Exclusion Column Research Articles

Crystal Structure of a Maltogenic Amylase Provides Insights into a Catalytic Versatility

Amylases catalyze the hydrolysis of starch material and play central roles in carbohydrate metabolism. Compared with many different amylases that are able to hydrolyze only alpha-D-(1,4)-glycosidic bonds, maltogenic amylases exhibit catalytic versatility: hydrolysis of alpha-D-(1,4)- and alpha-D-(1,6)-glycosidic bonds and transglycosylation of oligosaccharides to C3-, C4-, or C6-hydroxyl groups of various acceptor mono- or disaccharides. It has been speculated that the catalytic property of the enzymes is linked to the additional approximately 130 residues at the N terminus that are absent in other typical alpha-amylases. The crystal structure of a maltogenic amylase from a Thermus strain was determined at 2.8 A. The structure, an analytical centrifugation, and a size exclusion column chromatography proved that the enzyme is a dimer in solution. The N-terminal segment of the enzyme folds into a distinct domain and comprises the enzyme active site together with the central (alpha/beta)(8) barrel of the adjacent subunit. The active site is a narrow and deep cleft suitable for binding cyclodextrins, which are the preferred substrates to other starch materials. At the bottom of the active site cleft, an extra space, absent in the other typical alpha-amylases, is present whose size is comparable with that of a disaccharide. The space is most likely to host an acceptor molecule for the transglycosylation and to allow binding of a branched oligosaccharide for hydrolysis of alpha-D-(1,4)-glycosidic or alpha-D-(1,6)-glycosidic bond. The (alpha/beta)(8) barrel of the enzyme is the preserved scaffold in all the known amylases. The structure represents a novel example of how an enzyme acquires a different substrate profile and a catalytic versatility from a common active site and represents a framework for explaining the catalytic activities of transglycosylation and hydrolysis of alpha-D-(1,6)-glycosidic bond.

Partial characterization of mosquito larvae extract modulating mouse and human cell proliferation

Mosquito larvae crude extract have been found to alter the mitotic rate of several mouse epithelial cell populations such as enterocytes and tongue keratinocytes. Also, the dialysed fraction inhibits hepatocyte proliferation in hepatectomized males. These experiments suggested an inhibitory effect on the G1/S interphase. Consequently, we suggested the presence of some molecule or molecules related to the TGF-β superfamily. In the present paper, we have assayed the crude extract on human mononuclear cells and the dialysed fraction of the extract on tongue keratinocyte proliferation. Furthermore, different protein fractions obtained using a molecular exclusion chromatographic column were assayed on hepatocyte proliferation of hepatectomized mice. Three groups of proteins have been isolated. Results show a dose-dependent effect of crude extract on mononuclear cell proliferation and the dialysed extract caused an inhibitory effect on tongue keratinocyte proliferation. With regard to the hepatocyte mitotic rate, an inhibitory effect appeared only in animals receiving the fraction with lower molecular weight. These results suggest the presence in mosquito larvae of some peptidic molecule or molecules resembling the activity of members of the TGF-β superfamily.

Automation and integration of multiplexed on-line sample preparation with capillary electrophoresis for high-throughput DNA sequencing.

An integrated and multiplexed on-line instrument starting from DNA templates to their primary sequences has been demonstrated based on multiplexed microfluidics and capillary array electrophoresis. The instrument automatically processes eight templates through reaction, purification, denaturation, preconcentration, injection, separation, and detection in a parallel fashion. A multiplexed freeze/thaw switching principle and a distribution network were utilized to manage flow and sample transportation. Dye-labeled terminator cycle-sequencing reactions are performed in an eight-capillary array in a hot-air thermal cycler. Subsequently, the sequencing ladders are directly loaded into separate size exclusion chromatographic columns operated at approximately 60 degrees C for purification. On-line denaturation and stacking injection for capillary electrophoresis is simultaneously accomplished at a cross assembly set at approximately 70 degrees C. Not only the separation capillary array but also the reaction capillary array and purification columns can be regenerated after every run. The raw data allow base calling up to 460 bp with an accuracy of 98%. The system is scalable to a 96-capillary array and will benefit not only high-speed, high-throughput DNA sequencing but also genetic typing.

