| MMPs are enzymes that are largely responsible for degrading ECM proteins.MMPs have been proposed to participate in the degradation and autolysis of fish muscle after post-mortem and during storage process.To date,there have few reports about MMPs extracted from freshwater fish muscle.In the present study,we purified the metalloproteinase from silver carp muscle,and characterized the biochemical properties systematically.We cloned the full-length sequence of the purified enzyme DNA by partial acquired amino acid fragments and analyzed genetic information,which were expected to predict the protein structure.Further,we study the degrading activity of collagen and the activity during process of storage.The MMP activity from silver carp(Hypophthalmichthys molitrix)muscle was detected using gelatin zymography.The results showed that a 72-kDa matrix metalloproteinase,named sMP,was purified from silver carp skeletal muscle by 30-70%ammonium sulfate fractionation and the combination of chromatographic steps including ion exchange on DEAE-Sephacel,affinity chromatography on Gelatin Sepharose 4B and hydrophobic interaction on Phenyl Sepharose.After treated by APMA,as an activator of MMPs,two other active forms of low molecular weight were burn out.The purified MMP revealed high gelatinolytic activity at 30-60?and showed an optimal pH of 8.0.As the metalloproteinase inhibitor,EDTA could inhibit gelatinolytic activity greatly,while other inhibitors didn't show any inhibition effects.These results suggested the enzyme was a form of metalloproteinases.Besides,DTT?L-Cys and L-His could inhibit the activity of sMP as well.We found that Ca2+enhanced the proteinase activity,while a higher concentration of Zn2+ inhibited it.These studies displayed the purified proteinase was a kind of metallproteinases.Peptide mass fingerprinting revealed that 14 fragments were identical to MMP-2 from grass carp(Ctenopharyngodon idella),aiming at the MMP identity of the proteinase.The Km and Vmax values of the enyme were 0.6474 mg/mL and 0.2719 mg/(minˇmL),respectively.Further,sMP could effectively hydrolyze type I collagen even at 4?and its activity remained steady during the entire storage period at 4?.These results indicated that sMP might have been involved in the collagen degradation and the autolysis of fish muscle during postmortem stage.The entire gene encoding sMP was cloned from silver carp by the techniques of RT-PCR and RACE.The full-length cDNA of sMP was 3005 bps and the opening reading frame was 1977 bps,encoding a sMP protein consist of 658 amino acid residues.The predicted mW was 74.3 kD and the pI was 5.174.The enzyme from silver carp has a high similarity of 98%and a close relationship of phylogenetic tree compared with MMP-2 of grass carp(Ctenopharyngodon idella).The results demonstrated sMP was MMP-2.The acccession number on GenBank of silver carp MMP-2 gene sequence was KR822184.1,and the corresponding accession number of deduced protein sequence was ALL25777.1.The deduced MMP-2 protein contains four conserved domains,including a signal peptide,a propeptide domain,three repeats of fibronectin-type II domain located in the catalytic domain and a hemopexin-like domain.Using the spatial prediction software of Swiss-model,the 3D structure of MMP-2 based on homologous modeling were predicted,which showed that the 3D structure was comprised of catalytic domain,fibronectin-type II domian and hemopexin-like domain.These domains were aimed at its function of maintaining protein structural stability and exhibiting enzymatic property.On the whole,our present studies has purified MMP from silver carp skeletal muscle and analysed the characteristics of the enzyme.In addition,the gene information obtained by its full sequence and the sMP activity of degrading collagen and the process of storage was studied systematically as well.Clearly,the results may provide some theories for elucidating the biochemical role in fish muscle and the autolysis and tenderization process of fish skeletal muscle after death.And it can also give some useful suggestions for silver carp's storage and food processing. |
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