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PubMed1958

Studies on the antigenicity of beta- and alpha-lipoproteins of human serum.

The techniques of agar immunoelectrophoresis and agar double diffusion were applied to the study of the antigenicity of beta- and alpha(1)-lipoproteins separated by ultracentrifugation from normal human sera. The effects of delipidation were also investigated. It was shown that beta- and alpha(1)-lipoproteins are antigenically distinct. For each class of lipoprotein studied, a single antigenic component was demonstrated. In some, but not all, preparations of alpha(1)-lipoprotein a second, small antigenic component was detected, and identified as albumin. Absorption with lipoprotein-free serum or albumin removed this component without changing the lipoprotein band. Delipidation did not affect the antigenicity of either beta- or alpha(1)-lipoproteins. Immunoelectrophoresis, because of its high sensitivity and specificity, provides an additional criterion of purity for antigenic proteins in addition to the data that can be obtained from ultracentrifugal and free electrophoretic analysis.

Antigens↗

PubMed1968

Chemotoxis of mononuclear cells.

Chemotaxis of rabbit mononuclear cells was studied by the micropore-filter technique. Mononuclear cells obtained from mineral oil-induced peritoneal exudates respond chemotactically to rabbit serum treated with immune complexes or with streptokinase and plasminogen, to soluble factors produced by bacteria, and to lysates obtained from rabbit neutrophils. The first chemotactic factor requires heat-labile factors in serum for its generation, but, once formed, the chemotactic factor is relatively heat stable. This factor has been compared with the complement-associated factor in serum, C'(5, 6, 7)a, that is chemotactic for neutrophils. The ability to generate the mononuclear cell chemotactic factor in serum that has been treated with potassium thiocyanate suggests that complement is not required. The position of the chemotactic factor in preparative electrophoresis and density-gradient ultracentrifugation indicates that on the basis of physical-chemical criteria, this factor is not C'(5, 6, 7)a. The mononuclear cell chemotactic factor present in lysates of neutrophils sediments slowly in the ultracentrifuge and may be related, at least in part, to cationic peptides of lysosomal granules. A study in the time course of the chemotactic response of mononuclear cells reveals that the response begins to level off after 4 or 5 hr. This is in sharp contrast to the time course for the chemotactic response of neutrophils, in which the reaction is complete within 1.5 hr. Rabbit mononuclear cells obtained from a starch-induced peritoneal exudate respond to serum treated with plasminogen and streptokinase and to the soluble factor produced by bacteria, but no chemotactic response is elicited to serum treated with immune complexes. This indicates a functional difference between two populations of mononuclear cells. Rabbit alveolar macrophages respond poorly to all agents tested, although a weak chemotactic response to bacterial factors was found. The requirement of a serine esterase in the mononuclear cell for the cell to respond chemotactically was defined by the use of organophosphorus inhibitors. Pretreatment of mononuclear cells with several series of phosphonates renders them unresponsive in the chemotactic system. The effect is similar for mononuclear cells responding chemotactically to activated serum and to the bacterial chemotactic factor. Several points of contrast with inhibition profiles obtained in the chemotactic system of the neutrophil suggest that, while the mononuclear cell requires a serine esterase for chemotactic responsiveness, this enzyme is different from the one previously defined in the neutrophil.

Animals↗

PubMed1969

Relative inability to induce tolerance in adult NZB and NZB-NZW F1 mice.

Immunologic tolerance to ultracentrifuged bovine gamma globulin could not be induced in 6-wk old NZB or B/W mice, but developed readily in C3H, NZW, and C57Bl mice. NZB and B/W mice, as well as Balb/c mice, failed to become tolerant to ultracentrifuged human gamma globulin. The NZB and B/W mice also showed higher antibody titers to a standard antigenic challenge. This immunologic hyperreactivity and lack of experimental tolerance may be related to the lack of self-tolerance, autoimmunity, and lymphomas that develop in these mice at a later age.

Animals↗

PubMed1971

Characterization of immunoglobulin structures from the surface of chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia cells with relatively large amounts of mu and kappa immunoglobulin structures on the surface, and apparently very small amounts intracellularly, were subjected to homogenization or washing after freezing and thawing. After a light centrifugation, which sedimented the nuclei and unbroken cells, most of the immunoglobulin structures were found in the supernatant. Ultracentrifugation, which was performed to remove the membranes from the supernatant, sedimented only half the amount of the immunoglobulin structures. By sucrose density gradient ultracentrifugation and Sephadex G-200 filtration, the unsedimented immunoglobulin structures were shown to consist of 7S IgM and free kappa chains. About 80,000 7S IgM molecules were calculated to be present on each cell. The amount of kappa chains not associated with IgM was estimated to be equal to the amount of kappa chains in IgM. Inhibition of passive hemagglutination was used to detect and quantitate the immunoglobulin structures.

