Sephadex G-100 Gel Filtration Research Articles

Intravenous anesthetics inhibit human paraoxonase-1 (PON1) activity in vitro and in vivo

Objectives Here we evaluated the in vitro and in vivo effects of the intravenous anesthetics, etomidate, propofol, and ketamine, on the activity of human serum paraoxonase (hPON1). Design and methods hPON1 was purified from human serum using simple chromatographic methods, including DEAE–Sephadex anion exchange and Sephadex G-200 gel filtration chromatography. Results The three anesthetics dose-dependently decreased in vitro hPON1 activity. Inhibition mechanisms are: etomidate was noncompetitive, propofol was competitive, and ketamine was uncompetitive. In vivo studies were performed on five patients for each drug. PON1 was significantly inhibited by 0.3 mg/kg etomidate ( p < 0.05), 2 mg/kg propofol ( p < 0.001), and 1 mg/kg ketamine ( p < 0.05) for up to 5 min following intravenous administration. Conclusions Our results showed that anesthetics significantly inhibit hPON1 activity, both in vitro and in vivo, with rank order etomidate > propofol > ketamine in vitro, and propofol > etomidate > ketamine in vivo.

Bacillus amyloliquefaciens H41이 생산하는 Vibrio anguillarum 생육 저해인자의 정제

어류 질병 치료를 위한 probiont의 개발에 목적을 두고 어병균 Vibrio anguillarum NCMB1의 생육에 저해물질을 생산하는 Bacillus amyloliquefaciens H41균주를 분리하고 이 물질의 특성을 규명하기 위하여 정제를 시행하였다. 분리 균을 배양한 배양 상등액을 70% 염석, 투석하여 조 효소액으로 제작하고 조 효소액을 DEAE-sephadex, A-50 ion exchange chromatography, sephadex G-200 gel filtration column chromatography을 통하여 정제하고 SDS-PAGE 를 통하여 단일밴드를 확인하고 최종 회수율 2.9%을 얻을 수 있었으며 40.8배의 정제된 V. anguillarum NCMB1 생육저해물질을 얻을 수 있었다. 정제된 저해물질은 저해정도에 따라 단위를 설정하여 활성을 측정하였으며, 분자량은 48 kDa 로 확인되었으며 정제물질의 활성을 위한 최적 반응 pH와 온도는 pH 7.5와 <TEX>$30^{\circ}C$</TEX>로 확인되었다. 금속이온의 효과에 있어서는 <TEX>$CoCl_2$</TEX>, <TEX>$HgCl_2$</TEX>, <TEX>$ZnSO_4$</TEX>, <TEX>$AgNo_3$</TEX>에서는 완전히 저해되는 양상을 나타내었고 <TEX>$MgSO_4$</TEX>, <TEX>$MnSO_4$</TEX>에서 미미한 효소활성의 증가를 나타내었다. 그리고 염에 관한 안정성은 일반 해수의 농도인 3%의 농도에서도 활성을 나타내는 것으로 확인되었다. 정제된 저해물질을 현재 상업적으로 사용하고 있는 화학처리제나 항생제와 함께 효율성을 비교 해 보았을 때 저해물질은 약 78%의 저해 활성을 나타내는 것을 알 수 있었고 항생제보다는 효율성이 낮았으나 독성검사를 위해 정제물질을 살아있는 어류에 투여하였으나 어떤 해수어도 폐사하지 않는 것으로 보아 어류 자체엔 독성을 나타내지 않는 물질로 나타났다. 따라서 B. amyloliquefaciens H41 균주가 생산하는 정제 물질이 V. anguillarum 생육에 저해물질로 작용하는 것으로 밝혀졌으며 친환경적인 특성을 가진 물질로 밝혀졌다. To study the possible use of probiotics in fish farming, we evaluated antagonism of antibacterial strain Bacillus amyloliquefaciens H41 against the fish pathogenic bacterium Vibrio anguillarum NCMB1. The purification of growth inhibition factor produced by B. amyloliquefaciens H41 was achieved by obtaining supernatant of this bacterium. The growth inhibition factor was purified to homogeneity by 70% ammonium sulfate precipitation, DEAE-sephadex A-50 ion exchange chromatography, sephadex G-200 gel filtration column chromatography, and sephadex G-50 gel filtration column chromatography with 40.8 fold of purification and 2.9% yield. The molecular weight of the purified growth inhibition factor was 48 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH and temperature for the growth inhibition factor were pH 7.5 and <TEX>$30^{\circ}C$</TEX>, respectively. The activity of growth inhibition factor was enhanced slightly by some metal ions, such as <TEX>$Mg^{+2}$</TEX>, <TEX>$Mn^{+2}$</TEX>, but was inhibited by the addition of <TEX>$Co^{+2}$</TEX>, <TEX>$Hg^{+2}$</TEX>, <TEX>$Zn^{+2}$</TEX> and <TEX>$Ag^{+2}$</TEX>. NaCl stability of the growth inhibition factor was observed with 50% residual activity at 3% NaCl concentration. Toxicity test showed that the purified B. amyloliquefaciens H41 growth inhibition factor did not affect the live of Japanese flounder (Paralichthys olivaceus) and the effectiveness was 78% of residual lethality compared to commercial antibacterial agents.

