Cloning,Sequencing, Expression and Structural Investigation of Mnemiopsin from <Emphasis Type="Italic">Mnemiopsis leidyi</Emphasis>: An Attempt Toward Understanding Ca<Superscript>2+</Superscript>-Regulated Photoproteins

A comparison of the two most famous groups of calcium-regulated photoproteins, cnidarians and ctenophores, showed unexpectedly high degree of structural similarity regardless of their low sequence identity. It was suggested these photoproteins can play an important role in understanding the structural basis of bioluminescence activity. Based on this postulate, in this study the cDNA of mnemiopsin from luminous ctenophore Mnemiopsis leidyi was cloned, expressed, purified and sequenced. The purified cDNA, with 621 base pairs, coded a 206 residues protein. Sequence of mnemiopsin showed 93.5 and 51% similarity to other ctenophore proteins and cnidarians, respectively. The cDNA encoding apo-mnemiopsin of M. leidyi was expressed in Escherichia coli. The purified apo-protein showed a single band on SDS-PAGE (molecular weight ~27 kDa). A semi-synthetic mnemiopsin was prepared using coelenterazine and EDTA and its luminescence activity was measured in the presence of CaCl₂. The results showed an optimum pH of 9.0 and lower calcium sensitivity compared to aequorin. Comparison of amino acid residues in substrate binding site indicated that binding pocket of ctenophores contains less aromatic residues than cnidarians. This can lead to a decline in the number of stacking interactions between substrate and protein which can affect the stability of coelenterazine in binding cavity. Structural comparison of photoproteins with low sequence identity and high 3D structural similarity, can present a new insight into the mechanism of light emission in photoproteins.     

PURIFICATION OF L-GLUTAMIC ACID DECARBOXYLASE FROM CATFISH BRAIN

—L-Glutamic acid decarboxylase (GAD) from brain of the channel catfish (Ictalurus punctatus) has been purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, calcium phosphate gel and preparative polyacrylamide gel electrophoresis. The purity of the enzyme preparation was established by showing that on both 7.5% regular and 3.7–15% gradient polyacrylamide gel electrophoresis the enzyme migrated as a single protein band which contained all the enzyme activity. The molecular weight of the purified GAD was estimated by gel filtration and gradient polyacrylamide gel to be 84,000 ± 2000 and 90,000 ± 4000, respectively. SDS-polyacrylamide gel electrophoresis revealed three major proteins with molecular weights of 22,000 ± 2000, 40,000 ± 5000 and 90, 000 ± 6000 which may represent a monomer, dimer, and tetramer. Antibodies against the purified enzyme were obtained from rabbit after four biweekly injections with a total of 80 μg of the enzyme. A double immunodiffusion test using these antibodies and a crude extract from catfish brains showed only a single, sharp precipitin band which still retained the enzyme activity, suggesting that the precipitin band was indeed a GAD-anti-GAD complex. In an enzyme inhibition study, a maximum inhibition of 60–70% was obtained at a ratio of GAD protein/anti-GAD serum of about 1:1.6. Furthermore, the precipitate from the GAD-anti-GAD incubation mixture also contained the enzyme activity, suggesting that the antibody was specific to GAD and that the antigen used was homogeneous. Advantages and drawbacks of the purification procedures described here and those used for mouse brain preparations are also discussed.     

A unique EF-hand motif in mnemiopsin photoprotein from Mnemiopsis leidyi: implication for its low calcium sensitivity

Up to now, all reported Ca²⁺-regulated photoproteins, except for mnemiopsin, have been cloned and expressed in Escherichia coli. In this study, the cDNA for an isotype of mnemiopsin, from the ctenophore Mnemiopsis leidyi, has been cloned, sequenced, and functionally expressed. The full length cDNA encoding mnemiopsin of M. leidyi was 624 bp open reading frame encoding a protein of 207 amino acid residues with calculated molecular mass of ∼24 kDa. The deduced amino acid sequence showed 90% and 84% identity to berovine (from ctenophore Beroe abyssicola) and bolinopsin 2 (from the ctenophore Bolinopsis infundibulum) respectively. In contrast to all known EF-hand in photoproteins, a unique EF-hand motif was found in mnemiopsin, in which a conserved glycine is substituted with glutamic acid. According to the results, the optimum pH was 9.0, time course of regeneration was 15 h and its Ca²⁺ sensitivity was lower than aequorin. Results of pK_a calculation for ionizable residues, motif scan and hydrophobic interactions of cavity aromatic residues of mnemiopsin in comparison with aequorin showed different patterns in these two photoproteins. In addition, experimental results are confirmed with the theoretical studies.     

