Properties of lactate dehydrogenase from the isopod, Saduria entomon

An anionic trypsin from pyloric caeca of chum salmon (Oncorhynchus keta) was purified by ammonium sulfate and acetone fractionation followed by affinity chromatography, gel-filtration, and DEAE-anion exchange chromatography. The apparent molecular mass was about 24 kDa as determined by SDS-PAGE. The anionic chum salmon trypsin was moderately active toward esterase substrates such as tosyl-L-arginine methyl ester and tosyl-L-lysine methyl ester. Its amidase activity for benzoyl-L-arginine p-nitroanilide was comparative to those of bovine and Streptomyces griseus trypsins. Kinetic characteristics of anionic chum salmon, bovine, and Streptomyces griseus trypsins toward inverse substrate (p-amidinophenyl ester) were compared. Inverse substrate behaved as a specific substrate for anionic chum salmon trypsin with specific binding, efficient acylation, and relatively slow deacylation.     

Crystal structure and nucleotide sequence of an anionic trypsin from chum salmon (Oncorhynchus keta) in comparison with Atlantic salmon (Salmo salar) and bovine trypsin

The nucleotide sequence and crystal structure of chum salmon trypsin (CST) are now reported. The cDNA isolated from the pyloric caeca of chum salmon encodes 222 amino acid residues, the same number of residues as the anionic Atlantic salmon trypsin (AST), but one residue less than bovine beta-trypsin (BT). The net charge on CST determined from the sum of all charged amino acid side-chains is -3. There are 79 sequence differences between CST and BT, but only seven sequence differences between CST and AST. Anionic CST isolated from pyloric caeca has also been purified and crystallized; the structure of the CST-benzamidine complex has been determined to 1.8A resolution. The overall tertiary structure of CST is similar to that of AST and BT, but some differences are observed among the three trypsins. The most striking difference is at the C terminus of CST, where the expected last two residues are absent. The absence of these residues likely increases the flexibility of CST by the loss of important interactions between the N and C-terminal domains. Similarly, the lack of Tyr151 in CST (when compared with BT) allows more space for Gln192 in the active site thereby increasing substrate accessibility to the binding pocket. Lys152 in CST also adopts the important role of stabilizing the loop from residue 142 to 153. These observations on CST provide a complementary view of a second cold-adapted trypsin, which in comparison with the structures of AST and BT, suggest a structural basis for differences in enzymatic activity between enzymes from cold-adapted species and mammals.     

灰色链霉菌胰蛋白酶在变铅青链霉菌中的异源表达及酶学性质分析

胰蛋白酶作为一种重要的丝氨酸蛋白酶被广泛应用于食品、医药和皮革等工业领域.本文成功实现了灰色链霉菌来源的胰蛋白编码基因在变铅青链霉菌中的高效活性表达,并对其酶学性质进行分析比较.以灰色链霉菌ATCC10137基因组为模板,获得胰蛋白酶编码基因sprT并克隆至表达质粒pIJ86,成功构建了重组链霉菌工程菌TK24/pIJ86-sprT.以R2YE和SELF为发酵培养基,最高酶活分别达9.21 U/mL和8.61 U/mL.酶学性质分析表明,和牛胰蛋白酶(BT)相比,重组链霉菌胰蛋白酶(rSGT)的耐酸能力强,具有较广的pH;且rSGT对酰胺键具有更高的特异性;此外,Zn2+和有机溶剂分别对rSGT的酯酶活力和酰胺酶活力具有促进作用;本研究结果为rSGT的性质改造以及工业应用提供了依据.     

Affinophoresis of trypsins

We devised a new separation technique for the protein, "affinophoresis," which is based on its specific affinity and utilizes electrophoresis. This technique requires a carrier macromolecule, "affinophore," which contains both an affinity ligand for a certain protein and many charges, either positive or negative, in order to migrate rapidly in an electric field. When a mixture of proteins is electrophoresed in the presence of the affinophore, the protein having an affinity with the ligand will form a complex with the affinophore. This results in a change in the apparent electrophoretic mobility. If the protein is sufficiently accelerated, we can separate it from other materials. A cationic affinophore for trypsin was prepared. Soluble dextran MW approximately 10,000) was coupled with a DEAE-group and m-aminobenzamidine, a competitive inhibitor of trypsins. Electrophoresis of trypsins from several origins on agarose gel plates in the presence of the affinophore showed that affinophoresis actually occurred. The electrophoretic mobilities of trypsins increased towards the cathode, the same direction as the affinophore movement. The presence of leupeptin and treatment of the trypsins with TLCK suppressed the effect of the affinophore. Streptomyces griseus trypsin, contained in Pronase, was easily separated and detected. This procedure is distinct from affinity chromatography and so-called affinity electrophoresis in that the support of the affinity ligand moves, and has advantages especially for analytical purposes: for example, the detection of specific molecules regardless of their isoelectric points.     

