RS virus components. II. Antigenicity.

Rabbits were immunized with RS virus components, isolated as described previously. The sera were tested by neutralization, double diffusion and complement-fixation. RS virus components induced low titers of precipitating and complement-fixing antibodies, and failed to stimulate the neutralizing antibodies.     

Structural components of mouse mammary tumor virus. II. Isolation and purification of virion polypeptides.

Mouse mammary tumor virus (MMTV) glycoproteins and nonglycosylated polypeptides were purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Primary amino groups were labeled with fluorescamine to enable visualization of MMTV polypeptides in the gels. Protein bands were sliced from the gels and eluted with 90 to 95% recovery. Eight MMTV polypeptides, including three of the major viral components as well as five minor proteins, were routinely obtained. Double diffusion assays and immunoelectrophoresis confirmed the retention of antigenicity identical to that of untreated MMTV virions. Antisera obtained from MMTV-free BALB/c mice immunized with these purified proteins reacted with the polypeptide immunogen as well as with detergent-disrupted MMTV virions from mouse milk or cell culture. Double diffusion assays using the specific mouse antisera failed to detect any cross-reactivity among the isolated polypeptides. A hemagglutination-inhibition assay demonstrated that the ability of MMTV virions to inhibit the hemagglutinating properties of influenza virus resides in the glycosylated polypeptides gp52, gp37.7, and gp33.     

Lymphocytic Choriomeningitis virus. I. Concentration and purification of the infectious virus.

Two procedures for the purification of infectious lymphocytic choriomeningitis virus from cell culture fluid have been developed. If large quantities of very pure virus are to be prepared, infected L cells are maintained with a medium supplemented with calf serum, the proteins of which have been largely removed by pretreatment with polyethylene glycol. Two days after infection of the cultures, the media are collected and the virus is concentrated by treatment with polyethylene glycol 40,000. Purification with a 10,000-fold increase of specific infectivity is achieved with steric chromatography on controlled-pore glass beads with pore sizes of 42 to 44 nm and centrifugation in density gradients prepared with amido trizoate. An alternative method begins with precipitation of the virus from infected cell cuture medium with zinc acetate, followed by controlled-pore glass chromatography and density centrifugation in a discontinuous sucrose gradient. Purification thus obtained is 200-fold in terms of specific infectivity.     

Turkey acrosin. I. Isolation, purification, and partial characterization

Acrosin was extracted from turkey spermatozoa by use of urea together with sonication and freezing, and purified approximately 18-fold by sequential use of chromatofocusing and affinity chromatography. The use of chromatofocusing for the initial purification step proved to be superior to preparative isoelectric focusing. Similar to acrosin from many mammalian species, turkey acrosin was found to be a glycoprotein possessing characteristics of serine proteases. Polyacrylamide gel electrophoresis (PAGE) of the enzyme indicated the presence of two isozymes. Sodium-dodecyl sulfate PAGE under reducing conditions revealed three subunits with approximate molecular weights of 11,700, 13,900, and 15,900.     

An actin-modulating protein from Physarum polycephalum. I. Isolation and purification

High-speed centrifugation supernatants from slime mould plasmodia show considerable activities to inhibit the polymerization of actin as revealed by viscosity measurements. By following increasing inhibitory activities an actin modulating protein (AM-protein) has been isolated and purified which affects the polymer state of actin. AM-protein has a peptide chain weight of 42 000 and is thus indistinguishable from actin by SDS-electrophoresis, but can be clearly distinguished by isoelectric focussing. Peptide maps from partial proteolytic digests of AM-protein and Physarum actin reveal no similarities thereby excluding that AM-protein is a denatured or modified form of actin. The protein is isolated from crude extracts as a heterodimer with actin to which it strongly binds. This heterodimer affects the polymerization of large amounts of actin by inducing oligomeric or low-polymer actin complexes and thus inhibiting the formation of long actin filaments. The AM-protein/actin heterodimer has only a slight effect of F-actin. It partially depolymerized F-actin within several hours. By ion exchange chromatography in 8 M urea the AM-protein is separated from the actin. The purified AM-protein monomer is renatured and inhibits the polymerization of actin like the heterodimer but additionally, depolymerizes actin filaments very rapidly and effectively by breaking them into oligomer or low-polymer complexes. The addition of less than 1% AM-protein causes a decrease of the specific viscosity of an F-actin solution by 50%. The degree of polymerization inhibition and depolymerization of actin is strictly dependent on the amount of AM-protein added; therefore a catalytic type of reaction between both proteins can be excluded.     

