60 YEARS of POMC: Lipotropin and Beta-Endorphin: a Perspective

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d g smyth Lipotropin and beta-endorphin 56:4 T13–T25 Thematic Review

60 YEARS OF POMC Lipotropin and beta-endorphin: a perspective

Correspondence should be addressed to D G Smyth D G Smyth Email [email protected] Department of Endocrinology, William Harvey Research Institute, London, UK

Abstract

Many important fields of research had a humble origin. In the distant past, A J P Martin’s Key Words discovery that amino acids could be separated by paper chromatography and Moore ff lipotropin and Stein’s use of columns for quantitative amino acid analysis provided the first steps ff neurotransmitters towards the determination of structure in complex biologically active molecules. They ff beta-endorphin opened the door to reveal the essential relationship that exists between structure and ff POMC function. In molecular endocrinology, for example, striking advances have been made by chemists with their expertise in the identification of structure working with biologists who contributed valuable knowledge and experience. Advantage was gained from the convergence of different background, and it is notable that the whole is greater than the sum. In the determination of structure, it may be recalled that four of the world’s great pioneers (Archibald Martin, Rodney Porter, Fred Sanger and Vincent du Vigneaud) were acknowledged for their fundamental contributions when individually they were awarded the Nobel Prize. They foresaw that the identification of structure would prove of outstanding importance in the future. Indeed, study of the structures of β-endorphin Journal of Molecular Endocrinology Journal of Molecular and enkephalin and the different forms of opiate activity they engender has led to a Endocrinology transformation in our understanding of chemical transmission in the brain. (2016) 56, T13–T25

Lipotropin: precursor of β-endorphin It is well known, of course, that most if not all peptide unexpected discoveries: firstly, that a 31-residue fragment hormones are formed as fragments of larger peptides and of lipotropin shown to be present in pituitary and brain lipotropin is no exception. This 91-residue polypeptide, possessed potent opiate activity (Feldberg & Smyth 1976, first isolated byBirk and Li (1964) and sequenced by Bradbury et al. 1976a, Li & Chung 1976); and secondly, Li et al. (1965), possessed lipolytic activity, and it was that lipotropin was itself a fragment of a precursor, which provisionally assigned the title ‘lipotrophic hormone’. proved to be a multifunctional prohormone (Mains et al. The primary structure included a sequence of amino acids 1977, Roberts & Herbert 1977, Nakanishi et al. 1979). It that exhibited a marked similarity to α-melanotropin is of interest to recall the background to these advances (α-MSH), and it was generally assumed that ‘β-MSH’ was which stemmed from the study of prohormone processing the biologically active counterpart to α-MSH in lipotropin. and generation of their biologically active constituents. As a melanotrophic hormone, however, β-MSH was much less active than -MSH and alarm bells began to ring when α Lipotropin: from structure to function it was reported that β-MSH was not present in the human pituitary (Bloomfield et al. 1974), which raised question For several years, the principal interests of my on its physiological significance. This set the stage for two laboratory at the National Institute for Medical Research

http://jme.endocrinology-journals.org © 2016 Society for Endocrinology This paper is part of a thematic review section on 60 Years of POMC. DOI: 10.1530/JME-16-0033 Printed in Great Britain The Guest Editors for this section were Adrian Clark and Philip Lowry. Published by Bioscientifica Ltd. Downloaded from Bioscientifica.com at 09/30/2021 01:38:48PM via free access

10.1530/JME-16-0033 Journal of Molecular Endocrinology Copyright (1976),withpermissionfrom Elsevier. Biophysical ResearchCommunications , 69BradburyAF, SmythDG&SnellCR,Lipotropin:Precursortotwo biologicallyactivepeptides,950–956. corresponds tolipotropinresidues61–91, theCʹ-fragmenttolipotropinresidues61–87,β-MSH41–58.Reprintedfrom andBiochemical Alignment ofpeptidesisolatedfrom porcinepituitaryintheaminoacidsequenceoflipotropin(LPH).TheC-fragment oflipotropin(β-endorphin) Figure 1 the timewasbelievedtobebiologicallyactiveregion. forthereleaseofβ lipotropin necessary on theN-terminalsideofsequenceatcentre limited digestionwitheitherenzyme( could bereleasedfromtheC-terminusoflipotropinby that oneofthepeptidesweidentifiedinpituitary by enzymeswithbasicspecificity( peptide hormonesweregeneratedfromtheirprecursors isolated fromthepituitary, becauseitwasbelievedthat also byanenzymewithsimilarspecificitywhichwehad andthe sensitivity of lipotropin to cleavage by trypsin discovered. To obtainanindicationofthis,weexamined or they might possess an activity that was waiting to be fragments ‘left over’afteractivationof a prohormone, the EndocrineSociety(see possibility hadbeendiscussedinformallyatMeetingsof are formedfromacommonprohormone,althoughthe At thattime,itwasnotknownACTHandlipotropin hormone (ACTH)togetherwiththesequenceoflipotropin. accounted forthesequenceofadrenocorticotrophic . 1975,1976b et al a seriesofnovelpeptidesfromporcine ( pituitary its linearstructure.In1974,weweresuccessfulinisolating putative prohormoneandthenreassemblethemtoreveal to isolateandidentifytheconstituentfragmentsofa where theprohormonesaresynthesised,ourstrategywas As fragmentsofprohormonesmightberetainedintissues likely thatthiswouldprovetobeageneralphenomenon. insulin isformedfromaprohormone,anditseemed studies oninsulinprecursorshaddemonstratedthat its formationfromlipotropin.DonaldSteiner’s classical London)werefocusedonβ-endorphinand Hill, (Mill 1976c DOI: 10.1530/JME-16-0033 http://jme.endocrinology-journals.org Thematic Review The peptidesweisolatedcouldhavebeeninert ), butsurprisinglytherewasnegligiblecleavage ) and we observed thatagroupofthem ) andweobserved Lowry 2016 Lowry d

smyth ). -MSH, whichat Bradbury Bradbury Printed inGreatBritain ). We found Bradbury Bradbury t al. et on insulinsecretionfromthepancreas. effects onsodiumtransportinthekidneyandinfluence investigating potentialperipheralactivitiessuchas hormones intothecirculation, Edwardsonstartedby whichsecretesits had beenisolatedfromthepituitary C-terminal peptide1,named‘C-fragmentoflipotropin’, College, London,toinvestigateitsactivity. Asthe a collaborativestudywithJamesEdwardsonofImperial β This impliedthattheC-terminalpeptideratherthan Enkephalin 2: Enkephalin 1: C-Fragment oflipotropin: Tyr–Gly ( on digestion withenzymesbasicspecificityreleased contained thesequenceLys–Asp–Lys–Arg–Tyr–Gly, which release ofitsC-fragmenttakesplace.Themodelpeptide synthesised tomodeltheregionoflipotropinwhere ( alignment ofourpeptidesinthelipotropinsequence demonstrate this,Ishowedadiagramillustratingthe of lipotropin,mightpossessbiologicalactivity. To reason to believe that one of the peptides, the C-fragment the sequenceoflipotropin.Iexplainedthatwehad identified aseriesofnovelpeptidesthataccountedfor peptides we had isolated, and I described how we had I wasinvitedtoImperialCollegegiveatalkonthe In September1975,therewasanastonishingcoincidence. Discovery ofβ-endorphin Lipotropin andbeta-endorphin i. 1 Fig. -MSH mightbefunctionallysignificant,soIarranged Published byBioscientifica Ltd. ) andIdrewattentiontoapeptidewehad Bradbury Bradbury

et al. 1976b Downloaded fromBioscientifica.com at09/30/202101:38:48PM

). Tyr–Gly–Gly–Phe–Met... Tyr–Gly–Gly–Phe–Leu Tyr–Gly–Gly–Phe–Met ...His–Lys–Lys–Gly–Gln 56 : 4 T14 via freeaccess

