Characterising the association of latency with α1-antitrypsin polymerisation using a novel monoclonal antibody

α₁-Antitrypsin is primarily synthesised in the liver, circulates to the lung and protects pulmonary tissues from proteolytic damage. The Z mutant (Glu342Lys) undergoes inactivating conformational change and polymerises. Polymers are retained within the hepatocyte endoplasmic reticulum (ER) in homozygous (PiZZ) individuals, predisposing the individuals to hepatic cirrhosis and emphysema. Latency is an analogous process of inactivating, intra-molecular conformational change and may co-occur with polymerisation. However, the relationship between latency and polymerisation remained unexplored in the absence of a suitable probe. We have developed a novel monoclonal antibody specific for latent α₁-antitrypsin and used it in combination with a polymer-specific antibody, to assess the association of both conformers in vitro, in disease and during augmentation therapy. In vitro kinetics analysis showed polymerisation dominated the pathway but latency could be promoted by stabilising monomeric α₁-antitrypsin. Polymers were extensively produced in hepatocytes and a cell line expressing Z α₁-antitrypsin but the latent protein was not detected despite manipulation of the secretory pathway. However, α₁-antitrypsin augmentation therapy contains latent α₁-antitrypsin, as did the plasma of 63/274 PiZZ individuals treated with augmentation therapy but 0/264 who were not receiving this medication (p < 10⁻¹⁴). We conclude that latent α₁-antitrypsin is a by-product of the polymerisation pathway, that the intracellular folding environment is resistant to formation of the latent conformer but that augmentation therapy introduces latent α₁-antitrypsin into the circulation. A suite of monoclonal antibodies and methodologies developed in this study can characterise α₁-antitrypsin folding and conformational transitions, and screen methods to improve augmentation therapy.     

Purification and Partial Characterization of a Genetically-Defined Superoxide Dismutase (SOD-1) Associated with Maize Chloroplasts

The chloroplast-associated form of superoxide dismutase from maize (Zea mays L.) (SOD-1) has been purified by a stepwise procedure consisting of (NH₄)₂SO₄ fractionation, G-100 Sephadex gel filtration, DEAE-Sephacel chromatography, and hydroxylapatite chromatography. This procedure resulted in a single band on sodium dodecyl sulfate-polyacrylamide gels indicating that the preparation is homogeneous. The holoenzyme molecular weight was estimated at 31,000 to 33,000 by gel filtration. The subunit molecular weight of this dimeric protein was estimated at 14,500 on sodium dodecyl sulfate-polyacrylamide gels. Studies involving amino acid composition analysis, immunological cross-reactivity, in vitro subunit hybridizations, and H₂O₂ sensitivity indicate that SOD-1 differs significantly from SOD-2 and SOD-4, the other cupro-zinc forms of SOD from maize. The possible physiological role of SOD-1 within the chloroplast is discussed.     

Structure of a β-galactan isolated from the nuclei of Physarum polycephalum

A sulfated and phosphorylated β-D-galactan ([α]_D + 8°) was isolated from the nuclei of the acellular slime mould Physarum polycephalum. The polysaccharide was isolated from cesium chloride gradients during the preparation of ribosomal DNA and purified. The purified galactan contained 89% galactose, 2.5% phosphate and 9.6% sulfate groups and had an average degree of polymerisation of 560. Periodate degradation and permethylation studies indicated the presence of mainly (1 → 4)-, but also of (1 → 3)-, and (1 → 6)-linked galactose units with one branch every 13 units. These results suggested that the intranuclear galactan, apart from its higher sulfate content, is similar to the extra-cellular polysaccharide produced by P. polycephalum.     

Selectivity of the surface binding site (SBS) on barley starch synthase I

Starch synthase I (SSI) from various sources has been shown to preferentially elongate branch chains of degree of polymerisation (DP) from 6–7 to produce chains of DP 8–12. In the recently determined crystal structure of barley starch synthase I (HvSSI) a so-called surface binding site (SBS) was seen, which was found by mutational analysis to be essential for the activity of HvSSI on glycogen. We now show in binding studies using surface plasmon resonance that HvSSI has no detectable affinity for malto-triose and -tetraose, but clearly binds maltopentaose, -hexaose, -heptaose (M7) and β-cyclodextrin (β-CD) albeit with a measurable K _D for only β-CD and M7. Moreover, an HvSSI SBS mutant F538A lost the ability to bind β-CD and maltooligosaccharides. This behaviour suggests that a chain in the α-glucan molecule (amylopectin) that is undergoing extension attaches itself at the SBS and that the active site itself, likely working on a different end chain, has low affinity for both substrate and product.     