1,3-beta-D-glucanases from Pisum sativum seedlings. I. Isolation and purification.

Two buffer-soluble endo-1,3-beta-D-glucanases (EC 3.2.1.6) have been purified to within 1% of electrophoretic homogeneity from etiolated Pisum sativum stem tissues. Purified glucanase I and II differ in physical properties, such as electrophoretic mobility in sodium dodecyl sulfate polyacrylamide gels (Mr values were 22 000 and 37 000, respectively) and isoelectric focusing, (pI values were 5.4 and 6.8, respectively). Although the enzymes have similar pH optima (5.5--6.0), Km values for various substrates (0.6--7.4 mg/ml) and thermal inactivation profiles, they are localized in different tissues and they differ markedly in the rates with which they attack the internal linkages of long- vs. short-chain substrates. Glucanase I is concentrated in apical regions of the stem and is most effectively assayed reductometrically (as laminarinase), while glucanase II is localized in mature regions and is relatively more active in viscometric assays (as carboxymethyl-pachymanase).     

Isolation and purification of series bioactive components from Hypericum perforatum L. by counter-current chromatography

Counter-current chromatography (CCC) combined with pre-separation by ultrasonic solvent extraction was successively used for the separation of series bioactive compounds from the crude extract of Hypericum perforatum L. The petroleum ether extract was separated by the solvent system of n-heptane-methanol-acetonitrile (1.5:0.5:0.5, v/v) and n-heptane-methanol (1.5:1, v/v) in gradient elution, yielding a phloroglucinol compound, hyperforin with HPLC purity over 98%. The ethyl acetate extract was separated by using the solvent system composed of hexane-ethyl acetate-methanol-water (1:1:1:1 and 1:3:1:3, v/v) in gradient through both reverse phase and normal phase elution mode, yielding a naphthodianthrone compound, hypericin with HPLC purity about 95%. The n-butanol extract was separated with the solvent system composed of n-butanol-ethyl acetate-water (1:4:5 and 1.5:3.5:5, v/v) in elution and back-extrusion mode, yielding two of flavones, rutin and hyperoside, with HPLC purity over 95%. HPLC-MS, reference sample and UV spectrum were selectively used in separation to search for target compounds from HPLC-DAD profiles of different sub-extracts. The structures of isolated compounds were further identified by ESI-MS, 1HNMR and 13CNMR.     

Isolation and purification of viable Ureaplasma urealyticum cells free from medium components

A procedure was devised to produce Ureaplasma urealyticum preparations free of adsorbed components of the growth medium, which contains high concentrations of serum. The ureaplasmas were cultivated in a medium containing PPLO-serum fraction as a replacement for horse serum. High titres of ureaplasmas (greater than 10(7) c.f.u. ml-1) were obtained. Harvested cells were then purified by Urografin density gradient centrifugation. By use of 3H-labelled ureaplasma cells and 125I-labelled medium components, a distinct band of viable cells devoid of serum constituents was demonstrated. The absence of medium components was verified by immunoblotting cells from this band with antiserum to medium components. Medium components that had been present before the purification procedure were undetectable in the purified cell fraction obtained. The viability of the purified ureaplasma cells represented an 85% recovery rate and their antigenicity, examined with anti-serotype specific antiserum, remained intact. This easy and reproducible procedure can be used to prepare purified ureaplasmas for investigation of ureaplasmal antigens and their expression and/or role in disease.