Purification and properties of Aspartate aminotransferase (E.C. 2.6.1.1) from skeletal muscle of Cirrhina mrigala

Aspartate aminotransferase (E.C. 2.6.1.1.) from the skeletal muscle of fresh water fish Cirrhina mrigala has been purified 40 fold by ammonium sulphate fractionation, adsorption on alumina Csub(8) gel and chromatography using DEAE-cellulose column and the properties of the purified enzyme studied. The pH optimum of the enzyme is 7.8. The Km value of aspartic acid and 2-oxoglutaric acid are found to be 2.8 x 10sub(-3) M and 1.0 x 10sub(-4) M respectively. The activity of enzyme is inhibited by p-chloromercurybenzoate, hydroxylamine hydrochloride and sodium cyanide. The inhibition by pchloromercurybenzoate is reversed by reduced glutathione, B-mercaptoethanol and cysteine. Dicarboxylic acids such as maleic acid, malic acid and succinic acid inhibit the enzyme activity. The enzyme is not activated by any of the metal ions tested and heavy metal ions such as mercury and silver strongly inhibit the enzyme activity.

Purification and characterisation of phosphorylase from muscle of Tilapia (Tilapia mosambica)

Phosphorylase from muscle of tilapia (Tilapia mosambica) was extracted by water and purified by ammonium sulphate precipitation, centrifugation and repeated recrystallisation. Electro-phorogram of the enzyme preparation showed a single band near origin. The enzyme showed optimum pH and temperature at 6.1 and 37°C respectively. Glucose and glucose-6-phosphate were found to be competitive inhibitors of the enzyme. Maltose and starch acted as primers for the phosphorylase reaction like glycogen.

Extraction, purification and analysis of conotoxin of Conus textile captured from Persian Gulf and the investigation of analgesic effects of conotoxins in an animal model

Identification of venomous species of Persian Gulf cone snails and characterization of venom composition and their features is so important from the point of medical importance. Marine cone snails from the genus Conus are estimated to consist of up to 700 species. The venom of cone snails has yielded a rich source of novel neuroactive peptides or conotoxins. The present study was aimed to study the analgesic effect of Persian Gulf Conus textile and its comparison with morphine in mouse model. The specimens of Conus textile were collected of Larak Island from depth of 7 m. The collected samples were transferred to laboratory alive and were stored at -700 c. he veno s ducts were separated and ho ogenized with deionized water he ixture centrifuged at rp for inutes upernatant was considered as extracted veno and stored at - C after lyophylization. The protein profile of venom determined by using SDS-PAGE and HPLC used to investigate the extracted venom and to evaluate the analgesic activity, formalin test was carried out. SDS-PAGE indicated several bands ranged between 6 and 250 kDa. Chromatogram of the venom demonstrated more than 44 large and small fractions. The amount of 10 ng of Conus crude venom and analgesic peptide showed the best anti-pain activity in formalin test. No death observed up to 100 mg/kg, which is 250,000 times higher than the effective dose.Venom characterization of Persian Gulf Conus textile may be of medical importance and potential for new pharmaceutical drugs as well.

Venomics and biological analysis of Scatophagus argus argus (Spotted scat) venom isolated in Persian Gulf, extraction and purification of hemolytic protein

Green scat namely as Scatophagus argus argus is a venomous aquarium fish belonging to Scatophagidae family. It can induce painful wounds in injured hand with partial paralysis to whom that touch the spines. Dorsal and ventral rough spines contain cells that produce venom with toxic activities. According to unpublished data collected from local hospitals in southern coastal region of Iran, S. argus is reported as a venomous fish. Envenomation induces clinical symptoms such as local pain, partial paralysis, erythema and itching. In the present study green scat (spotted scat) was collected from Persian Gulf coastal waters. SDS-PAGE indicated 12 distinct bands in the venom ranged between 10-250 KDa. The crude venom had hemolytic activity on human erythrocytes (1%) with an LC100 (Lytic Concentration) of about 1.7 μg. The crude venom can release 813 μg proteins from 0.5% casein. Phospholipase C activity was recorded at 3.125 μg of total venom. Our findings showed that the edematic activity remained over 48 h after injection. The purification of the venom was done by HPLC and 30 peaks were obtained within 80 min but only one peak in 68 min retention time showed hemolytic activity at 90% acetonitril was isolated. The area percentage of the hemolytic protein showed that this hemolytic protein consist of 32 percent of total proteins and its molecular weight was 72 KDa in SDS_PAGE. The results demonstrated that crude venom extracted from Iranian coastal border has different toxic and enzymatic activities.