Two L‐amino acid oxidase isoenzymes from Russell’s viper (Daboia russelli russelli) venom with different mechanisms of inhibition by substrate analogs

Two isoforms, L(1) and L(2), of L-amino acid oxidase have been isolated from Russell's viper venom by Sephadex G-100 gel filtration followed by CM-Sephadex C-50 ion exchange chromatography. The enzymes, with different isoelectric points, are monomers of 60-63 kDa as observed from size exclusion HPLC and SDS/PAGE. Partial N-terminal amino acid sequencing of L(1) and L(2) showed significant homology with other snake venom L-amino acid oxidases. Both the enzymes exhibit marked substrate preference for hydrophobic amino acids, maximum catalytic efficiency being observed with L-Phe. Inhibition of L(1) and L(2) by the substrate analogs N-acetyltryptophan and N-acetyl-L-tryptophan amide has been followed. The initial uncompetitive inhibition of L(1) followed by mixed inhibition at higher concentrations suggested the existence of two different inhibitor-binding sites distinct from the substrate-binding site. In the case of L(2), initial linear competitive inhibition followed by mixed inhibition suggested the existence of two nonoverlapping inhibitor-binding sites, one of which is the substrate-binding site. An inhibition kinetic study with O-aminobenzoic acid, a mimicking substrate with amino, carboxylate and hydrophobic parts, indicated the presence of three and two binding sites in L(1) and L(2), respectively, including one at the substrate-binding site. An inhibitor cross-competition kinetic study indicated mutually excluding binding between N-acetyltryptophan, N-acetyl-L-tryptophan amide and O-aminobenzoic acid in both the isoforms, except at the substrate-binding site of L(1). Binding of substrate analogs with different electrostatic and hydrophobic properties provides useful insights into the environment of the catalytic sites. Furthermore, it predicts the minimum structural requirement for a ligand to enter and anchor at the respective functional sites of LAAO that may facilitate the design of suicidal inhibitors.

Purification and biochemical characterization of a novel α-amylase from Bacillus licheniformis NH1 : Cloning, nucleotide sequence and expression of amyN gene in Escherichia coli

A thermostable α-amylase from a newly isolated Bacillus licheniformis NH1 was purified, characterized and the gene was isolated, sequenced and expressed in Escherichia coli BL21. The enzyme (BLA.NH1) was purified to homogeneity by 40–60% ammonium sulphate precipitation, Sephadex G-100 gel filtration and Sepharose mono Q anion exchange chromatography, with a 3.08-fold increase in specific activity and 15.9% recovery. The molecular weight of the BLA.NH1 was estimated to be 58 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and gel filtration. The enzyme was highly active over a wide range of pH from 5.0 to 10.0. The relative activities at pH 5.0, 9.0 and 10.0 were about 89, 96.6 and 90%, of that at pH 6.5, respectively. The optimum temperature of the purified enzyme was 90 °C. BLA.NH1 belonged to the EDTA-sensitive α-amylase, but its activity was not stimulated by the presence of Ca 2+ ions. The purified enzyme showed extreme stability towards surfactants (SDS, Tween 20 and Triton X-100) and excellent compatibility with a wide range of commercial solid and liquid detergents at 40 °C, suggesting potential application in the detergent industry. In addition, BLA.NH1 was relatively stable towards oxidizing agents, retaining 57% of its initial activity after 1 h incubation in the presence of 1% (w/v) sodium perborate. The amyN gene, which encodes the α-amylase from B. licheniformis NH1, was isolated and its DNA sequence was determined. It showed 92% homology to the sequence encoding α-amylase from B. licheniformis NCIB 8061. The sequence of the BLA.NH1 differs from that of B. licheniformis NCIB 8061 by 21 amino acids. The region, encoding the mature α-amylase was heterologously expressed in E. coli cells using the pDEST17 expression system. The recombinant (His) 6-tag enzyme was purified in a single affinity chromatography step and biochemical properties of the recombinant enzyme were determined and compared to those of the native-type enzyme. Interestingly, the recombinant α-amylase showed improved thermostability compared to the native enzyme. At 85 °C, recombinant BLA.NH1 and native BLA.NH1 showed half-lives of 60 and 8 min, respectively.