Collagenolytic activity of crustacean midgut serine proteases: comparison with the bacterial and mammalian enzymes.

1. We have investigated the collagenolytic activity of the following serine proteases: proteinase K, subtilisin Novo, Staphylococcal endoproteinase Glu-C, Streptomyces pronases, the trypsins and chymotrypsins from shrimp midgut and bovine pancreas. 2. By assays on both the insoluble 3H-collagen fibrils and the soluble type I collagen, it was demonstrated that the shrimp midgut serine proteases, and less efficiently, the pronases from Streptomyces griseus, could hydrolyze collagen while the other serine proteases tested could not. 3. Our data indicate that the trypsins and chymotrypsins of shrimp (Penaeus monodon) directly and indirectly digest native collagen, and that the indirect pathway probably involves activation of procollagenase in the native collagen by these serine proteases.     

Interactions of derivatives of guanidinophenylalanine and guanidinophenylglycine with Streptomyces griseus trypsin

The rates of hydrolysis of the ester, amide and anilide substrates of p-guanidino-L-phenylalanine (GPA) by Streptomyces griseus trypsin (S. griseus trypsin) were compared with those of arginine (Arg) substrates. The specificity constant (kcat/km) for the hydrolysis of GPA substrates by the enzyme was 2-3-times lower than that for arginine substrates. The kcat and Km values for the hydrolysis of N alpha-benzoyl-p-guanidino-L-phenylalanine ethyl ester (Bz-GPA-OEt) by S. griseus trypsin are in the same order of magnitude as those of N alpha-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt), although both values for the former when hydrolyzed by bovine trypsin are higher by one order of magnitude than those for the latter. The specificity constant for the hydrolysis of Bz-GPA-OEt by S. griseus trypsin is much higher than that for N alpha-benzoyl-p-guanidino-L-phenylglycine ethyl ester (Bz-GPG-OEt). As with the kinetic behavior of bovine trypsin, low values in Km and kcat were observed for the hydrolysis of amide and anilide substrates of GPA by S. griseus trypsin compared with those of arginine substrates. The rates of hydrolysis of GPA and arginine substrates by S. griseus trypsin are about 2- to 62-times higher than those obtained by bovine trypsin. Substrate activation was observed with S. griseus trypsin in the hydrolysis of Bz-GPA-OEt as well as Bz-Arg-OEt, whereas substrate inhibition was observed in three kinds of N alpha-protected anilide substrates of GPA and arginine. In contrast, no activation by the amide substrate of GPA could be detected with this enzyme.     

Amino acid sequence of crayfish (Astacus fluviatilis) trypsin If

The complete amino acid sequence of trypsin from the crayfish Astacus fluviatilis has been determined. The protein was fragmented with cyanogen bromide after S-carboxymethylation of the reduced disulfide bonds and by trypsin after S-carboxymethylation as well as after succinylation of lysine residues and aminoethylation of the reduced disulfide bonds. Peptides were purified by gel filtration and by reversed-phase high-performance liquid chromatography. Stepwise degradation was performed in a spinning cup sequencer. The enzyme contains 237 amino acid residues and has a molecular weight of 25 030. In contrast to bovine trypsin, it contains three rather than six disulfide bonds which are paired in the same fashion as those in trypsin from Streptomyces griseus. The constituents of the active site of bovine trypsin are present in corresponding positions in the crayfish enzyme. Crayfish trypsin shows 43.6% sequence identity with the bovine enzyme as compared to 40.0% identity with the S. griseus enzyme. The present analysis affords the first detailed view into the evolution of trypsins at the invertebrate level.     

Anhydrotrypsin and trypsin: subtle difference in the active-site conformations detected by chemical modification and CD spectroscopy.