Liver cytochrome oxidase P4501A1 purification of Acipenser persicus and designing ELISA for measuring

Moon oxygenases related to cytochrome P450s are the molecular Biomarkers which have important role in Biotransformation of endogenous and exogenous compounds and catalazyin of many biological reactions. One of the important isoenzyme is cytochrome P4501A. This isoenzyme involved in metabolism of environment pollutnts such as PAHs. Because of its inducibility, it has a key tool for impact assesment of contaminants in aquatic environment. In this study, at first, that fractions containing Acipenser persicus and Huso huso isoenzyme were purified, and after that Antibodies against them were prepared. For isolation of isoenzyme fraction, Microsomes were prepared from fish liver using differential centrifugation at high speeds. microsomes were solubiized by cholat sodium and Emulgen. Extraction of this isoenzyme was done with the combinatuion of ionexchange chromatography and gelfiltration or chromatofocusing chromatography. Ion exchange chromatography and gel filtration were applied in DEAE sepharose fast flow and sephacryl S200 respectively and chromatofocusing was done at poly buffer 74 and 94 exchanger. The results of SDS-PAGE Showed that the molecular weight of isoenzyme was about 58±1 KDa. Furthermore the inmunoblotting results confirmed this subject. Isoenzyme activigy based on EROD (Ethoxyresorofin o-deethylase) reaction showed about 20-26 fold increase in enzyme activity of treated fish than control fish. The results of Elisa, Using monoclonal anti cod P4501A demonstrated the inducibility and highly elevated of its activity in treated sample more than the control fish. Mean while, the fish sample were showed the strong reaction to polyclonal antibody against beluga P4501A1 prepared in our Lab compared to monoclonal anti body.

Purification, characterization and primary structure of a chymotrypsin inhibitor from Naja atra venom

A chymotrypsin inhibitor, designated NA-CI, was isolated from the venom of the Chinese cobra Naja atra by three-step chromatography. It inhibited bovine (x-chymotrypsin with a K-i of 25 nM. The molecular mass of NA-CI was determined to be 6403.8 Da by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) analysis. The complete amino acid sequence was determined after digestion of S-carboxymethylated inhibitor with Staphylococcus aureus V8 protease and porcine trypsin. NA-CI was a single polypeptide chain composed of 57 amino acid residues. The main contact site with the protease (PI) has a Phe, showing the specificity of the inhibitor. NA-CI shared great similarity with the chymotrypsin inhibitor from Naja naja venom (identities = 89.5%) and other snake venom protease inhibitors. (C) 2003 Elsevier Inc. All rights reserved.

Separation and purification of ovulation-inducing factors in the seminal plasma of the Bactrian camel (Camelus bactrianus)

Ovulation in the Bactrian camel depends upon ovulation-inducing factors in the seminal plasma. The present study was conducted to isolate and purify the bioactive fractions from the seminal plasma of these camels. The seminal plasma was fractionated by anion-exchange chromatography, and six fractions were obtained. The bioactive potential of each fraction was estimated from its effect on rat pituitary tissue cultured in vitro and by the effect of an intramuscular injection of the fraction into female camels in vivo. Both the third fraction (F3) and the fifth fraction (F5) stimulated the release of LH in vitro and in vivo. In addition, female camels ovulated within 48 h after intramuscular injection of F3. However, neither F3 nor F5 had any significant effect on the secretion of FSH, either in vitro or in vivo. When F3 was further fractionated into four subfractions, the third subfraction (F3-3) still stimulated the in vitro release of LH, but not of FSH. An attempt to further purify the ovulation-inducing factors in F3-3 failed owing to the similarity of the molecular characters.

