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1、J.Biochem.125,1137-1143(1999)CharacterizationofNewFluorogenicSubstratesfortheRapidandSensitiveAssayofCathepsinEandCathepsinD1YoshiyukiYasuda,*'TakashiKageyama,1AkifumiAkamine,'MasahiroShibata,'EikiKominami,"YasuoUchiyama,1andKenjiYamamoto'2Departmentsof'Pharmacologyand'ConservativeDentistryU,Kyu
2、shuUniversityFacultyofDentistry,Fukuoka812-8582;^DepartmentofCellularandMolecularBiology,PrimateResearchInstitute,KyotoUniversity,Inuyama;^DepartmentofCellBiologyandAnatomy,OsakaUniversityMedicalSchool,Suita;and"DepartmentofBiochemistry,JuntendoUniversitySchoolofMedicine,TokyoReceivedFebruary17,
3、1999;acceptedMarch18,1999CathepsinEandcathepsinDaretwomajorintracellularasparticproteinasesimplicatedinthephysiologicalandpathologicaldegradationofintra-andextracellularproteins.InthisDownloadedfromstudy,wedesignedandconstructedhighlysensitivesyntheticdecapeptidesubstratesforassaysofcathepsinsEa
4、ndDbasedontheknownsequencespecificitiesoftheircleavagesites.Thesesubstratescontainahighlyfluorescent(7-methoxycoumarin-4-yl)acetyl(MOCAc)moietyandaquenching2,4-dinitrophenyl(Dnp)group.WhenthePhe-Phebondiscleaved,thefluorescenceatanexcitationwavelengthof328nmandemissionwavelengthof393increasesdue
5、todiminishedquenchingresultingfromtheseparationofthefluores-http://jb.oxfordjournals.org/centandquenchingmoieties.Thefirstsubstrate,MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)/-NH2,inwhichtheLys-ProcombinationatpositionsP5andP4wasdesignedforspecificinteractionwithcathepsinE,ishydrolyzedeq
6、uallywellbycathepsinsEandD(Atat/-Km=10.9pM~l-s"1forcathepsinEand15.6/tM'1-s"1forcathepsinD).AveryacidicpHoptimumof4.0wasobtainedforbothenzymes.Thesecondsubstrate,MOCAc-Gly-Lys-Pro-ne-Ile-Phe-Phe-Arg-Leu-Lys(Dnp)7-NH2,inwhichtheisoleucineresidueatpositionP2wasmeanttoincreasethespecificityforcathe
7、psinE,isalsohydrolyzedequallybybothenzymes(kcnl/Km=12.2^M.~l-B~1forcathepsinEand16.3pM'1-s"1forcathepsinD).Thekm/KmvaluesforbothsubstratesaregreaterthanthoseforatYorkUniversityLibrariesonMarch15,2016thebestsubstratesforcathe
