Partial Purification and Some Properties of Thyroid Peroxidase Solubilized by Extraction with n-Butanol

Thyroid peroxidase has been extracted In 100% yield from suspensions of frozen pig thyroid “microsomes” by treatment with aqueous n-hutanol at pH 8.9. The solubilized enzyme does not sediment at 105, 000 × g in 1 hour, is retarded on a Sephadex G-200 gel and contains no membrane-like structures detectable by electron microscopy. Additional purification of the aqueous butanol extract is achieved by isoelectric precipitation and ion-exchange chromatography on DEAE cellulose. The partially purified TPO is recovered in 90% yield, with a 55 fold increase in specific TPO activity over the homogenate and a final nucleic acid content of about 4%. The phospholipid content is reduced to 10–15% of the 105.000 × g particles.

The partially purified preparations catalyze the peroxidation of guaiacol and the iodination of moniodotyrosine. The preparation solubilized by butanol is subject to aggregation as either protein or ionic concentration is increased. Aggregation is partialis reversed by dilution of protein, reduction of ionic strength or by the addition of detergents.     

Solubilization,stabilization and isoelectric focusing of human liver neuraminidase activity

Homogenate preparations of human liver have been prepared and over 75% of the particulate neuraminidase activity (which comprises approx. 90% of the total activity) has been solubilized using 0.85% (w/v) Triton X-100 in 25 mM phosphate buffer (pH 6.8). The solubilized neuraminidase activity is extremely labile, but can be stabilized for at least 4 weeks at 2–4°C, using 10 mM N-acetylneuraminic acid. Kinetic characterization of homogenate and solubilized supernatant fluid neuraminidase activities indicated comparable pH optimum curves (maximum activity at pH 4.5–4.7) and apparent K_m values (0.2–0.4 mM) for the synthetic fluorometric substrate $\text{4-}\text{methylbelliferyl}\text{-α-}\text{D}\text{-N-}\text{acetylneuraminic}$ acid. Isoelectric focusing has been performed on human liver homogenates and Triton X-100-solubilized neuraminidase activities, and the presence of several forms (4–6) with isoelectric points (pI values) between 4.4 and 5.2 has been demonstrated in both preparations. The similar kinetic and isoelectric focusing properties of the two preparations suggest that the solubilized enzyme activity is representative of the homogenate activity and that the solubilized enzyme is suitable for purification purposes.     

Properties of solubilized prostaglandin synthetase from sheep vesicular glands.

Prostaglandin synthetase activity associated with the microsomal fraction from sheep vesicular glands has been solubilized by treatment with the non-ionic detergents Tween 20, Lubrol Px and Lubrol Wx. Approx.8 fold purification from microsomes is obtained and over 90% of the activity is recovered in the detergent solubilized fraction. The solubilized synthetase activity is stable at pH 5.0 but is gradually lost at pH 8.0; it is also heat and acid labile. The relative amounts of prostaglandins E₂, D₂ and F_2α formed by the microsomal-bound synthetase and by the solubilized synthetase are similar. Also similar are the pH optima (7.9–8.5) of the two synthetase preparations. The solubilization process appears to yield a fully active enzymatic preparation which could be employed for further purification and characterization of the prostaglandin synthetase complex.     

Solubilization and partial purification of GABAB receptor from bovine brain

gamma-Aminobutyric acid (GABA)B receptor has been solubilized and partially purified by an affinity column chromatography. GABAB receptor was solubilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in the presence of asolectin. The solubilized GABAB receptor was adsorbed on baclofen-coupled epoxy-activated Sepharose 6B. The affinity matrix adsorbed 80% of the solubilized [3H]GABA binding activity to GABAB receptor, and approximately 75% of the adsorbed activity could be eluted with 1 M KC1. GABAB receptor binding in the fraction eluted from affinity column was displaced by GABA, baclofen and 2-hydroxy saclofen in a dose-dependent manner. Furthermore, the purified GABAB receptor showed approximately 2800-fold purification as compared with the original solubilized fraction and possessed the specific binding activity of 17.68 p mol/mg of protein. This binding consisted of a single binding site with a dissociation constant of 64.4 nM. The present results indicate that affinity column chromatographic procedures using baclofen-coupled epoxy-activated Sepharose 6B are suitable for the partial purification of GABAB receptor from cerebral tissues.     