Purification, characterization and biological activities of the L-amino acid oxidase from Bungarus fasciatus snake venom

L-Amino acid oxidases (LAAOs) are widely distributed in snake venoms, which contribute to the toxicity of venoms. However, LAAO from Bungarus fasciatus (B. fasciatus) snake venom has not been isolated previously. In the present study, LAAO from B. fasciat

Expression, purification and characterization of recombinant Jerdonitin, a P-II class snake venom metalloproteinase comprising metalloproteinase and disintegrin domains

Jerdonitin is a P-II class snake venom metalloproteinase comprising metalloproteinase and disintegrin domains. In this study, we established a high-level expression system in Pichia pastoris and developed a purification strategy for the recombinant Jerdonitin. This recombinant Jerdonitin degraded fibrinogen at a level of activity comparable with its wild type. The effects of recombinant Jerdonitin on inhibiting ADP-induced human platelet aggregation were in a dose-dependent manner with an IC50 of 248 nM. In addition, we reported here that Jerdonitin can significantly inhibit the growth of several cell lines, including human liver cancer cells (Bel7402), human leukemia cells (K562) and human gastric carcinoma cells (BGC823). This study offers recombinant Jerdonitin that will be valuable for further functional and structural studies of Jerdonitin. (C) 2009 Elsevier Ltd. All rights reserved.

Estimation of the natural purification rate of a eutrophic lake after pollutant removal

Pollution resulting from increased human activities is threatening Lake Donghu, its effects being characterized by serious eutrophication. A steady increase of phosphorus loading is the most important factor of the lake eutrophication. Pollution external control projects are being implemented and will be accomplished before the year 2010. In order to predict the restoration rate by the lake's self-purification after the projects of external control, a model of predicting the removal rate of total phosphorus (TP) from lake water is developed, and a brief method of estimating the release and export rate of sediment phosphorus is suggested. Results show that, on the premise of external loading fully controlled. The restoration needs about 55 years or more. Obviously, the great P pool in the sediment will be a limiting factor of preventing the improvement of water quality after the external loading is under control. Based on the estimation we conclude that after the external control projects before 2010, in order to restore the lake in a few years, although highly cost, the first step must be the sediment dredging to remove internal loading. The second step is diverting water of River Changjiang into the lake to accelerate the improvement of lake water. Otherwise, removal of pollutant sources will become meaningless. (c) 2006 Elsevier B.V. All rights reserved.

Purification and characterization of hydrosoluble components from the sap of Chinese lacquer tree Rhus vernicifera

Continuous gradient elution chromatography (CGEC) was employed to purify and separate enzymes and polysaccharides from the sap of Rhus vernicifera Chinese lacquer tree. There are three different molecules with laccase enzyme activity. Two are enzymes of each other (L1, and L2), whereas the third (RL) is an entirely separate entity. Two polysaccharides (GP1 and GP2) were also found. The Rhus laccase (RL), and isoenzymes L1 and L2, have peak molecular masses of 109,100, 120,000, 103,000 respectively; each has four copper atoms per molecule, and the pI values were 8.2, 8.6, and 9.1, respectively. The structure of the laccases was studied by Fourier-transform infrared (FT-IR) and Matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) mass spectrometry. The typical amide I (1646 cm(-1)) and amide II (1545 cm(-1)) bands were observed. The results from MALDI-TOF were similar to those from CGEC, but the molecular mass from the MALDI-TOF was significantly different from that obtained from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). (c) 2006 Elsevier B.V. All rights reserved.