Determination and evaluation of secondary structure content derived from calcium-induced conformational changes in wild-type and mutant mnemiopsin 2 by synchrotron-based Fourier-transform infrared spectroscopy

Mnemiopsin 2 from a luminous ctenophore with two functional EF-hand motifs is a calcium-regulated photoprotein that is responsible for emitting a bright blue bioluminescence upon reacting with coelenterazine and calcium ions in Mnemiopsis leidyi. Synchrotron radiation-based Fourier-transform infrared (SR-FTIR) spectroscopy was applied to analyze the distribution of secondary structures, the conformational changes resulting from calcium binding and the structural stabilities in wild-type mnemiopsin 2, as well as its mutant type that possesses three EF-hand motifs. The distribution of secondary structures of these proteins indicates that mutant apo-mnemiopsin 2 has a more stable secondary structure than the wild-type. Analyses of the SR-FTIR spectra revealed that the conformational changes at the secondary structures of both mnemiopsin 2 depend on the calcium concentrations, such that the most noticeable changes in structures of wild-type and mutant mnemiopsin 2 occur at optimum concentrations 6 and 2 mM of calcium chloride, respectively. The addition of calcium to both proteins increases the proportions of their secondary structures in the amide I and II regions. The major amide I bands in the IR spectra of both mnemiopsin‑calcium complexes shift towards smaller wavenumbers, whereas their main amide II bands are identified at larger wavenumbers.     

Purification and Partial Characterization of an Antimicrobial Peptide Produced by a Novel <Emphasis Type="Italic">Bacillus</Emphasis> sp. Isolated from the Amazon Basin

An antimicrobial peptide produced by a new Bacillus species isolated from the Amazon Basin was purified and characterized. The antimicrobial peptide was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography, and after the final purification step, one active fraction was obtained, designated BLS P34. Direct activity on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was observed. A single band on SDS-PAGE suggested that the peptide was purified to homogeneity and had a molecular mass of about 5 kDa. The molecular weight (MW) was accurately determined by mass spectroscopy as 1456 Da. The purified BLS P34 remained active over a wide temperature range and was susceptible to all proteases tested.     

On the Metabolism of Organic Acids by Clostridium acetobutylicum

Racemiase, an enzyme which catalyzed racemization of lactic acids, was isolated from culture filtrate of Clostridium acetobutylicum by salting-out, and its purification was attained to about twenty-fold by treating with calcium phosphate gel. It was shown that racemiase requires for its full activity cofactors, pyridoxamine phosphate and ferrous ions. These substances were detected in the racemiasc preparation. Several other properties of racemiase were also investigated.     

Isolation and Characterization of a Sialic Acid-specific Lectin from Hemolymph of the Southeast Asian Horseshoe Crab Tachypleus gigas

A lectin was isolated from hemolymph of the Southeast Asian horseshoe crab Tachypleus gigas by using glycophorin HA affinity chromatography and Sephacryl S-300 gel filtration. The purified lectin had a molecular mass of approximately 396 kDa and was composed of 13 identical subunits with molecular masses of 31 kDa. The serological specificity of the purified lectin was specifically inhibited by sialic acids sialoglycoproteins, but not by neutral sugars, hexosamines, N-acetylhexosamines, or asialoglycoproteins. Although the N-terminal amino acid sequence of the lectin from T. gigas was identical to that from American horseshoe crab (liphemin) by the same purification method and cross reacted with the anti-liphemin serum, the calcium concentration of hemagglutinating activity of the purified lectin showed a smaller optimal concentration than that of liphemin.     

Purification and Characterization of Amidase which Participates in Nitrile Degradation

Amidase was purified from the cell-free extract of acetonitrile-grown Arthrobacter sp. J-1 by a procedure involving protamine sulfate precipitation, ammonium sulfate fractionation, and column chromatographies on DEAE-cellulose, hydroxyapatite and Sephadex G-200. The overall purification was 47-fold. The purified enzyme was homogeneous as judged by ultracentrifugal analysis and disc gel electrophoresis. The molecular weight of the enzyme was estimated to be about 300,000 and 320,000 by disc gel electrophoresis and gel filtration, respectively. The enzyme was possibly composed of eight identical subunits of a molecular weight of 39,000. The isoelectric point was 3.8. The enzyme catalyzed the stoichiometric hydrolysis of acetamide to form acetic acid and ammonia. The enzyme was active toward acetamide, acrylamide and propionamide and the Km values were 0.97, 23.3 and 8.05 mm, respectively. The enzyme showed acyltransferase activity.     