The stereospecific d-glucose transport activity of cholate extracts from human erythrocyte membranes

The glucose transport protein of human erythrocyte membranes was solubilized with cholate to facilitate rapid reconstitution and direct glucose transport measurements. This may simplify the isolation of the native glucose transporter. In most experiments the membranes were prepared from fresh blood within 8 h, frozen in liquid nitrogen and stored at ?70°C to minimize proteolytic degradation. Solubilization with 25 mM cholate in the presence of 200 mM NaCl at pH 8.4 for 12 min at room temperature gave a high d-glucose transport activity. The solubilized mixture contained 20% of the total membrane protein, only 6% of the polypeptides of molecular weight around 90 000, 23% of the polypeptides of molecular weight around 55 000, 30% of the phospholipids and at least 6% of the stereospecific d-glucose transport activity. At cholate concentrations up to 22 mM the ratio of solubilized phospholipids to cholate increased steeply, concomitant with an increase in solubilized activity. Above 30 mM cholate the activity diminished. At 4°C the activity of the extrac decreased rapidly within the first day and slowly during the next few days. The initial changes seem to have produced a fairly stable, but not native form or fragment of the transporter. When 20 mM EDTA and 5 mM dithioerythritol were included in the solubilization mixture a high activity was preserved for about one day.     

A liver factor that interconverts multiple forms of tyrosine aminotransferase.

Three activity peaks of rat liver soluble tyrosine aminotransferase have been resolved using hydroxyl-apatite chromatography. These peaks interconvert during storage of the soluble enzyme preparation in ice for 20 h. A component of a particulate fraction of liver which will interconvert the forms of tyrosine aminotransferase in vitro with no alteration of total enzyme activity has been detected. This factor is present in a 31, 000 gh pellet of liver and is solubilized by sonication. When the factor is subjected to dialysis or incubation at 25°C for 30 min. its effect on tyrosine aminotransferase is greatly diminished.     

Genetic identification and purification of the respiratory NADH dehydrogenase of Escherichia coli

Escherichia coli membrane particles were solubilized with potassium cholate. An NADH:ubiquinone oxidoreductase was resolved by hydroxylapatite chromatography of the solubilized material. This enzyme has been identified as the respiratory NADH dehydrogenase since it is absent in chromatograms of solubilized material from an ndh mutant strain. Such mutants lack membrane-bound NADH oxidase activity and have previously been shown to have an inactive NADH dehydrogenase complex [Young, I. G., & Wallace, B. J. (1976) Biochim. Biophys. Acta 449, 376-385]. The respiratory NADH dehydrogenase was amplified 50- to 100-fold in vivo by using multicopy plasmid vectors carrying the ndh gene and then purified to homogeneity on hydroxylapatite. Hydroxylapatite chromatography of cholate-solubilized material from genetically amplified strains purified the enzyme approximately 800- to 100-fold relatively to the activity in wild-type membranes. By use of a large-scale purification procedure, 50-100 mg of protein with a specific activity of 500-600 mumol of reduced nicotinamide adenine dinucleotide oxidized min-1 mg-1 at pH 7.5, 30 degrees C, was obtained. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme showed that the enzyme consists of a single polypeptide with an apparent Mr of 45 000.     

Spatio-Temporal Characteristics of Global Warming in the Tibetan Plateau during the Last 50 Years Based on a Generalised Temperature Zone - Elevation Model

Temperature is one of the primary factors influencing the climate and ecosystem, and examining its change and fluctuation could elucidate the formation of novel climate patterns and trends. In this study, we constructed a generalised temperature zone elevation model (GTEM) to assess the trends of climate change and temporal-spatial differences in the Tibetan Plateau (TP) using the annual and monthly mean temperatures from 1961–2010 at 144 meteorological stations in and near the TP. The results showed the following: (1) The TP has undergone robust warming over the study period, and the warming rate was 0.318°C/decade. The warming has accelerated during recent decades, especially in the last 20 years, and the warming has been most significant in the winter months, followed by the spring, autumn and summer seasons. (2) Spatially, the zones that became significantly smaller were the temperature zones of −6°C and −4°C, and these have decreased 499.44 and 454.26 thousand sq km from 1961 to 2010 at average rates of 25.1% and 11.7%, respectively, over every 5-year interval. These quickly shrinking zones were located in the northwestern and central TP. (3) The elevation dependency of climate warming existed in the TP during 1961–2010, but this tendency has gradually been weakening due to more rapid warming at lower elevations than in the middle and upper elevations of the TP during 1991–2010. The higher regions and some low altitude valleys of the TP were the most significantly warming regions under the same categorizing criteria. Experimental evidence shows that the GTEM is an effective method to analyse climate changes in high altitude mountainous regions.     