Journal of Molecular Endocrinology to letmeknowwhatthesecrecywasabout. a pharmacologygroupinAberdeen,Scotland,telephoned discuss, butthenextmorningHansKosterlitz,headof but confidentialcollaborationwhichhewasnotfreeto possibly functionalrelationshipbetweenthesepeptides. the C-fragment of lipotropin, pointing to a structural and been identifiedcorrespondedtothisN-terminalsequenceof the Leu-enkephalinandMet-enkephalinpeptideswhichhad became apparentthatthefourresiduesatN-terminusof http://jme.endocrinology-journals.org DOI: 10.1530/JME-16-0033 Thematic Review Morris told me that he was involved in an important Morris toldmethathewasinvolvedinanimportant In discussionwithHowardMorrisaftermylecture,it d

smyth Printed inGreatBritain but highly significant overlap in our research, so it was of lipotropin.Itwasclearthatthereanunexpected the enkephalinsandourinvestigationofC-fragment background andprogressmadeintheiridentificationof to Londonforthemeeting,andwediscussed later, KosterlitzandHughescamedownfromAberdeen myself, totakeplaceinhisofficeattheNIMR.Afewdays his colleagues,JohnHughesandHowardMorris, at once arranged a meeting with Kosterlitz and two of Institute forMedicalResearch (NIMR),MillHill,London, Lipotropin andbeta-endorphin Published byBioscientifica Ltd. Sir ArnoldBurgen,thedirectorofNational Copyright (1977),withpermission from Elsevier. the actionofaminopeptidases,476–482. The NH2-terminusofC-Fragmentis resistantto Communications, from determined byaminoacidanalysis. Reprinted pH 2.2andtheliberatedtyrosinewas solutions wereaddedto1 mLof0.2 Mcitrateat internal standard.Atintervals,aliquotsofthe at pH7.4.dl-Norleucinewasincludedasan (upper figure)in0.1 Mammoniumbicarbonate of anaminopeptidasefromratbrain (lower figure,0.02 mg/mL)orwithapreparation incubated at37˚ peptides. Thepeptides(0.34mM)were β Rate ofreleaseN-terminal Figure 2 -endorphin andstructurallyrelatedsynthetic Biochemical andBiophysicalResearch Downloaded fromBioscientifica.com at09/30/202101:38:48PM C withaminopeptidaseM 76, AustinBM&SmythDG, 56 : 4 tyrosine from

T15 via freeaccess Journal of Molecular Endocrinology Li 1967 isolation andcharacterisationofγ-lipotropin( ofthecamel( the pituitary more potentthanmorphine.Liisolatedβ-endorphinfrom the rat,itwasdemonstratedthatβ-endorphinmuch ( the 31-residuepeptideexhibitedstronganalgesicactivity method ofsolid-phasesynthesisandtheyreportedthat biochemistry. lipotropin 10 years before( lipotropin 10 years Thus, Li’s achievementindeterminingthesequenceof and Wendlandt, we wentontoshowthatβ-endorphin Ree showed thatthiswasnotto be( not producetoleranceanddependence, butexperiments had hopedthatasitoccurs naturally, β-endorphinwould profound centraleffectscharacteristicofmorphine.We that β-endorphin produced a range of other observed was themostpotentanalgesicagentknown. for severalhours.Feldbergconcludedthatβ-endorphin ( times morepotentthanmorphineasananalgesicagent that ( reversed bynaloxone,aclassical antagonist oftheopiates for opiate receptors in the brain, and the binding was C-fragment oflipotropin(β-endorphin)hasahighaffinity With BirdsallandHulme,wedemonstrated thatthe β-endorphin andopiate activity al.1975 et Aberdeen on the identification of theenkephalins ( (β-endorphin) andtheworkofKosterlitzgroupfrom of lipotropinwhichledustoisolatetheC-fragment biological activityofβ collaboration withEdwardsonweweresearching forthe β H Li). Thus, at that time we had isolated and identified was laterrenamed‘β the physiologicalroleofC-fragment(ourpeptide receptors fortheenkephalins,whilewewouldinvestigate NIMR) shouldcontinuetheirworkbyconcentratingon part bytheMedicalResearch Council(MRC)(aswasthe decided thatKosterlitzandHugheswhowerefundedin and it isabundantlyclearthatidentificationofenkephalin Loh Feldberg &Smyth1976,1977 Bradbury -endorphin andarelated27residuepeptide,in DOI: 10.1530/JME-16-0033 http://jme.endocrinology-journals.org Thematic Review Li andhiscolleaguespreparedβ-endorphinbythe In additiontoitsanalgesicproperties,Feldberg Looking back, it isclear that ourworkontheprocessing t al.1978 al.1976,1979,Jacquet et et β-endorphin administeredin cat ventricles was 100 β-endorphin openedupanexcitingareaofbrain . 1976 et al ) ledontotheidentificationofβ-endorphin. t al.1976a et )

had convergedbyserendipity. Inanycase, ). Using the hot plate assay performed on -endorphin’ byEricSimonsandC -endorphin. ). Alongsidethis,Feldbergfound Li &Chung1976 Li ) andtheanalgesiapersisted . 1965 et al d Gispen

smyth ) andhisfurther . 1976,Van et al Printed inGreatBritain ). With Deakin ) andtheox. Chretien & Hughes