Arabinogalactan from Western Larch, Part I; Effect of Uronic Acid Groups on Size Exclusion Chromatography

Arabinogalactan (AG) from Western larch has sometimes been reported to contain a small proportion of uronic acid units. This concept is supported and extended by the present work, which shows that these units occur in some AG molecules. Molecular assemblies which contain such molecules are consequently partially excluded from the stationary phase of size exclusion chromatography (SEC) columns when low ion strength eluent is used, and the degree of exclusion is a function of the number of acid units per assembly. In this way the ion exclusion effect provides a sensitive means of measuring low levels of uronic acid content in larch AG. Recognition of this effect helps resolve inconsistencies in the earlier AG literature. The dramatic effect which a few uronic acid groups have on the chromatographic behavior of AG implies possible differences in solution properties and biological activities between the charged and uncharged species.

Expression, purification and properties of recombinant E. coli ribonuclease III.

Cloned RNase III gene in a T7 RNA polymerase promoter system was expressed in Escherichia coli cells lacking endogenous RNase III, and the over-expressed recombinant RNase III was purified to homogeneity using ion exchange, exclusion and affinity column chromatography. The overexpressed RNase III was found to separate with the membrane fraction after sonication, which was solubilized, fractionated with (NH)2SO4 and the active fractions used for further purification. The properties of the purified recombinant RNase III were studied using the synthetic RNA substrate, 3[H]poly[A].poly[U], and the natural substrates, 7S and p10Sa RNAs, and compared with the partially purified RNase III from wild-type E. coli cells. The recombinant RNase III showed maximal activity at 37 degrees C and at a pH range of 6.9 to 7.4, which was similar to the RNase III purified from the wild-type cells. Recombinant RNase III efficiently hydrolyzed 3[H].poly[A].poly[U] in the presence of Mg2+. However, the recombinant RNase III cleaved natural RNA substrates efficiently and accurately in the presence of Mn2+. A concentration of Mn2+ ranging from 150 to 300 microM was found to be optimal; concentrations higher than 0.5 mM were inhibitory. Other divalent cations did not support RNase III activity. Monovalent cations, Na+, K+ and NH4+ at 20 mM were equally effective in stimulating RNase III activity although they were not absolutely required for the activity. The thermal stability of the recombinant RNase III was examined at two temperatures, 37 degrees and 50 degrees C. Incubation of RNase III at 37 degrees C for 30 min did not affect activity, but it lost almost 50% of its activity when incubated at 50 degrees C for 30 min. Thus, the recombinant RNase III prefers Mn2+ for the cleavage of natural substrates and exhibits several properties similar to the wild-type RNase III.

A new type of highly polymerized yolk protein from the cochineal insect Dactylopius confusus.

A female specific protein was isolated from eggs and female hemolymph of cochineal insects, using density gradient ultracentrifugation, ammonium sulfate precipitation, and size exclusion column chromatography. The protein was found to consist of four different subunits with apparent molecular weights (Mr) 45,000, 49,000, 53,000, and 56,000, respectively. All four subunits were found to be glycosylated; no association of lipids was detected. Size exclusion column chromatography and non-denaturing polyacrylamide gel electrophoresis demonstrated that the native yolk protein exists as large polymers. Electron microscopy showed that these molecules are long, helical ribbons of variable size which are found in both hemolymph and eggs. Using cryo-electron microscopy, it was shown that the ribbons were 14.6 +/- 1.5 nm wide; the helix they form has a repeat distance of 104.9 +/- 11.3 nm and a diameter of 42.1 +/- 5 nm. A clear substructure of the ribbons was recognized. The newly identified protein is the major yolk protein of Dactylopius confusus and no other proteins resembling the more familiar vitellins of other insect species were detected. Moreover, the D. confusus yolk protein appears to be unique both in its subunit structure and in its polymerizing qualities. Thus, the cochineal yolk protein (CYP) is suggested to represent a new type of insect yolk protein.

