Phylogenetic relationship between common carp myofibril-bound serine protease and trypsin

Purification and partial characterization of a myofibril-bound serine protease from ostrich skeletal muscle

Purification of a Novel Serine Proteinase Inhibitor from the Skeletal Muscle of White Croaker (Argyrosomus argentatus)

Genome wide identification of taste receptor genes in common carp (Cyprinus carpio) and phylogenetic analysis in teleost

Recently, a myofibril-bound serine proteinase (MBSP) in the skeletal muscle of silver carp was identified. MBSP could be dissociated from myofibrils by treatment at pH 4.0. Following ultrafiltration concentration and chromatography on Sephacryl S-200, High Q ion-exchange and affinity column of Arginine Sepharose-4B, MBSP was partially purified. The enzyme with an estimated molecular weight of 28 kDa cleaves synthetic fluorogenic substrates specifically at the carboxyl sites of arginine and lysine residues. MBSP activity is suppressed by serine proteinase inhibitors such as Pefabloc SC, lima bean trypsin inhibitor and benzamidine; it is insensitive to Pepstatin, l-3-carboxy-trans-2, 3-epoxypropionyl-l-leucine-4-guanidinobutylamide and ethylenediaminetetraacetic acid, suggesting MBSP is a trypsin-like serine proteinase. Optimal profiles of pH and temperature of the enzyme are 8.5 and 55C, respectively. Hydrolysis of myofibrillar proteins such as myosin heavy chain, actin and tropomyosin by purified MBSP occurred especially at around 55C, consistent with our proposal that MBSP plays a significant role in the Modori phenomenon.

A novel type of myofibril-bound serine protease from white croaker ( Argyrosomus argentatus)

A myofibril-bound serine proteinase (MBSP) was highly purified from the skeletal muscle of crucian carp (Carasius auratus) by acidic treatment of myofibril solution and chromatographies on Q-Sepharose and benzamidine-Sepharose 6B. MBSP revealed a main protein band of approximately 28 kDa on SDS-polyacrylamide gel electrophoresis (PAGE) and was particularly inhibited by serine proteinase inhibitors. Substrate-specificity analysis revealed that the enzyme specifically cleaved at the carboxyl side of arginine and lysine residues, suggesting the characteristics of a trypsin-type serine proteinase. MBSP gene was cloned on the basis of the N-terminal sequence and the conserved active site peptide of serine proteinases together with 5'-rapid amplification of cDNA ends (5'-RACE) and 3'-RACE. The coding region gave an amino acid sequence of 242 residues including the initiation methionine and a signal peptide of 20 residues. Amino acid residues of His60, Asp106, and Ser196 consisting of the catalytic triad of serine proteinases were conserved in the sequence. Crucian carp MBSP shared relatively high identities with other serine proteinases, especially in well-conserved regions.

Proteolysis of a myofibril‐bound serine proteinase (MBP) from carp Cyprinus carpio on myofibrillar proteins and their gel formation ability were investigated. MBP readily decomposed myosin heavy chain as indicated by SDS‐PAGE. In the preparation of kamaboko, the gel formation ability was diminished by addition of MBP. The optimum degradation temperatures of MBP to myosin heavy chain in myofibril and kamaboko gel were 55°C and 60°C, respectively. The degradation effects of MBP on actin, α‐actinin and tropomyosin were studied by the immunoblotting method. Because of its myofibril‐bound and myofibrillar protein degradation characteristics, MBP was regarded as the proteinase most probably involved in the modori effect.