was highlypotentintherat( acids fromtheNH leucine aminopeptidase,anenzymethatremovesamino digesting methionineenkephalinandβ to enzymaticattack.We obtainedevidenceforthisby adopt aconformationthatwouldmakeitlessvulnerable 31-residue peptidecouldbeattributedtoitstendency the brain.Atstructurallevel,stabilityof β enkephalins mightbeduetoresistanceexhibitedby actions ofβ be demonstratedunequivocallyinopiateassaysvitro. noted, however, thattheactivityofenkephalins can half-lives inbrainareonlyafewseconds.Itshouldbe The enkephalins act at natural enkephalins( little ornoanalgesiceffectscouldbedetectedwiththe From thehypothalamus,long beadedaxonsextended also be seen along the walls of the third ventricle. of thethirdventricle,but axonsandterminalscould the medianeminenceand theventromedialborder the hypothalamuswasconfined tothearcuate nucleus, of thedorsalcolliculi.Theimmunoreactivityin intheregionDense fluorescencewasalsoobserved Zakarian 1982,&Smyth1982a amygdala, hippocampusand brainstem ( was seenwerethehypothalamus,thalamus-midbrain, showed thattheprincipalareaswherefluorescence Immunohistochemical stainingofrattissuesections β-endorphin inthebrain the peptideispresentinitsbiologicallyactiveform. fluid (CSF).Itisalsoimportant,ofcourse,toestablishthat analgesia or to determine the levels in the cerebrospinal β-endorphin intheregionsofbrainthatareinvolved it is important therefore to determine the levels of receptors. Instudyingpainmechanisms( peptide doesnotappeartohaveaccesstheanalgesic can participateinanalgesicmechanisms:thepituitary This impliedthatitisonlyinthebrainβ-endorphin activity wasobtainedbyintravenousadministration. weakornoanalgesic applied intheventricles,very stable ( the sameN-terminalsequenceasenkephalinremained enkephalin wasrapidlydegraded,β rat brain.Inbothcases,underconditionswherethe peptides withamembrane-boundaminopeptidasefrom Lipotropin andbeta-endorphin -endorphin todegradationbyproteolyticenzymesin Published byBioscientifica Ltd. The strikingdifferencebetweenthelong-lasting In contrasttothehighpotencyofβ-endorphin Fig. 2 -endorphin andthetransienteffectsof ) ( Austen &Smyth1977,et al. 1979 2

terminus. We alsodigestedthetwo . 1977 et al Smyth 1976,Bradbury Downloaded fromBioscientifica.com at09/30/202101:38:48PM δ-opioid receptors, and their Bradbury Bradbury -endorphin whichhas 56 : -endorphin with 4 t al.1977 et Smyth 1983 , b ) ( Smyth & Fig. 3 ), but T16 via freeaccess ). ). ). ). ),

Journal of Molecular Endocrinology lipotropin wasonlyaminor component;themajority extracted fromregionsof ratbrainshowedthat of definedlocations. axons andendinginbundles ofterminalsatanumber the hypothalamic cell bodies, ramifying through long revealed ahighlyorganisednetworkcommencingwith and hippocampus.Ingeneral,immunofluorescence at more distant locations in the amygdala, colliculi dense inthethalamusandterminalswerepresent dorsally andlaterally, thefluorescence becoming less http://jme.endocrinology-journals.org DOI: 10.1530/JME-16-0033 Thematic Review Gel exclusionofβ -endorphin-related peptides d

smyth Printed inGreatBritain determined byradioimmunoassay( ion-exchange chromatography, theamounts being were identifiedbyextractionfromthetissuesand contained predominantly β brainstem and colliculi ( and amygdala,thesecond ofthehippocampus, was characteristicofthe hypothalamus,midbrain distinct processing patterns were apparent: the first 1982, Zakarian&Smyth1982b of of thepeptideshadapproximatemolecularsize Lipotropin andbeta-endorphin Published byBioscientifica Ltd. β -endorphin. The 202, 561–571.CopyrighttheBiochemicalSociety. Zakarian S&SmythDG,1982,BiochemicalJournal, thalamus. Reproduced,withpermission,from dorsolaterally fromtheventricletowards ventricle. (D’)Longbeadedaxonsextending fibres alongthelengthofwallthird (C’) Densefluorescenceintheformofaxonsand and fibresventromedialtothethirdventricle. the regionofarcuatenucleus.(B’)Cellbodies β-endorphin andcontainingfibresin hypothalamus. (A’) Cellbodiessynthesising Different formsofβ-endorphinfluorescenceinrat throughout thebrainstemarevisible.(Plate2) sparse buthighlyorganisedfibresandaxon terminals intheamygdaloidnucleus.(D) Relatively thalamus andmidbrain.(C)Densefibres beaded axonstypicaloftheregion by positivelystainedcytoplasm.(B)Numerous bodies withdarkunstainednucleiaresurrounded in themedianeminenceandarcuatenucleus;cell synthesising cellbodiesandbeadedfibresareseen immunofluorescence scanning.(A)β-endorphin β-endorphin inregionsofratbrainby (Plate 1)Localisationofpeptidesrelatedto Figure 3 Downloaded fromBioscientifica.com at09/30/202101:38:48PM β -endorphin-related peptides Fig. 4 -endorphin(1–31), and . 1977 , Snellet al ). The hypothalamus Smyth &Zakarian 56 : 4 ). Two T17 via freeaccess Journal of Molecular Endocrinology forms ofβ brainstem andcolliculicontainedprincipallyN of acetylatedpeptides.Incontrast,thehippocampus, these regions,therewerenegligibleconcentrations would alsocontainglycyl-glutaminereleasedfromthe contained C-terminallyshortenedformsofβ phenomenon. It is significant, however, that the cells that neurotransmission would seem to be a less common forms ofβ opiate activity. Intheregionsofbrainwhereinactive associated with these regions may involve regulation by and itispossiblethatthephysiologicalfunctions regions ofthebrainstorepotentformβ which aredevoidofopiateactivity. by the midbrainandamygdalabutaccompaniedthere this was the major DOI: 10.1530/JME-16-0033 http://jme.endocrinology-journals.org Thematic Review The results showed that the nerve terminalsincertain The resultsshowedthatthenerve β -endorphin(1–27) andβ -endorphin predominated,opiate-controlled -endorphin(1–27) andβ β-endorphin-related peptide in d