Purification of murine thymus leukemia antigen (TL). A quantitative assessment of limited proteolysis.

The murine thymus leukemia antigen (TL) has been solubilized from the tumor ASL1 and from an established cell line ASL1W, by papain digestion. When a 15-min digest was chromatographed on Sephadex G-200, two peaks of TL activity were eluted with apparent molecular weights of approximately 58,000 and 31,000. Chromatography of a 30-min digest under the same conditions resulted in elution of a single peak of activity with an apparent molecular weight of 58,000. Additional purification was carried out on the 58,000 molecular weight material by absorption to, and elution from DEAE-cellulose. The combination of gel filtration and ion exchange chromatography resulted in approximately a 150-fold purification.     

Isolation of Cellulolytic Anaerobic Extreme Thermophiles from New Zealand Thermal Sites

Avicel enrichment cultures from 47 thermal-pool sites in the New Zealand Rotorua-Taupo region were screened for growth and carboxymethyl cellulase activity at 75°C. Eight anaerobic cellulolytic cultures were obtained. The effect of temperature on carboxymethyl cellulase activity was measured, and bacteria were isolated from the five best cultures. Bacteria from two sources designated TP8 and TP10 grew at 75°C, accumulated reducing sugar in the growth medium and gave free cellulases with avicelase activity. Bacteria from sources designated Tok4, Tok8, and Wai21 grew at 75°C, accumulated no free sugars in the medium, and gave free carboxymethyl cellulases with virtually no avicelase activity. All were obligate anaerobic nonsporeforming rods which stained gram negative, grew on pentoses as well as hexoses, and gave ethanol and acetate as major fermentation end products. The isolated strain which produced the most active and stable cellulases (trivially designated TP8.T) had lower rates of free endocellulase accumulation at 75°C than did Clostridium thermocellum at 60°C, but its cellulase activity against avicel and filter paper in culture supernatants was comparable. Tested at 85°C, TP8.T carboxymethyl cellulases included components which were very stable, whereas C. thermocellum carboxymethyl cellulases were all rapidly inactivated. The TP8.T avicelase activity was relatively unaffected by Triton X-100, EDTA, and dithiothreitol. Evidence was obtained for the existence of unisolated, cellulolytic extreme thermophiles producing cellulases which were more stable and active than those from TP8.T.     

A simple, quick, and high-yield preparation of the human thromboxane A2 receptor in full size for structural studies

Human thromboxane A2 receptor (TP), a G protein-coupled receptor (GPCR), is one of the most promising targets for developing the next generation of anti-thrombosis and hypertension drugs. However, obtaining a sufficient amount of the full-sized and active membrane protein has been the major obstacle for structural elucidation that reveals the molecular mechanisms of the receptor activation and drug designs. Here we report an approach for the simple, quick, and high-yield preparation of the purified and active full-sized TP in an amount suitable for structural studies. Glycosylated human TP was highly expressed in Sf-9 cells using an optimized baculovirus (BV) expression system. The active receptor was extracted and solubilized by different detergents for comparison and was finally purified to a nearly single band with a ratio of 1:0.9 +/- 0.05 (ligand:receptor molecule) in ligand binding using a Ni column with a relatively low yield. However, a high-yield purification (milligram quantity) of the TP protein, from a modulate scale of transfected Sf-9 cell culture, has been achieved by quick and simple purification steps, which include DNA digestion, dodecyl-maltoside detergent extraction, centrifugation, and FPLC purification. The purity and quantity of the purified TP, using the high-yield approach, were suitable for protein structural studies as evidenced by SDS-PAGE, Western blot analyses, ligand binding assays, and a feasibility test using high-resolution one-dimensional and two-dimensional (1)H NMR spectroscopic analyses. These studies provide a basis for the high-yield expression and purification of the GPCR for the structural and functional characterization using biophysics approaches.     

