction and (3Me)histidyl-proline diketopiperazir~e (cycIo-~3Me-His)TRH-OH)in t h e particulate fraction. C~mparing the inactivation of the two peptides, (3Me-His)TRH would appear to b~~only slightly more resistant to CNS degradation than TRH. From the,~e results, the technique of HPLC can clearly be used to separate and identi ~y the principal brain raetabolites formed from TRH and one of its hyperactive analog~es~ (3Me-His)TRH. The presence of proline endopeptidase and pyrogluta~yl peptida~ in .supernatan¢ and partlc,~late,-fractmns, ~respect~ ely, cant,be defii,,ed by their prod~action of appropriatedearaidated:peptides and diketQpiperazine~ frem the parent tl:ipepfides~ thus showing ~that TRH and)(3h4e-His)TgH inactivated by s~flar pathways in rat braim tn the case of the d~ei:op~perazme
63
1
_j! ~n
3g~n
CT P
w
Fig. 1. HPLC profiles of products formed from TRH after incubation ~ith sepematant (S) and particulate (P) fractioes of rat cortex (CT] far ~5-~g80 rain. Pe:~?; ] ~ TRI~]; peak 2 = TRH-OH; peak 3 = cyclo-His-Pro. Vertical arrow shews the injection point.
formation, the pyroglutamyl peptida,se first yield5 His-ProNH2 from TRH, which then spontaneously ¢yd~es at neu~rat pH to cyclo-lk]is-Pro [t2]; a similar mechanism obviously occurs for (3Me-His)TRH. Since b~th TRH-OH arid cyck> His-Pro have their own biologieai activity [1~ 1 I], the ability to separate these metabolites from their parent peptides cou!d be very important; b~th metabelites have beer~ detected by radioimmunoassay in rat brain [3, 15], so their separatioa and measurement could now be achieved with e~se by ¢ombiaing the rapid reprodudbility of HPLC system described here with ~he respective radioimmunoassayso This would provide information not only on TRH turaover in the brain bu~ also o~ the relative rates of formatm, and Nays~olog~cN significaace of the two metabolites
[5]. From the HPLC rezt~Rs (3Me-His}TRH seems to be .~igb~iy ~cs.s readi!y degrada~ hie than TRH .in-the CNS, which would in part explain this analog~e's e~.har~ced biologiea~ activity and coafkm previous radioimmm~eas~ay data on the anategue's inactivation 1t012 However, m~ altemative explaaatioa would be that its i~creased activity is d~e to a comb ir~ation of i~:reased s~abiiity to m~ct~v,~t~or~ with ~he
r$0mm
•~
t,$n
:ii
Fig. 2. HPLC profiles of products formed from (3Me-HLs)TRH after ~ncubation with supematant (S) and partkulate (P) fractions of rat cortex (C~) for 30-189 rain. Peak 1 = (3Me.His)TRH; peak 2 = C~Me-}-Iis.)'I'RH-OH; peak 3 = cydo-(3Me-His)Pro. Vertical arrow shows/he injection point.