The reactivities of the active-site histidine residue in bovine trypsin and its anhydro-derivative, as well as in Streptomyces griseus trypsin and its anhydro-derivative have been compared. The reactivity with TLCK was found to be lost in both of the anhydrotrypsins. On the other hand, alkylation by iodoacetamide either in the presence or absence of 1-methylguanidine proceeded faster in anhydrotrypsins than in trypsins. These differential responses to alkylating reagents are discussed in terms of a subtle change in the active-site conformation which occurs during the conversion of trypsin into anhydrotrypsin. The examination of difference CD spectra, produced by interaction with benzamidine or beta-naphthamidine, also suggested a conformational difference of the active-site between the proteins of bovine origin.     

响应面法优化灰色链霉菌产纤维素酶的工艺研究

以灰色链霉菌为原料,在单因素试验的基础上,采用响应面法试验,优化灰色链霉菌产纤维素酶活性的发酵条件。结果表明,单因素试验灰色链霉菌产纤维素酶活性的最适发酵条件:碳源为CMC-Na,氮源为明胶,温度为28℃,pH为7.0,转速为130 r/min。响应面法试验优化灰色链霉菌产纤维素酶活性最佳发酵条件为:温度27.7℃,pH值6.9,转数130.3 r/min,在此优化条件下,灰色链霉菌产纤维素平均酶活性为6.103 U/mL(n=3),与模型的预测值(6.217 U/mL)比较接近,误差为1.83%,证明了该响应面模型具有可靠性。     

Molecular cloning of tetracycline resistance genes from Streptomyces rimosus in Streptomyces griseus and characterization of the cloned genes.

Two tetracycline resistance genes of Streptomyces rimosus, an oxytetracycline producer, were cloned in Streptomyces griseus by using pOA15 as a vector plasmid. Expression of the cloned genes, designated as tetA and tetB was inducible in S. griseus as well as in the donor strain. The tetracycline resistance directed by tetA and tetB was characterized by examining the uptake of tetracycline and in vitro polyphenylalanine synthesis by the sensitive host and transformants with the resultant hybrid plasmids. Polyphenylalanine synthesis with crude ribosomes and the S150 fraction from S. griseus carrying the tetA plasmid was resistant to tetracycline, and, by a cross-test of ribosomes and S150 fraction coming from both the sensitive host and the resistant transformant, the resistance directed by tetA was revealed to reside mainly in crude ribosomes and slightly in the S150 fraction. However, the resistance in the crude ribosomes disappeared when they were washed with 1 M ammonium chloride. These results suggest that tetA specified the tetracycline resistance of the machinery for protein synthesis not through ribosomal subunits, but via an unidentified cytoplasmic factor. In contrast, S. griseus carrying the tetB plasmid accumulated less intracellular tetracycline than did the host, and the protein synthesis by reconstituting the ribosomes and S150 fraction was sensitive to the drug. Therefore, it is conceivable that tetB coded a tetracycline resistance determinant responsible for the reduced accumulation of tetracycline.     

Temporal differentiation and conditions of reproduction of chum salmon <Emphasis Type="Italic">Oncorhynchus keta</Emphasis> (Salmoniformes,Salmonidae) from the Tugur River basin (Khabarovsk Krai)

Chum salmon Oncorhynchus keta from the Tugur River of Khabarovsk krai was studied. It was found that, in the river basin, chum salmon of two ecological groups reproduces: the first spawns at river sections with a pronounced underflow and the second spawns at river sections with a groundwater outlet. In the first anadromous migration, usually three maxima of the number of approaches of migrating fish are observed. The first in time maximum is represented by early (or summer) chum salmon, the second includes migrants of early and late (or autumn) form, and the third is represented by chum salmon of only late form. Forms of chum salmon from the Tugur River basin different in dates of the run and the spawning sites differ not only in the ecology of reproduction but also in biological indices. The later chum salmon is larger; its gonads at entry into the river for spawning are less mature as compared with early chum salmon. The autumn form of chum salmon reproducing at key spawning grounds is distributed more widely than the summer form. It is found along the Asian coast from Chukotka in the north to Japan and Korea in the south. Summer chum salmon occupies only part of the range of the autumn form.     