Macromolecular Complexation between Bovine Serum Albumin and the Self-Assembled Hydrogel Nanoparticle of Hydrophobized Polysaccharides

Macromolecular complexation between bovine serum albumin (BSA) and self-assembled hydrogel nanoparticle formed by the self-aggregation of cholesterol-bearing pullulan (CHP) was studied by high performance size exclusion column chromatography (HPSEC) and circular dichroism (CD). The CHP self-aggregates complexed with one BSA molecule to give colloidally stable nanoparticles (RG = 17 nm) at pH 7.0 and 25 °C. This was almost irrespective of the substitution degree of the cholesterol group of CHP. The helical content of BSA decreased upon complexation. Unfolding of BSA by either heating or a denaturant such as urea was largely suppressed upon complexation. BSA would be incorporated inside into the hydrogel matrix of the CHP nanoparticle. Kinetic analysis of the complexation suggested a two-step process: namely, the fast pre-equilibrium of looser binding of BSA to the CHP self-aggregate followed by the slower process of tighter inclusion into the hydrogel network. The substitution degree of the cholesterol group in CHP significantly affected the complexation kinetics.

Synthesis and structural characterization of the N2G-mitomycin C-N2G interstrand cross-link in a model synthetic 23 base pair oligonucleotide DNA duplex.

Mitomycin C (MMC) is a genotoxic cancer chemotherapeutic agent that reacts principally at the N2 position of guanine to form one of two predominant monoadducts, or a G-G interstrand cross-link at CpG sites, or a G-G intrastrand cross-link at GpG sites. Previous studies of MMC adduction have principally used very short duplex oligonucleotides (5-15 bp) or very long native duplex DNAs. We examined the formation and structural features of the MMC CpG interstrand cross-link on a model 23 bp synthetic oligonucleotide duplex having the (upper strand) sequence 5'-ATAAATACGTATTTATTTATAAA-3'. MMC was reacted with the duplex oligonucleotide in the presence of sodium dithionite at ratios of 6 mM dithionite: 1.5 mM MMC:0.03 mM duplex. The yield of cross-link in the reaction was determined to be approximately 4.8% by denaturing gel electrophoresis, which represented approximately 75% of the total bound MMC. The cross-linked DNA was isolated to greater than 97% purity in a single step by high temperature size exclusion column chromatography. Characterization of the purified product confirmed that the complex contained exclusively the N2G-MMC-N2G cross-link at the single central CpG site. CD spectroscopy demonstrated a negative band at approximately 290-320 nm which has previously been shown to be characteristic of the MMC cross-link. The relative intensity of this band compared to those reported for shorter duplexes suggested that the majority of the duplex is in a normal B-DNA helical configuration. Base-specific chemical footprinting techniques also indicated that there were subtle but distinct structural perturbations principally within the central four to six base pairs containing and adjacent to the cross-link.

Size exclusion and adsorption column chromatographic studies on functionality distribution of HTPB

AbstractAdsorption column chromatography and size exclusion chromatography have been used to determine the functionality distribution of hydroxyl terminated polybutadiene (HTPB), a modern solid propellant binder. The resin after conversion to 3,5‐dinitrobenzoyl ester was separated into various fractions on a silica column. The fractions were analysed using a dual detector (UV and differential refractive index) analytical gel permeation chromatograph. The functionality of each fraction calculated from the chromatogram showed a continuous increase with increase in the molecular weight. The adsorption chromatography method is unable to fractionate the polymer into fractions with specific functionality. All the fractions have molecules ranging from monofunctional to multifunctional ones and therefore the apportionment method followed in the conventional adsorption chromatography method yields conflicting results.

Separation of two forms of neuropathy target esterase in the soluble fraction of the hen sciatic nerve