Aged↗

PubMed1961

Chromatographic separation, and characteristics of nucleic acids from HeLa cells.

The application of the phenol-duponol method to extraction of nucleic acids from HeLa cells is described. Chromatography of the phenol extract on an esterified bovine serum albumin column with a salt gradient of sodium chloride gives separation of soluble RNA, DNA, and two different high molecular RNA fractions. Ultracentrifugation of the DNA eluted from the column gives a sedimentation coefficient (s(20) (o),w) of 38, which agrees with ultracentrifugation data on the phenol extract. The eluted RNA appears polydisperse at low ionic strength, but at high ionic strength and after alcohol precipitation two fractions with the sedimentation coefficients of 16 and 25 to 29, respectively, were obtained.

Chromatography↗

PubMed2003

Size-exclusion chromatography purification of high-titer vesicular stomatitis virus G glycoprotein-pseudotyped retrovectors for cell and gene therapy applications.

Vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped replication-defective retroviral particles are pantropic and amenable to concentration to high titer by ultracentrifugation. These features have allowed development of effective retroviral transduction protocols for stem cells in vitro as well as for tissue engineering in vivo. However, retroparticle ultracentrifugation protocols will also copellet cellular and subcellular debris released from retroviral producer cell lines during vector manufacture. We have analyzed concentrated vector preparations by chromatography and have found that a significant amount of genomic DNA released from producer cells coconcentrates with retroviral particles. In an effort to generate high-purity retroparticle preparations, devoid of subcellular contaminants and contaminating genomic DNA, we have developed a process using size-exclusion chromatography combined with host cell nucleic acid digestion and concentration by ultrafiltration. The procedure allowed for a final recovery of 19 +/- 0.4% infectious viral particles from unfractionated starting material, with an average retroparticle concentration of 7.7 x 10(7) +/- 1.5 x 10(6)/ml. The intact virus is of high purity, >90% as determined by anion-exchange high-performance liquid chromatography. Retroparticle structure appeared intact as determined by negative stain electron microscopy and purified virus was functional and allowed for efficient transduction of primary human bone marrow stromal cells in vitro. In conclusion, we have developed a VSV-G retrovector purification process that can be applied to large-scale retroviral production ideal for cell and gene therapy applications.

Cells, Cultured↗

PubMed1985

Casein content of human milk.

Three methods for estimating the casein content of human milk were tested; isoelectric precipitation with washing and correction for co-precipitating proteins, sedimentation by ultracentrifugation, and indirect analysis (ie analyzing for the content of the major whey proteins and subtracting these from the total protein content). Gel electrophoresis and amino acid analysis were used to confirm some of the results. The casein content (mg/ml) of mature human milk (n = 9) was 2.33 +/- 1.69 by isoelectric precipitation, 1.80 +/- 0.48 by sedimentation and 2.96 +/- 1.08 by the indirect approach. A probable partition of nitrogen in breast milk would be casein N: whey protein N: non-protein N of 20:50:30; ie the correct ratio of casein nitrogen: whey nitrogen is approximately 20:80. Analysis of trace elements and minerals demonstrates that of total Ca 10%, Mg 5%, Zn 28%, Cu 17%, and Fe 27% is bound to casein when prepared by ultracentrifugation while isoelectric precipitation causes a redistribution of some of these elements. Since the protein ratio of human milk is considered a guideline when manufacturing infant formulas, these findings should be considered with regard to infant nutrition.

Amino Acids↗

PubMed1996

Sequence analysis of hypervariable region of hepatitis C virus (HCV) associated with immune complex in patients with chronic HCV infection.

Immune complex (IC) frequently exists in sera of patients with hepatitis C virus (HCV) infection. When circulating HCV particles are fractionated by differential flotation ultracentrifugation, HCV in the bottom fraction appears to be in the form of IC. Flotation ultracentrifugation of serum was done in 7 patients positive for serum anti-C3d-binding circulating IC and in 7 negative patients. In all IC-positive patients, HCV RNA was detected in the bottom fraction but not in the top fraction of 3 patients. Amino acid sequences of hypervariable region (HVR) of HCV in the bottom fraction of IC-positive patients were almost the same as those of whole serum, while those of the top fraction considerably differed from those of whole serum except for 1 case. These data suggest that the major population of HCV forms IC in IC-positive patients, and antibody response to the HVR of HCV is responsible for IC formation.