微卫星标记对黑龙江流域大麻哈鱼遗传多样性的研究

采用 12个微卫星标记 ,对中国 3个大麻哈鱼洄游群体 (乌苏里江、黑龙江和绥芬河 )的遗传多样性进行了检测。计算出各个种群的基因杂合度、遗传多样性和各个座位的多态信息含量。结果表明 ,3个大麻哈鱼洄游种群的平均基因杂合度分别为 :0 .6 732、0 5 995、0 .6 917,种群遗传多样性分别为 0 .70 82、0 .6 5 11、0 .76 16。这些结果表明大麻哈鱼遗传多样性还比较丰富 ,其资源的恢复具有良好的前景 ,说明当前中国大麻哈鱼资源数量下降并非由遗传因素引起 ,主要原因可能是由于过度捕捞和水域环境污染等人为因素造成。人工增殖放流为恢复中国大麻哈鱼资源起到了重要作用 ,但目前大麻哈鱼的小种群极易产生遗传瓶颈的现状也应引起人们高度重视     

Differential rejection of salmon lice by pink and chum salmon: disease consequences and expression of proinflammatory genes

The consequences of high (735 copepodids fish-1) and low (243 copepodids fish-1) level exposures of size-matched juvenile pink and chum salmon to Lepeophtheirus salmonis copepodids were examined. At both levels of exposure the prevalence and abundance of L. salmonis was significantly higher on chum salmon. In addition, the weight of exposed chum salmon following the high exposure was significantly less than that of unexposed chum salmon. At both exposures, the haematocrit of exposed chum salmon was significantly less than that of unexposed chum. Neither weight nor haematocrit of pink salmon was affected by exposures at these levels. Despite the presence of microscopic inflammatory lesions associated with attachment of L. salmonis on the epithelium of gill and fin of both salmon species, there were no mortalities following either exposure. A transient cortisol response was observed in chum salmon 21 d after low exposure. An earlier and quantitatively higher expression of the proinflammatory genes interleukin-8 (IL-8), tumour necrosis factor alpha-1 (TNFalpha-1) and interleukin-1beta (IL-1beta) in fin and head kidney of pink salmon suggested a mechanism of more rapid louse rejection in this species. Together, these observations indicate a relatively enhanced innate resistance to L. salmonis in the juvenile pink salmon compared with the juvenile chum salmon.     

Effect of Actinomycin D and Cycloheximide on Gonadotropin-Induced 17α, 20β-Dihydroxy-4-pregnen-3-one Production by Intact Ovarian Follicles and Granulosa Cells of the Amago Salmon,Oncorhynchus rhodurus*

The effect of actinomycin D and cycloheximide on gonadotropin (partially purified chum salmon gonadotropin, SGA)-induced 17α, 20β-dihydroxy-4-pregnen-3-one (17α, 20β-diOHprog, a maturation-inducing steroid in amago salmon) production was examined in intact ovarian follicles and granulosa cells of postvitellogenic amago salmon, Oncorhynchus rhodurus. Both actinomycin D and cycloheximide blocked gonadotropin-induced 17α, 20β-diOHprog production by intact follicles. In contrast, gonadotropin-induced 17α-hydroxyprogesterone production by intact follicles was not abolished by actinomycin D, but was abolished by cycloheximide, suggesting that postvitellogenic amago salmon ovarian follicles already contain the RNAs necessary for the synthesis of 17α-hydroxyprogesterone. In isolated granulosa cells, chum salmon gonadotropin was able to stimulate 17α, 20β-diOHprog production only when a precursor, 17α-hydroxyprogesterone was provided in the incubation medium, indicating that gonadotropin acts directly on granulosa cells to enhance the activity of 20β-hydroxysteroid dehyrogenase (20β-HSD). Total inhibition of 20β-HSD enhancement in granulosa cells, judged by 17α, 20β-diOHprog production, was achieved when actinomycin D was added between 1 hr before the start of incubation with 17α-hydroxyprogesterone and gonadotropin to 6 hr after. With cycloheximide total inhibition was observed when added in the period of 1 hr before to 9 hr after the start of the incubation. These results suggest that chum salmon gonadotropin acts on granulosa cells to enhance the de novo synthesis of 20β-HSD by a mechanism involving RNA synthesis.     