间羟苯甲酸4－羟化酶纯化及部分性质研究

经超声破碎、硫酸铵分级沉淀、凝胶过滤、磷酸钙胶层析和离子交换层析等步骤, 从Comamonas testosteroni菌株中获得了SDS-PAGE单一条带, 相对分子质量为62×10³的间羟苯甲酸4-羟化酶比活提高21倍, 产率为30%.此酶为FAD加单氧酶, 催化间羟苯甲酸转变为3,4二羟苯甲酸.     

Purifcation and properties of multiple forms of brain acetylcholinesterase (EC 3.1.1.7)

—Approximately 70 per cent of the total AChE of bovine brain tissue was solubilized by repeated homogenization and centrifugation in 0.32 m sucrose containing EDTA. After ammonium sulphate fractionation, application of the enzyme preparation to an agarose affinity gel column effected a 700-fold purification. Subsequent molecular filtration separated three active forms of AChE with molecular weights of 130,000, 270,000 and 390,000 with an average specific activity of 575 mmol of acetylthiocholine hydrolysed/mg of protein/h. The complete procedure represented an approximate 23,000-fold purification of the enzyme from that in the original tissue homogenate. The three forms of AChE exhibited certain differences in properties, including apparent K_m values, pH optima and sensitivity to inhibitory agents. Ancillary studies on less purified enzyme preparations by use of polyacrylamide gel electrophoresis and isoelectric focusing techniques also suggested that brain AChE exists in multiple forms.     

Transcription of chromatin from mouse fibroblasts

Chromatin purified from mouse fibroblasts can be fractionated after shearing by sedimentation in a sucrose gradient into an extended «light and a compact «heavy component. Further purification of these classically described components can be achieved by a second cycle of centrifugation of the light and heavy components through an equilibrium density gradient of metrizamide. The light component purified from sucrose gradient sediments faster (peak I) on metrizamide than its heavy counterpart (peak II).Template activity for DNA directed RNA synthesis in the presence of E. coli RNA polymerase is negligible in peak II but very pronounced in the peak I fraction. The difference in template activity appears to be connected with differences in propagation rather than initiation rates. Comparison of gel electrophoresis patterns of proteins indicate that the active subcomponent includes high molecular weight components not present in the inactive one, but that its histone content is somewhat lower. Using a very highly sensitive automatic recording apparatus for the measurement of melting profiles, no clear cut difference has been observed in the behaviour of active and inactive chromatin subcomponents nor in that of their total DNA.     

Improved purification of Candida boidinii formate dehydrogenase

Formate dehydrogenase (FDH, EC 1.2.1.2) from Candida boidinii was purified to homogeneity. The two step procedure comprised anion exchange chromatography (2.9-fold purification, 85% step yield, elution with 35 mM KCl), followed by dye-ligand affinity chromatography on immobilized Cibacron Blue 3GA (1.4-fold purification, 75% step yield, elution with 0.15 mM NAD⁺/2 mM Na₂SO₃). The procedure afforded FDH at 63.8% overall yield and a specific activity of 7.2 units/mg. The purity of the final FDH preparation was evaluated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), high performance gel filtration liquid chromatography (gfHPLC) and N-terminal amino acid sequencing. The analytical techniques showed the presence of a single polypeptide chain that corresponds to the molecular weight of 41 kDa (as determined by SDS-PAGE) and 81 kDa (as determined by gfHPLC).     