Identification of a myofibril-bound serine proteinase (MBSP) in the skeletal muscle of lizard fish Saurida wanieso which specifically cleaves the arginine site

BackgroundSerine proteases (SPs) and serine proteases homologs (SPHs) are a large group of proteolytic enzymes, with important roles in a variety of physiological processes, such as cell signalling, defense and development. Genome-wide identification and expression analysis of serine proteases and their homologs in the silkworm might provide valuable information about their biological functions.ResultsIn this study, 51 SP genes and 92 SPH genes were systematically identified in the genome of the silkworm Bombyx mori. Phylogenetic analysis indicated that six gene families have been amplified species-specifically in the silkworm, and the members of them showed chromosomal distribution of tandem repeats. Microarray analysis suggests that many silkworm-specific genes, such as members of SP_fam12, 13, 14 and 15, show expression patterns that are specific to tissues or developmental stages. The roles of SPs and SPHs in resisting pathogens were investigated in silkworms when they were infected by Escherichia coli, Bacillus bombysepticus, Batrytis bassiana and B. mori nucleopolyhedrovirus, respectively. Microarray experiment and real-time quantitative RT-PCR showed that 18 SP or SPH genes were significantly up-regulated after pathogen induction, suggesting that SP and SPH genes might participate in pathogenic microorganism resistance in B. mori.ConclusionSilkworm SP and SPH genes were identified. Comparative genomics showed that SP and SPH genes belong to a large family, whose members are generated mainly by tandem repeat evolution. We found that silkworm has species-specific SP and SPH genes. Phylogenetic and microarray analyses provide an overview of the silkworm SP and SPHs, and facilitate future functional studies on these enzymes.

利用微卫星标记技术对3个鲤鱼群体(DP、WS和SY)的遗传多样性及亲缘关系进行研究.结果表明.3个群体的遗传多样性总体水平较高,其中SY群体最高,WS群体次之,DP群体最低.根据基因型频率(P)检验了各位点的Hardy- Weinberg平衡情况.Fis值表明3个群体中共有32个微卫星位点的杂合度观测值过剩.3个群体间,WS和SY群体间的遗传相似系数最大(0.8320),遗传距离最小(0.1680),表明这两个群体亲缘关系较近;WS和DP群体间的遗传相似系数最小(0.8288),遗传距离最大(0.1712),表明这两个群体亲缘关系较远.;The genetic diversity and the phylogenetic relationships of three Cyprinus carpio L. populations were studied by using microsatellite marker technology. It showed that the genetic diversity level of three Cyprinus carpio L. populations was high, population was the highest, WS population was lower,and DP population was the lowest. In order to check the depature of microsatellite loci from HWE, the genotypic frequencies were calculated. Furthermore,32 cases of observed heterozygosity excess(Fis <0) were observed in three populations. The genetic similarity index between WS population and SY population was the highest(0.8320) among the three populations, and their genetic distance was the lowest (0.1680),which indicated that the phylogenetic relationship between these two populations was quite close. On the other hand, the genetic similarity index between WS population and DP population was the Iowest(0.8288),and their genetic distance was the highest(0.1712),which indicated that the phylogenetic relationship between ihese two populations was quite far.

Degradation of myofibrillar proteins by a myofibril-bound serine proteinase in the skeletal muscle of crucian carp ( Carasius auratus)

Purification and characterization of myofibril-bound serine protease from lizard fish ( Saurida undosquamis) muscle

Site-directed mutagenesis of myofibril-bound serine proteinase from Crucian carp: possible role of Pro95, A127 and I130 on thermal stability

Glucose-6-phosphate isomerase (GPI) was purified to homogeneity from the skeletal muscle of crucian carp ( Carassius auratus ) by ammonium sulfate fractionation, column chromatographies of Q-Sepharose, SP-Sepharose, and Superdex 200 with a yield of 8.0%, and purification folds of 468. The molecular mass of GPI was 120 kDa as estimated by gel filtration, while on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two subunits (55 and 65 kDa) were identified, suggesting that it is a heterodimer. Interestingly, GPI revealed specific inhibitory activity toward a myofibril-bound serine proteinase (MBSP) from crucian carp, while no inhibitory activity was identified toward other serine proteinases, such as white croaker MBSP and crucian carp trypsin. Kinetic analysis showed that GPI is a competitive inhibitor toward MBSP, and the K(i) was 0.32 microM. Our present results indicated that the multifunctional protein GPI is an endogenous inhibitor to MBSP and may play a significant role in the regulation of muscular protein metabolism in vivo.

Genetic diversity of the common carp black strain population based on mtDNA (D-loop and cytb)