smyth -endorphin(1–26) Printed inGreatBritain -endorphin -endorphin -acetyl

β regulating neurotransmission. messenger may actasalow-molecularweightinhibitory should therefore be consideredthat glycyl-glutamine brainstem neurons( demonstrated thatthisdipeptideinhibitedthefiringof 1982 C-terminus ofthe31-residuepeptide( 125 using carrier-free radio-labelled β We investigatedtheopiate-likebindingpropertiesof peptide thanforanyofitsnaturallyoccurringrelatives, sites thathadahigheraffinityforthe31-residue we wereabletodemonstratetheexistenceofbinding position 27ofthehumansequence.Usingthissystem, Lipotropin andbeta-endorphin -endorphin(1–31) tosynaptosomesfromratcortex -endorphin andopiate receptors Published byBioscientifica Ltd. iodine attachedspecificallytothetyrosineresidueat ). Inarelatedelectrophysiologicalstudy, Wolstencroft 561–571. CopyrighttheBiochemical Society. Smyth DG,1982,BiochemicalJournal , Reproduced, withpermission,from ZakarianS& β β right bythearrows.α β α β the radio-labelledreferencepeptides(α β The peptidesweredetectedbyRIAwitha Sephadex C-25chromatography(Sigma-Aldrich). obtained bygelexclusionwereresolvedSP rat brain.Theβ related peptidesextractedfromtheregionsof Ion-exchange chromatographyofβ Figure 4 -endorphin(1–27) arenotindicated bymarkers. -endorphin(1–27) anddes-histidine -endorphin(1–31) areindicatedfromleftto -endorphin(1–27), -endorphin antiserum.Theelutionpositionsof - N -acetyl Parish Downloaded fromBioscientifica.com at09/30/202101:38:48PM β -endorphin(1–31) and t al.1983 et -endorphin-containing fractions β -endorphin(1–27), - N -acetyl des-histidine β 56:4 -endorphin with ). Thepossibility Parish &Smyth -endorphin- 202, - N -acetyl T18 via freeaccess Journal of Molecular Endocrinology ( receptor’ (possiblytheεreceptorpresentinratvasdeferens β opioidpeptides, receptors interactwithcomplementary sites ( Goldstein andhiscolleagueondynorphinbinding addition toanalgesia. functions other than or in receptors in the brain subserve potencies. Thiscouldsuggestthattheβ considerably higherthanthecorrespondinganalgesic binding affinitiesoftheshorterformsβ and arangeofclassicalligands( β β including Smyth 1979,1982a present specificallyinthecorticotrophs( In ratpituitary, weshowed thatβ processing β at adifferentreceptor, permittingadiversityofresponse. the hypothesisthateachfunctionalopiatepeptideacts ledto specific pharmacologicalassays.Theseobservations the fragmentsprovedtobesignificantlymorepotentin binding affinitiesthantherespectiveparentpeptide,yet of receptor issituated. opiate activityisfocusedatthesitewhereappropriate is targettedatacorrespondingreceptor, ensuringthat hypothesis proposedthateachfunctionalopioidpeptide present inmousevasdeferens( enkephalin atan‘enkephalinreceptor’(theδreceptor receptor’ (theκreceptor( ileum ( Hammonds -endorphin actingpreferentiallyata‘β -endorphin(1–17) (γ -endorphin(1–26) (des-histidineCʹ -endorphin inpituitary:tissue-specific http://jme.endocrinology-journals.org DOI: 10.1530/JME-16-0033 Thematic Review β -endorphin(1–31) anddynorphin(1–12)showedlower Our results,takentogetherwiththestudiesof thatrelativetoβ It wasobserved It isnotablethatcertain naturally occurring fragments Chavkin &Goldstein1981 Paterson β t al.1984 et -endorphin(1–27) (theCʹ . 1983 et al ) ( -endorphin) aswellenkephalin Fig. 5 )

Chavkin &Goldstein 1981 or theμreceptoringuineapig )), dynorphinata‘dynorphin ). Thiswasinlinewiththe Wuster d Toogood ), suggestedthatopiate

smyth . 1979 et al -fragment) and -endorphin was -endorphin, the -endorphin were Printed inGreatBritain et al. 1986 Zakarian & -fragment), -endorphin -endorphin )). The )) and ).

C-terminal proteolysisorN-terminalacetylation( ( the resultsofexperimentsconductedinXenopuslaevis ( in itspituitary to significantchangesintheprocessingofβ-endorphin immune systembytissuetransplantationintheratled tissues. Ofinterestwasthefindingthatactivationof isms thatgeneratedifferentbiologicalactivitiesin multifunctional prohormoneofβ-endorphin,mechan exist forfragmentationofpro-opiomelanocortin,the determining theirconcentrationsbyradioimmunoassay. by gelexclusionandion-exchangechromatography by extractingthepeptidesfromtissues,resolvingthem 1982a 1980, Smyth1981,et al. 1981a,bZakarian&Smyth and/or truncatedattheirC-terminus( in the pars intermedia were acetylated at their N-terminus was pituitary Deakin for itspotentanalgesicactivity( N- andC-terminalsequencesofβ potent analgesicactivity. Thisshowedthatboththe al.1979 et 1978, 1981a six immunoreactive forms ofβ in theposteriorpituitary. We hadbythistimeidentified present inallthecellsofparsintermediabutnone Herbert 1977 from acommonprecursor( Portland, Oregon,thatlipotropinandACTHareformed madeinHerbert’simportant discovery in laboratory pars intermedia,α-MSHisaccompaniedbyinactiveforms variable amountsofβ is sometimes accompanied by in the anteriorpituitary physiological signals. of pro-opiomelanocortinisdynamic:itrespondsto Lipotropin andbeta-endorphin Maruthainar Published byBioscientifica Ltd. We foundthatalternative processing mechanisms It is of interest that, depending on the species, ACTH The predominantformofβ ) ( et al. 1980 Fig. 6 ), andonlyβ , ). Theprocessingpatternsweredemonstrated b β t al. 1992 et , Smyth1984 ). -endorphin(1–31), but the principal forms Zakarian Biochemical Society. Biochemical Journal,202561–571.Copyrightthe permission, fromZakarianS&Smyth DG,1982, antibody toβ-endorphin.Reproduced,with brain fromLewisratswerestainedwith the scanning. Serialsectionsofperfused andfixed intermedia oftheratbyimmunofluorescence (A) in theanteriorpituitaryand(B)inpars Localisation ofpeptidesrelatedtoβ-endorphin Figure 5 ). β -endorphin immunoreactivitywas -endorphin(1–31), whereasinthe Downloaded fromBioscientifica.com at09/30/202101:38:48PM . 1989 et al ), it is clear that the processing -endorphin(1–31) possessed Mains ), whichweregeneratedby -endorphin inratanterior -endorphin ( -endorphin areessential ). Taken togetherwith . 1977, Roberts & . 1977,Roberts & et al Geisow 56:4 Smyth &Zakarian : t al.1977, et Smyth Smyth T19 . et al via freeaccess

Journal of Molecular Endocrinology indicate theelutionpositionsof I (A) anteriorpituitaryand(B)parsintermedia. ThearrowsfromLtoR obtained bygelexclusionwereresolvedexchangechromatography: from theregionsofratpituitary. Theβ-endorphin-containingfractions Ion-exchange chromatographyofβ-endorphin-relatedpeptidesextracted Figure 6 system thatcouldbeinvestigatedtoadvantage.An a commonprecursorpresenteduswithphysiological this processingreaction. two activitiesisalteredandpossiblyregulatedinvivoby ( the N-terminusofβ activates thismelanotropichormonebutacetylationat of Biochemical Society. Smyth DG,1982,BiochemicalJournal , 202561–571.Copyrightthe β-endorphin(1–31). Reproduced,with permission,fromZakarianS& β-endorphin(1–27), α-N-acetyl β-endorphin(1–31)and Smyth DOI: 10.1530/JME-16-0033 http://jme.endocrinology-journals.org Thematic Review β -endorphin. Acetylation at the N-terminus of α-MSH The presenceofacascadepeptidesoriginatingfrom t al.1979 et ). Consequently, the‘balance’of -endorphin leadstoitsinactivation 125 -labelled peptides,α-N-acetyl d