Synthesis of 1-(2,6,6-Trimethyl-4-hydroxy-1-cyclohexen-1-yl)-1,3-butanedione

Streptococcus dysgalactiae IID 678, belonging to group C of the streptococci, secreted a large amount of hyaluronidase (hyaluronate lyase, EC 4.2.2.1) into a culture medium containing hyaluronic acid. The purification procedures of hyaluronidase were 70% ammonium sulfate precipitation, ECTEOLA-cellulose chromatography, phospho-cellulose chromatography, and gel filtration on Sephacryl S-300. The hyaluronidase was purified approximately 27,000-fold from the culture filtrate. The purified enzyme was homogeneous by SDS-poIyacrylamide gel electrophoresis. The enzyme degradated only hyaluronic acid and chondroitin to zl 4,5-unsaturated disaccharides and did not act on other glycosaminoglycans containing sulfate groups, while the degradation rate of chondroitin was about 1/60 of that of hyaluronic acid. The optimum pH was wide, from pH 5.8 to pH 6.6, and the optimum temperature was 37°C. Fe^{2 +}, Cu^{2 +}, Pb^{2 +}, and Hg^{2 +} ions inhibited the activity strongly and Zn²⁺ inhibited it by half. The molecular weight of the enzyme was estimated to be 125,000 by gel filtration and 117,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was different immunochemically from the hyaluronidase from Streptococcus pyogenes belonging to group A.     

Partial purification and characterization of NADPH-linked aldehyde reductase from monkey brain

Abstract— The activity of NADPH-linked aldehyde reductase (EC 1.1.1.2) in various regions of monkey brain was determined in vitro. The highest specific activity of the enzyme was found in areas of the brain stem; including the pons, medulla and midbrain. A greater than 500-fold purification of the monkey brain enzyme was obtained by a combination of ammonium sulphate fractionation and subsequent chromatography on calcium phosphate gel cellulose and DEAE-cellulose. The aldehyde metabolites of the biogenic amines, norepinephrine, serotonin, dopamine and octopamine, were readily reduced by the NADPH-linked aldehyde reductase. The K_m values for 3,4-dihydroxyphenylglycolaldehyde, 3,4-dihydroxyphenyl-acetaldehyde, and 5-hydroxyindoleacetaldehyde were 12.0 μm , 6.1 μm and 27 μm , respectively. The maximum velocity (V_max) for 3,4-dihydroxyphenylglycolaldehyde was, respectively, five-fold or three-fold greater than that determined for 3,4-dihydroxyphenylacetaldehyde or 5-hydroxyindoleacetaldehyde. The highly purified enzyme derived from monkey brain was markedly inhibited by barbiturates, diphenylhydantoin, and chlorpromazine, but not by pyrazole. From data obtained by sucrose density gradient centrifugation and Sephadex chromatography the molecular weight of aldehyde reductase was determined to be about 70,000 daltons.     

Properties of trehalase from different organs of alfalfa, <Emphasis Type="Italic">Medicago sativa</Emphasis>

Trehalase activity (THA) was identified in the cell-free extracts of various organs of Medicago sativa: roots, roots nodules, stems and leaves, as well as in seedlings and seeds. It showed the high activity at acid pH and optimal temperature ranged from 45° to 55°C. It was also differently affected by ions, i.e. the presence of calcium stimulated this activity but it was inhibited by Zn²⁺ and NH ₄ ⁺ . After separation by DEAE-cellulose chromatography and purification procedures, trehalase from alfalfa was purified 800 times, and Superose 12 HR gel filtration allowed to determine the molecular weight of 120 kDa for the native enzyme from the stems of alfalfa.     

Purification and Characterization of Isocitrate Lyase from Ethanol-grown Euglena gracilis

Isocitrate lyase was purified to homogeneity from ethanol-grown Euglena gracilis. The specific activity was 0.26 μmol/min/mg protein. The molecular mass of the enzyme was calculated to be 380 kDa by gel filtration on a Superose 6 column. The subunit molecular mass of the enzyme was 116 kDa as determined by SDS-polyacrylamide gel electrophoresis. These results showed that the native form of this enzyme was a trimer composed of three identical subunits. The pH optimum for cleavage and condensation reactions was 6.5 and 7.0, respectively. The K_m values for isocitrate, glyoxylate and succinate were 3.8, 1.3 and 7.7 mM, respectively. Isocitrate lyase absolutely required Mg for enzymatic activity. This is the first report of the purification of isocitrate lyase to homogeneity from Euglena gracilis.     