The yeast mitochondrial adenosine triphosphatase complex. Purification, subunit composition, and some effects of protease inhibitors.

(i) The method of preparing the oligomycin-insensitive F₁-ATPase by chloroform treatment of mitochondrial membranes (Beechey et al., 1975, Biochem. J.148, 533–537) has been modified such that a five-subunit protein is obtained from yeast with an activity of 140 μmol of ATP hydrolyzed/min/mg of protein. Repetition of this procedure in the presence of protease inhibitors (in particular, p-aminobenzamidine) allows isolation of a four-subunit protein with an activity of 243 μmol of ATP hydrolyzed/min/ mg of protein, (ii) A modified procedure is described for the preparation of the yeast oligomycin-sensitive F₁-F₀ ATPase complex, making use of protease inhibitors throughout and solubilization of the ATPase from mitochondrial membranes using Triton X-100 and sodium deoxycholate simultaneously. Two polypeptides Of 42,000 and 29,000 molecular weight are eliminated, the largest corresponding to the missing band of the F₁ sector. The complex retains oligomycin- and uncoupler-sensitive ATP-³²P_i exchange and ATP-driven proton uptake, indicating the retention of a complete coupling mechanism. (iii) F₁-ATPase is released from the F₁-F₀ complex by brief heating at 50 °C in the presence of ATP. The remaining hydrophobic polypeptides aggregate and are isolated by centrifugation. The F₁ sector can be isolated containing either four or five subunits depending on whether the starting F₁-F₀ complex contained the 42,000 and 29,000 molecular weight polypeptides. (iv) Sensitivity of the F₁-F₀ ATPase complex to oligomycin and dicyclohexylcarbodiimide varies considerably depending on the activity measured and whether the complex was first reconstituted with phospholipids. The degree of inhibitor sensitivity is considered a poor guide to intactness of the complex.     

Rapid isolation of RNA using proteinase K and sodium perchlorate.

A simple, efficient procedure for the isolation of cellular nucleic acids is described. It combines the use of sodium dodecyl sulfate, proteinase K, sodium perchlorate, and isopropanol precipitation. The yields and purity of RNA extracted from a variety of sources are comparable or superior to those obtained by phenol extraction. High molecular weight RNA (ribosomal as well as nonribosomal) is recovered intact and in high yield. Fibroin messenger RNA (M_r 5.8 × 10⁶) isolated by this procedure is biologically active.     

Cellulase from Fusarium solani: Purification and properties of the C1 component

The C₁ component from Fusarium solani cellulase was purified extensively by molecular-sieve chromatography on Ultrogel AcA-54 and ion-exchange chromatography on DEAE-Sephadex. The purified component showed little capacity for hydrolysing highly ordered substrates (e.g., cotton fibre), but poorly ordered substrates (e.g., H₃PO₄-swollen cellulose), and the soluble cello-oligosaccharides cellotetraose and cellohexaose, were readily hydrolysed; cellobiose was the principal product in each case. Attack on O-(carboxymethyl)cellulose, a substrate widely used for measuring the activity of the randomly acting enzymes (C_x enzymes) of the cellulase complex, was minimal, and ceased after the removal of a few unsubstituted residues from the end of the chain. These observations, and the fact that the rate of change of degree of polymerisation of H₃PO₄-swollen cellulose was very slow compared with that effected by the randomly acting endoglucanases (C_x, CM-cellulases), indicate that C₁ is a cellobiohydrolase. Fractionation by a variety of methods gave no evidence for the non-identity of the cellobiohydrolase and the component that acted in synergism with the randomly acting C_x enzyme when solubilizing cotton fibre.     

