Purification,characterization and crystallization of a group of earthworm fibrinolytic enzymes from Eisenia fetida

Seven fibrinolytic enzymes were purified from the earthworm Eisenia fetida. The molecular weights of the enzymes were 24663, 29516, 29690, 24201, 24170, 23028 and 29595, and the respective isoelectric points were 3.46, 3.5, 3.5, 3.68, 3.62, 3.94 and 3.46. All the proteases showed different fibrinolytic activity on fibrin plates. Studies on substrate specificity and inhibition indicated that they belonged to different types of serine proteases. N-Terminal sequencing indicated their high homology to those from the earthworm Lumbricus rubellus. All the enzymes have been crystallized.     

Purification and properties of alcohol oxidase from Tanacetum vulgare

Alcohol oxidase (alcohol: O₂ oxidoreductase) from leaves of Tanacetum vulgare has been purified 5150-fold to homogeneity on disc electrophoresis and gel electrofocussing. The enzyme which is probably flavoprotein, has molecular weight 180 000 daltons and is comprised of two sub-units of 94 000 and 75 000 daltons. It is active over a broad range (pH 5–9) and best accepts primary aliphatic alcohols with 6 to 10 carbons, especially those with a 2-ene group. K_m values for hex-trans-2-ene-1-ol, geraniol (3,7-dimethylocta-trans-2,6-dien-1-ol) and n-octanol were 0.19, 1.56 and 0.49 mM respectively. The significance of the enzyme in the formation of leaf aldehyde (hex-trans-2-ene-1-al) and in terpene metabolism is discussed.     

Glutamyl Endopeptidases: Structure,Function, and Practical Application

Special features of the structural organization of serine proteases belonging to a new subfamily of glutamyl-specific endopeptidases, which possess an extremely strict substrate specificity, are discussed. Some areas of the practical application of these enzymes are considered.     

Phosphatidate Kinase,A Novel Enzyme in Phospholipid Metabolism (Characterization of the Enzyme from Suspension-Cultured Catharanthus roseus Cells)

Phosphatidate kinase (adenosine 5[prime]-triphosphate:phosphatidic acid phosphotransferase), a novel enzyme of phospholipid metabolism, was detected recently in the plasma membranes of suspension-cultured Catharanthus roseus cells and purified (J.B. Wissing, H. Behrbohm [1993] Plant Physiol 102: 1243-1249). In the present work the properties of phosphatidate kinase are described. The enzyme showed a pH optimum of 6.1 and an isoelectric point of 4.8, and was rather stable in the presence of its substrates. Although the kinase accepted both ATP and GTP, with Km values of about 12 and 18 [mu]M, respectively, the only lipid substrate was phosphatidic acid; neither lysophosphatidic acid nor any other lipid tested was phosphorylated. With 32P- and 14C-labeled diacylglycerol pyrophosphate, the product of the enzyme, it was shown that the kinase catalyzes a reversible reaction. The activity of the extracted enzyme depended on the presence of surfactants such as Triton X-100 or [beta]-octylglucoside, whereas deoxycholate was strongly inhibitory. Kinetic analysis with Triton X-100/phosphatidate mixed micelles performed according to the "surface dilution" kinetic model showed saturation kinetics with respect to both bulk and surface concentration of phosphatidate. The interfacial Michaelis constant for phosphatidate was determined as 0.6 mol %.     

A new kumamolisin-like protease from Alicyclobacillus acidocaldarius: an enzyme active under extreme acidic conditions

A new serine-carboxyl proteinase, called kumamolisin-ac, was purified from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius. The enzyme is a monomeric protein of 45?kDa, active over a wide temperature range (5.0–70°C) and extremely acidic pHs (1.0–4.0), showing maximal proteolytic activity at pH?2.0 and 60°C. Interestingly, kumamolisin-ac displayed a significant proteolytic activity even at 5°C, thus suggesting a sort of cold-adaptation for this enzyme. The protease was remarkably stable at high temperatures (t_1/2 at 80°C, 10?h, pH?2.0) and over a broad range of pH (2.0–7.0). Substrate analysis indicated that kumamolisin-ac was active on a variety of macromolecular substrates, such as haemoglobin, hide powder azure, and azocoll. In particular, a high specific activity was detected towards collagen. The corresponding gene was cloned, expressed and the recombinant protease, was found to be homologous to proteases of the ‘S53’ family. From the high identity with kumamolisin and kumamolisin-As, known as collagenolytic proteases, kumamolisin-ac can be considered as the third collagenolytic affiliate within the ‘S53’ family. Cleavage specificity investigation of kumamolisin-ac revealed a unique primary cleavage site in bovine insulin B-chain, whereas a broad specificity was detected using bovine α-globin as substrate. Thus, kumamolisin-ac could represent an attractive candidate for industrial-scale biopeptide production under thermoacidophilic conditions.     