Root zone microbial populations, urease activities, and purification efficiency for a constructed wetland

In order to investigate the effects of microorganisms and their urease activities in macrophytic root zones on pollutant removal, four small-scale plots (SSPs) of vertical/reverse-vertical flow wetlands were set up to determine: a) the relationship between the abundance of microorganisms in the root zones and water purification efficiency; and b) the relationship between urease activities in the root zones and pollutant removal in a constructed wetland system. Total numbers of the microbial population (bacteria, fungi, and actinomyces) along with urease activities in the macrophytic root zones were determined. In addition, the relationships between microbial populations and urease activities as well as the wastewater purification efficiencies of total phosphorus (TP), total Kjeldahl nitrogen (TKN), biochemical oxygen demand in 5 days (BOD5), and chemical oxygen demand (COD) were also analyzed. The results showed that there was a highly significant positive correlation (r = 0.9772, P < 0.01) between the number of bacteria in the root zones and BOD5 removal efficiency and a significant negative correlation (r = -0.9092, P < 0.05) between the number of fungi and the removal efficiency of TKN. Meanwhile, there was a significant positive correlation (r = 0.8830, P < 0.05) between urease activities in the root zones and the removal efficiency of TKN. Thus, during wastewater treatment in a constructed wetland system, microorganism and urease activities in the root zones were very important factors.

Roles of substrate microorganisms and urease activities in wastewater purification in a constructed wetland system

A vertical/reverse-vertical flow constructed wetland system was set up in Wuhan, China, to study its treatment efficiency of polluted lake water. The numbers of substrate microorganisms and urease activities in the substrate of the constructed wetland were determined by plate counts and colorimetric analysis, respectively. The removal efficiencies of biochemical oxygen demands (BOD5). chemical oxygen demands (COD), total phosphorus (TP), total Kjeldahl nitrogen (TKN), and total suspended solids (TSS) were measured by EPA approved methodology. The results showed there were significant positive correlations (P < 0.05) between numbers of microorganism in the substrate and removal rates of TKN and CODCr. Meanwhile, there was significant positive correlation (P < 0.05) between urease activities and removal efficiencies of TKN and negative correlation between urease activities and removal efficiencies of BOD5. Substrate microorganisms and urease activities played key factors during purification processes and they could be utilized as indicator of wastewater treatment performances in the constructed wetland system. (C) 2004 Elsevier B.V. All rights reserved.

Purification and characterization of White Spot Syndrome Virus (WSSV) produced in an alternate host: crayfish, Cambarus clarkii

Penaeid shrimp is the natural host of White Spot Syndrome Virus (WSSV) that can cause high mortality in the infected hosts. Attempts to obtain sufficient amounts of purified intact WSSV for characterization have been unsuccessful. Using crayfish, Cambarus clarkii as a proliferation system, a large amount of infectious WSSV was reproduced and intact WSSV viral particles were purified with a new isolation medium by ultra-centrifugation. Purified WSSV particles were very sensitive to organic solvents and the detergent, Triton X-100. The size of the rod-shape, somewhat elliptical, intact WSSV was 110-130 x 260-350 mm with a long, tail-like envelope extension. The naked viral nucleocapsid was about 80 x 350 nm, and it possessed 15 spiral and cylindrical helices composed of 14 globular capsomers along its long axis, and a 'ring' structure at one terminus. Distinct WSSV genome DNA patterns were obtained when the purified genomic dsDNA of WSSV was digested with five different restriction enzymes (HindIII, XhoI, B(BamHI, SalI, and SacI). In addition, at least 13 major and distinct protein bands could be observed when purified intact WSSV viruses were separated by SDS-PAGE followed by Coomassie Brilliant R-250 staining. The estimated molecular weights of these proteins were 190, 84, 75, 69, 68, 58, 52, 44, 28, 27.5, 23, 19, and 16 kD, respectively. Both the 44 and 190 kD proteins were easily removed if the hemolymph from the: WSSV infected crayfish was transiently treated with 1%, Triton X-100 before it was subjected to gradient centrifugation, indicating that both of them are located on the surface of the viral envelope. These characteristics are consistent with WSSV isolated from the penaeid shrimp. (C) 2001 Elsevier Science B.V. All rights reserved.