Cloning, expression, and substrate specificity of a fungal chymotrypsin. Evidence for lateral gene transfer from an actinomycete bacterium

Unlike trypsins, chymotrypsins have not until now been found in fungi. Expressed sequence tag analysis of the deuteromycete Metarhizium anisopliae identified two trypsins (family S1) and a novel chymotrypsin (CHY1). CHY1 resembles actinomycete (bacterial) chymotrypsins (family S2) rather than other eukaryote enzymes (family S1) in being synthesized as a precursor species (374 amino acids, pI/MW: 5.07/38,279) containing a large N-terminal fragment (186 amino acids). Chy1 was expressed in Pichia pastoris yielding an enzyme with a chymotrypsin specificity for branched aliphatic and aromatic C-terminal amino acids. This is predictable as key catalytic residues determining the specificity of Streptomyces griseus chymotrypsins are conserved with CHY1. Mature (secreted) CHY1 (pI/MW: 8.29/18,499) shows closest overall amino acid identity to S. griseus protease C (55%) and clustered with other secreted bacterial S2 chymotrypsins that diverged widely from animal and endocellular bacterial enzymes in phylogenetic trees of the chymotrypsin superfamily. Conversely, actinomycete chymotrypsins are much more closely related to fungal proteases than to other eubacterial sequences. Complete genomes of yeast, gram eubacteria, archaebacteria, and mitochondria do not contain paralogous genes. Expressed sequence tag data bases from other fungi also lack chymotrypsin homologs. In light of this patchy distribution, we conclude that chy1 probably arose by lateral gene transfer from an actinomycete bacterium.     

Comparative kinetic studies of the primary specificity of bovine and salmon trypsin]

A comparative kinetic analysis of Pacific salmon and bovine trypsins revealed that the former hydrolyzes p-nitroanilide-N,L-benzoyl-D,L-arginine (BApNA) with a far greater efficiency in comparison with bovine trypsin due to the decrease in Km. The inhibition constants for the BApNA hydrolysis by bovine and salmon trypsin with glycine, beta-alanine, L-lysine, L-arginine and benzamidine were determined. With an increase in the length of the hydrocarbon chain in the inhibitor molecule (i.e., in the order of glycine-beta-alanine-L-lysine) the inhibiting effect increased both with salmon and bovine trypsins. The Ki values for benzamidine and L-arginine appeared to be by one order of magnitude higher with salmon trypsin than with bovine trypsin. L-arginine was a much more effective inhibitor compared to L-lysine when both salmon and bovine trypsins were used.     

Purification and characterization of recombinant Streptomyces griseus aminopeptidase

Recombinant Streptomyces griseus aminopeptidase (SGAP) was produced using Cangene's expression system, CANGENUS. This heat-stable aminopeptidase with an N-terminal Ala-Pro-Asp-Ile-Pro-Leu-Ala-Asn-Val-Lys-Ala sequence was purified from 16L of Streptomyces lividans fermentation supernatant with high purity and 19.5% recovery rate. This was achieved by the combination of hydrophobic-interaction and size-exclusion chromatographic procedures. The calcium-activated zinc metalloprotein demonstrated no loss of activity at -20 degrees C for at least 8 weeks in both liquid and freeze-dried formulations. The recombinant SGAP showed an apparent molecular mass of 31 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and 26.8 kDa by gel filtration. The simple, high-yield, inexpensive purification method with few intermediate steps provides a novel and practical procedure for large-scale production of active recombinant S. griseus aminopeptidase.     

Evidence for competition at sea between Norton Sound chum salmon and Asian hatchery chum salmon

Increasing production of hatchery salmon over the past four decades has led to concerns about possible density-dependent effects on wild Pacific salmon populations in the North Pacific Ocean. The concern arises because salmon from distant regions overlap in the ocean, and wild salmon populations having low productivity may compete for food with abundant hatchery populations. We tested the hypothesis that adult length-at-age, age-at-maturation, productivity, and abundance of a Norton Sound, Alaska, chum salmon population were influenced by Asian hatchery chum salmon, which have become exceptionally abundant and surpassed the abundance of wild chum salmon in the North Pacific beginning in the early 1980s. We found that smaller adult length-at-age, delayed age-at-maturation, and reduced productivity and abundance of the Norton Sound salmon population were associated with greater production of Asian hatchery chum salmon since 1965. Modeling of the density-dependent relationship, while controlling for other influential variables, indicated that an increase in adult hatchery chum salmon abundance from 10 million to 80 million adult fish led to a 72% reduction in the abundance of the wild chum salmon population. These findings indicate that competition with hatchery chum salmon contributed to the low productivity and abundance of Norton Sound chum salmon, which includes several stocks that are classified as Stocks of Concern by the State of Alaska. This study provides new evidence indicating that large-scale hatchery production may influence body size, age-at-maturation, productivity and abundance of a distant wild salmon population.