广西沿海裸体方格星虫纤维蛋白溶解酶的研究

采用离心、硫酸铵分级盐析、离子交换、凝胶过滤层析等方法进行分离纯化,从裸体方格星虫内脏得到纤维蛋白溶解酶(纤溶酶),SDS-PAGE聚丙烯酰胺凝胶电泳测定为单一组分,相对分子质量为33250;酶学分析结果最适反应温度约为45℃,最适反应pH值为7.5 Ba2+离子对该酶活力有一定的抑制作用,而Mg2+、Ca2+、K+、Na+和Ag+不影响该酶的活性。裸体方格星虫纤溶酶纤溶活性完全被苯甲基磺酰氟(PMSF)抑制,为丝氨酸蛋白酶;此外,裸体方格星虫纤溶酶具有直接溶解纤维蛋白和激活纤溶酶原的双重作用,对预防和治疗血栓性疾病具有一定的药用价值。     

长白山白眉蝮蛇毒类凝血酶Gussurobin基因的克隆、表达与纯化

根据同源性 ,在高度保守的上游信号肽区域设计引物 ,通过RT PCR反应 ,从长白山白眉蝮蛇 (Gloydiusussurensis)毒腺总RNA中克隆得到类凝血酶 gussurobincDNA ,双向测序得到 gussurobin基因的全序列并由此推测出相应的氨基酸序列。与其他已知的类凝血酶不同 ,gussurobin只含有一个可能的糖基化位点 ,即Asn12 4 Ser12 5 Thr12 6。将gussurobin基因克隆到表达载体 pPIC9K中 ,电极转化至毕氏酵母菌株GS115中 ,经G418抗性筛选和营养缺陷型筛选获得重组子。经摇瓶培养 ,获得表达。经过柱层析分离 ,获得SDS PAGE电泳纯的重组gussurobin。     

Substrate Specificity of a Novel Alcohol Resistant Metalloproteinase,Vimelysin, from Vibrio sp. T1800

Vimelysin is a novel alcohol resistant metalloproteinase from Vibrio sp. T1800. The substrate specificity of vimelysin was studied by using natural and furylacryloyl dipeptide substrates. Vimelysin cleaved mainly Pro⁷-Phe⁸ bond and slightly Tyr⁴-Ile⁵ bond in human angiotensin I. Vimelysin also cleaved mainly Phe²⁴-Phe²⁵ and Tyr¹⁶-Leu¹⁷ bonds, and slightly His⁵-Leu⁶, His¹⁰-Leu¹¹, Ala¹⁴-Leu¹⁵, and Gly²³-Phe²⁴ bonds in oxidized insulin B-chain. The substrate specificity of vimelysin, by using furylacryloyl (Fua) dipeptides were also studied. The ratio of k_cat/K_m for Fua-Gly-Phe-NH₂/Fua-Gly-Leu-NH₂, Fua-Phe-Leu-NH₂/Fua-Gly-Leu-NH₂, and Fua-Phe-Phe-NH₂/Fua-Gly-Leu-NH₂ were 15.9, 27.8, and 59.0, respectively. These results indicate that vimelysin easily recognizes phenylalanine in P1′ positions, which is different from thermolysin.     