smyth Printed inGreatBritain

competitive, that is, they do not take place simultaneously. to lipotropin toβ its C-terminus. The processing reactions that convert rise toβ concluded thatduringbiosynthesis,lipotropingives that containsthefullC-terminalsequence.Itcanbe from the 26-and27-residueforms. of C-terminal proteolysis: they were present principally as directly fromtheparsintermediaexhibitedahighdegree peptides withasimilarsizetoβ 27-residue forms was generated. In marked contrast, 31-residue sequenceofβ the sameC-terminalpeptide:itcontainedfull andtheparsintermediawasfoundtogiverise pituitary C-terminus. and with an intact C-terminal region, but strategy wastoreleaseβ groups oflysinereversiblybycitraconylation.Overall,the POMC →lipotropin(1–91) lipotropin(61–91)→ commences: sequence, eachstepgoingto completionbeforethenext processing indistinctstagesthatoccuratime-ordered the prohormoneofβ release ofβ sequence doesnotactattheLys–Lys forthe sitenecessary enzyme thatreleasesβ Furthermore, itisimplicitthatthepairedbasicresidue et al. 1988 exclusion andcationexchangechromatography( andthenresolvingthembygel intermedia ofthepituitary to obtained byextractinglipotropinandpeptidesrelated independently inaregimentedmanner?Evidencewas occur or do the different processing reactions take place granulewheretheprocessingreactions in thesecretory from theirprecursorsremaininthesamecompartment interesting questionwas:dothedifferentpeptidesreleased the lipotropinsequence,achievedbyprotectingε-NH restricted totheCOOHgroupofarginineatposition60 cleavagebeing its C-terminalfragmentreleasedbytrypsin, the structureoflipotropinwouldberevealedbyidentifying Lipotropin andbeta-endorphin Published byBioscientifica Ltd. lipotropin(61–87) β The experimentrevealedthatlipotropindiffers The lipotropinobtainedfromboththeanterior In summary, theinvestigation demonstratedthat β -endorphin from the anterior pituitary andpars -endorphin fromtheanteriorpituitary β -endorphin(1–27) arethusorderedandnot β -endorphin(1–26) fromlipotropintruncatedatthe -endorphin inthatitoccursexclusivelyaform -endorphin beforeproteolysistakesplaceat ). Itwasenvisagedthatpossibleheterogeneityin -endorphin(1–27). -endorphin(1–31) andβ → lipotropin(61–86) -endorphin undergoesproteolytic -endorphin(1–31) fromlipotropin -endorphin. None of the 26-and Downloaded fromBioscientifica.com at09/30/202101:38:48PM -endorphin(1–31) ataLys–Arg -endorphin isolated 56:4 β -endorphin(1–31) -endorphin(1–27) : Smyth T20 via freeaccess 2

Journal of Molecular Endocrinology located in different cells or secretory granules. located indifferentcellsorsecretory processing enzymeswithdifferentspecificityappeartobe In conclusion, the experimental results indicated that on theβ showed thatthecleavagereactiontookplaceexclusively this same sequence in lipotropin. The experiments sequence inβ same compartment,theenzymethatactsatLys–Lys different compartments.Iftheywerepresentinthe that theprocessingenzymesmustbesequesteredin basic N-terminalNH environment wasthe‘non-acetylated’peptidewitha offrogsmaintainedinadaylightMSH inthepituitary form ofthehormone.Incontrast,main frogs was the acetylated peptide, which is the potent form ofMSHintheparsintermediadark-adapted with theirsurroundings.We foundthattheprincipal skin toconcealthemselvesfrompredatorsbyblending Against adarkbackground,frogsusedarkeningofthe and obtained bystudyingtheacetylationofβ logically atthelevelofprohormoneprocessingwas Evidence that hormone activity is regulated physio Processing of molecules, anditwasinteresting toseethatmorphine activity toβ Morphine, analkaloid, exhibits similarbiological sensitivity to physiological signals β against thelightbackground. and non-acetylated proved to be the principal peptide on the dark background also proteolysis.Theacetylformofβ environment, withrespectnotonlytoacetylationbut β mechanisms. We alsofoundthattheprocessing of adaptation islikelytoinvolverelativelyslowbiosynthetic certainly notinstantaneous,whichindicatesthatcolour place overthecourseofseveraldays.Theresponseswere lation reaction. mone, probablybytheenzymethatcatalysesacety- by specificprocessingoftheACTH–endorphinprohor- activity of melanotropic activity( -endorphin changedinresponsetochangethe -endorphin andmorphine: spatial structure http://jme.endocrinology-journals.org DOI: 10.1530/JME-16-0033 Thematic Review The resultssuggestedanadditionalpointofinterest It isinterestingthattheprocessingchangestook α -MSH inXenopus -endorphin andnotatallonthelipotropin. α -MSH in background adaptation is governed -endorphin. We constructed modelsofthese -endorphin wouldbeexpectedtoactat β -endorphin andmelanotropin: β 2 -endorphin(1–31) (the potent form)

group, whichpossessesonlyweak laevis ( Fig. 7 ). Thus,themelanotropic Maruthainar d

smyth -endorphin(1–8) Printed inGreatBritain . 1992 et al -endorphin ). ).

Schwyzer whenheappliedittoACTH( This concept of message and address was proposed by the continuing chain of aminoacids(Smyth1980). linked toan‘addresssequence’whichisformedfrom site or‘messageregion’,theN-terminaltetrapeptide, a hypothesisthatopioidpeptidescompriseanactive profound influenceonanalgesicactivityledustopropose that extendsbeyondtheN-terminalpentapeptidehas a the conformationofN-terminalTyr–Gly–Gly–Phe. receptor,complementary aninteractionthatinfluences of the31-residuepeptidewithinenvironmenta an interactionbetweentheN-andC-terminalregions β that thecontributionofC-terminalregion but farlessinothers.Dynorphin, forexample,isfar exceptionally highpotency incertainopiateassays thatsomeopioidpeptideshaveexplain theobservation address confersspecificity andpotency. Thiswould the bindingaffinityfor opiate receptor, whereasthe The message region of and magnesium, whichindicatesthatβ was inhibitedbysodiumandaugmented to brainreceptors,likethatofβ we foundthatthebindingof27-residuepeptide contains theintactC-terminalsequence.Inaddition, β than itschemistry. morphine dependontheshapeofmoleculerather implied thattheopiateactivitiesofβ β β similartotheN-terminalregionof fused ringswasvery which hasarigidmolecularstructureimposedby a potentsyntheticderivative of morphine ( There was an even closer similarity with benzyloripavine, of theN oninvestigation effectswereobserved no inhibitory bindingsiteforanalgesicreceptors,buta secondary that theintactC-terminal region ofβ not forthedurationofanalgesia.Ithasbeensuggested of ( but thedurationofanalgesicactivityisundiminished is 2 orders ofmagnitudelessthanthe31-residuepeptide, lacks the four C-terminal residues of The analgesicpotency of β Lipotropin andbeta-endorphin Feldberg &Smyth1977) -endorphin totheproductionofanalgesiaisrelated -turn predictedfromitssequence( -endorphin whenthepeptidechainwasfoldedin -endorphin: message andaddress regions Published byBioscientifica Ltd. β -endorphin arethusimportantforthepotencybut The findingthatthepeptidechainofβ β -endorphin(1–31) actasagonists.Itispossible -acetyl derivativeofβ β Downloaded fromBioscientifica.com at09/30/202101:38:48PM -endorphin appears to provide . ThefourC-terminalresidues -endorphin(1–27), which -endorphin(1–31), which Bradbury Bradbury β 56:4 -endorphin(1–31), -endorphin(1–31), -endorphin(1–27) -endorphin offers : Schwyzer 1977 -endorphin and et al. 1976d Fig. 8 -endorphin ). This T21 via freeaccess ). ). ).

