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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,

ALBERTO

MALESCI,

ANDROSALYN S. YALOW

VeteransAdministrationHospital,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- cificproteases

capabfe

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 thepresumedparent

CCK-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 porcine

cholecystokinin (pCCK-33)

tosmaller immunoreactiveforms but which fails

toconvertgastrin-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. Animalswerekilled

by

injection of lethaldoses of sodiumpentathol.

Immediately

after injection theentirebrainwasremoved,placedin a

plastic bag,

andrapidlyfrozenondryice. Pieces of frozen cerebral corticaltissueweresubsequentlycutand extractedat40C with

aTeflon

grinder

indistilledwater,whichwasaddedtoacon- centrationof 0.1gofwetweighttissueperml. Nodifferences inactivitywereobservedif theextractantwas0.9%NaClor

0.25 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.

(2)

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

were

boiled for5 minto inactivatetheenzyme.The

substrates,

before andafter enzymaticdigestion,werethenfractionated

by

starch gel electrophoresisorSephadexgelfiltration. Radioimmuno- assayofthe eluateswasthen usedtodeterminealterationsin

substrate inducedbyenzymaticconversion.Thesamesubstrates werelabeled with125Iand fractionated

similarly

aswellas on paperchromatoelectrophoresis, andthe enzymatic

degradation

oflabeledand unlabeled substrateswas

compared.

Theactivityandspecificityofthe brainenzymewere com- pared with thoseof

chymotrypsin-free

trypsin

(Sigma,

bovine trypsin,DCC-treatedtypeXI)at a concentrationof1

mg/ml.

Theactivefractionfrom the

Sephadex purification

of the brain extract wasused ataconcentrationof

40,gl

ofactivefraction per ml of incubation volume. The effect of temperatureon enzymatic activitywastested at25°, 379,

450,

and 55'C

by

preincubating each of theenzymesfor 15 min to

bring

it to temperature,thenadding each ofthetwo125I-labeledsubstrates and

incubating

for60 min

longer

atthefour temperatures.The pH rangeofactivityofbothenzymes wasevaluated

by

incu- bation of enzymeand substrateatpH4.5-9. For somestudies excesslimabeantrypsin inhibitor(1

mg/ml,

sufficienttoinhibit 3.2mgof trypsin per

ml)

wasaddedtoboth enzymesbefore addition of eachofthe labeledand unlabeled substrates.In some studies anextract from the

spleen prepared similarly

tothe brainextract wastested forenzymatic activitywith bothun-

labeledsubstrates.Inallcasesthe

samples

wereboiledfor5min toinactivate theenzymesbefore fractionationinthe various physicochemicalsystemstotestfor enzymaticconversion.

Radioimmunoassay. The gastrinandCCK

peptides

were

measured by radioimmunoassay

according

to

published

methods (12-14). 125I-Gastrin-17 (porcine

heptadecapeptide

gastrinwas agift fromR. A.Gregory

through

thecourtesyof MortonGrossman,

Wadsworth,

VA)wasusedas tracerfor both assay systems. The guinea pig antiserumusedfor the gastrin assaydoesnot crossreactwithCCK orCCK-8(13).Theanti- serumobtainedatthecurrent

bleedings

ofrabbitBcrossreacts

identicallywith CCK and CCK-8 andsomewhatmore

strongly

withgastrin-17. Thecrossreactivitywith gastrin-17doespermit useof

125I-gastrin-17

asa tracerbut presentsno

problem

since pure CCKsubstrateisused inthe CCK assays.Starch

gel

and Sephadex eluateswere

assayed

asdescribed(12, 13).

RESULTS

125I-pCCK,

in commonwithmany

peptide

hormonessuchas

insulin,

corticotropin,and

parathyroid hormone,

remains atthe siteof

application

when

applied

topaperfor chromatoelec-

trophoresis

while theserumproteins,

iodide,

and othermore

acidic

peptides

suchasgastrin,CCK-8, and

fragments

suchas

the

iodotyrosines

migrate

anodally (12). Therefore,

in this systemit isdifficultto

distinguish

total

proteolytic degradation

from

specific cleavage

to a

fragment

ofCCKsuchasCCK-8.

However, paper

chromatoelectrophoresis

doesserve as a

rapid

screeningtestfor

degradative

activity.ShowninFig. 1arein- tact

125I-pCCK-33

beforeand after incubationat25°Cfor60 minwithafractioncontainingactiveenzyme.After

Sephadex

G-50and G-75

gel

filtrationof thebrainextracts,

only

thevoid volume regioncontained active

fractions;

i.e., thoseable to

fragment

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(0.5

Mg/ml) each

in 0.25 M

phosphate

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.Thesamefoursubstratesinthe

samebufferwerealsoincubatedwith 1 mgof

trypsin

per ml.

The starch

gel electrophoretic

patternsof the four substrates before and aftertreatmentwith thetwoenzyme

preparations

isshownin

Fig.

2.

Trypsin

converted labeledand unlabeled

gastrin-34

to a

peptide having

the starch

gel electrophoretic

behavior of

gastrin-17 (Fig.

2

right, bottom).

Italso converted labeledandunlabeledCCK-33to a

peptide resembling

CCK-8

(Fig.

2

left, bottom).