Structure of the Bacillus anthracis Sortase A Enzyme Bound to Its Sorting Signal: A FLEXIBLE AMINO-TERMINAL APPENDAGE MODULATES SUBSTRATE ACCESS*

The endospore forming bacterium Bacillus anthracis causes lethal anthrax disease in humans and animals. The ability of this pathogen to replicate within macrophages is dependent upon the display of bacterial surface proteins attached to the cell wall by the B. anthracis Sortase A (^BaSrtA) enzyme. Previously, we discovered that the class A ^BaSrtA sortase contains a unique N-terminal appendage that wraps around the body of the protein to contact the active site of the enzyme. To gain insight into its function, we determined the NMR structure of ^BaSrtA bound to a LPXTG sorting signal analog. The structure, combined with dynamics, kinetics, and whole cell protein display data suggest that the N terminus modulates substrate access to the enzyme. We propose that it may increase the efficiency of protein display by reducing the unproductive hydrolytic cleavage of enzyme-protein covalent intermediates that form during the cell wall anchoring reaction. Notably, a key active site loop (β7/β8 loop) undergoes a disordered to ordered transition upon binding the sorting signal, potentially facilitating recognition of lipid II.     

Identification and Characterization of IgdE,a Novel IgG-degrading Protease of Streptococcus suis with Unique Specificity for Porcine IgG

Streptococcus suis is a major endemic pathogen of pigs causing meningitis, arthritis, and other diseases. Zoonotic S. suis infections are emerging in humans causing similar pathologies as well as severe conditions such as toxic shock-like syndrome. Recently, we discovered an IdeS family protease of S. suis that exclusively cleaves porcine IgM and represents the first virulence factor described, linking S. suis to pigs as their natural host. Here we report the identification and characterization of a novel, unrelated protease of S. suis that exclusively targets porcine IgG. This enzyme, designated IgdE for immunoglobulin G-degrading enzyme of S. suis, is a cysteine protease distinct from previous characterized streptococcal immunoglobulin degrading proteases of the IdeS family and mediates efficient cleavage of the hinge region of porcine IgG with a high degree of specificity. The findings that all S. suis strains investigated possess the IgG proteolytic activity and that piglet serum samples contain specific antibodies against IgdE strongly indicate that the protease is expressed in vivo during infection and represents a novel and putative important bacterial virulence/colonization determinant, and a thus potential therapeutic target.     

Purification and Characterization of Two Endo-β-1,4-glucanases from Mollusca, Ampullaria crossean

Two novel endo-β-1,4-glucanases, EG45 and EG27, were isolated from the gastric juice of mollusca, Ampullaria crossean, by anion exchange, hydrophobic interaction, gel filtration and a second round of anion exchange chromatography. The purified proteins EG45 and EG27 appeared as a single band on sodium dodecylsulfate polyacrylamide gel electrophoresis with a molecular mass of 45 kDa and 27 kDa, respectively. The optimum pH for CMC activity was 5.5 for EG45 and 4.4-4.8 for EG27. The optimum temperature range for EG27 was broad, between 50℃ and 60 ℃; for EG45 it was 50 ℃. The analysis on the stability of these two endo-β-1,4-glucanases showed that EG27 was acceptably stable at pH 3.0-11.0 even when the incubation time was prolonged to 24 h at 30 ℃, whereas EG45 remained relatively stable at pH 5.0-8.0. About 85% of the activity of EG27 could be retained upon incubation at 60 ℃ for 24 h. However, less than 10% residual activity of EG45 was detected at 50 ℃. Among different kinds of substrates, both enzymes showed a high preference for carboxymethyl cellulose. EG45, in particular, showed a carboxymethyl cellulose hydrolytic activity of 146.5 IU/mg protein. Both enzymes showed low activities to xylan （from oat spelt） and Sigmacell 101, and they were inactive to p-nitrophenyl-β-D-cellobioside, salicin and starch.     

Ketomethylureas. A New Class of Angiotensin Converting Enzyme Inhibitors

Abstract

The design rationale for a new series of tripeptide derived angiotensin converting enzyme (ACE) inhibitors, which we term “ketomethylureas”, is described. Analogs of tripeptide substrates (i.e. N-benzoyl-Phe-Ala-Pro) in which the nitrogen atom of the scissile amide bond and the adjacent asymmetric carbon atom of the penultimate amino acid residue are formally transposed give rise to this novel class of inhibitors. The most potent ketomethylureas inhibit ACE wtih I₅₀ values in the nM range.     