Journal of Molecular Endocrinology Maruthainar et al.(1992). adaptation toablackbackground.Reproduced, withpermission,from adaptation toadaylightbackground and(BC)after24 weeks Sephadex C-25werelocatedbyRIA with MSHantibodyafter(A)2 weeks exclusion onSephadexG-50andion-exchange chromatographyonSP adapted toblackorwhitebackgrounds. Thepeptidespurifiedbygel des-acetyl α-MSHextractedfromtheparsintermediaofXenopuslaevis Resolution byion-exchangechromatographyofacetylα-MSHand Figure 7 DOI: 10.1530/JME-16-0033 http://jme.endocrinology-journals.org Thematic Review d

smyth Printed inGreatBritain for identification( gland, wheretheyarepresentinsufficientquantity occur asmajorpeptidesinthemedullaofadrenal species, C-terminallyextendedformsofenkephalin ( separated byadifferentsequenceofaminoacids pentapeptide andonecopyofLeu-enkephalin,each precursor thatcontainsseveralcopiesofthe of analgesicproperties. β and of Similarly, itappearsthattheshorteraddresssequences their N-terminusbuthavedifferentaddresssequences. at thesereceptorspossessthesamefourresidues in athird,becausethepeptidesthatactpreferentially β gene duplicatedprohormones. to thebiologicallyactivepeptidesderivedfromother credible andithasanaestheticattraction.Itmayapply single prohormone, each with its preferred receptor, is concept ofarangeopioidpeptidesderivedfrom properties intermsofspecificityandpotency. The but different address sequences and possess different active peptidesthathavethe same message region findings pointtotheexistenceofaspectrumopiate intermediates formedenroute toanenkephalin.These their ownrightandareunlikelytobemerelytransient potent activities,thesepeptidesseemlikelytoexistin enkephalin inspecificopiateassays.Becauseoftheir it wasreportedthattheyexhibithigherpotencythan opioid receptors,δ rationalise thefindingofκ properties. Themessageandaddresshypothesismayalso ileum assay, butitdoesnothaveoutstandinganalgesic more potentthanleucineenkephalinintheguineapig rates) andarebrokendown rapidly, whichexplainstheir have relativelylowaffinity forreceptors(rapidon/off They actovershortdistances acrosssynapticgaps, resemble classicaltransmitters such as acetylcholine. actions atthetwolocations. enkephalin indicatesthatsimilarprinciplesgoverntheir act ashormones.Ourexperiencewithβ neurotransmitters, whereaspeptidesintheperiphery Peptides inthebrainaregenerallyconsideredtoact as and neurotransmitter Lipotropin andbeta-endorphin Comb -endorphin(1–31) mayaccountfortheirrelativelack -endorphin andenkephalin: neuromodulator Published byBioscientifica Ltd. β -endorphin(1–27), Methionine enkephalinisbiosynthesisedfroma In thebrain,smallpeptides suchasenkephalin α -endorphin (β t al.1982 et -specificity inanotherandμ Kilpatrick ). Ithasbeenshownthatinsome -endorphin(1–16)) comparedwith γ -endorphin (β Downloaded fromBioscientifica.com at09/30/202101:38:48PM -specificity inonegroupof . 1981 et al 56:4 -endorphin(1–17)) -endorphin and ). Interestingly, -specificity T22 via freeaccess

Journal of Molecular Endocrinology properties anddistinctivefunctionalroles. ‘neurotransmitter’, eachwithcharacteristicallydistinct neuroactive peptide,thatistosay, ‘neuromodulator’and and enkephalinappeartorepresentdifferentclassesof to havecounterpartsintheperiphery. Evenso,β both smallandlargepeptidesinthebraincanbeseen transmitting signalsthatmodulatehormoneactivity. do inthebrain:theycanactlocallyasparacrineagents, astransmitters peptides maybehaveintheperiphery to performtheirfunctionashormones,butsmall viathecirculationare transportedfromthepituitary the periphery, largepeptidesbeingrelativelystable that expresstheirfunctionsatmoredistanttargets.In consistent withtheirfunctioningasneuromodulators are muchmorestableandproducelong-lastingeffects, brain suchasβ-endorphinhavehigherreceptoraffinities, short-lasting evanescent effects. Larger peptides in the pituitary that ledthewaytodemonstrationofpotent pituitary sequence andouridentification ofβ of & Li1967 and laterthecharacterisation ofγ of theaminoacidsequencelipotropin( system. transmission inthecentralnervous advancing ourknowledgeandunderstandingofchemical and event, identificationofthestructuresenkephalin be ofoutstandingimportanceinthefuture.In that thedeterminationofstructurewouldprove to The earlypioneershadtheforesighttoanticipate structure Opioid peptides:ontheidentification of http://jme.endocrinology-journals.org DOI: 10.1530/JME-16-0033 Thematic Review β In respectoftheiractivities,themodesaction In adifferentfieldofinvestigation,Li’s identification -endorphin. Itwasknowledge ofthelipotropin β -endorphin laidthefoundationsforanewera, ) served as a signpost pointing to the discovery asa signpostpointingtothediscovery ) served d

-lipotropin ( smyth -endorphin inthe Printed inGreatBritain Li -endorphin . 1965 et al Chretien )