Partial purification of plant plasma membrane K+, Mg2+-ATPase by ion exchange chromatography

A purification procedure is presented which differs in three respects from other procedures for the purification of plant plasma membrane H⁺-pumping ATPase (EC 3.6.1.35) from various plants. Soybean ( Glycine max L. cv. Williams) hypocotyls were homogenized in the presence of physiological ionic strength and plasma membrane vesicles were purified by aqueous polymer two-phase partitioning. Plasma membrane vesicles were then solubilized in one step by using non-ionic detergent (either Triton X-100 or C₁₂E₈). The Mg-ATPase was separated by ion exchange chromatography from other solubilized membrane proteins. ATPase molecules bound to phosphocellulose fibers were eluted by a 0–1 M gradient of NaCl. The NaCl-eluted fractions contained a Mg-ATPase which showed the characteristics of Mg-ATPase present in the plasma membranes. The specific activity of the partially purified enzyme was 2–5 μmol mg⁻¹ min⁻¹ when it was reconstituted into proteoliposomes. This value is in good agreement with data obtained by other purification methods in the literature.     

Saccharification of Complex Cellulosic Substrates by the Cellulase System from Clostridium thermocellum

True cellulase activity has been demonstrated in cell-free preparations from the thermophilic anaerobe Clostridium thermocellum. Such activity depends upon the presence of Ca²⁺ and a thiol-reducing agent of which dithiothreitol is the most promising. Under these conditions, native (cotton) and derived forms of cellulose (Avicel and filter paper) were all extensively solubilized at rates comparable with cellulase from Trichoderma reesei. Maximum activity of the Clostridium cellulase was displayed at 70°C and at pH 5.7 and 6.1 on Avicel and carboxymethylcellulose, respectively. In the absence of substrate at temperatures up to 70°C, carboxymethylcellulase was much more unstable than the Avicel-hydrolyzing activity.     

Solubilization and Characterization of a Membrane-Bound Auxin-Binding Protein from Cell Suspension Cultures of Nicotiana tabacum

A membrane-bound auxin-binding protein (MABP) was solubilized by Triton X-100 from cell suspension cultures of Nicotiana tabacum L. Solubilization of MABP was dependent on the detergent concentration and more than 80% of naphthalene-1-acetic acid (NAA)-binding activity was recovered by an optimum concentration of 0.2%. The solubilized MABP was highly heat-unstable and sensitive to protease. The properties of MABP (affinity, temperature dependence, pH optimum, and analog specificity for auxin binding) did not significantly change after solubilization, e.g. the solubilized MABP showed no or very low levels of NAA-binding at 0 to 4°C but showed a high-affinity binding (dissociation constant K_d = 2.7 ± 0.3 × 10⁻⁷m) at 25°C at an optimum pH of 5.0. NAA-binding of the solubilized MABP proceeded very slowly, i.e. a time of half-maximum binding was at least 15 minutes, although the solubilized MABP showed higher rates of association (k₁ = 1.3 versus 0.9 × 10⁵m⁻¹ min⁻¹) and dissociation (k₋₁ = 2.2 versus 1.6 × 10⁻² min⁻¹) with NAA than the bound MABP. These results show that specific, saturable, and reversible auxin binding to MABP from dicotyledonous N. tabacum differs from that from monocotyledonous Zea mays, and confirm that MABP is distinct from a soluble auxin-binding protein which also is present in N. tabacum.     

Solubilization and some properties of a semicarbazide-sensitive amine oxidase in brown adipose tissue of the rat.