Determination of Some Kinetic and Characteristic Properties of Glutathione S-transferase from Bovine Erythrocytes

Glutathione S-transferase was purified from bovine erythrocytes and some kinetic and characteristic properties of the enzyme were investigated. The purification procedure was composed of preparation of homogenate and Glutathione-Agarose affinity chromatography. Thanks to the procedure, the enzyme was purified 6,800 fold with 97% yield and a specific activity of 136 EU/mg proteins. On sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE), one band with a mass of 27 kDa was found. The native molecular weight of the enzyme was found to be approximately 53 kDa by Sephadex G-100 gel filtration chromatography. Optimum pH, stable pH, optimum temperature, and optimum ionic strength were determined as 7.0, 6.5 in K-phosphate buffer, 20 degrees C, 0.1 M K-phosphate, respectively. The best activity was obtained with 1-chloro-2,4-dinitrobenzene (CDNB) in a study performed with different substrates. Vmax, Km, and kcat values were calculated as 402.63 +/- 4.99 EU/mg proteins, 0.7447 +/- 0.0007 mM, and 11436 min(-1) for CDNB, and 88.00 +/- 2.30 EU/mg proteins, 0.3257 +/- 0.0012 mM, and 477 min(-1) for GSH, respectively, by using Lineweaver-Burk graphs obtained from 1/V versus 1/[CDNB] and 1/[GSH].

Dantrolene Inhibits Human Erythrocyte Glutathione Reductase

Dantrolene, a drug used to treat malignant hyperthermia, inhibits abnormal Ca2+ release from the sarcoplasmic reticulum. Glutathione reductase (Glutathione: NADP+ oxidoreductase, EC 1.8.1.7), a member of the pyridine-nucleotide disulfide oxidoreductase family of flavoenzymes, catalyzes the reduction of glutathione disulfide (GSSG) to reduced form (GSH) in the presence of nicotinamide adenine dinucleotide phosphate (NADPH). In the present study, the in vitro effects of dantrolene on human erythrocyte glutathione reductase were investigated. For this purpose, initially, human erythrocyte glutathione reductase was purified 2555.56 fold in a yield of 29.74% using both 2',5'-ADP Sepharose-4B affinity gel chromatography and Sephadex G-200 gel filtration chromatography. The purity of the enzyme was controlled by sodium dodecyle sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) which showed a single band. A constant temperature (+4 degrees C) was maintained during the purification process. Enzyme activity was determined with the Beutler method at 340 nm by means of a spectrophotometer. Dantrolene showed remarkable in vitro inhibitory effects on the enzyme. Ki constant and 50% inhibitory concentration (IC50) value for dantrolene were determined by Lineweaver-Burk graphs and plotting activity % vs. [I], respectively. Ki constant for dantrolene was found to be 0.1116+/-0.04 mM; IC50 value was 0.0523 mM. Dantrolene displayed non-competitive inhibition.