activity later. HappilyallroadsleadtoRome! followed. With β of thepredictedopiatewasanticipated,thenstructure and breakthrough camewiththeidentificationofenkephalin the InternationalNarcotic Research Conference.The Hughes, Kosterlitz,SimonsandSnyderatMeetingsof morphine had been discussed regularly by Goldstein, that thebrainmightcontainanendogenousformof identifying peptides with opiate activity, the possibility of biologicallyactivepeptides.Inthecompetitiveracefor offers examplesof different approaches used intheisolation profound significanceinbrainfunction. opiate activityofthisinterestingpeptideandrevealedits Austen BM & Smyth DG 1977 TheNH2-terminusofC-fragmentis &Smyth DG Austen BM References concepts presented. Marta Korbonits(WilliamHarveyResearchInstitute)fordiscussingthe Clark for assistance in the preparation of this manuscript and Professor The authorwishestoexpresshisindebtednessProfessorAdrian Acknowledgements the public,commercialornot-for-profit sector. This articledidnotreceiveanyspecificgrantfromfundingagency in Funding perceived asprejudicingtheimpartialityofthisreview. The authordeclaresthatthereisnoconflictofinterestcouldbe Declaration ofinterest Lipotropin andbeta-endorphin Published byBioscientifica Ltd. 291X(77)90749-5) Biophysical Research Communications resistant totheactionofaminopeptidases. Biochemicaland The identificationofenkephalinandβ β -endorphin. Inthecaseofenkephalin,activity -endorphin, thestructurecamefirst, . with permission,fromBradburyet al.(1976d (B) oripavine and(C)morphine.Reproduced, β Spatial modelsof(A)residues1–5 Figure 8 -endorphin (methionineenkephalin), Downloaded fromBioscientifica.com at09/30/202101:38:48PM 76 477–482 56:4 : (doi:10.1016/0006- -endorphin T23 ). via freeaccess Journal of Molecular Endocrinology Hughes J, Kosterlitz HW, Fothergill LA, Morgan BA & Morris HR 1975 &Morris HR Morgan BA Kosterlitz HW, Fothergill LA, Hughes J, 1984beta-endorphin-(1-27)isan & Li CR Nicolas P Hammonds RG, Wiegant Smyth DG, VM,Bradbury AF,Gispen WH, Hulme EC, Geisow MJ, Deakin JFW, Dostrovsky JFW & Smyth DG 1977Analgesic DeakinJFW, &Smyth DG Geisow MJ, Dostrovsky JFW 1977C-fragmentoflipotropin–an &Smyth DG Feldberg WS Feldberg WS&SmythDG1976TheC-fragmentoflipotropin–a 1980InfluenceofN-terminal &Smyth DG Deakin JFW, Dostrovsky JO Comb M, Seeberg PH, Adelman J, Eiden L & Herbert E 1982 Primary EidenL&HerbertE1982Primary Adelman J, Seeberg PH, Comb M, 1967 Isolation, purification, and characterization &Li CH Chretien M 1981Specificreceptorfortheopioidpeptide &Goldstein A Chavkin C &Wendlandt S 1977 Deakin JFW Snell CR, Bradbury AF, Smyth DG, 1976d &Snell CR Bradbury AF, Smyth DG 1976c &Snell CR Bradbury AF, Smyth DG 1976bProhormonesofbeta- &Snell CR Bradbury AF, Smyth DG 1976a &Hulme EC Birdsall NJ Snell CR, Bradbury AF, Smyth DG, 1975Biosynthesisofbeta-MSHand &Snell CR Bradbury AF, Smyth DG 1974 &Rees LH Scott AP, Gilkes JJH Bloomfield GA, Lowry PJ, 1964Isolationandpropertiesofanew, &Li CH Birk Y 1979Susceptibilityofneuroactive &Smyth DG Evans CR Austen BM, DOI: 10.1530/JME-16-0033 http://jme.endocrinology-journals.org Thematic Review (doi:10.1038/258577a0) potent opiateagonistactivity. Nature Identification oftworelatedpentapeptides fromthebrainwith antagonist ofbeta-endorphinanalgesia. PNAS (doi:10.1038/264794a0) rat byfragmentsoflipotropin.Nature &deWied D1976Inductionofexcessivegroominginthe Snell CR tetrapeptide. activity oflipotropinC-fragmentdependsoncarboxylterminal 60 445–453. ofPharmacology endogenous potentanalgesicpeptide.BritishJournal ofPhysiology potent analgesic.Journal bj1890501) of beta-endorphin.BiochemicalJournal acetylation andC-terminalproteolysisontheanalgesicactivity 1211–1217. mRNA. structure ofthehumanMet-andLeu-enkephalinprecursorits o67-133) ofBiochemistry Canadian Journal glands.of gamma-lipotropichormonefromsheeppituitary dynorphin: structure-activityrelationships.PNAS 291X(77)90365-5) Research Communications and stabilizedenkephalinsintherat.BiochemicalBiophysical Comparison oftheanalgesicpropertieslipotropinC-fragment (doi:10.1038/260165a0) conformation ofmethionineencephalin.Nature Research Communications 69 two biologicallyactivepeptides.BiochemicalandBiophysical Elsevier/Excerpta Medica. Aspects. CIBAFoundationSymposium, MolecularandCellular and activation.InPolypeptideHormones, and corticotropin(adrenocorticotropichormone,ACTH):structure melanotropin (betamelanocyte-stimulatinghormone,beta-MSH) opiate receptors.Nature C fragmentoflipotropinhasahighaffinityforbrain Science PublishersInc. Eds RWalter &JMeienhofer. AnnArbor, MI,USA:AnnArbor ACTH. (doi:10.1038/252492a0) A reappraisalofhumanbetaMSH.Nature www.jbc.org/content/239/4/1048.long) ofBiologicalChemistry Journal glands.biologically activepeptidefromsheeppituitary size. peptides toaminopeptidasedigestionisrelatedmolecular Biochemical andBiophysicalResearch Communications In Peptides,Chemistry, andBiology,609–615. Structure Nature (doi:10.1016/0006-291X(79)91196-3) Nature 295 663–666. 269 167–168. 74 748–753. 260 (doi:10.1038/295663a0) 950–956. 793–795. 239 1048–1052. 45 1163–1174. (doi:10.1038/269167a0) 41 61–75.NewYork, NY, USA: Biosynthetic origin and receptor Biosyntheticoriginandreceptor 258 577–580. 260 30P–31P. d (doi:10.1016/0006- 189 501–506. 264 794–795.

smyth 252 492–493. 81 1389–1390. 165–166. 260 165–166. (available at:http:// 78 6543–6547. Printed inGreatBritain (doi:10.1139/ (doi:10.1042/