Aged↗

PubMed1986

The stiffness of dsRNA: hydrodynamic studies on fluorescence-labelled RNA segments of bovine rotavirus.

The sedimentation coefficients of dsRNA segments of bovine rotavirus were determined in the analytical ultracentrifuge. The eleven segments were separated by preparative gel electrophoresis, and isolated by elution from gel pieces. The RNA was labelled by the intercalating fluorescent dye ethidium bromide at a ratio bound dye per base pair between 0.003 to 0.018. The analytical ultracentrifuge was equipped with a fluorescence recording optics. Sedimentation coefficients could be determined with amounts of RNA as little as 8 ng. All sedimentation coefficients were extrapolated to zero-concentration, zero-dye binding, and zero-impurities from the preparative gel electrophoresis. The hydrodynamic model of flexible cylinders was applied for the interpretation of the sedimentation coefficients. All dsRNA segments of rotavirus (663-3409 base pairs) and the dsRNA5 of cucumber mosaic virus (335 base pairs) fit the model of a "worm-like" or flexible cylinder with a persistence length of 1125 A and a hydrated diameter of 30 A. The results are compared with data from the literature on the persistence lengths of the B- and Z-forms of dsDNA and of viroids.

Animals↗

PubMed1977

Physical properties of nucleoprotein cores from adenovirus type 5.

Analytical ultracentrifugation, thermal denaturation, and electron microscopy have been used to study nucleoprotein core particles, obtained from disrupted type 5 adenovirus and partially purified on glycerol density gradients. Electron microscopy at low salt concentrations has shown that the cores are homogeneous particles with characteristic structures, which vary with conditions of observation from a fairly loose network of fibers to a highly condensed, compact particle. Sedimentation measurements in the analytical ultracentrifuge, both by boundary and by band techniques, show that the cores are relatively homogeneous in solution and have sedimentation coefficients near 185 S at low salt concentrations, about 243 S in 1 or 2 M NaCl, and 376 S in 1 mM MgCl2. Correlation of sedimentation data with electron microscopic observations suggests that the 185 S particle has a loose, fibrous structure, while the faster species are more highly condensed particles. The melting temperature of the cores in 5 mM Tris/HCl is 79 degrees C, which is 10 degrees C higher than the Tm for purified, viral DNA. This indicates that the protein enhances the stability of DNA in the nucleoprotein complex.

Adenoviruses, Human↗

PubMed1982

Behavior of apolipoprotein C-II in an aqueous solution.

The behavior of human apolipoprotein C-II (apoC-II; molecular weight, 8,837) has been studied in an aqueous solution by using gel permeation chromatography, sedimentation equilibrium ultracentrifugation, and circular dichroic (CD) spectroscopy. The elution volume of apoC-II was equivalent to that of standard proteins with molecular weights of 30,000 and 17,800 by high performance liquid chromatography (HPLC) on TSK-Gel G3000SW and by gel permeation chromatography on Sephadex G-75, respectively. Sedimentation equilibrium experiments in a table-top high-speed air turbine centrifuge and in an analytical ultracentrifuge showed values of 9,400 and 7,900, respectively, for the molecular weight of apoC-II in the absence of any denaturants or surfactants. The CD spectrum of apoC-II in the far-ultraviolet region indicated that it had a highly disordered structure. These results showed that the apoC-II molecule, when in dilute solution, is dominantly monomeric with an extended and loosely folded structure. The concentration dependence of the ellipticities at 220 nm and of the molecular weight in the sedimentation equilibrium experiment suggests that apoC-II self-associates weakly in an aqueous solution. The disordered state of apoC-II was highly stable. However, helical conformation was induced by sodium dodecylsulfate, trifluoroethanol, and phosphatidylcholine vesicles.

Apolipoprotein C-II↗

PubMed1998

Development of a method to separate lipoprotein-bound and lipoprotein-depleted tissue factor pathway inhibitor. Measurement of free tissue factor pathway inhibitor activity.