Effects of some antibiotics on human erythrocyte glutathione reductase: an in vitro study

Inhibitory effects of some antibiotics on purified human erythrocyte glutathione reductase were investigated. Human erythrocyte glutathione reductase was purified 2800-fold (29% yield) at 4°C using 2′, 5′-ADP Sepharose 4B affinity gel and Sephadex G-200 gel filtration chromatography. SDS polyacrylamide gel electrophoresis showed a single band for the enzyme. Imipenem, rifamycin, sulfanylacetamide, ceftazidime, chloramphenicol, seftriaxon, vancomycin, cefuroxime and ornidazole exhibited inhibitory effects but clindamycin, lincomycin, amoxicillin, amikacin exhibited activatory effects on the enzyme in vitro. The IC50 values of imipenem, rifamycin, sulfanylacetamide, ceftazidime, chloramphenicol, seftriaxon, vancomycin, cefuroxime and ornidazole were 0.030, 0.146, 0.59, 2.476, 2.36, 2.88, 4.83, 15.43 and 19.632 mM, respectively, and the Ki constants were 0.06 ± 0.01, 0.275 ± 0.10, 0.85 ± 0.05, 3.59 ± 0.51, 3.85 ± 0.40, 3.71 ± 0.60, 15.11 ± 2.50, 23.50 ± 2.94 and 28.49 ± 6.50 mM, respectively. While imipenem, rifamycin, sulfanylacetamide, ceftazidime, chloramphenicol and seftriaxon cefuroxime and ornidazole showed competitive inhibition, vankomycine displayed noncompetitive inhibition.

Multiplex PCR for Rapid Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Isolated from Bandung, Indonesia

Alarge number of microorganism especially filamentous fungi has ability to degrade cellulose. The purpose of this study was to conduct screening, purification, and characterization of cellulase from fungi which was isolated from used paddy straw mushroom substrate. Screening of cellulytic activity using CMC medium shown that 11 out of 20 isolates of fungi produced a clearing zone surrounding fungal colony. Among them isolate number JMF 12 showed the highest cellulase activity and was further used for purification and characterization. The cellulase was purified to electrophoretical homogeneity by ammonium sulfate precipitation, dialyzed by Novagen D-Maxi TubeTM Dialyzer, and Sephadex G-100 gel filtration chromatography. The recovery and purification fold was 3.82 % and 1.98 respectively, after Sephadex G-100 gel filtration chromatograph. The o purified cellulase had an optimal pH and temperature at 6 and 45 C. The Km and Vmax of cellulase was 11.43 mM and 0.006mmol/min respectively. The purified cellulase was activated by Na+ and Zn++ but inhibited by Ca++, Co++, Fe++, and Hg++ .

A fast and gentle method for the isolation of myrosinase complexes from Brassicaceous seeds

Myrosinase is a β-thioglucosidase glucohydrolase that catalyses the hydrolysis of the thioglucoside bond in glucosinolates, allelochemicals present in Brassicaceous plants. These isoenzymes have been found to form complexes with other proteins; however, traditional isolation procedures involving ammonium sulphate precipitation and/or ion exchange chromatography do not allow for the isolation of these complexes. The present paper reports a fast and gentle procedure for the isolation of myrosinases in the complex form. Partial purification by Con A affinity chromatography followed by Sephadex G-200 gel filtration allowed for the isolation of myrosinase complexes from seeds of Brassica carinata, B. oleracea var. capitata, B. napus and Sinapis alba. Myrosinases in the Brassicas formed complexes of different molecular weight (500–600 kDa, 270–350 kDa and 140–200 kDa) whereas in seeds of S. alba it was only possible to isolate and detect 140–200 kDa complexes. In all species the complexes were formed by isoenzymes with isoelectric points between 4.8 and 5.6 and in some cases up to 6.8. SDS-PAGE confirmed that the myrosinase isoenzymes were composed by several protein subunits of molecular weights ranging between 10 and 110 kDa. The relative amount and enzymatic activity of the myrosinase complexes varied amongst the species studied. The isolation of myrosinase complexes in their native form is of great importance for the study of the hydrolysis of glucosinolates under autolysis conditions.

