Proc.Nati.Acad. Sci. USA
Vol. 75, No.11, pp. 5711-5714, November1978 Medical Sciences
Characterization of a nontrypsin cholecystokinin converting enzyme in mammalian brain
(cholecystokinin fragments/hormonespecificity/speciesspecificity/gastrin/radioimmunoassay) EUGENE
STRAUS,
ALBERTOMALESCI,
ANDROSALYN S. YALOWVeteransAdministrationHospital,Bronx,NewYork10468; and TheMountSinaiSchoolofMedicine,CityUniversityofNewYork,NewYork,NewYork10029 ContributedbyRosalynS.Yalow,August 28,1978
ABSTRACT Anenzymehas been partiallypurifiedfrom canineand porcinecerebral corticalextractsthatdiffersfrom trypsin inthat itmanifestssomedegree of hormonespecificity sinceitconvertsporcine cholecystokinintosmallerimmuno- reactive forms, i.e., the COOH-terminal dodecapeptide and octapeptidefragments, but failsto convertbiggastrin(34amino acids) to heptadecapeptide gastrin. This enzyme is distin- guishable fromtrypsinnotonlyinsubstratespecificity,butalso inseveralphysicochemicalproprties. Itis notinhibitedinthe presence ofconcentrations oflimabeantrypsininhibitor suf- ficienttoinhibit1 mg of trypsin rmlof incubationmixture.
It isinactivated whenincubate with substrateat450Cfor1 hr., whereastrypsin remainsfullyactivewhen incubated under the same conditions at550C.Theenzyme elutes inthevoid volumeonSephadexG-50andG-75
gel
filtration.On sucrose gradientcentrifugation,theproteolyticactivity associated with trypsin isrecovered aboveabuminbutthatofthesolubilized brainenzymeisrecoveredbelowgammaglobulin.Theenzyme isnotdetectableinsplenicextracts,whichdocontainnonspe- cificproteasescapabfe
ofcompletelydegrading cholecystokinin.Further investigationisrequiredtodeterminewhether theen- zymeinthegutthat convertscholecystokinintothebioactive andimmunoactiveCOOH-terminalfragments resemblesoris different from the brainconverting enzyme.
Overthepastdecadeithas become evident that many, ifnot all, peptide hormonesarefoundintheirtissuesof origin in more than one form(seeref. 1 forreview).Following the discovery that proinsulinistheprecursorof insulin(2) ithasgenerally been assumed that, if the larger hormonal form contains a peptidewith amino acidsequenceidentical to or if it is con- vertibleby enzymatic conversion to asmaller, well-charac- terized, andmorebiologicallyactiveform,thelarger molecule is a prohormone, whether or notthe biosynthetic precursor relationship has been established.Thus, aprecursor relationship has beendefinitely established forseveral peptidehormones, including proinsulin (2)and proparathyroidhormone (3-5), but it has only been assumed for others, such as big (34- amino-acid)gastrin(gastrin-34)(6, 7) andthe39-amino-acid cholecystokininvariant(CCK-39)(8).Similarly,cholecystokinin (CCK-33)canbeconsideredtobe aprecursorfor thechole- cystokinin
dodecapeptide
(CCK-12)andoctapeptide (CCK-8), the COOH-terminal fragmentswhich have biologicpotencies greaterthan thatof thepresumedparentCCK-33
molecule.Althoughseveral groups have reported on the existence in the tissues of origin oftryptic-like enzymes involved in the conversion ofproinsulin (9) andproparathyroidhormone (10, 11), precursors to insulin and parathyroid hormone, respec- tively, noevidence has as yetbeen presented to determine whetherthe sameenzymemay beinvolved inprocessingmore than onehormonalfamily. In this report wecharacterizeand partially purifyanenzyme,obtainedfrom extracts ofmam-
malian
brain,
which converts porcinecholecystokinin (pCCK-33)
tosmaller immunoreactiveforms but which failstoconvertgastrin-34to
heptadecapeptide
gastrin(gastrin-17).
We demonstrate that this enzyme is distinguishable from trypsinin avariety ofphysicochemicalsystems.
MATERIALS AND METHODS
PreparationandFractionationofBrain Extracts as Source of
Enzyme.