Neuropathy target esterase (NTE) activity is operatively defined in this paper as the phenyl valerate esterase activity resistant to 40 μM paraoxon but sensitive to 250 μM mipafox. Molecular exclusion column chromatography with Sephacryl S-300 of the soluble (S) fraction from chick sciatic nerve demonstrated two NTE activity peaks. The first eluted with the front, thus indicating a mol. wt. of over 700 kDa (peak V 0), while the second peak eluted with kd = 0.36, suggestting a mol. wt. of about 100 kDa. The curve of total phenyl valerate (PVase) activity inhibition with paraoxon (0.19–200 μM) shows that at a concentration of 40 μM the esterases highly sensitive to paraoxon are inhibited in the V 0 and 100-kDa peaks. The NTE activity in these two peaks in turn represented 31% and 44% of the 40 μM paraoxon resistant activity, respectively. The mipafox inhibition curves (1.0μ-250 μM) revealed different sensitivities to mipafox, with I 50 values ( t = 30 min) of approximately 1.47 and 63 μM, for V 0 and 100-kDa peaks respectively. Mipafox sensitivity of the V 0 and 100-kDa peaks correlates with the two components, that had been deduced from the kinetic properties of the S-fraction.

Neuronal Cyclin‐Dependent Kinase‐5 Phosphorylation Sites in Neurofilament Protein (NF‐H) Are Dephosphorylated by Protein Phosphatase 2A

Neurofilament (NF) protein [high molecular mass (NF-H)] is extensively phosphorylated in vivo. The phosphorylation occurs mainly in its characteristic KSP (Lys-Ser-Pro) repeat motifs. There are two major types of KSP motifs in the NF-H tail domain: KSPXKX and KSPXXX. Recent studies by two different laboratories have demonstrated the presence of a cdc2-like kinase [cyclin-dependent kinase-5 (cdk5)] in nervous tissue that selectively phosphorylates KSPXKX and XS/TXK motifs in NF-H and lysine-rich histone (H1). This article describes the identification of phosphatases dephosphorylating three different substrates: histone (H1), NF-H in a NF preparation, and a bacterially expressed C-terminal tail domain of NF-H, each containing KSPXKX repeats phosphorylated in vitro by cdk5. Among various phosphatases identified, protein phosphatase (PP) 2A from rabbit skeletal muscle appeared to be the most effective phosphatase in in vitro assays. Three phosphatase activity peaks--P1, P2, and P3--were partially purified from frozen rat spinal cord by ion exchange and size exclusion column chromatography and then characterized on the basis of biochemical, pharmacological, and immunochemical studies. One of the three peaks was identified as PP2A, whereas the others were mixtures of both PP2A and PP1. These three peaks could dephosphorylate cdk5-phosphorylated 32P-histone (H1), 32P-NF-H in the NF preparation, and 32P-NF-H tail fusion protein. These studies suggest the involvement of PP2A or a PP2A-like activity in the regulation of the phosphorylation state of KSPXKX motifs in NF-H.

Characterization of a protein which binds phosphatidylinositol 3,4,5-trisphosphate and 4,5-bisphosphate

Protein(s) which bind polyphosphatidylinositol phosphates ( RI 3,4,5-P 3 and PI 4,5-P 2 ) were identified in the wheat-germ agglutinin bound fraction of cells and tissues. The binding of this protein(s) to the phospholipid could be demonstrated in two ways, either by a shift in the migration of the lipid by size exclusion column chromatography or directly by binding to the protein after capture on wheat-germ agglutinin-coupled beads. Of the rat tissues tested (muscle, spleen, brain, heart, kidney and liver), the activity was highest in liver. The protein(s) was purified more than 500-fold by sequential chromatography on columns of wheat-germ agglutinin, phosphocellulose, Blue-Sepharose, Mono Q and Superose 6. The peak of activity appeared to have a molecular weight on this latter column of approx. 240 000. The protein(s) bound PI 3,4,5-P 3, PI 3,4-P 2, and PI 3-P in the ratio of 4:2:1. The binding of 3-phosphorylated PI phosphates to the protein(s) was not significantly inhibited by 36 μg/ml of either phosphatidylinositol or phosphatidylcholine, but was inhibited 10% and 65% by 36 μg/ml of PI 4-P and PI 4,5-P 2 , respectively. Since these results suggested that the binding protein(s) could also bind I4,5-P 2 , binding of this lipid was directly tested and found to be comparable to that of PI 3,4,5-P 3 . These results suggest that this protein(s) could be involved in the signaling mechanism elicited by these polyphosphoinositides.