Putative reaction mechanism of heterologously expressed octopine dehydrogenase from the great scallop, Pecten maximus (L)

cDNA for octopine dehydrogenase (ODH) from the adductor muscle of the great scallop, Pecten maximus, was cloned using 5'- and 3'-RACE. The cDNA comprises an ORF of 1197 nucleotides and the deduced amino acid sequence encodes a protein of 399 amino acids. ODH was heterologously expressed in Escherichia coli with a C-terminal penta His-tag. ODH-5His was purified to homogeneity using metal-chelate affinity chromatography and Sephadex G-100 gel filtration. Recombinant ODH had kinetic properties similar to those of wild-type ODH isolated from the scallop's adductor muscle. Site-directed mutagenesis was used to elucidate the involvement of several amino acid residues for the reaction catalyzed by ODH. Cys148, which is conserved in all opine dehydrogenases known to date, was converted to serine or alanine, showing that this residue is not intrinsically important for catalysis. His212, Arg324 and Asp329, which are also conserved in all known opine dehydrogenase sequences, were subjected to site-directed mutagenesis. Modification of these residues revealed their importance for the catalytic activity of the enzyme. Conversion of each of these residues to alanine resulted in strong increases in K(m) and decreases in k(cat) values for pyruvate and L-arginine, but had little effect on the K(m) and k(cat) values for NADH. Assuming a similar structure for ODH compared with the only available structure of a bacterial opine dehydrogenase, these three amino acids may function as a catalytic triad in ODH similar to that found in lactate dehydrogenase or malate dehydrogenase. The carboxyl group of pyruvate is then stabilized by Arg324. In addition to orienting the substrate, His212 will act as an acid-base catalyst by donating a proton to the carbonyl group of pyruvate. The acidity of this histidine is further increased by the proximity of Asp329.

Purification and biochemical characterization of a thermostable extracellular glucoamylase produced by the thermotolerant fungus Paecilomyces variotii

An extracellular glucoamylase produced by Paecilomyces variotii was purified using DEAE-cellulose ion exchange chromatography and Sephadex G-100 gel filtration. The purified protein migrated as a single band in 7% PAGE and 8% SDS-PAGE. The estimated molecular mass was 86.5 kDa (SDS-PAGE). Optima of temperature and pH were 55 degrees C and 5.0, respectively. In the absence of substrate the purified glucoamylase was stable for 1 h at 50 and 55 degrees C, with a t (50) of 45 min at 60 degrees C. The substrate contributed to protect the enzyme against thermal denaturation. The enzyme was mainly activated by manganese metal ions. The glucoamylase produced by P. variotii preferentially hydrolyzed amylopectin, glycogen and starch, and to a lesser extent malto-oligossacarides and amylose. Sucrose, p-nitrophenyl alpha-D-maltoside, methyl-alpha-D-glucopyranoside, pullulan, alpha- and beta-cyclodextrin, and trehalose were not hydrolyzed. After 24 h, the products of starch hydrolysis, analyzed by thin layer chromatography, showed only glucose. The circular dichroism spectrum showed a protein rich in alpha-helix. The sequence of amino acids of the purified enzyme VVTDSFR appears similar to glucoamylases purified from Talaromyces emersonii and with the precursor of the glucoamylase from Aspergillus oryzae. These results suggested the character of the enzyme studied as a glucoamylase (1,4-alpha-D-glucan glucohydrolase).

Purification and characterization of chitosanase from Bacillus cereus D-11

A chitosanase-producing bacterium was isolated from Taiwan soils and identified as Bacillus cereus D-11 based on the biochemical properties and 16S rRNA gene sequence. The optimal medium for enzyme production consisted of 0.7% colloidal chitosan, 1% yeast extract, and 1% NaCl at an initial pH of 7.0 with 0.5% inoculation concentration (1.4 × 10 8 CFU/ml). After cultivation at 30 °C for 3 days, the maximal activity of 4.85 U/ml was observed. An extracellular chitosanase produced from B. cereus D-11 was concentrated by lyophilization and purified by Sephadex G-150 gel filtration and CM-Sephadex ion exchange column chromatography. The molecular weight of the purified chitosanase was estimated to be 41 kDa by 12.5% SDS–PAGE. The optimal pH and temperature for the chitosanase were 6.0 and 60 °C, respectively. The enzyme was stable below 50 °C and from pH 5 to 10. N-terminal amino acid sequence exhibited highest homology to the chitosanases belonging to glycoside hydrolase family 8. The enzyme was inhibited by 10 mM 2-hydroxy-5-nitrobenzyl bromide but activated by phenylglyoxal and chloramine T. The K m and V max values were 7.5 mg/ml and 2.15 × 10 −7 mol/mg/s for soluble chitosan (degree of deacetylation, DD 86%) as substrate. The D-11 chitosanase degraded chitosan with DD ranging from 70% to 100%, but did not degrade chitin. The most susceptible substrate was 86% deacetylated chitosan. Furthermore, the D-11 chitosanase inhibited the mycelial growth of Rhizoctonia solani on PDA medium.