The aim of this study was to develop a method to separate lipoprotein-bound from lipoprotein-free tissue factor pathway inhibitor (TFPI) and to measure the TFPI chromogenic activity and antigen in both fractions. This was performed by ultracentrifugation of plasma, after increasing its density to 1.21 g/ml with potassium bromide. Blood was taken from nine volunteers before and after an injection of low-molecular-weight heparin. The ultracentrifugation procedure was adequate, since the mean cholesterol recovery was 91% and only 2% of the cholesterol remained in the lipoprotein-depleted fraction. No free TFPI protein was found in the lipoprotein-rich fraction. Moreover, the amount of free TFPI in the lipoprotein-depleted fraction was close to that found in plasma. Using this method, we confirmed that heparin does not induce an increase in bound TFPI and that the moderate increase in total TFPI antigen in plasma is entirely caused by the enhancement of free TFPI. We then applied the TFPI chromogenic assay to the lipoprotein-depleted fraction to assess the activity of free TFPI. The activity was 0.11+/-0.03 and 0.36+/-0.08 U/ml before and after heparin, respectively (a 3.6-fold increase) while the activity of bound TFPI did not increase at all. We suggest that this method may be an alternative to gel filtration for measuring free TFPI activity, and might be of value in the search for TFPI abnormalities in patients with thrombosis.

Anticoagulants↗

PubMed1999

Monomer-trimer equilibrium of the ectodomain of SIV gp41: insight into the mechanism of peptide inhibition of HIV infection.

The monomer-trimer equilibrium of the ectodomain of SIV gp41 (residues 27-149, e-gp41) has been characterized by analytical ultracentrifugation, circular dichroism (CD), and NMR spectroscopy. Based on analytical ultracentrifugation experiments performed at different rotor speeds and protein concentrations, the equilibrium association constant for the SIV e-gp41 trimer is 3.1 x 10(11) M(-2). The presence of intermolecular nuclear Overhauser effects in a mixture of 12C and 13C-labeled e-gp41 prepared under nondenaturing conditions unambiguously demonstrates that there is a dynamic equilibrium between the monomer and trimer. The CD spectra taken as a function of SIV e-gp41 concentration suggest that the helical content of the monomeric state does not change significantly relative to that of the trimeric state. The relevance of the monomer-trimer equilibrium is discussed with respect to gp41 function and the inhibitory properties of gp41 peptides.

Anti-HIV Agents↗

PubMed1984

Rapid purification of extracted bacterial lipopolysaccharides by continuous free-flow electrophoresis.

The use of continuous free-flow electrophoresis for the purification of extracted lipopolysaccharides ( LPSs ) was investigated. Commercial (nucleic acid contaminated) LPS preparations, isolated by the hot phenol-water method of Westphal from Salmonella typhimurium and Escherichia coli 0111: B4, were analyzed. Continuous free-flow electrophoresis for purification of crude LPSs proved to be a rapid and useful means for the continuous purification of large amounts of LPS (more than 45 mg crude LPS per hr) and it showed good reproducibility and pure LPS. The electrophoretic profile of both crude LPSs obtained by continuous free-flow electrophoresis showed two distinct, sharp peaks; one representing the nucleic acid fraction and the other the LPS fraction. Under the continuous free-flow electrophoresis conditions employed, nucleic acid in the crude LPSs possessed low electrophoretic mobility, whereas LPS migration was negligible. Thus for both preparations pure LPS (no detectable nucleic acid) was obtained. Electrophoretic profiles of these purified LPSs on sodium dodecylsulfate-polyacrylamide gel electrophoresis were similar in both cases to those of crude LPS and of LPS purified by repeated ultracentrifugation. By immunological analysis using double immunodiffusion and immunoelectrophoresis, it was found that two components of crude E. coli 0111: B4 LPS were eliminated by continuous free-flow electrophoresis, but each component of purified E. coli 0111: B4 LPS was immunologically identical to the corresponding component in its crude LPS. In S. typhimurium LPS, none of its components were influenced by continuous free-flow electrophoresis but not by ultracentrifugation. In spite of these results, both purified LPSs possessed stronger mitogenic activity than each crude LPS. These results indicated that continuous free-flow electrophoresis is a useful means of purifying extracted crude LPS.

Animals↗

PubMed1976

Determination of molecular weight and molecular structure of rat-liver pyruvate carboxylase.

The molecular weight of pyruvate carboxylase isolated from pigeon and rat liver mitochondria was examined using analytical ultracentrifugation and electron microscopy. The enzyme molecule appeared as a tetramer with the four subunits arranged at the corners of a square. Sedimentation studies in the analytical ultracentrifuge, extrapolated to infinite dilution, showed the tetramer to have a molecular weight Mc=0r of 280 000 and an So20,w of 12.7 S. The tetramer could be dissociated into trimers and dimers of lower specific enzymic activity by storage at 4 degrees C or incubation at -- 20 degrees C at low protein concentrations. The isolated trimers and dimers had a molecular weight Mc=0r of 210 000 and 140 000, respectively, and an So20,w of 10.85 S and 7.55 S, respectively. Incubation with 2 M urea at 20 degrees C yielded enzymically inactive subunits (Mc=0r = 70 000; So20,w = 4.95 S). The molecular weights (for pyruvate carboxylase and its subunits), as calculated from the subunit diameter observed in the electron microscope, were consistent with the values obtained from sedimentation studies.

