Studies Of Effects And Mechansim Of Nitric Oxide On Pork During Postmortem Aging

Nitric oxide(NO),as a signaling molecular,can participant in many physiological and biochemical processes.Protein S-nitrosylation is termed as the the covalent attachment to the sulfhydryl group of protein cysteines to form S-nitrosothiol.Protein S-nitrosylation is involved in regulating protein activity,structure,function and protein interaction.In living skeletal muscle,NO and protein S-nitrosylation are capable to mediate many metabolic processes including glucose uptake,muscle contraction and calcium homeostasis.Progress has been made based on the pre-slaughter and post-slaughter researches by manipulation of the NO level in the muscle cell.NO could affect the meat quality and biochemical parameters in postmortem meat.However,there is no report concerning the effect of NO on meat aging and pocrine meat quality.As the NO is formed by the catalysis of nitric oxide synthase(NOS)from L-arginine to L-citrulline,NOS activity is detected with a duration of 24 h during postmortem aging in pork.Thus,this study is to investigate how NO and protein S-nitrosylation affect the postmortem aging and in which pathways they are involved.This will give a better understanding of the effect of NO on meat quality and the behind mechanism.The details are as follows:1.The changes of NOS in pork muscles during postmortem agingThe objective of this study was to investigate the biochemical changes of NOS in pork skeletal muscles during postmortem storage.Six Longissimus thoracis(LT),psoas major(PM)and semimembranosus(SM)muscles were obtained from pork carcasses within 45 min after slaughter and stored under vacuum condition at 4? for 0,1 and 3 d.Results showed that NOS activity in LT and PM muscles were gradually decreased during postmortem aging and could not be detected at 3 d postmortem aging(P<0.05).The SM exhibited the greatest NOS activity which was stable across 3 of aging.As the predominant isoforrn of NOS,neuronal NOS(nNOS)in LT and PM muscles was degraded during postmortem aging and the bands of nNOS disappeared at 3 d of postmortem aging.The content of nNOS in SM muscle was stable across 3 d of storage(P>0.05)and presented the greatest nNOS values among three muscles(P<0.05).The NOS activity and the nNOS expression were positively correlated with the relative value of type ?A/X fibers and negatively correlated with the proportions of type IIB fibers in pork skeletal muscles(P<0.05).Immunstaining showed that nNOS was located at not only sarcolemma but also cytoplasm at 0 and 1 d of storage.The relative intensity of fluorescence gradually decreased corresponding to the changes of nNOS expression during postmortem aging.Based on the evidences from the model of muscle extract in vitro and the incubation of LT muscle with calpain-1 inhibitor in vivo,calpain-1 was.proved to be endogenous enzyme which was responsible for degradation of nNOS in LT muscle during postmortem aging.2.Identification of S-nitrosylated proteins and cysteines sites in pork muscle during postmortem agingBovine serum albumin was reduced by tris(2-carboxyethyl)phosphine and utilized as the substrate to verify the efficacy of modified biotin switch method(BSM).The biotin swtich method comprised three main steps including the alkylation of free sulfhydryl by N-theylmaleimide(NEM),the reduction of S-nitrosothiol(SNO)group to free thiol and transiently the labeling with Na-(3-malemidylpropionyl)biocytin(MPB),enrichment of the biotin-labeled peptides by high capacity streptavidin agarose.Results showed that NEM could sufficiently block the protein free thiol while the selected biotin and streptavidin agarose had high efficacy to bind the reduced sulfhydryl from S-NO bond and enrich the S-nitrosylated peptides,respectively.The current BSM could precisely identify the S-nitrosylated cysteines in S-nitrosoglutathione(GSNO)-incubated BSA and evidence to be applicable to identify the subsequent muscle protein samples.The muscle extracts were prepared from the aging samples of pork muscle(aged 0 and 3 d)and exogenous GSNO(concentration at 10 and 100 ?M)treatments of aged 0 d sample.After suffering the procedures of BSM,the samples were labeled with tandem mass tags(TMT126-129)for the LC-MS/MS analysis.The total content of S-nitrosothiol and the S-nitrosylated protein bands were also determined.Results showed that S-nitrosothiol at 3 d was significantly greater than that of 0 d of postmortem aging(P<0.05).GSNO treatment stepwisely increased the S-nitrosothiol content of AO samples along with the concentration of 10 and 100 ?M(P<0.05).A total of 381 cysteine-sites were identified to be S-nitrosylated corresponding