Purification and characterisation of a novel alkalophilic α-D-mannosidase from Pseudomonas fluorescens

An extracellular alkaline α-D-mannosidase in the cell culture of a marine bacterium Pseudomonas fluorescens JK-02 was purified to homogeneity with a 30.7-fold by ammonium sulphate fractionation, anion-exchange chromatography and gel-filtration chromatography. The molecular weight of the purified enzyme was estimated to be 50.5 kDa based on the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and temperature of the purified enzyme were 8.5 and 30°C. The K_m and V_max values of the purified enzyme towards p-nitrophenyl-α-D-mannopyranoside were determined to be 77 µM and 0.23 µM min^?1mg^?1 of protein, respectively. The α-D-mannosidase showed higher substrate specificity to α-1,3-mannobiose than other isomeric substrates such as α-1,2- and α-1,6-mannobiose. In addition, molecular characterisation of this enzyme reveals that it belongs to a class II α-mannosidase from the glycosyl hydrolase family 38. To the best of our knowledge, this is the first report on the alkalophilic α-1,3 D-mannosidase of Pseudomonas species, which has selective algal-lytic activity against Alexandrium tamarense, Akashiwo sanguine, Gymnodinium catenatum, Gymnodinium mikimotoi and Prorocentrum dentatum.     

大连蛇岛蝮蛇类凝血酶基因在毕赤酵母中的克隆表达及分离纯化

根据同源性分析设计引物,通过RT-PCR方法从大连蛇岛蝮蛇毒腺总RNA中合成扩增出类凝血酶基因,之后将该基因克隆到表达载体pPIC9K中,经电激转化后整合至毕赤酵母细胞基因组中.经筛选得到甲醇快速生长型转化子His+Mut+在500 ml摇瓶中培养,甲醇诱导分泌表达.上清液中重组类凝血酶是通过两步柱层析得到:Q Sepharose FF和Benzamidine-Sepharose 4BCL.与天然蛇毒类凝血酶一致,分泌表达的重组类凝血酶具有较强的酯酶活性,但精氨酸甲酯如TAME的水解活性较弱.此重组类凝血酶在37℃中性溶液中保存过夜将分解成小肽,但在0℃下很稳定.该酶的最适pH为8.0.     

Tryptophan decarboxylase from Catharanthus roseus cell suspension cultures: purification,molecular and kinetic data of the homogenous protein

The purification of tryptophan decarboxylase from Catharanthus roseus (TDC, E.C.:4.1.1.27), to apparent homogeneity, is described. The enzyme represents a soluble protein with a molecular weight of 115 000±3 000, consisting of 2 identical subunits of 54 000±1 000. The pI was estimated to be 5.9 and the K_m for L-tryptophan was found to be 7.5×10^-5 M. Phenylalanine, tyrosine and DOPA were not decarboxylated by tryptophan decarboxylase from Catharanthus cells. Similar to the aromatic amino acid decarboxylase from hog kidney the enzyme does not appear to be obligatorily dependent on exogenously supplied pyridoxal phosphate, as it seems to contain a certain amount of this cofactor. The average percentage of TDC in the cells was found to be 0.002% in the growth medium while the level increased up to 0.03% when indole alkaloid biosynthesis was induced. The role of the protein as a bottleneck enzyme of indole alkaloid biosynthesis is discussed.     

Properties and substrate specificities of an extracellular lipase purified from Ophiostoma piceae