Coldwaterextractsofporcineand canine cerebral corticaltissueswerepreparedasfollows. Animalswerekilledby
injection of lethaldoses of sodiumpentathol.Immediately
after injection theentirebrainwasremoved,placedin a
plastic bag,
andrapidlyfrozenondryice. Pieces of frozen cerebral corticaltissueweresubsequentlycutand extractedat40C withaTeflon
grinder
indistilledwater,whichwasaddedtoacon- centrationof 0.1gofwetweighttissueperml. Nodifferences inactivitywereobservedif theextractantwas0.9%NaClor0.25 Mphosphatebuffer(pH7.5). Theextracts were centri-
fuged
at10,000Xgfor15min.Thesupernatants weretested forability toconvertCCK-33 and gastrin-34 tosmallerim- munoreactiveformsasdescribedbelow. Theywerealsochro- matographedon1 X50cmcolumnscontainingSephadex G-50 or G-75. Thecolumns werecalibratedbyapplication ofra-dioactivemarkerstoestablish thepositionsofthe void volume and theradioiodidepeak. Afterapplication of brainextract, thecolumns wereeluted with0.9% NaCl and 1-mlfractions werecollected and tested forenzymaticactivity.
Theactivefractions from the Sephadex column were also centrifugedat190,000Xgfor2hr. Portions of thesupernatant andresuspended pelletswerethenlayered aboveacontinuous 10-40% sucrose gradient in a 5-ml polyethylene tube and centrifugedat-45,000 rpmfor22hrinaSpincomodel L ul- tracentrifuge fitted with a Beckman swinging bucket rotor (SW50.1). After thecentrifuge came to restwithoutbraking, successive0.4-mlsampleswereremovedfrom the bottom of eachtube and studied forenzymeactivity. Forcomparison,
125I-labeled
albumin, 125I-labeledgammaglobulin, andpan- creatictrypsin weresimilarlylayeredinduplicatetubesand centrifuged along with the activefractions of the brain ex- tracts.Substrates andEnzymatic Assay. Twosubstrateswereused:
purified pCCK-33, whichwas agift from Victor Mutt (Gas- trointestinal Hormone ResearchUnit,Stockholm) and wasre- ceivedthroughtheNIAMDDGastrointestinal HormoneRe- source, Bethesda, MD,and gastrin-34, whichwas agiftfrom R. A.Gregory (University of Liverpool, England) and which had beenpurifiedfromahumangastrinoma.
Enzymaticactivity wastestedbyincubatingthe brainextract Abbreviations:CCK-33,the 33-amino-acidcholecystokinin;CCK-12, COOH-terminal cholecystokinin dodecapeptide; CCK-8, COOH- terminalcholecystokinin octapeptide;gastrin-34,the 34-amino-acid biggastrin;gastrin-17,heptadecapeptidegastrin.
5711 Thepublicationcosts of this article weredefrayed in part by page chargepayment. This article musttherefore be hereby marked "ad- vertisement" inaccordancewith 18 U. S. C. §1734 solely to indicate thisfact.
5712 Medical Sciences: Straus et al.
orthevariousfractions from the separation systems with pCCK orgastrin-34,usuallyat concentrationsof500ng/mlin0.25 M phosphatebuffer (pH 7.5), fortified with1mgof humanserum albuminper ml.The incubation wasgenerallycarriedout at 250C for 60 min. At the end of that time the
samples
wereboiled for5 minto inactivatetheenzyme.The
substrates,
before andafter enzymaticdigestion,werethenfractionatedby
starch gel electrophoresisorSephadexgelfiltration. Radioimmuno- assayofthe eluateswasthen usedtodeterminealterationsinsubstrate inducedbyenzymaticconversion.Thesamesubstrates werelabeled with125Iand fractionated
similarly
aswellas on paperchromatoelectrophoresis, andthe enzymaticdegradation
oflabeledand unlabeled substrateswascompared.
Theactivityandspecificityofthe brainenzymewere com- pared with thoseof
chymotrypsin-free
trypsin(Sigma,
bovine trypsin,DCC-treatedtypeXI)at a concentrationof1mg/ml.