Analysis of both chitinase and chitosanase produced by Sphingomonas sp. CJ-5

A novel chitinolytic and chitosanolytic bacterium, Sphingomonas sp. CJ-5, has been isolated and characterized. It secretes both chitinase and chitosanase into surrounding medium in response to chitin or chitosan induction. To characterize the enzymes, both chitinase and chitosanase were purified by ammonium sulfate precipitation, Sephadex G-200 gel filtration and DEAE-Sepharose Fast Flow. SDS-PAGE analysis demonstrated molecular masses of chitinase and chitosanase were 230 kDa and 45 kDa respectively. The optimum hydrolysis conditions for chitinase were about pH 7.0 and 36 degrees C, and these for chitosanase were pH 6.5 and 56 degrees C, respectively. Both enzymes were quite stable up to 45 degrees C for one hour at pH 5~8. These results show that CJ-5 may have potential for industrial application particularly in recycling of chitin wastes.

Effects of Some Metal Ions on Human Erythrocyte Glutathione Reductase:An In Vitro Study

In this study, we investigated inhibitory effects of some metal ions on human erythrocyte glutathione reductase. For this purpose, initially human erythrocyte glutathione reductase was purified 1051-fold in a yield of 41% by using 2', 5'-ADP Sepharose 4B affinity gel and Sephadex G-200 gel filtration chromatography. SDS polyacrylamide gel electrophoresis was done in order to control the purification of enzyme. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. A constant temperature (4 degrees C) was maintained during the purification process. Enzyme activity was determined with the Beutler method by using a spectrophotometer at 340 nm. Hg(2+), Cd(2+), Pb(2+), Cu(2+), Fe(3+) and Al3+ exhibited inhibitory effects on the enzyme in vitro. K(i) constants and IC(50) values for metal ions were determined by Lineweaver-Burk graphs and plotting activity % vs. [I]. IC(50) values of Pb(2+), Hg(2+), Cu(2+), Cd(2+), Fe(3+) and Al(3+) were 0.011, 0.020, 0.0252, 0.0373, 0.209 and 0.229 mM, and the Ki constants 0.0254+/-0.0027, 0.0378+/-0.0043, 0.0409+/-0.0048, 0.0558+/-0.0083, 0.403+/-0.043 and 1.137+/-0.2 mM, respectively. While Pb(2+), Hg(2+), Cd(2+) and Fe(3+) showed competitive inhibition, others displayed noncompetitive inhibition.

Pepsinogen and pepsin from the stomach of smooth hound ( Mustelus mustelus): Purification, characterization and amino acid terminal sequences

Pepsinogen from the stomach of smooth hound ( Mustelus mustelus) was purified to homogeneity by 20–70% ammonium sulphate precipitation, Sephadex G-100 gel filtration and DEAE-cellulose anion exchange chromatography with a 9.4-fold increase in specific activity and 38.36% recovery. Upon activation at pH 2.0, M. mustelus pepsinogen was converted to active form in one-step pathway. Molecular weights of the purified pepsinogen and the active pepsin were estimated to be 40,000 and 35,000 Da using SDS-PAGE and gel filtration, respectively. The optimum pH and temperature for the pepsin activity were pH 2.0 and 40 °C, respectively, using haemoglobin as a substrate. Activity was completely inhibited by Pepstatin A but not by phenylmethylsulphonyl fluoride, a serine-protease inhibitor and ethylenediaminetetraacetic acid, a metalloenzyme inhibitor. The N-terminal amino acid sequences of the first 15 amino acids of the activation segment of the pepsinogen and the first 20 amino acids of the active pepsin were LLRVPLRKGKSTLDV and ATEPLSNYLDSSYFGDISIG, respectively. M. mustelus pepsinogen, which showed high homology to rat C pepsinogen, had Thr-Leu-Asp sequence at amino acid positions 12–14 not found in all pepsinogen sequences. A remarkable substitution was found in the activation segment of M. mustelus pepsinogen: the Arg-13 conserved in all gastric proteinases, whose sequences are known, is replaced by Leu-13.