Removal of endotoxin from antibody preparations for clinical use

Despite attempts to maintain asepsis, good manufacturing practices, and the use of terminal sterilization by millipore filtration, the nuclear practitioner is always worried about the possibility of endotoxin contamination. Methods, such as ion-exchange chromatography, have been tried for removing endotoxins during the preparation of radiolabeled antibodies, and so on. As suggested by Stevenson (1990), we evaluated the Issekutz technique (1) of endotoxin removal by affinity chromatography using a polymyxin cyanogen bromide (CNBr) Sepharose column. The endotoxin content of millipore filtrates of heat killed/sonicated suspensions of Pseudomonas pyocyaneus, E. coli were measured using a Sigma (St. Louis, MO) Endotoxin Assay Kit before and after filtration through such columns and compared with the results obtained using gel exclusion and ion-exchange columns of the same length and diameter. Reduction of endotoxin content to undetectable levels by the polymyxin column was observed. The use of such columns for terminal endotoxin removal analogous to terminal sterilization is advocated especially when developing a radiopharmaceutical such as radiolabeled antibodies for in house use.

Purification and Characterization of B2 Bradykinin Receptor from Rat Uterus

A B2 bradykinin (BK) receptor was solubilised and partially purified from rat uterine membranes by a combination of ammonium sulphate precipitation, desalting on Sephadex G-50, and hydroxyapatite and wheat germ agglutinin affinity chromatography. The partially purified BK receptor, enriched 1,500-fold, was then cross-linked to 125I-Tyr0-BK using disuccinimidyl suberate and purified to homogeneity as a single protein species on two-dimensional gel electrophoresis with a molecular mass of 81 kDa. This molecular size was in agreement with the value of 80-120 kDa estimated from Sephacryl 300 size exclusion column chromatography of the B2 receptor. The partially purified and the crude solubilised B2 BK receptor from rat uterus showed similar affinities for BK and the BK analogues iodo-Tyr0-Bk, D-Phe7-BK, and des-Arg9-BK, indicating that the ligand binding specificity of the receptor had been retained during the purification procedures. The biochemical properties of the solubilised B2 BK receptor correspond to those of a hydrophobic acidic glycoprotein (isoelectric focusing gave a value of 4.5-4.7) that binds specifically to wheat germ agglutinin but has no affinity for either concanavalin A or lentil lectin, suggesting the absence of terminal mannose or glucose residues.

High level production of hybrid potyvirus-like particles carrying repetitive copies of foreign antigens in Escherichia coli.

Synthesis in E. coli of native coat protein of Johnsongrass mosaic virus, and hybrid protein molecules containing foreign antigens, resulted in the intracellular formation of potyvirus-like particles (PVLPs). The foreign antigens used were an octapeptide epitope from Plasmodium falciparum and a decapeptide hormone (luteinizing hormone releasing hormone) at the N- or at both N- and C-terminal regions of the coat protein molecule, and a full length protein antigen (Sj26-glutathione S-transferase of 26 kD from Schistosoma japonicum) replacing the N-terminal 62 amino acids of the coat protein. Electron microscopy of ultrathin sections of E. coli revealed that PVLPs resulting from coat protein molecules containing peptide fusions appeared in vast arrays of parallel strands within the cytoplasm sometimes extending the length of the cell and at times the cells were strung together, with "threads" of PVLPs appearing to connect individual bacterial cells. PVLPs resulting from the fusion of the 26 kD antigen Sj26 to coat protein were shorter and wider. The physical form of the high molecular weight PVLPs enabled purification by simple size exclusion column chromatography. The Sj26-PVLPs administered to mice without adjuvant elicited antibody responses comparable to monomeric Sj26 administered with Freund's Complete Adjuvant.

A peptide from Schumanniophyton magnificum with anti-cobra venom activity.

Using the chick biventer cervicis preparation as a bioassay for antivenom activity, a peptide has been isolated from the aqueous extract of the bark of Schumanniophyton magnificum. The peptide was isolated and purified by a combination of reverse-phase and exclusion column chromatography, preparative TLC, and HPLC. The peptide has a relative molecular mass of about 6000 daltons and is similar in amino acid composition to the cardiotoxins present in snake venom. The isolated peptide showed dose-related inhibitions of the effects of cardiotoxin and total venom of cobra species using the chick biventer cervicis preparation.