Theactivefractionfrom the
Sephadex purification
of the brain extract wasused ataconcentrationof40,gl
ofactivefraction per ml of incubation volume. The effect of temperatureon enzymatic activitywastested at25°, 379,450,
and 55'Cby
preincubating each of theenzymesfor 15 min tobring
it to temperature,thenadding each ofthetwo125I-labeledsubstrates andincubating
for60 minlonger
atthefour temperatures.The pH rangeofactivityofbothenzymes wasevaluatedby
incu- bation of enzymeand substrateatpH4.5-9. For somestudies excesslimabeantrypsin inhibitor(1mg/ml,
sufficienttoinhibit 3.2mgof trypsin perml)
wasaddedtoboth enzymesbefore addition of eachofthe labeledand unlabeled substrates.In some studies anextract from thespleen prepared similarly
tothe brainextract wastested forenzymatic activitywith bothun-labeledsubstrates.Inallcasesthe
samples
wereboiledfor5min toinactivate theenzymesbefore fractionationinthe various physicochemicalsystemstotestfor enzymaticconversion.Radioimmunoassay. The gastrinandCCK
peptides
weremeasured by radioimmunoassay
according
topublished
methods (12-14). 125I-Gastrin-17 (porcineheptadecapeptide
gastrinwas agift fromR. A.Gregorythrough
thecourtesyof MortonGrossman,Wadsworth,
VA)wasusedas tracerfor both assay systems. The guinea pig antiserumusedfor the gastrin assaydoesnot crossreactwithCCK orCCK-8(13).Theanti- serumobtainedatthecurrentbleedings
ofrabbitBcrossreactsidenticallywith CCK and CCK-8 andsomewhatmore
strongly
withgastrin-17. Thecrossreactivitywith gastrin-17doespermit useof125I-gastrin-17
asa tracerbut presentsnoproblem
since pure CCKsubstrateisused inthe CCK assays.Starchgel
and Sephadex eluateswereassayed
asdescribed(12, 13).RESULTS
125I-pCCK,
in commonwithmanypeptide
hormonessuchasinsulin,
corticotropin,andparathyroid hormone,
remains atthe siteofapplication
whenapplied
topaperfor chromatoelec-trophoresis
while theserumproteins,iodide,
and othermoreacidic
peptides
suchasgastrin,CCK-8, andfragments
suchasthe
iodotyrosines
migrateanodally (12). Therefore,
in this systemit isdifficulttodistinguish
totalproteolytic degradation
fromspecific cleavage
to afragment
ofCCKsuchasCCK-8.However, paper
chromatoelectrophoresis
doesserve as arapid
screeningtestfor
degradative
activity.ShowninFig. 1arein- tact125I-pCCK-33
beforeand after incubationat25°Cfor60 minwithafractioncontainingactiveenzyme.AfterSephadex
G-50and G-75gel
filtrationof thebrainextracts,only
thevoid volume regioncontained activefractions;
i.e., thoseable tofragment
the 125I-CCK-33.The void
volulme
eluate was incubated underthe same con- ditions also with'25I-gastrin-34,
gastrin-34(0.5 Mg/ml), and
pCCK-33(0.5Mg/ml) each
in 0.25 Mphosphate
buffer(pH 7.5)
Serum Anode
Origin proteins
FIG. 1. Paper chromatoelectrophoresis of 125I-CCK before
(Upper)and after(Lower)treatmentwith active fraction(voidvol-
umeeluate afterSephadexG-50gelfiltration)ofextractofpigcere-
bral cortex. Intact 125I-CCK remains at the site of application;
the125I-labeledfragmentsmigratewith theserumproteins.
fortifiedwith 1 mgofhumanserumalbuminper ml.Thebrain enzymewasusedat aconcentrationof40
Ml
ofactivefraction per mlof incubationmixture.Thesamefoursubstratesinthesamebufferwerealsoincubatedwith 1 mgof
trypsin
per ml.The starch
gel electrophoretic
patternsof the four substrates before and aftertreatmentwith thetwoenzymepreparations
isshownin
Fig.
2.Trypsin
converted labeledand unlabeledgastrin-34
to apeptide having
the starchgel electrophoretic
behavior of
gastrin-17 (Fig.
2right, bottom).
Italso converted labeledandunlabeledCCK-33to apeptide resembling
CCK-8(Fig.
2left, bottom).
However, theSephadex
G-50void
volume eluateof the brain extract, whichconvertedCCK-33tosmaller immunoreactivepeptides resembling
CCK-12and CCK-8(Fig.
2
left, middle),
didnotaltergastrin-34 (Fig.