Purification of oat and rye phytochrome

A purification procedure employing normal chromatographic techniques is outlined for isolating phytochrome from etiolated oat (Avena sativa L.) seedlings. Yields in excess of 20% (25 milligrams or more) of phytochrome in crude extract were obtained from 10- to 15-kilograms lots. The purified oat phytochrome had an absorbance ratio (A₂₈₀ nm/A₆₆₅ nm) of 0.78 to 0.85, comparable to reported values, and gave a single major band with an estimated molecular weight of 62,000 on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. A modification of the oat isolation procedure was used to isolate phytochrome from etiolated rye Secale cereale cv. Balbo) seedlings. During isolation rye phytochrome exhibited chromatographic profiles differing from oat phytochrome on diethylaminoethyl cellulose and on molecular sieve gels. It eluted at a higher salt concentration on diethylaminoethyl cellulose and nearer the void volume on molecular sieve gels. Yields of 5 to 10% (7.5-10 milligrams) of phytochrome in crude extract were obtained from 10- to 12-kilogram seedling lots. The purified rye phytochrome had an absorbance ratio of 1.25 to 1.37, significantly lower than values in the literature and gave a single major band with an estimated molecular weight of 120,000 on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. It is suggested that the absorbance ratio and electrophoretic behavior of rye phytochrome are indices of purified native phytochrome, and that oat phytochrome as it has been described is an artifact which arises as a result of endogenous proteolysis during isolation. A rationale is provided for further modifications of the purification procedure to alleviate presumed protease contaminants.     

Purification and properties of an extracellular endo-1,4-β-glucanase fromLenzites trabea

An extracellular endo-1,4--glucanase (EC 3.2.1.4) has been isolated and purified from the culture solution of the basidiomyceteLenzites trabea grown on glucose and cellulose. Besides-glucosidase activity (EC 3.2.1.21) no evidence for C₁-activity (EC 3.2.1.91) in the culture solution was found.The endoglucanase has been purified in a four-step procedure including chromatography on Sepharose 6-B and DEAE-Sephadex A-50, adsorption on hydroxylapatite and gel filtration on Bio-Gel P-100. The enzyme showed maximum activity at pH 4.4 and 70°C. A molecular weight of 29000 Daltons was estimated by calibration on Bio-Gel P-100. The enzyme hydrolyses carboxymethyl cellulose (CMC) as well as xylan.List of Abbreviations CMC carboxymethyl cellulose - D.S. degree of substitution - D.P. degree of polymerisation - MW molecular weight     

The reactivity of ruthenium mono(bipyridine) carbonyl complexes in an alcoholic solution of alkali metal carbonates

Chlorine containing ruthenium bipyridine carbonyl compounds react readily in dilute alkaline solutions under a CO atmosphere affording a poorly soluble and air sensitive product that is suggested to have a polymeric nature. Various analysis methods (MS and TPD) were used in the characterisation of the product. The replacement of the axial chloride ligands in trans(Cl), cis(CO)[Ru(bpy)(CO)₂Cl₂] and [Ru(bpy)(CO)₂Cl]₂ is proposed to be the initial step in the polymerisation. The replacement of chlorides in methanolic solution was confirmed by isolating and characterising the dimeric intermediate [Ru(bpy)(CO)₂(COOCH₃)]₂.     

Preparation of dehydrobenzylisoquinolines by immobilized (S)-tetrahydroprotoberberine oxidase from plant cell cultures

(S)-Tetrahydroprotoberberine oxidase (STOX) has been isolated in enriched (7.4-fold) form from a high yielding cell suspension of Berberis wilsoniae var. subcaulialata in a three step procedure and was immobilized by several different methods. The properties of immobilized STOX were compared with immobilized Berberis cells and with the soluble enzyme. Although pH and temperature optima were shifted by immobilization, K_m-values with (S)-norreticuline remained unaffected. The stability of immobilized STOX was 50 times better than the free enzyme. A cyclic process is described using the stereospecific enzymatic oxidation of (S)norreticuline to 1,2-dehydronorreticuline followed by sodium borohydride reduction for the transformation of racemic norreticuline to the (R)-enantiomer.     

Discovery of novel quinoline-based analogues of combretastatin A-4 as tubulin polymerisation inhibitors with apoptosis inducing activity and potent anticancer effect

A new series of quinoline derivatives of combretastatin A-4 have been designed, synthesised and demonstrated as tubulin polymerisation inhibitors. These novel compounds showed significant antiproliferative activities, among them, 12c exhibited the most potent inhibitory activity against different cancer cell lines (MCF-7, HL-60, HCT-116 and HeLa) with IC₅₀ ranging from 0.010 to 0.042 µM, and with selectivity profile against MCF-10A non-cancer cells. Further mechanistic studies suggest that 12c can inhibit tubulin polymerisation and cell migration, leading to G₂/M phase arrest. Besides, 12c induces apoptosis via a mitochondrial-dependant apoptosis pathway and caused reactive oxygen stress generation in MCF-7 cells. These results provide guidance for further rational development of potent tubulin polymerisation inhibitors for the treatment of cancer.