Determination of Dioxins and Furans in Foods and Biological Tissues: Review and Update

Determination of trace residues of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDDs and PCDFs) in various matrixes is carried out by a limited number of laboratories in the United States, Canada, and other countries. Current methods for analysis of foods and biological tissues include a combination of preparation, extraction, cleanup, isolation, determination, and identity confirmation procedures. Soxhlet, liquid/liquid, solid-phase, and column extraction procedures are used as well as treatment with acid or base before solvent extraction. Cleanup and isolation steps include sulfuric acid partitioning; adsorption chromatography on Florisil, silica gel, or alumina; gel permeation chromatography; multi-stage column chromatography on sulfuric acid silica and alkali silica; carbon column chromatography; and liquid chromatography fractionation with size exclusion, normal-phase, and reverse-phase columns. Activated carbon and multistage chromatographic columns are widely used in cleanup schemes. Isomer-specific identification and quantitation of PCDD and PCDF congeners at parts-per-trillion levels or lower are carried out by high resolution (capillary) gas chromatography (HRGC) and multiple ion detection mass spectrometry. In addition to chemical methods, bioassay procedures have been recommended (e.g., use of monoclonal antibodies, for immunoassay determination of PCDDs and PCDFs).

Purification and characterization of cyclosporine and FK-506 binding proteins from a human T-helper cell line

Cytosolic proteins that specifically bind cyclosporine A and FK-506 were isolated and purified from the JURKAT human T-helper cell line. These binding proteins were purified by affinity, molecular weight exclusion and weak cation exchange column chromatography. Radiolabeled cyclosporine A specifically bound to a approximately 17 kDa molecule which is cyclophilin and also bound to a approximately 50 kDa protein(s). Radiolabeled FK-506 did not bind to the approximately 17 kDa molecular weight protein, but specifically bound to soluble approximately 10 kDa and approximately 50 kDa proteins.

酸性ａｃｔｉｎｏｍｙｃｉｎ類縁物質Ｂ‐１６２５ＦＡ３βのａｃｔｉｎｏｍｙｃｉｎ Ｘ２への変換

The active component of acidic actinomycin congener B-1625 FA was obtained by exclusion column chromatography with Diaion HP-20 SS and aluminium oxide TLC, and was considered to be the B-1625 FA3β already reported. After repeated fractionations by aluminium oxide TLC, its FAB-MS spectrum showed the ion peak at m/z M+1 1269, C62H85O17N12 except at m/z M+1, 1343, C64H87O20N12. During the purification procedure, FA3β was found to be converted to another active product in alkaline solution. The conversion product FA-n1 obtained as a bright orange rhombic crystal was identified with actinomycin X2 (m/z M+1, 1269, C62H85O17N12). Thus FA3β was presumed to be weakly acidic by the substitution of one proton of actinomycin X2 by the group of C2H2O3.

Immunoreactive Human Epidermal Growth Factor in Human Seminal Plasma

We measured immunoreactive epidermal growth factor (EGF) by a homologous RIA in seminal plasma (SP) from 31 fertile and 52 infertile men to determine the relationship between SP EGF levels and total sperm count in the ejaculates. The mean SP EGF levels in fertile and infertile men were 41.7 +/- 21.5 (+/- SD) and 53.1 +/- 30.8 micrograms/L, respectively. Infertile men with sperm-associated immunoglobulin G (n = 9), immunoglobulin A (n = 6), or both (n = 8) had mean SP EGF levels of 48.9 +/- 26.1, 47.9 +/- 17.5, and 56.5 +/- 32.1 micrograms/L, respectively. Seven men with severe oligospermia had a mean SP EGF level of 58.5 +/- 35.9 micrograms/L. There was no correlation (r = 0.14; P greater than 0.05) between SP EGF levels and total sperm counts in these men. Fractionation of SP by high performance liquid chromatography on a size exclusion (TSK G2000 SW) column revealed a single immunoreactive peak with an approximate mol wt of 8000, slightly higher than the mol wt of circulating human EGF (6000). We conclude that SP EGF may be distinct from peripheral plasma EGF.