A semicarbazide-sensitive clorgyline-resistant amine oxidase (SSAO) was solubilized from membrane fractions of rat brown adipose tissue by the non-ionic detergent, Triton X-100. Alteration of ionic strength or addition of chelating agents alone failed to release the enzyme from its membrane. Lipid-depletion led to loss of enzyme activity and alteration of substrate affinity. Over 80% of the activity of the solubilized enzyme was found in gel filtration fractions corresponding to an Mr of between 160 000 and 180 000. The glycoprotein nature of SSAO was established from affinity chromatography with either immobilized concanavalin A or Lens culinaris lectin. Elution of over 50% SSAO activity from the lentil lectin was achieved with 0.25M-alpha-methyl D-mannoside to give 80-90-fold purification of the enzyme. Irradiation inactivation gave a value for Mr of around 183 000 for both soluble and membrane-bound SSAO. Substrate affinity and inhibitor sensitivity of the enzyme were unaltered by solubilization. The copper-chelating agent, diethyldithiocarbamate, did not affect the enzyme, shedding doubt on the suggestion that SSAO is a copper-requiring enzyme. The significance of these findings in relation to the nature of SSAO and to its disposition within the cell membrane is discussed.     

Membrane ATPase of Bacillus subtilis. I. Purification and properties

The membrane ATPase (EC 3.6.1.3) of Bacillus subtilis can be solubilized by a shock-wash process. Two procedures for purifying the solubilized enzyme are reported. A protease inhibitor, phenylmethane sulfonylfluoride, was introduced in the solubilization and purification step.The resultant ATPase purified by density gradient centrifugation has a molecular weight of 315 000, an s_20,w of 13,4 and an ámino acid composition very similar to bacterial ATPases already studied.After exposure to polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate (SDS), or 8 M urea or SDS-urea, the purified ATPase can be dissociated in two non-identical subunits of molecular weights 59 000 (α) and 57 000 (β) with different charges.Kinetic studies showed that Ca²⁺ or Zn²⁺ are required for ATPase activity, although Mg²⁺ was uneffective. At optimal Ca²⁺ concentration, the Mg²⁺ has an inhibitory effect. The K_m for ATP is 1.3 mM. Inhibitors of the oxydative phosphorylation, of the mitochondrial ATPase and of the (Na⁺ + K⁺)-ATPase are studied.     

Production,purification, characterization,antioxidant and antiproliferative activities of extracellular L-asparaginase produced by Fusarium equiseti AHMF4

L-Asparaginase is an antileukemic agent that depletes L-asparagine “an important nutrient for cancer cells” through the hydrolysis of L-asparagine into L-aspartic acid and ammonia leading to leukemia cell starvation and apoptosis in susceptible leukemic cell populations. Moreover currently, bacterial L-asparaginase has been limited by problems of lower productivity, stability, selectivity and a number of toxicities along with the resistance towards bacterial L-asparaginase. Then the current work aimed to provide pure L-asparaginase with in-vitro efficacy against various human carcinomas without adverse effects related to current L-asparaginase formulations. Submerged fermentation (SMF) bioprocess was applied and improved to maximize L-asparaginase production from Fusarium equiseti AHMF4 as alternative sources of bacteria. The enzyme production in SMF was maximized to reach 40.78 U mL⁻¹ at the 7th day of fermentation with initial pH 7.0, incubation temperature 30 °C, 1.0% glucose as carbon source, 0.2% asparagine as nitrogen source, 0.1% alanine as amino acid supplement and 0.1% KH₂PO₄. The purification of AHMF4 L-asparaginase yielded 2.67-fold purification and 48% recovery with final specific activity of 488.1 U mg⁻¹ of protein. Purified L-asparaginase was characterized as serine protease enzyme with molecular weight of 45.7 kDa beside stability at neutral pH and between 20 and 40 °C. Interestingly, purified L-asparaginase showed promising DPPH radical scavenging activity (IC₅₀ 69.12 μg mL⁻¹) and anti-proliferative activity against cervical epitheloid carcinoma (Hela), epidermoid larynx carcinoma (Hep-2), hepatocellular carcinoma (HepG-2), Colorectal carcinoma (HCT-116), and breast adenocarcinoma (MCF-7) with IC₅₀ equal to 2.0, 5.0, 12.40, 8.26 and 22.8 μg mL⁻¹, respectively. The enzyme showed higher activity, selectivity and anti-proliferative activity against cancerous cells along with tiny cytotoxicity toward normal cells (WI-38) which indicates that it has selective toxicity and it could be applied as a less toxic alternative to the current formulations.     