Smyth DG 1975Thepeptidehormones:molecularandcellularaspects. Smyth DG review. 1977ACTH:ashortintroductory Schwyzer R AnnalsoftheNew Smyth DG & Zakarian S 1980 Selectiveprocessingofbeta-endorphinin &Zakarian S Smyth DG 1984Chromatographyofpeptidesrelatedtobeta-endorphin. Smyth DG 1983Opioidpeptidesandpain.ClinicsinAnaesthesiology Smyth DG oftheopiatepeptides:enkephalins and 1981Chemistry Smyth DG 1976Searching fortheendogenousanalgesic.Lancet Smyth DG Smyth DG & Zakarian S 1982 Beta-endorphininbrain.Chemical &Zakarian S Smyth DG structureandspecificity inendogenous 1980Primary Smyth DG Roberts JL & Herbert E 1977Characterizationofacommonprecursor &Herbert E Roberts JL 1983Classificationofopioid &Kosterlitz HW Robson LE Paterson SJ, &Wolstencroft JH 1983 Normanton JR Smyth DG, Parish DC, 1982Isolationofglycylglutamine,the &Smyth DG Parish DC Chang AC, Tanaka L, Kita T, Nakamura M, Inoue A, Nakanishi S, 1992Theprocessing &Smyth DG Peng-Loh Y Maruthainar K, 1977Commonprecursorto &Ling L Eipper BA Mains RE, 201660YEARSOFPOMC:Purificationandbiological Lowry P Tseng LF,Loh HH, 1976beta-endorphinisapotent Wei E &Li CH 1965Isolationandamino-acid &Chung D Chretien M Barnafi L, Li CH, 1976Isolationandstructureofanuntriakontapeptide &Chung D Li CH 1981Ahighly Tamiguchi T, &Udenfriend S Kilpatrick DL, Jones BN Jacquet YF, 1978beta-endorphin:modulation Klee WA &Smyth DG Lipotropin andbeta-endorphin Published byBioscientifica Ltd. tb41843.x) Nature York AcademyofSciences regions ofporcine pituitary. Nature 2697(84)90314-2) Analytical Biochemistry 201–217. pp 56–69.EdKBrunfeld.Copenhagen,Denmark:Scriptor. endorphins. InProceedings ofthe6thEuropean PeptideSymposium , 2 Transmission intheBrain, theRoleofAmines,AminoAcidsandPeptides, USA: PergamonPress. Opiate AgonistsandAntagonists,pp1–4.EdELWei. NewYork, NY, opioid peptides.InAdvancesinEndogenousandExogenous joe.0.1350469) 8993(78)90527-9) molecule. precursor andidentificationofcorticotropinpeptidesinthe to corticotropinandbeta-lipotropin:cell-freesynthesisofthe receptors. 306267a0) beta-endorphin. neuropeptidederivedfromGlycyl glutamine,aninhibitory Biochemical SocietyTransactions C-terminal dipeptideoftheß-endorphincorticotropinprohormone. 423–427. for bovinecorticotropin-beta-lipotropinprecursor. Nature 1979NucleotidesequenceofclonedcDNA &Numa S Cohen SN adaptation. intermedia ofXenopuslaevisisinfluencedbybackground of beta-endorphinandalpha-melanotrophininthepars corticotropins andendorphins.PNAS Molecular Endocrinology of characterisation ofmelanotrophinsandcorticotrophins.Journal analgesic agent.PNAS 1093–1094. glands.Nature sequence ofbeta-LPHfromsheeppituitary 1145–1148. glands.PNAS with opiateactivityfromcamelpituitary (doi:10.1073/pnas.78.5.3265) [Met]- and[Leu]enkephalinsequence.PNAS potent 3200-daltonadrenalopioidpeptidethatcontainsbotha systems. of acutetoleranceandantagonismbyendogenousbrain 665. 257 89–90. Brain Research (doi:10.1038/278423a0) British MedicalBulletin PNAS Journal ofEndocrinology Journal 74 4826–4830. Nature (doi:10.1038/257089a0) 136 127–135. 73 2895–2898. (doi:10.1016/B978-0-08-025488-3.50006-4) 56 T1–T12. 156 396–401. 297 3–26. 306 267–270. Downloaded fromBioscientifica.com at09/30/202101:38:48PM

10 (doi:10.1073/pnas.74.11.4826) 39 31–36.

221. (doi:10.1111/j.1749-6632.1977. (doi:10.1530/JME-15-0260) 288 613–615. 135 469–478. (doi:10.1016/0003- 74 3014–3018. (doi:10.1073/pnas.73.8.2895)

(doi:10.1016/0006- (doi:10.1038/ 78 3265–3268. 56:4 : (doi:10.1677/

73 278 208

T24

1 via freeaccess Journal of Molecular Endocrinology Snell CR, Jeffcoate W, Lowry PJ, Rees LH & Smyth DG 1977Preparation &Smyth DG Rees LH Jeffcoate W, Lowry PJ, Snell CR, 1988Sequentialformation &Maruthainar K Darby NJ Smyth DG, 1981b &Massey DE Deakin JFW Zakarian S, Smyth DG, 1981a &Zakarian S Smith CCF Smyth DG, 1979Endorphinsare &Finnie MDA Zakarian S Massey DE, Smyth DG, 1978Isolationofthe &Massey CR Snell CR Smyth DG, http://jme.endocrinology-journals.org DOI: 10.1530/JME-16-0033 Thematic Review 5793(77)80570-X) lipotropin. and characterisationofaspecificantiserumtotheC-Fragment Neuroendocrinology of beta-endorphin-relatedpeptidesinporcine pituitary. Medical. pp 79–100.EdsGLawrenson&DCEvered.London,UK:Pitman , identification anddistribution.InPeptidesoftheParsIntermedia gland:isolation,endorphin relatedpeptidesinthepituitary 0.50053-X) Kodansha/Elsevier. Ed HTakagi. Tokyo, Japan/Amsterdam,TheNetherlands: In AdvancesinEndogenousandExogenousOpioids,pp145–148. identification oftwonewpeptidesrelatedtobeta-endorphin. acetyl peptides.Nature stored inbiologicallyactiveandinactiveforms:isolationofa-N- (doi:10.1042/bj1750261) and C-fragmentfrombrain.BiochemicalJournal C-fragment andC'-fragmentoflipotropinfrompigpituitary & DFSwaab.NewYork, NY, USA:ElsevierBiomedicalPress. Progress inBrainResearch , vol55,pp FEBS Letters 47 317–322. (doi:10.1016/B978-0-444-80402- 279 252–254. 81 427–430. 123–132. EdsRMBuius,PPevet (doi:10.1016/0014- (doi:10.1038/279252a0) d

smyth Printed inGreatBritain 175 261–270.

beta-

Accepted Preprint published online 22 February 2016 Accepted Preprintpublishedonline22February 2016 Accepted 22February 2016 Received infinalform16February Zakarian S, Eleazar M & Silvers WK 1989Regulationofpro- &Silvers WK Eleazar M Zakarian S, 1982bDistributionofbeta-endorphin-related &Smyth DG Zakarian S 1982a &Smyth DG Zakarian S 1979Distributionofactiveandinactiveforms &Smyth DG Zakarian S 1979Specificityofopioidstowardsthe &Herz A Sculz R Wuster M, 1979Dependence &Colpaert FC Smyth DG Van Ree JM, & Smyth DG Bradbury AF, deWied D, Van Hulme EC, Ree JM, 1986Useof &Smyth DG Hulme EC Toogood CIA, McFarthing KG, Lipotropin andbeta-endorphin Published byBioscientifica Ltd. immunity. opiomelanocortin biosynthesisandprocessingbytransplantation (doi:10.1042/bj2020561) 202561–571. andbrain.BiochemicalJournal peptides inratpituitary Nature 296250–252. andbrain.differently inspecificregionsofratpituitary (doi:10.1073/pnas.76.11.5972) andbrain.PNAS of endorphinsinratpituitary 193–198. mu-, delta-andepsilon-opiatereceptors.Neuroscience Letters 24 495–502. evidence foritsinternalcontrolofbehaviour. LifeSciences creating propertiesoflipotropinC-fragment(beta-endorphin): (doi:10.1038/264792a0) lipotropin C-Fragment.Nature 1976Inductionoftolerancetotheanalgesicaction Snell CR (doi:10.1159/000124592) sites inratcortex.Neuroendocrinology 125I-Tyr27 beta-endorphinforthestudyofbinding Nature (doi:10.1016/0024-3205(79)90170-X) 339 553–556. (doi:10.1038/296250a0) Downloaded fromBioscientifica.com at09/30/202101:38:48PM beta-endorphinisprocessed 264 792–794. (doi:10.1038/339553a0) 43 629–634. 56:4 : 76 5972–5976.

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