Highlights

• A novel series of quinoline derivatives of combretastatin A-4 have been designed and synthesised.
• Compound 12c showed significant antiproliferative activities against different cancer cell lines.
• Compound 12c effectively inhibited tubulin polymerisation and competed with [³H] colchicine in binding to tubulin.
• Compound 12c arrested the cell cycle at G₂/M phase, effectively inducing apoptosis and inhibition of cell migration.

     

A 2-step synthesis of Combretastatin A-4 and derivatives as potent tubulin assembly inhibitors

A series of combretastatin derivatives were designed and synthesised by a two-step stereoselective synthesis by use of Wittig olefination followed by Suzuki cross-coupling. Interestingly, all new compounds (2a-2i) showed potent cell-based antiproliferative activities in nanomolar concentrations. Among the compounds, 2a, 2b and 2e were the most active across three cancer cell lines. In addition, these compounds inhibited the polymerisation of tubulin in vitro more efficiently than CA-4. They caused cell cycle arrest in G₂/M phase further confirming their ability to inhibit tubulin polymerisation.     

Preparation of high purity monodisperse oligosaccharides derived from mannuronan by size-exclusion chromatography followed by semi-preparative high-performance anion-exchange chromatography with pulsed amperometric detection

Oligosaccharides of ([4)-β-d-ManpA-(→]_n) with a degree of polymerisation (DP) of 5, 10 and 15 were generated by partial acid hydrolysis of alginate mannuronan. These were subsequently purified by a combination of size-exclusion chromatography and semi-preparative high-performance anion-exchange chromatography with pulsed amperometric detection. The purity of the isolated oligosaccharides was greater than 96%. With automated operation of the chromatography system, milligram quantities can be generated over a period of a few days. Thus, our methodology now offers some significant advantages over earlier, including our own, protocols focused on uronic acid oligomers, where the final products are either not as pure or more starting material is needed to generate an equivalent yield of product. Removal of ammonium ions in collected fractions after size-exclusion chromatography and prior to freeze-drying was found to be essential to prevent the formation of imines and subsequent Maillard browning products.     

The effects of sodium and potassium on ouabain binding by human erythrocytes

Ouabain binding by the human erythrocyte membrane is reversible, exhibits a high degree of chemical specificity, and can be detected at ouabain concentrations as low as 1 x 10^-10 M. The relation between ouabain binding and ouabain concentration can be described by a rectangular hyperbola permitting determination of the maximal binding (B _max) and the ouabain concentration at which ouabain binding is half-maximal (K_B). Reducing the external sodium concentration increased K_B, while reducing the external potassium concentration decreased K_B. Neither cation altered B _max The reciprocal of K_B was a linear function of the sodium concentration at sodium concentrations ranging from 0 to 150 mM. Conversely, the relation between the reciprocal of K_B and the external potassium concentration was nonlinear, and raising the potassium concentration above 4 mM produced no further increase in K_B. These results are compatible with a model which postulates that the erythrocyte membrane contains a finite number of receptors each composed of a glycoside-binding site and a cation-binding site. When sodium occupies the cation-binding site, the affinity of the glycoside site for ouabain is increased; when potassium occupies the cation-binding site the affinity of the glycoside site for ouabain is decreased.     

Discovery of new quinolines as potent colchicine binding site inhibitors: design,synthesis, docking studies,and anti-proliferative evaluation

Discovering of new anticancer agents with potential activity against tubulin polymerisation is still a promising approach. Colchicine binding site inhibitors are the most relevant anti-tubulin polymerisation agents. Thus, new quinoline derivatives have been designed and synthesised to possess the same essential pharmacophoric features of colchicine binding site inhibitors. The synthesised compounds were tested in vitro against a panel of three human cancer cell lines (HepG-2, HCT-116, and MCF-7) using colchicine as a positive control. Comparing to colchicine (IC₅₀ = 7.40, 9.32, and 10.41 µM against HepG-2, HCT-116, and MCF-7, respectively), compounds 20, 21, 22, 23, 24, 25, 26, and 28 exhibited superior cytotoxic activities with IC₅₀ values ranging from 1.78 to 9.19 µM. In order to sightsee the proposed mechanism of anti-proliferative activity, the most active members were further evaluated in vitro for their inhibitory activities against tubulin polymerisation. Compounds 21 and 32 exhibited the highest tubulin polymerisation inhibitory effect with IC₅₀ values of 9.11 and 10.5 nM, respectively. Such members showed activities higher than that of colchicine (IC₅₀ = 10.6 nM) and CA-4 (IC₅₀ = 13.2 nM). The impact of the most promising compound 25 on cell cycle distribution was assessed. The results revealed that compound 25 can arrest the cell cycle at G2/M phase. Annexin V and PI double staining assay was carried out to explore the apoptotic effect of the synthesised compounds. Compound 25 induced apoptotic effect on HepG-2 thirteen times more than the control cells. To examine the binding pattern of the target compounds against the tubulin heterodimers active site, molecular docking studies were carried out.     

