Matrixmetalloproteinases(MMP)areZn2+-andCa2+-dependentendopeptidases[1].MainsubfamiliesofMMPareCollagenases,gelatinases,stromelysinsandmembrane-typematrixmetalloproteinases[2].Threehomologouscollagenaseshavebeenidentifiedinhumantissues:interstitialcollagenase,neutrophilcollagenaseandcollagenase-3.Thethreeenzymescleavefibrillarcollagensatasinglesite,generatingfragmentsofapproximately¾and¼thesizeoftheoriginalmolecules.
Procollagenase-3consistsof452aminoacidswithacalculatedMrof51.680Da[3].DuetoN-linkedglycosylationtheapparentMrisabout60.000Da[4].Withintheproteinthefollowingdomainsandsequenceregionscanbedistinguished[3,4]:anN-terminalpropeptide,whichconferslatencytotheproenzyme,aCa2+-andZn2+-ionsbindingcatalyticdomain,ahingeregion,andaC-terminalhemopexin-likedomain.
Procollagenase-3canbeactivatedbyproteasesasstromelysin[4],gelatinaseA,membranetype1matrixmetalloproteinaseandplasmin[5]orbyincubationwithorganomercurials(e.g.APMA)[4].
Activecollagenase-3beginswiththeN-terminalsequenceYNVFPRTL[4].Collagenase-3hydrolyzestypeIIcollagen5-to6-timesfasterthantypeIandtypeIIIcollagens.TheenzymeexhibitsalsohighactivitytowardsgelatinanditdegradesSERPINSas1-antichymotrypsinandplaminogenactivatorinhibitor-2[4].Collagenase-3isinhibitedina1:1stoichiometricfashionbyTIMP-1,TIMP-2andTIMP-3.Collagenase-3isexpressedduringfetalbonedevelopment[6]. Inadulthumantissuescollagenase-3hasbeendetectedonlyinpathologicalconditions:inmalignanttumors[3],inchroniculcers[7],inarthriticcartilage[8]andsynovium[9].
Purity:Recombinantprocollagenase-3appearsasasingleproteinbandofabout60.000DainSDS-PAGE(>95%oftotalprotein).Duetoautoproteolyticactivityminorbandsofactivatedcollagenase-3maybevisIBLeintheenzymepreparation.
SpecificActivity:Thespecificactivityofcollagenase-3is≥200mU/mg.1Uistheactivitythathydrolyzes1molpeptide(7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L-2,3-diaminopropionyl)-Ala-Arg-NH2(Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg)within1minundertheassayconditionsdescribedbelow.
Inhibitors:Collagenase-3isinhibitedbytissueinhibitorsofmatrixmetalloproteinases(TIMP)andbychelatorsofdivalentcationsasEDTAoro-phenanthroline.
StABIlity&Storage:Recombinantprocollagenase-3tableuntiltheexpirydategivenonthelabelifstoredat-70°C.Theproenzymecanbekeptat-20°Cforseveralweeks.Repeatedfreezingandthawingshouldbeavoided.
Applications:Recombinantprocollagenase-3canserveasantigenstandardinimmunochemicalanalyses. Theactiveenzymemaybeusedtostudythedegradationofextracellularmatrixproteins,toscreeninhibitorsofmatrixmetalloproteinasesandtocharacterizeinhibitoractions.