Purification and Characterization of the Antitumor Protein from Chinese Tartary Buckwheat (Fagopyrum tataricum Gaertn.) Water-Soluble Extracts

A novel antitumor protein, coded as TBWSP31, was isolated from tartary buckwheat water-soluble extracts and purified by DEAE-Sepharose Fast Flow anion exchange, Sephadex G-100 gel filtration, and Sephacryl S-200 gel filtration column chromatography. TBWSP31 was identified to a homogeneous fraction by native PAGE. The antitumor effect of TBWSP31 against human mammary cancer cell Bcap37 was measured by an MTT assay. TBWSP31 showed higher antitumor activity, and time- and concentration-dependent effects were observed. SDS-PAGE analysis showed that TBWSP31 is composed of a single polypeptide with an approximate molecular weight of 57 kDa. TBWSP31 was rich in Glx (Gln+Glu), Arg, and Asx (Asp+Asn) according to amino acid analysis. Secondary structural analysis by CD spectroscopy revealed that TBWSP31 has the following: alpha-helix (33.9%), beta-sheet (22.8%), beta-turn (11.3%), and random coil (32.0%).

Purification and characterization of an alkaline serine-protease produced by a new isolated Aspergillus clavatus ES1

An extracellular bleach stable protease from the fungus Aspergillus clavatus ES1, isolated from wastewater, was purified and characterized. The protease of ES1 strain was purified to homogeneity using acetone precipitation, Sephadex G-100 gel filtration and CM-Sepharose ion exchange chromatography, with a 7.5-fold increase in specific activity and 29% recovery. The molecular mass was estimated to be 32 kDa on SDS-PAGE. The optimum pH and temperature for the proteolytic activity were pH 8.5 and 50 °C, respectively. The enzyme was stable in the pH range of 7.0–9.0. The protease was activated by divalent cations such as Ca 2+ and Mg 2+. The alkaline protease showed extreme stability towards non-ionic surfactants (5% Tween 80 and 5% Triton X-100). In addition, the enzyme was relatively stable towards oxidizing agents, retaining more than 71 and 53% of its initial activity after 1 h incubation in the presence of 1 and 2% (w/v) sodium perborate, respectively. The N-terminal sequence of the first 15 amino acids of the purified alkaline protease of A. clavatus ES1 showed high similarity with other fungal alkaline proteases. The activity was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine-protease.

Inhibitory Effects of Ofloxacin and Cefepime on Enzyme Activity of 6-Phosphogluconate Dehydrogenase from Chicken Liver

In this study, effects of some antibiotics, namely, ofloxacin, cefepime, cefazolin, and ampicillin on the in vitro enzyme activity of 6-phosphogluconate dehydrogenase have been investigated. For this purpose, 6-phosphogluconate dehydrogenase was purified from chicken liver 535-fold with a yield of 18% by using ammonium sulphate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. In order to check the purity of the enzyme, SDS polyacylamide gel electrophoresis (SDS-PAGE) was performed. This analysis revealed a highly pure enzyme band on the gel. Among the antibiotics, ofloxacin and cefepime exhibited inhibitory effects, but cefazolin and ampicillin showed neither important inhibitory nor activatory effects on the enzyme activity. The measured I(50) values by plotting activity percent vs. inhibitor concentration, [I(50)] were 0.1713 mM for ofloxacin and 6.0028 mM for cefepime. Inhibition constants, K(i), for ofloxacin and cefepime were also calculated as 0.2740 +/- 0.1080 mM and 12.869 +/- 16.6540 mM by means of Lineweaver-Burk graphs, and inhibition types of the antibiotics were found out to be non-competitive and competitive, respectively. It has been understood from the calculated inhibitory parameters that the purified chicken enzyme has been quite inhibited by these two antimicrobials.

The Interaction of Prothrombin and Its Activation Products with Platelets.