2right, middle).
Other
experiments
overarange of enzymeconcentrations(40-800 Ml
of voidvolumeeluateperml)
and substratecon- centrations(0.2-5.0
,gofCCKperml)
confirm theability
of the brain enzymeto convertCCKtosmallerimmunoreactivefragments
anditsfailuretoconvertgastrin-34
togastrin-17.
Thus,
the substratespecificity
for the brain enzymedoesnotresembletrypsin.
Furthermore,
limabeamtrypsin
inhibitor doesnotinhibitthe brainenzymeatconcentrationssufficientto inhibit
completely
1 mgoftrypsin
per ml of incubation mixture. Thebrain enzyme also differs fromtrypsin
in its temperature sensitivity.Trypsin
remainedfully
active inconvertingCCK-33tothesmaller
fragments
whenmaintained for 1hrattemperatures upto550Cwhile the brain enzymewasinactivewhen incubatedwith substrate for 1 hrat
450C.
The
proteolytic
activityof both the brain enzyme andtrypsin
wasmaintainedoverthe
pH
range from6.5to9. Asyetnoat-tempthas beenmadetodetermine
pH optimum
foractivity
ofthe brainenzyme;it is inactiveat
pH
6and below.The brainenzymediffers
significantly
fromtrypsin
notonly
insubstrate
specificity
andtemperaturesensitivity,
but alsoin Proc.Nati. Acad.Sci. USA75(1978)
Proc.Natl.Acad.Sci. USA 75(1978)
125I-pCCK-33 pCC K-33
Bromophenol Albumin blue
1 ~~~~E_
'I-Gastrin-34
I.J E
v
IT x
dw
u C
V
13:m
6-
0
co 8-
y -1
0
cr C:
E 8-
0
ECD
u
IT 3- x -
> -
.
4 0
-7Cr
30
0o
Gastrin-34 8-
E8-
E
Anode
FIG. 2. Starch gelelectrophoresis of1251-labeledandunlabeledCCK andof125I-labeledand unlabeled gastrin-34 before(Top)and after treatment withbrainconverting enzyme (Middle)orwithtrypsin(Bottom).Trypsin converts labeled and unlabeledCCK to fragments with starchgelelectrophoretic mobility resembling CCK-8andlabeled and unlabeled gastrin-34 to fragmentsresemblinggastrin-17. Thebrain con- vertingenzymedoes not alter gastrin-34 but converts CCK to fragments with starch gel electrophoretic mobility resembling CCK-12 and CCK-8.
0,origin.
molecularsize.Thus, the brainenzymeactivity isfound in the void volume eluates on Sephadex G-75 fractionation while trypsinappears ineluatesatabout 34% of the volume between the voidvolume and the radioiodide peak. The void volume fractionwascentrifugedat190,000Xgfor2hr.Thepelletwas resuspendedintheoriginal volume ofsaline.The enzyme ac- tivityof thesupernatantequalled that of the resuspended pellet.
Portionsof both fractions werethen subjectedtodensitygra- dientultracentrifugation and their sedimentationpattern was compared with that of
125I-albumin,
125I-gammaglobulin,and trypsin. Consistentwithitsknown molecularsize,thetryptic proteolyticactivity isrecovered above albumininthe ultra- centrifuge tube while the brainenzymeactivityboth of the supernatant and of the resuspended pellet is found below gammaglobulin.The speciesspecificityofthe brainenzyme was also inves- tigated. The potency of canine cerebral cortex extracts in converting pCCK tothe smallerpeptidedid not differsignifi- cantly from the potency of the porcinecerebral cortex ex- tracts.
Anenzymesimilar to the brain enzymecouldnotbe found inthespleen. Splenicextractspreparedsimilarlytothebrain extractscompletelydestroyed theimmunoreactivityof both CCK-33andgastrin-34,suggestingthepresenceofnonspecific proteolyticenzymes.Thevoid volumeeluates ofSephadexG-75 filtrationof the spleenextracts, prepared similarly with the brain extracts,didnotalterCCK-33.