to 343 proteins.Comparison of total intensity and individual S-nitrosylated sites between aging samples revealed that S-nitrosylation did occur in pork muscle during postmortem aging through possible pathways of denitrosylation and transnitrosylation.It was deduced that S-nitrosylation could be involved in the postmortem metabolic process possibly through the regulation of activity or function of glycolytic enzymes,calcium release,heat shock proteins,antioxidant enzymes and myofibrillar proteins.3.Characteristics and metabolic pathways of protein S-nitrosylation in postmortem pork muscleThe 343 S-nitrosylated proteins corresponding to 381 cysteines sites were regarded as database to investigate the potential characteristics of protein S-nitrosylation in postmortem muscle using bioinfonnatics analysis.The S-nitrosylated proteins of porcine were blasted to mouse,rat or human proteins(http://blast.ncbi.nlm.nih.gov/Blast.cgi)setting threshold of E-value<10-5 and similarity>60%.A total of 139 proteins were derived for the differential protein between aging 0 and 3 d samples setting the fold change of>1.3 or<0.67.Those proteins were used to detect the significant pathways and cellular functions using integrity pathway analysis(IPA).Results showed that S-nitrosylated proteins had a broad range of molecular weight and pI value and were mainly located in the functional region of secondary structure including alpha helix,turn,beta sheet with subtle distributions in coil.The motif revealed the lysine(K)positioned at-5,-7,+1 and+5 through the S-nitrosocysteine while "C-X-X-C" was identified as non-SNO modified cysteine motif.The S-nitrosylated proteins were mostly belonged to enzymes participating in the metabolic process.Glycolysis was the most significant pathways of S-nitrosylated proteins in postmortem muscle(P<0.01).The cell death of muscle cells was predicted to be inhibited by protein S-nitrosylation with the suggestive influence on the apoptosis during aging.4.Effect of NO and calpastatin on the inhibition of calpain-1 activity,autolysis and proteolysis of myofibrillar proteinsThe calpain-1 and calpastatin were purified from porcine semimembranosus which was obtained from the carcass within 45 min after slaughter.Gradient concentration of NO donor NOR-3 and different amount of calpastatin were incubated with calpain-1 and fluorescent casein was employed to detect the calpain-1 activity.The inhibition of 0.125 U calpain-1 activity were 50%and 30%by the 1000 ?M NOR-3 and 0.051 U calpastatin treatments,respectively.Those were used to assess the cross effect of NOR-3 and calpastatin on calpain-1 activity,autolysis and proteolyisis by the following five treatments:calpain-1,calpain-1+calpastatin,calpain-1+NOR-3,calpain-1+calpastatin+NOR-3,and calpain-1+NOR-3+calpastatin.Results showed that NOR-3 could modify calpain-1 in presence of calpastatin and increase the S-nitrosyaltion and change the redox state of myofibrillar proteins to form cross-linkage.The combined treatment of NOR-3 and calpastatin exerted a further inhibitory effect on calpain-1 activity,autolysis and proteolysis which was affected by the addition order of NOR-3 and calpastatin(P<0.05).It is proposed that S-nitrosylation could change the binding extent of calpain-1/calpastatin complex.5.Effect of NO on myofibrillar proteins and the susceptibility to calpain-1 proteolysisThe calpain-1 and myofibrils were purified from porcine semimembranosus which was obtained from the carcass within 45 min after slaughter.Isolated myofibrils were reacted with NO donor S-nitrosoglutathione(GSNO)at 0,20,50,250 and 1000 ?M for 30 min at 37? to induce protein S-nitrosylation and then incubated with calpain-1 to detect the susceptibility to calpain-1 proteolysis.Resulted showed that the incubation of GSNO significantly reduced the thiol content of myofibrillar proteins(P<0.05)without affecting the protein surface hydrophobicity(P>0.05).The intensity and amount of S-nitrosylated proteins were increased in a manner consistent with GSNO concentration(P<0.05).Different myofibrillar proteins were found to have variable reactivity in response to GSNO treatments to form S-nitrosylation.GSNO caused the formation of cross-linkage proteins through intermolecular disulfide among myofibrillar proteins.More desmin and titin(T2,the degraded fragment of original titin)were degraded at 1000?M GSNO group while the cleavage of troponin-T by calpain-1 was suppressed at 250 and 1000 ?M GSNO groups(P<0.05).Our data suggest that nitric oxide could significantly change the redox state of myofibrillar proteins and subsequently affect the extent of protein degradation by the calpain-1 as well as meat postmortem aging.