Application of immobilized thrombin for production of S-thanatin expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

S-thanatin, a small antimicrobial peptide with 21 amino acid residues, was expressed as a fusion protein containing thrombin cleavage site in Escherichia coli BL21 (DE3). To reduce the production cost, immobilization of thrombin in polyacrylamide gel for cleavage was studied in this work. The immobilized thrombin exhibited excellent activity within wider ranges of pH value and temperature for reaction than free enzyme, and the residual activity could remain above 75% after ten times of usage. Tricine–SDS–PAGE result showed that the immobilized thrombin could cleave the S-thanatin fusion protein effectively. After cleavage, recombinant S-thanatin was purified by preparative reversed-phase high-performance liquid chromatography and mass spectrum showed that the molecular weight (2,448.86) was close to the theoretical value (2,448.98). After purification, about 7 mg of S-thanatin was obtained from 1 l of culture and the recombinant exhibited excellent bioactivity to E. coli ATCC 25922, with the minimum inhibitory concentration of 12 μg/ml. The purification method could be applied to prepare other peptides with similar properties at low cost.     

l-Myoinositol-1-phosphate synthase from Neurospora crassa: Purification to homogeneity and partial characterization

The purification procedure of milligram quantities of stable myoinositol-1-phosphate synthase (EC 5.5.1.4) from Neurospora crassa is reported. The procedure includes: (a) (NH₄)₂SO₄ and protamine sulfate precipitations, (b) gel filtration in Ultrogel AcA-34 (LKB), (c) DEAE-cellulose chromatography, (d) AH-Sepharose 4B chromatography, and (e) calcium phosphate gel chromatography. The enzyme is considered pure according to the following criteria: (a) gel filtration, (b) sucrose density gradient centrifugation, (c) polyacrylamide gel electrophoresis, and (d) isoelectric focusing technique. The molecular weight estimated by gel filtration chromatography and sucrose density gradient centrifugation is 345,000. The subunit molecular weight is 59,000. The active enzyme seems to posses an hexameric structure. The isoelectric point estimated for the pure enzyme is 5.2. The enzyme was optimally stimulated by 10 mm (NH₄)₂SO₄ and by 50 mm KCl, while NaCl had a minor inhibitory effect at higher concentrations. The divalent cations Mg²⁺ and Mn²⁺ were inhibitory only at nonphysiological concentrations. The enzymatic activity after the salt fractionation steps was about 33% NAD⁺ independent; but with purification the resulting homogeneous enzyme showed less than 5% NAD⁺-independent activity.     

Partial purification and characterization of nitrous oxide reductase fromParacoccus denitrificans

We report the first partial purification of nitrous oxide reductase, a unique and labile enzyme of denitrifying bacteria. The procedure, which required anaerobic conditions throughout, resulted in a 60-fold purification relative to crude lysate in the case ofParococcus denitrificans. The molecular weight was estimated by gel exclusion chromatography to be about 85,000. The partially purified enzyme is inactivated rapidly by O₂, dithionite, and mercaptoethanol and is reversibly inhibited by moderate concentrations of common salts. Up to 80% of the original activity can be reconstituted following O₂ inactivation by incubating the enzyme with reduced benzyl viologen for 2 to 3 h. TheV _max pH profile shows a broad maximum at pH 8. The enzyme is irreversibly retained by common anion exchangers in the range pH 7 to 8 but can be eluted in acceptable yield as one of the last components from an imidazole-based anion exchange material by means of a pH gradient. This behavior implies that nitrous oxide reductase is very acidic. Among the several peptides observed by sodium dodecyl sulfate slab electrophoresis, only two, with apparent molecular weights of 58,000 and 25,000, correlated closely with the activity of fractions eluted from the imidazole column. These two peptides together comprised about 30% of the total protein in the fractions with highest specific activity.     

A Crystalline Aminopeptidase from Streptomyces peptidofaciens: Physico-chemical Properties and Characteristics as a Ca-Metalloprotease

A crystalline aminopeptidase obtained from the culture filtrate of Streptomyces peptidofaciens KY 2389 appeared to be homogeneous on ultracentrifugation and acrylamide gel electrophoresis. The sedimentation coefficient, s_{20, w}., was determined to be 2.6 S. The molecular weight was estimated to be approximately 19,000 by sedimentation equilibrium studies. The amino acid analyses indicated that the enzyme was composed of 147 amino acid residues and contained no sulfhydryl group. The isoelectric point was found to be around pH 7.4 by isoelectric focusing on ampholites.

The enzyme required Ca²⁺ for its maximal activity and was strongly inhibited by some metal-chelating agents such as ethylenediaminetetraacetic acid (EDTA) and o-phenanthroline. The EDTA-inactivated enzyme restored its activity almost completely by the addition of Ca²⁺ The crystalline preparation of aminopeptidase contained 1 g-atom of calcium and about 2 g-atoms of magnesium per mole of enzyme protein, and the calcium was essential for the activity of the enzyme.