The interaction of human 125I-prothrombin and its activation products with unactivated and thrombin-stimulated human platelets was studied. 125I-prothrombin binding to unactivated platelets was found to be a reversible and calcium-dependent process (n=48,000 sites/platelet, Kd=3.0μM). Thrombin stimulation of platelets resulted in increased prothrombin binding both in the presence and absence of calcium (n=84,000 sites/platelet, Kd=6.0μM and n=53,000 sites/platelet, Kd=8.0μM, respectively). Thrombin stimulation of platelets also increased the calcium-independent binding of prethrombin-1 compared with unstimulated cells. Using thrombin-stimulated platelets as a membrane surface for prothrombinase, 125I-prothrombin was converted exclusively to 125I-Fragment 1.2 and 125I-thrombin when the reactions were carried out in the presence of the reversible thrombin inhibitor, 5-dimethylaminonapthalene-1-sulfonylarginine-N-(3-ethyl-1,5-pentanediyl)amide (DAPA). 125I-thrombin was the major prothrombin activation product that remained bound to the thrombin-stimulated platelets (50,000 molecules/platelet). Platelet-bound 125I-thrombin either added directly or generated from 125I-prothrombin by the action of prothrombinase was found to be inacessible to human antithrombin (AT)/heparin-induced inactivation. Utilization of phosphatidylcholine/phosphatidylserine (PCPS) vesicles as a membrane surface for prothrombinase in the presence of DAPA demonstrated that 125I-prothrombin activation proceeded initially through a meizothrombin intermediate that was replaced at later times by Fragment 1.2 and the A and B chains of α-thrombin. 125I-Fragment 1.2 was the main prothrombin activation product that remained bound to the PCPS vesicles after Sephadex G-150 gel filtration chromatography. The above results indicate that human prothrombin binds specifically to unstimulated human platelets via its Fragment 1 domain only in the presence of calcium and thrombin-stimulation results in the exposure of additional calcium-dependent and -independent prothrombin binding sites. In addition, thrombin was the major prothrombin activation product that remains associated with the thrombin-stimulated human platelets and this platelet-bound thrombin is protected from AT/heparin- induced inactivation. During prothrombin activation on platelets, large amounts of thrombin remain bound and Fragment 1.2 is released from the platelet surface. In contrast, Fragment 1.2 was the major prothrombin activation product that binds PCPS vesicles. Therefore, although both thrombin-stimulated human platelets and PCPS vesicles may each serve as an effective membrane component of prothrombinase, they appear to differ both in their prothrombin activation pathway as well as their ability to bind the products of prothrombin activation.

Partial characterization of giant extracellular hemoglobin of Glossoscolex paulistus: A MALDI-TOF-MS study

In this work, MALDI-TOF-MS analysis was performed to obtain information on the molecular mass of the different subunits from the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) in the oxy-form. Experiments were performed for the whole protein at pH 7.0, for the partially dissociated protein at pH 9.0, and for the fraction obtained from gel filtration in Sephadex G-200, at pH 9.0, corresponding to the isolated monomer d. Besides that, experiments were performed for the whole protein treated with 2-mercaptoethanol in order to monitor the effects of reduction of the disulfide bonds, which are expected to maintain the trimer ( abc) in the native molecule. The results are compared to those reported for the homologous hemoglobin of Lumbricus terrestris (HbLt) and some tentative assignments are made for the observed polypeptides. The monomer d is found to exist in, at least, two major forms of identical proportions with masses of 16,355 ± 25 and 16,428 ± 24 Da, respectively. Two minor forms were also observed around 16 kDa for the monomers. Upon disulfide bonds reduction the peak associated to the trimer is absent in the mass spectrum, and new peaks assigned tentatively to the monomers a, b and c on the basis of comparison with Lumbricus terrestris hemoglobin literature data are observed. Their molecular masses were 18,258 ± 30, 16,492 ± 24 and 17,363 ± 17 Da, respectively. Two linker chains for HbGp were also observed at 25,817 ± 50 and 26,761 ± 16 Da, and this result is different from HbLt, where four linker chains were reported in the range 24–32 kDa. Finally, trimers ( abc) were observed at 51–52 kDa. This partial characterization, performed for the first time, is an important step in the characterization of subunits of this giant extracellular hemoglobin.