Characterization and partial purification of indole-3-butyric acid synthetase from maize (Zea mays)

In previous work it has been shown that the route from indoleacetic acid (IAA) to indolebutyric acid (IBA) is likely to be a two-step process with an unknown intermediate designated ‘product X′. Our objective was to characterize and purify enzyme activities that are involved in these reactions. Indole-3-butyric acid synthetase was isolated and characterized from light-grown maize seedlings (Zea mays L.), which were able to synthesize IBA from indole-3-acetic acid (IAA) with ATP and acetyl-CoA as cofactors. The enzyme activity is most likely located on the membranes of the endoplasmic reticulum, as shown by means of aqueous two-phase partitioning and sucrose density gradient centrifugation, with subsequent marker enzyme analysis. It was possible to solubilize the enzyme from the membranes with a detergent (CHAPS) and high concentrations of NaCl. The molecular mass of solubilized IBA synthetase was ca 31 kDa and its isoelectric point was at pH 4.8. The enzyme forming the reaction intermediate had a molecular mass of only 20 kDa and it seemed to be located on different membranes. Inhibition experiments with reducing agents and sulfhydryl reagents indicated that no sulfhydryl groups or disulfide bridges were present in the active centre of IBA synthetase. KCN inhibited the enzyme activity completely, and sodium azide by about 50%. Substrate analogs. such as 1-IAA, 2,4-dichlorophenoxyacetic acid, phenylacetic acid, and naphthaleneacetic acid, inhibited IBA formation to a high extent. Experiments with tunicamycin gave evidence that the enzyme is not a glycoprotein. These findings were confirmed by affinity chromatography with Concanavalin A. where the enzyme did not bind to the matrix. Further purification of the IBA synthetase on an ATP-affinity column resulted in a more than 1 000-fold purification compared to the microsomal membranes. IBA synthetase activity was also present in other plant families. Our results present further evidence that IBA is synthesized by a two-step mechanism involving two different enzyme activities.     

Specific elution from hydroxylapatite of the mitochondrial phosphate carrier by cardiolipin

The role of cardiolipin in the purification of the mitochondrial phosphate carrier by hydroxylapatite has been investigated. Without added cardiolipin, the reconstituted phosphate-transport activity in the hydroxylapatite eluate is small and only confined to the first fraction. With cardiolipin added to the extract, the eluted activity is much higher and present until fraction 6. The activity retained by hydroxylapatite in the absence of cardiolipin is eluted after addition of this phospholipid to the column. The requirement of added cardiolipin diminishes on increasing the concentration of solubilized mitochondria. The hydroxylapatite eluate contains five protein bands in the Mr-region of 30 000-35 000, which are differently distributed in the various fractions. Among these, only the presence and the relative amount of band 3 of Mr 33 000 corresponds to the phosphate transport activity. Cardiolipin is the only phospholipid tested which causes elution of band 3 from hydroxylapatite; on the other hand, it prevents the elution of band 2 and retards that of band 5 (the ADP/ATP carrier). Band 1 starts to appear in the second fraction even without cardiolipin. On increasing the concentration of cardiolipin, in the first fraction of the hydroxylapatite eluate band 3 increases and the contamination of band 4 decreases. Under optimal conditions a preparation of band 3 about 90% pure and with high reconstituted phosphate transport activity is obtained. It is concluded that the elution of the phosphate carrier from hydroxylapatite requires cardiolipin and that the phosphate carrier is identical with (or with part of) band 3 of the hydroxylapatite eluate.     

A constitutive, plasma-membrane bound β-glucosidase in Trichoderma reesei

Abstract Plasma membranes of Trichoderma reesei QM 9414, isolated from protoplasts by means of the concanavalin A procedure, contained β-glucosidase activity, which appeared constitutively upon growth on glucose. The enzyme had a pH optimum around 6, and was active on p -nitrophenyl-β- d -glucoside, cellobiose and sophorose ( K _m 0.7, 3.9 and 3.1 mM, respectively). Glucose was only weakly inhibitory ( K _i 7 mM). Treatment of the plasma membranes with Triton X-100, Tween 80 or digitonin solubilized more than 60% of the membrane-bound β-glucosidase activity. The enzyme so solubilized exhibited an M _r of 70 000 ± 5000 and an isoelectric point at pH 8.2 ± 0.3.