DISCUSSION
Steinerandassociates(9)have
hypothesized
thattrypsin-like andcarboxypeptidase B-likeproteinaseshaveimportantroles intheconversionofproinsulinintoinsulinaswellas inthein- tracellularprocessingof a variety ofotherprecursorforms and wereabletodemonstrate theexistenceofsimilarenzymaticactivitiesinthe Islets ofLangerhans.However,theywereun- able toimplicate those enzymesdirectly in the endogenous conversionprocess. MacGregoretal. (10)and later Habener etal. (11)have demonstrated that subcellular fractions of bo- vineparathyroidtissuecontainenzymatic activitycapableof specificallyconvertingtheprohormoneto intactparathyroid hormone. None of these groups haspresentedevidence indi- catingwhetherthe converting enzymestudiedwasactive in converting two or moreprohormonestosmaller forms.
The presentstudywasundertakenbecause of the demon- strationthat the cerebral cortical tissuesfrom severalmam- malian species contain CCKpeptidesinamountscomparable to those found in the gastrointestinal mucosa and that the fractionof the immunoreactivity in componentssmaller than CCK-33 isgreaterinthe brain thaninthe gut(15).These ob- servationssuggestedtousthatbrain extractsmightcontainan enzymeofhighpotencyfor thespecificconversionof CCK-33 tothe smaller hormonal forms. The question answeredbythese studies isthat the brainenzyme,which iscapableofcleaving CCK-33attheArg-Ile bondtoyieldCCK-12andatthe Arg- AspbondtoyieldCCK-8, is morespecific thansimplytryp- tic-likesince,unlike trypsin, it failstocleavebiggastrinatthe Lys-Lys-Glu bonds. Furthermore, this enzyme can also be distinguishedfrom trypsin notonlyintermsof substrate spec- ificity, but alsointermsofmolecularsize, temperaturestability, and behaviorinthepresenceoftrypsininhibitor.
Although the brain enzyme appearstomanifest hormone specificity,itdoesseem toeffectcleavageat twodifferent sites, asshownbyitsabilitytogeneratebothCCK-12andCCK-8.
This raises thepossibility thatthere aretwodifferent butclosely relatedenzymesbetween which we have been unabletodis-
tinguish
becauseourpurification methodsare asyetrelatively crude. Thereis noevidence thatthe enzymeisspeciesspecific inthat bothcanineandporcinecortical extracts appeartobe equally effectiveincleavingporcineCCK-33. Thus,thereisMedicalSciences: Strausetal. 5713
5714 Medical Sciences: Straus et al.
noreason to believe that the failure of porcine brain enzyme toconverthumangastrin-34 to gastrin-17 was due to species specificity. The enzyme has some degree of organ specificity inthatadegradativeenzyme of the same molecular size was not detectable in splenic extracts, which contain nonspecific proteases capable of completely degrading CCK.
In ourstudies we have had no problem solubilizing a major fractionofthe converting enzyme. The supernatant fraction, aftercentrifugation for 2 hr at 190,000Xg,contained half the activity and thisactivity was shown to be associated with a molecule somewhatlarger than gamma globulin. We have not yetdetermined whetherthepackagingofconverting enzyme andtheCCKhormonalsubstrateis differentin the brain from inglandulartissue orwhether thereis aconverting enzyme in gutthatis aseasily solubilized.
It isof interest toconsidersomepossible comparisons between the CCK-converting enzyme systemdescribed hereand the kidney renin-brainisorenin systems(see ref. 16 for review). The converting enzymes of therenin-angiotensin and the CCK systemsdonothavethedibasicpeptidespecificitycharacteristic ofmanyotherprohormone-hormonesystems. The brain and kidneyrenins aresimilarinmolecularsizeand certainother physical chemical properties, including sufficient structural identitysothat antibodiesagainstdogkidneyreninalso have aninhibitory effect onbrainisoreninactivity. Furthermore, bothreninsfromonespeciesare active onsubstratesfroman- otherspecies.
Nonetheless,
thebrain andkidney
enzymesare notidenticalinthatratbrainisoreninhashigh affinity
fordog
substrate whereasratkidneyrenindoesnot. Thus,brain and kidneyrenins aresimilar butnotidentical.Further investigation isrequiredtodetermine whether theenzymeinthe gutthat converts CCKtothebioactiveandimmunoactiveCOOH-ter- minalfragments resemblesor isdifferentfrom that foundinthebrain.
Supported bythe Medical ResearchProgramof the Veterans Ad-
ministration. A.M. is afellow ofthe Solomon A. Berson Fund for MedicalResearch.
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