A Study On Proteinases And Inhibitor From The Skeletal Muscle Of Red Sea Bream (Pagrus Major)

Protei nases and their endogenous i nhi bi tors are extremely important proteins thatcomprise life and they are also widely applied in practice for different purposes.Fishare abundant species which supply high quality proteins for human beings.Musclemetabolism and postmortem changes of animals are relative to proteinases andinhibitors.Fish muscle postmortem tenderization is a combination of physical,chemical,biochemical and microbial processes and is also one of the most importantindicators of quality change.The initial changes occurring during postmortem of fishmuscle are due to endogenous enzymes promoting proteolysis of musde proteins andconnective tissues as well as fat hydrolysis.Thus,a detailed study on protei nases andendogenous inhibitors from fish muscle is essential both for theoretical purpose andapplication.In the present paper,three proteinases (leucine aminopeptidase,serineproteinase and metalloproteinase) and a tissue inhibitor of metalloproteinase 2(TIMP-2) from the skeletal muscle of red sea bream were studied.A leucine aminopeptidase (LAP) was purified from red sea bream (pagrus major)skeletal musdeto homogeneity with 4,850-fold and ayield of 7.4 %.The purificationprocedure consisted of ammonium sulfate fractionation and chromatographiesi ncl udi ng D EA E-Sephacel,Sephacryl S-200,Hydroxyapati te,and Phenyl-Sepharose.The molecular mass of the enzyme was approximately 96 kDa as estimated bySDS-PAGE and gel-filtration and it preferentially hydrolyzed substrate Leu-MCA.The enzymatic activity was optimal at 45℃and pH 7.5.The K_m and k_cat values ofLAP for Leu-MCA were 1.55 pmol/L and 26.4 s^-1 at 37℃,respectively.Activationenergy (E_α) of the enzyme was 59.6 kJ/mol.LAP was specifically inhibited bymetal-chelating agents,Zn²⁺ and (or) Mn²⁺ are quite possibly its metal cofactor(s).Inaddition,bestatin strongly inhibited its activity,and K_I was 1.44μmol/L.Zn²⁺ andCd²⁺ both were mixed type inhibitors to LAP.their inhibition constants (K_I) weredetermined to be 3.54 mmol/L and 0.92 mmol/L,while enzyme-substrate complexes (K_IS) were 4.49 mmol/L and 5.9 mmol/L,respectively.L-Leu,L-Trp,L-Phe werecompetitive inhibitiors,and their inhibition constants were 0.54,0.21 and 1.19mmol/L.L-Cys was a mixed type inhibitor to LAP.the inhibition constant (K_I) andenzyme-substrate complexes (K_IS) were 0.26 mmol/L and 9.43 mmol/L,respectively.Using a highly specific polydonal antibody,the location of LAP was detectedintracellularly and distributed in different tissues of red sea bream.The existence ofLAP was also detected in the skeletal muscle of different fish species,includingcommon carp,crucian carp,grass carp,silver carp,striped catfish,and round scad.A serine proteinase (SP) was purified from red sea bream skeletal muscle tohomogeneity by ammonium sulfate fractionation and chromatographies includingDEAE-Sephacel,PhenyI-Sepharose and Hydroxyapatite.The molecular mass of SPwas approximately 85 kDa as estimated by SDS-PAGE and gel-filtration and itpreferentially hydrolyzed substrate Boc-Leu-Lys-Arg-MCA.The enzymatic activitywas optimal at 40℃and pH 8.0.The enzyme was specifically inhibited by serineproteinase inhibitors,while inhibitors to other type proteinases did not show muchinhibitory effects.The K_m and k_cat values of SPfor Boc-Lea-Lys-Arg-M CA were 3.58μmol/L and 0.13 s^-1 at 37℃,respectively.Furthermore,SP effectively hydrolyzedgelatin,native typeâ… collagen and myofibrillar proteins such as myosin heavy chain(MHC).These data suggest that SP is different from muscle sarcoplasmic serineproteinase (MSSP) and myofibril-bound serine proteinase (MBSP).SP is quitepossibly a novel serine proteinase which play important role during postmortemtenderization of fish muscle.A metalloproteinase (MP) was also purified from red sea bream skeletal muscleto homogeneity by ammonium sul fate fracti onati on and chromatographi es includingDEAE-Sephacel,Phenyl-Sepharose and Gelatin-Sepharose.The molecular mass ofMP was approximately 52 kDa as estimated by SDS-PAGE and gel-filtration.Theenzymatic activity was optimal at 40℃and pH 8.0.The enzyme was almostcompletely inhibited by metalloproteinase inhibitors such as EDTA,EGTA and1,10-phenanthroline,while other proteinase inhibitors did not show any inhibitory effect.Divalent metal ion Ca²⁺ is essential for the gelatinolytic activity.Furthermore,MP effectively hydrolyzed gelatin and native typeâ… collagen,and also digestedmyofibrillar proteins such as myosin heavy chain (MHC),actin,α-actinin and nebulin,strongly suggesting its involvement in the texture softening of fish muscle during thepostmortem stage.A TIMP-2 encoding 195 amino acid residues was doned from red sea breamskeletal muscle.The identity of the amino acid sequence of TIMP-2 is 100%,91%,80%,72% and 70% to embryo cell of red sea bream,flounder,zebra fish,salmon andhuman,while only about 40% to other three types of TIM P from human.Furthermore,a recombinant TIM P-2 yeast expression system was constructed for the first ti me and10 mg/L reombinant TIMP-2 could be obtained under flask culture.ReombinantTIMP-2 was purified by Ni-NTA affinity column and inhibited metalloproteinasesfrom red sea bream and a gelatinase A like metalloproteinase from common carpmuscle to different degrees.Proteinases and endogenous inhibitors participate in muscle metabolism andprotein degradati on during postmortem.In the present study,purification methods ofthree different types of protei nase were established and thei r enzymatic characteristicswere investigated.A TIMP-2 gene was also cloned from red sea bream skeletalmuscle and successfully expressed in yeast GS115.The biological activity of therecombinant TIMP-2 to metalloprotei nase and gelatinase A like metalloprotei nase wasdetected.This study is beneficial for understanding the biochemical role ofproteinases and endogenous inhibitors in muscle metabolism and especially related topostmortem tenderization of fish muscle.It also provides some new information forenzymology study and potential applications of these proteinases and inhibitor foraquatic food processing.