An extracellular lipase produced by the sapstaining fungus Ophiostoma piceae 387N in a liquid medium was purified to homogeneity using ammonium sulphate and acetone fractionation, hydrophobic interaction and anion exchange chromatography. The overall purification based on lipase activity was 5200-fold with a yield of 26%. The molecular mass of the lipase was 35kDa, as determined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), and 37 kDa, as measured by size exclusion chromatography. The purified enzyme was resolved as three bands at pI values of 4.3, 4.1 and 3.8 in IEF (isoelectric focusing) gels. Lipolytic stain demonstrated that all three bands were lipolytically active. The N-terminal amino acid sequence was determined asD¹-V²-S³-V⁴-T⁵-T⁶-T⁷-D⁸-I⁹-D¹⁰-A¹¹-L¹²-A¹³-F¹⁴-F¹⁵-T¹⁶-Q¹⁷-W¹⁸-A¹⁹-G²⁰ . The lipase was shown to be glycosylated, containing 10.1% carbohydrate. The lipase was stable between pH 4 and pH 8 and at temperatures below 40°C. The lipase activity had a pH optimum of approximately 5 and a temperature optimum of 30°C. The enzyme activity was not influenced by N-ethylmaleimide, -mercaptoethanol or dithiothreitol, was enhanced by Ca²⁺ or Mn²⁺, but was severely inhibited by Hg²⁺, Fe³⁺, butyric acid, caproic acid, diethyl pyrocarbonate, and diethyl p-nitrophenyl phosphate. The lipase hydrolysed mainly triglycerides, although some activity was measured on waxes and cholesteryl esters. It belongs to a group of 1 (3) positional specific lipases. It showed little activity for substrates with short chain fatty acids (C2–C6), but demonstrated high specificity for substrates with intermediate and long chain fatty acid residues (C10–C18).     

Ribonucleotide reductases: the evolution of allosteric regulation.

Ribonucleotide reductases catalyze in all living organisms the production of the deoxyribonucleotides required for DNA replication and repair. Their appearance during evolution was a prerequisite for the transition from the "RNA world," where RNA sufficed for both catalysis and information transfer, to today's situation where life depends on the interplay among DNA, RNA, and protein. Three classes of ribonucleotide reductases exist today, widely differing in their primary and quaternary structures but all with a highly similar allosteric regulation of their substrate specificity. Here, I discuss the diversities between the three classes, describe their allosteric regulation, and discuss the evidence for their evolution. The appearance of oxygen on earth provided the likely driving force for enzyme diversification. From today's characteristics of the three classes, including their allosteric regulation, I propose that the anaerobic class III reductases with their iron-sulfur cluster and the requirement for S-adenosylmethionine for the generation of a glycyl protein free radical are the closest relatives to an ancestor ribonucleotide reductase.     

Structural comparison of phosphorylases from different sources

All of the -glucan phosphorylases so far purified from diverse origins have similar molecular and catalytic properties, whereas they differ in regulatory properties and glucan specificities. The activity of the rabbit muscle enzyme is regulated by phosphorylation-dephosphorylation and activated by AMP. On the other hand, the potato and Escherichia coli enzymes exist only in the active form, and are unaffected by the nucleotide. To elucidate the structural bases for these differences, we have determined the complete amino acid sequence of potato phosphorylase and compared it with those of the rabbit muscle and E. coli enzymes. The monomer of the potato enzyme contains 916 amino acids with a molecular weight of 103,916. About one-fourth of the amino-terminal threonine is blocked by an acetyl group. Sequence comparison among these enzymes reveals the presence of a characteristic 78-residue insertion in the middle of the polypeptide chain of the potato enzyme. Except for the large inserted portion, 51 and 40% of the amino acids in the potato enzyme are identical with the rabbit muscle and E. coli enzymes, respectively. The regions relevant to the regulation of the activity are completely different among the three enzymes, whereas those involved in the catalytic reaction are well conserved. The potato enzyme sequence is consistent with the tertiary structure of the rabbit muscle enzyme. The 78-residue insertion is located at the junction of the amino- and carboxyl-terminal domains on the molecular surface near the glycogen-storage site. This insertion could account for the substrate discrimination of the potato enzyme. The molecular evolution of phosphorylase is discussed based on the structural comparison among the three enzymes.     

An acid ribonuclease from rye germ cytosol

Acid ribonuclease from rye germ cytosol was purified 1200-fold. The enzyme is homogeneous on polyacrylamide gel. The optimum pH for ribonuclease activity is 5.8, its MW is 28 500. The enzyme is an endonuclease yielding in the first step of its activity oligonucleotides with a free —OH group in the 3′ position. The end products of RNA hydrolysis are cyclic purine and pyrimidine nucleoside phosphates and the corresponding nucleoside 3′-phosphates. This ribonuclease preferentially attacks sites close to the adenine base and shows a lag in the release of the cytosine base. Specificity tests on natural and synthetic substrates are in good agreement.