Simple ITC method for activity and inhibition studies on human salivary α-amylase

Isothermal titration calorimetry (ITC) has an increasing significance in enzyme kinetic studies owing to its general applicability and sensitivity. In the present work, we aimed at developing a simple ITC-based screening procedure for the measurement of human salivary α-amylase (HSA) activity. Reaction of two substrates was studied with three independent methods (ITC, HPLC and spectrophotometry). ITC experiments were made using free and chromophore-containing maltooligomers of different length as substrates. Detailed studies revealed that maltoheptaose or longer oligomers could model properly starch and the presence of aromatic chromophore group did not affect the K_M values considerably. It is the first time, when ITC was used to investigate of HSA-catalysed hydrolysis of different substrates (2-chloro-4-nitrophenyl-4-O-α-D-galactopyranosyl-maltoside, maltoheptaose and starch) in the presence of acarbose inhibitor. All measured IC₅₀ values are in micromolar range (0.9, 18.6 and 29.0?μM, respectively) and increased in parallel with the degree of polymerisation of substrates.     

Mechanochemical Phosphorylation and Solubilisation of β-D-Glucan from Yeast Saccharomyces cerevisiae and Its Biological Activities

To obtain a water-soluble β-D-glucan derivative cleanly and conveniently, a highly efficient mechanochemical method, planetary ball milling, was used to phosphorylate β-D-glucan isolated from yeast Saccharomyces cerevisiae in solid state. Soluble β-D-glucan phosphate (GP) with a high degree of substitution (0.77–2.09) and an apparent PEAK molecular weight of 6.6–10.0 kDa was produced when β-D-glucan was co-milled with sodium hexametaphosphate at 139.5–186.0 rad/s for 12–20 min. The energy transferred was 3.03–11.98 KJ/g. The phosphorylation of GPs was demonstrated by Fourier transform infrared spectroscopy and ¹³C and ³¹P Nuclear magnetic resonance spectroscopy. Three GP products with different degree of substitution (DS) and degree of polymerisation (DP) were able to upregulate the functional events mediated by activated murine macrophage RAW264.7 cells, among which GP-2 with a DS of 1.24 and DP of 30.5 exerted the highest immunostimulating activity. Our results indicate that mechanochemical processing is an efficient method for preparing water-soluble and biologically active GP with high DS.     

High-sensitivity sequence determination of proteins quantitatively recovered from sodium dodecyl sulfate gels using an improved electrodialysis procedure

An improved electrodialysis procedure has been developed to recover quantitatively proteins from sodium dodecyl sulfate (SDS)-polyacrylamide gels. The efficiency of the method has been demonstrated successfully on H-2K^k, β₂-microglobulin, complement factor D, and viral structural protein p27. The results indicate that yields exceeding 93% are obtainable, and that extended amino acid sequences of the eluted proteins in microgram quantities can be obtained in the presence of SDS without intrinsic and/or extrinsic labeling with radioisotopes.     

Purification and properties of poly(ADP-ribose) synthetase from mouse testicle

Poly(ADP-ribose) synthetase has been purified to apparent homogeneity from mouse testicle by a rapid and simple procedure using column chromatography on DNA-agarose and on Cibacron blue F₃G-A-Sephadex G-150. The purified enzyme absolutely requires DNA for activity, and half-maximal activation occurs at a DNA concentration of 25 μg/ml. The K_m for NAD and V at pH 8.0 and 25 °C are 47 μm and 1400 nmol/min/ mg, respectively. The molecular weight is 116,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analysis indicates that the mouse testicle enzyme is very similar to calf thymus enzyme, but there is a difference in the contents of several amino acid residues between the two enzymes. This difference appears to reflect species or tissue specificity of poly(ADP-ribose) synthetase.