However, the

Sephadex

G-50

void

volume eluateof the brain extract, whichconvertedCCK-33tosmaller immunoreactive

peptides resembling

CCK-12and CCK-8

(Fig.

2

left, middle),

didnotalter

gastrin-34 (Fig.

2

right, middle).

Other

experiments

overarange of enzymeconcentrations

(40-800 Ml

of voidvolumeeluateper

ml)

and substratecon- centrations

(0.2-5.0

,gofCCKper

ml)

confirm the

ability

of the brain enzymeto convertCCKtosmallerimmunoreactive

fragments

anditsfailuretoconvert

gastrin-34

to

gastrin-17.

Thus,

the substrate

specificity

for the brain enzymedoesnot

resembletrypsin.

Furthermore,

limabeam

trypsin

inhibitor doesnotinhibitthe brainenzymeatconcentrationssufficient

to inhibit

completely

1 mgof

trypsin

per ml of incubation mixture. Thebrain enzyme also differs from

trypsin

in its temperature sensitivity.

Trypsin

remained

fully

active in

convertingCCK-33tothesmaller

fragments

whenmaintained for 1hrattemperatures upto550Cwhile the brain enzymewas

inactivewhen incubatedwith substrate for 1 hrat

450C.

The

proteolytic

activityof both the brain enzyme and

trypsin

wasmaintainedoverthe

pH

range from6.5to9. Asyetnoat-

tempthas beenmadetodetermine

pH optimum

for

activity

ofthe brainenzyme;it is inactiveat

pH

6and below.

The brainenzymediffers

significantly

from

trypsin

not

only

insubstrate

specificity

andtemperature

sensitivity,

but alsoin Proc.Nati. Acad.Sci. USA75

(1978)

(3)

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 theexistenceofsimilarenzymatic

activitiesinthe 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,thereis

MedicalSciences: Strausetal. 5713

(4)

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 and

kidney

enzymesare notidenticalinthatratbrainisoreninhas

high affinity

for

dog

substrate whereasratkidneyrenindoesnot. Thus,brain and kidneyrenins aresimilar butnotidentical.Further investigation isrequiredtodetermine whether theenzymeinthe gutthat converts CCKtothebioactiveandimmunoactiveCOOH-ter- minalfragments resemblesor isdifferentfrom that foundin

thebrain.

Supported bythe Medical ResearchProgramof the Veterans Ad-

ministration. A.M. is afellow ofthe Solomon A. Berson Fund for MedicalResearch.

1. Yalow, R. S. (1974) Recent Prog. Hormone Res. 30, 597-633.

2. Steiner, D. F.,Cunningham, D.,Spigelman, L. & Aten, B. (1967) Science 157, 697-700.

3. Hamilton, J.W., MacGregor, R. R., Chu, L. L. H. & Cohn, D.V.

(1971) Endocrinology 89, 1440-1447.

4. Cohn, D. V.,MacGregor, R. R., Chu, L. L. H.,Kimmel,J.R.&

Hamilton,J. W. (1972)Proc.Natl.Acad. Sci. USA 69,1521- 1525.

5. Kemper,B., Habener, J. F., Potts, J. T., Jr. & Rich, A. (1972) Proc.

Natl.Acad. Sci. USA69,643-647.

6. Berson, S. A. & Yalow, R. S. (1971)Gastroenterology 60,215- 222.

7. Gregory, R.A. &Tracy, H. J.(1973)M. SinaiJ.Med.N.Y. 40, 359-364.

8. Mutt, V.& Jorpes, J. E. (1968) Eur. J. Biochem. 6, 156-162.

9. Zuhlke, H., Steiner, D. F.,Lernmark,A. &Lipsey, C.(1976)in Polypeptide Hormones: Molecular and Cellular Aspects, Ciba FoundationSymposium 41 (North-Holland, Amsterdam), pp.

183-195.

10. MacGregor, R.R., Chu, L. L. H. & Cohn, D. V. (1976) J. Biol.

Chem. 21,6711-6716.

11. Habener,J. F.,Chang,H. T.&Potts, J. T., Jr.(1977) Biochemistry 16,3910-3917.

12. Berson, S. A. & Yalow, R. S. (1973) in Methods in Investigative and DiagnosticEndocrinology,PartI-GeneralMethodology, eds. Berson, S. A.&Yalow, R. S.(North-Holland,Amsterdam), pp.84-120.

13. Yalow, R. S. & Berson, S. A. (1973) in Methods in Investigative andDiagnosticEndocrinology, PartIII-Non-PituitaryHor- mones,eds.Berson, S. A. & Yalow, R. S.(North-Holland,Am- sterdam),pp. 1043-1050.

14. Muller,J. E.,Straus, E.&Yalow,R. S.(1977)Proc. Natl. Acad.

Sci.USA 74,3035-3037.

15. Straus,E. &Yalow,R. S.(1978)Proc. Natl. Acad. Sci. USA 75, 486-489.

16. Ganten, D.,Fuxe, K.,Phillips,M.I.,Mann,J.F. E. &Ganten,U.

(1978)inFrontiersinNeuroendocrinology,eds.Ganong,W. F.

&Martini, L. (Raven, New York), Vol. 5, pp. 61-99.

Proc.Natl. Acad.Sci. USA75

(1978)

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