iacreased affinity of (3Me-His)TRH. fo~ TRH binding sites at pituitary and CNS levels shown recently by Taylc~r arm Burr [14], F~om the ap.~rent success with separating (3Me-His)TRH from its metabolites theHFLC method described may be eql~.~ally.applicable to the identification of brain metabolites formed from other TRH an~logues as well and could provide useful information on their relative ~smbilitie~ to ~te proline e_udopepti/dasearid pyro#ytmnyt aminopept~d~e present in the two subcel~utar fracfioas used. The ~p~d.and ease of metabolite separation from TRH ~nd (3Me-His)TRH by H P L C sure, is that this technique, could be widely, used for stud,ring, the mechanis~as of inactivation of other neuropeptides in vitro, The identification of the pahtways involved couM then be applied to investigating the m~hani,sms, of in vivo i~ac~iva.tion t~ deter~fine how ~europeptide turnover.at sites .~)fproduction and release, a~d how biotransformafi~":Of-;neurepeP~des:m,-other::::biolOgically~a~tive : Prel!mtaary resu]~ with lu~em~z!ng/h~tm~e~!eieas~ag hormone confirm that this HPLC method will ~deed fu!ffl ~ueh a;tr~ise~ [2],
65
C " ~ : ~ s"s fo~" technical assis~ace, the Wei~ce~e and We wish to thank Miss ~' " 6" o . ", L~wenste~n Trusts for ~mancm! :~ pport to ~~:.C.O. ~md Miss E. Whittaker for assistance iu preparing this manuscript, r
i ~ ¢ h i , G;, Lauhay, No a~ad Rips, R~ Induction of we~ dog shakes by intracereb~l 'acid' TRH in rats, Neurosci. Left., ~60980) 209-2~2, 21Edward~n. J:A:; ~Griffi~S,!E.C~ McDermotL LR. and Slrfith, A,I.) lnactivaaon of luteinizing < h0rmone'rdeasing h0rm0n¢ byrat br~, peptidases, J, Physiol, 0~ond.), in pres3. 3 Emerson, C.H,)Vogel, w , a n d cutn:ie:~ B.L;, Concentrations of thyro~rophin releasing hormone (TRH).and d ~ d o . T R H , and TRHdeamidase activity ~n brain, Endocrinology, 107 (~980) 4 Gfiffiths' E.C, and KeLly, J.A., Mechmaisms of inactivation of hypoth~lamic regutmory hormones, Mo!ec. cell. Endoer., 14(1980) 3-17. 5 Griffiths, E.C, and Webster, V.A.D., Thyrotrophhl re!~a~ing hormone and i~ me~abolites as modulators of CNS activity, Lancet, 1.(198!) 834-835. 6 Gfiffiths, E.C., Kelly, J.A., Klootwijk, W. and Visser, T.J., Enzyn~Jc formation of TRHoOH from TRH by rat hypothalamus, Molec. ceil. Endocr., 18 (1980) 59--67. 7 Matstfi, T., Prasad, C. and Peterkofsky, A., Metabdisa~ of thyrotropin°releasing hormone: isolafion arid characterization of an imidopeptidase for hisfidyl-prolineamide, J. biol. ,Chem., 254 (1979) 2439-2445. 8 McDermott, J.R., Smith, A.I., Biggins, J.A., AI-NoaemJ, M.C. and Edwardson, J.A., Characterization and determination of neuro~ptides by high-perfo~xnance liquid chromatography and radio~inmaunoassay, J. Chromatogr., 222 098t) 371-379. 9 Morley, J.E., Extra-hylmth~darr~c thyrotropin releasir~g hormone (TRH): its distribution and ffs functions, Life Sei., 25 (1979) 1539-d550. I0 Oliver, C., Gillioz, P., Girand, P. and Coute-Devo~x, B., Degradation of TRH and its analogues by rat serum and brain homogenate, Biochem. Biophys. rcs. Congnun., 84 (1978) 1097-1102. 11 Peterkofsy, A. and Battain~, F., The biological acclivities of the neuropepfide histidybproline diketopiperazine, Neuropeptides, I (1'980) 105-115. t2 Prasad, C. and Peterkofsky, A., Demonstration of pyroglutamyt ~eptidase and amidase activb ties in hamster hypothatamic extracts, Jl. biol. Chem., ~51 (1976) 322~- 3234. 13 Rupnow, 3.H., Taylor, W.L. and D~xon, J.E., Pm~ficafion and ch~:-acterizat:,on of a thyrotrop~n-releasing hormone deamidase from rat brain, Biochemist,'y, 18 (1979) ~2(~6-]2|2. 14 Taylor, R.L. and Bu~rt, D,R., Preparation of [-~H-3-Me-HisZ]TRH as an improved ligand for T~,H receptors, Neuroend~verinology, 32 (1981) 3 t0-316. 15 Yanagisawa, T., Prasad, C. and Peterkofsky, Ao, The subcdlular a~d organ distribution and natural form of t~istidyI-prolitnediketopiperaziine in rat brain de~:ermined by a specific radioimmunoassay, 3. biol. Chem., 255 (19?~)) 10290-10298.