Animals↗

PubMed1982

Isolation, purification, and chemical analysis of the lipopolysaccharide and lipid A of Acinetobacter calcoaceticus NCTC 10305.

The lipopolysaccharide of Acinetobacter calcoaceticus NCTC 10305 (London) was obtained by a modified phenol/chloroform/light petroleum method from the bacterial cells and from the culture medium in yields of 1.6% and 2.2% respectively (based on the bacterial dry weight). On chemical analysis, both preparations proved to be identical. The lipopolysaccharide obtained from the cells was purified by repeated ultracentrifugation, electrodialysis, and precipitation with sodium chloride. It was free of nucleic acids, proteins, and glycans. In the analytical ultracentrifuge, the triethylamine and sodium salt forms of the lipopolysaccharide showed a s20 value of 8.9 S and 51 S, respectively. The lipopolysaccharide consisted of glucosamine, 3-deoxy-D-manno-octulosonic acid, D-glucose, fatty acids, and phosphate in a molar ratio of 2:1:7:6:4. The fatty acids were predominantly lauric acid, 2-hydroxy, and 3-hydroxylauric acid in a molar ratio of 1:1:2. Only 3-hydroxylauric acid was found in amide linkage. On mild acid hydrolysis of the lipopolysaccharide, 65% lipid A were obtained, to which glucosamine was retained quantitatively. It still contained 50% of the original glucose, while one third (15%) of the liberated glucose was in monomeric form.

Acinetobacter↗

PubMed1986

Purification and characterization of propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium.

The enzyme propylamine transferase, catalyzing the transfer of the propylamine moiety from S-adenosyl(5')-3-methylthiopropylamine to several amine acceptors, has been purified 643-fold in 20% yield from Sulfolobus solfataricus, an extreme thermophilic archaebacterium optimally growing at 87 degrees C. The purified enzyme (specific activity 2.05 units/mg protein), is homogeneous by criteria of gel electrophoresis, gel filtration, isoelectric focusing and ultracentrifugation analysis. The molecular mass of the native enzyme was estimated to be about 110 kDa by gel permeation and ultracentrifugation analysis. The protein migrates on SDS/polyacrylamide gel electrophoresis as a single band of 35 kDa, suggesting that the enzyme is a trimer composed by identical subunits. An optimum pH of 7.5 and an acidic isoelectric point of 5.3 have been calculated. The optimum temperature was 90 degrees C and no loss of activity is observable even after exposure of the purified enzyme to 100 degrees C for 1 h. No reducing agents are required for enzymatic activity. Substrate specificity towards the amine acceptors is rather broad in that 1,3-diaminopropane (Km = 1675 microM), putrescine (Km = 3850 microM), sym-norspermidine (Km = 954 microM) and spermidine (Km = 1539 microM) are recognized as substrates. Conversely S-adenosyl(5')-3-methylthiopropylamine is the only propylamine donor (Km = 7.9 microM) and the deamination of the sulfonium compound prevents the recognition by the enzyme. The reaction is irreversible and initial-rate kinetic studies indicate that the propylamine transfer is operated through a sequential mechanism. 5'-Methylthioadenosine, a product of the reaction, acts as a powerful competitive inhibitor with a Ki of 3.7 microM. Enzyme-substrate binding sites have been investigated with the aid of several substrate analogs and products. Among the compounds assayed, 5'-methylthiotubercidin, S-adenosyl(5')-3-thiopropylamine and S-adenosyl-3-thio-1,8-diaminooctane are the most active inhibitors.

Archaea↗

PubMed1991

The promotion of self-association of horse-heart cytochrome c by hexametaphosphate anions.

In the presence of the highly charged hexametaphosphate anion, horse heart cytochrome c aggregates to form stable protein complexes. The formation of protein aggregates has been detected by high-resolution 1H-NMR spectroscopy from an increase in the linewidth of resolved ferricytochrome c resonances with hexametaphosphate concentration. Alternatively, analytical ultracentrifugation reveals protein association from the increase in apparent sedimentation coefficients of cytochrome c in the presence of equimolar hexametaphosphate. Protein aggregation is dependent on the concentration of background electrolyte since in the range 10-150 mM sodium cacodylate alternative stabilisation of dimeric and trimeric complexes was observed by both NMR and analytical ultracentrifugation. A model is proposed for the mechanism of protein aggregation caused by polyphosphate binding to the surface of cytochrome c.

Animals↗