Separation and characterization of the troponin components from bovine cardiac muscle.

The three major components of bovine cardiac troponin were separated by successive chromatography on sulfopropyl-Sephadex and DEAE-Sephadex columns in the presence of 6 M urea. All three of the bovine cardiac troponin subunits were necessary to restore full troponin activity in both skeletal and cardiac actomyosin ATPase assay systems. The 38,000-dalton subunit bound tropomyosin, and the 20,000-dalton subunit bound calcium, like skeletal TN-T and TN-C, respectively. The 28,000 component, although presumably analogous to skeletal TN-I, gave very little inhibition of actomyosin ATPase activity. Differences between cardiac and skeletal troponin subunits were also found in the elution patterns from ion exchange columns and in amino acid composition, thus demonstrating a significant muscle-type specificity.     

Separation and characteristics of the components of the antibiotic virenomycin

The composition of virenomycin, a new antitumor antibiotic was studied. Two components V and M were detected with high resolution liquid chromatography and thin layer chromatography on siluphol (Czechoslovakia) and silica gel (Merk, BRD). A preparative method for separation of the antibiotic components with the use of chromatography on columns with silica gel was developed. Biological and physicochemical properties of separate components were studied to show that they significantly differed by their antibacterial action in vitro: virenomycin V was 2 to 4 times more active than virenomycin M against a number of microbes. The physicochemical properties of the components are similar. It was shown with mass spectrometry that the molecular weight of virenomycin is 12 units higher than that of virenomycin M. The PMR spectra showed that this difference is due to the presence of a vinyl group in the chromophore moiety of the virenomycin V molecule and a methyl group at the similar site of the virenomycin M molecule.     

Separation and identification of different refolding components

A dual-gradient ion-exchange chromatography (IEC) refolding process has been successfully applied to renature lysozyme at high concentration. The different refolding components were separated by size-exclusion chromatography with low concentrations of urea and NaCl added into elution buffer. The hydrodynamic characteristics during separation and the hydrophobic properties of these components were also tested.     

Separation of the epithelial and mesenchymal components of the newt limb regenerate with salt.

After incubation of isolated forelimb regenerates of Notophthalmus (Triturus) viridescens at all developmental stages for 60 minutes at 37 degrees C in a salt medium containing 111 mM sodium chloride, 5.6 mM potassium chloride and 100 mM sodium phosphate buffer at pH 7.5, the wound epithelium of each regenerate was removed intact from its underlying mesenchymal component. The suggestion is made that the salt medium is an effective epithelial-mesenchymal separating agent due to a combination of its hypertonicity, high ionic strength and the fact that the medium precipitates calcium as calcium phosphate. Attempts to dissect away the epithelium from the mesenchyme after incubation of isolated regenerates in sodium phosphate containing 1% or 3% Difco 1:250 trypsin, 10 mM EDTA or 150 units collagenase/ml medium were unsuccessful. Epidermis of adult newt forelimb skin was removed only after extended incubation of the forelimbs in the salt medium for three hours at 37 degrees C or after freezing isolated forelimbs in buffer and subsequent thawing.     

Separation of nucleic acid components on polyacrylamide gel columns.

The exclusion and sorptive properties of the polyacrylamide gel Bio-Gel P-2 for low-molecular-weight anionic species allows separations of nucleic acid components to be effected in dilute alkaline borate buffer at pH 8.8. The degree of ionization, determined by pH, fixes the elution position of the compounds chromatographed in this system. Optimum conditions are given for making group separations as well as many other useful separations of nucleic acid components and related compounds.     

Separation of the glycoprotein and ganglioside components of thyrotropin receptor activity in plasma membranes.

The two components of thyroid plasma membranes known to interact with thyrotropin, i.e., a glycoprotein with specific thyrotropin binding activity and the gangliosides of the thyroid membranes, are shown to segregate differently when membranes are solubilized with lithium diiodosalicylate. Individually examined, the interaction of each component with thyrotropin exhibits a different sensitivity to salts. The data suggest that the thyrotropin receptor on the thyroid membrane is a complex which is composed of both glycoprotein and ganglioside components and that its properties are derived from each component.     

Separation procedures for the pharmacologically active components of rhubarb

Rhubarb, as an important Chinese medicine, has many functions owing to containing anthraquinone derivatives. The analysis of anthraquinone derivatives in Chinese rhubarb is reviewed. The analytical techniques include high performance liquid chromatography, capillary electrophoresis, thin-layer chromatography and so on. The main operation parameters in every technique were given. The structures of anthraquinone derivatives and the classification of Chinese rhubarb were summarized too.     

The regulatory proteins of the myofibril. Separation and biological activity of the components of inhibitory-factor preparations

1. Inhibitory-factor preparations isolated from myofibrils were shown to consist principally of proteins with molecular weights of 37000 and 23000. Under certain preparative procedures an additional component of molecular weight 14000 was present. 2. The 23000-dalton protein, the inhibitory factor, was the major active component. Its activity was enhanced by tropomyosin. 3. The 14000-dalton component also possessed inhibitory activity, although less than that of the 23000-dalton component when compared on a molar basis. Its activity was not always enhanced by tropomyosin. The 14000-dalton component could not be detected in whole fresh myofibrils and the limited evidence available is compatible with its formation during the preparation of the troponin complex. 4. The 37000-dalton component could not replace the inhibitory factor, calcium-sensitizing factor or tropomyosin as components of the relaxing-protein system. 5. All three components had distinctive amino acid compositions, particularly in their cysteine content.     

Separation of active enzyme components from the fatty acid synthetase of chicken liver.

Fatty acid synthetase (EC 6.2.1.-) of chicken liver was dissociated into half-size subcomplexes and then separated into three protein fractions by the preparative disc-gel electrophoresis technique. The anodal protein (Fa) of a molecular weight of approx. 6000 contains the prosthetic group, 4'-phosphopantetheine. It binds acetyl group from acetyl-CoA and is identified as the acyl carrier protein component. The slower moving proteins (FI and FII) correspond to the subcomplexes resolved by the analytical method (Y.n, S.L. and Hsu, R.Y. (1972) J. Biol. Chem 247, 2689--2698). Both contain acetyl transacylase and palmityl-CoA deacylase activities, but only FI contains beta-ketoacyl reductase activity. Active acetyl transacylase and palmityl-CoA deacylase components were obrained by the sucrose density centrifugation technique in a broad 3 S protein band from the FI fraction, following dissociation at 4 degrees C for 12 days. Slight modification of the electrophoresis conditions yields a homogeneous 1.55 S beta-ketoacyl reductase component.     

Separation and characterization of the soluble and insoluble components of insoluble laminaran

Insoluble laminaran, a (1→3)-β-D-glucan from Laminaria hyperborea (L. cloustoni), has been fractionated by differential solubility into soluble and insoluble fractions. These fractions were degraded with a purified exo-(1→3)-β-D-glucanase from Basidiomycete sp. QM806 giving, as primary hydrolysis products, D-glucose, gentiobiose, laminarabiose, and 1-O-β-laminarabiosylmannitol. Gentiobiose was obtained in only trace amounts from the insoluble fraction of laminaran, suggesting the absence of branching. Successive application of periodate oxidation, reduction, mild acid hydrolysis, and enzymic degradation indicated that the branch in the soluble fraction consists of a single β-(1→6)-linked D-glucosyl residue. The results indicate that “insoluble” laminaran is apparently an aggregate of three closely related polysaccharide species: a soluble, branched, reducing component (soluble laminarose); an insoluble, unbranched, reducing component (insoluble laminarose); and an unbranched, nonreducing component (laminaritol) that has a monosubstituted mannitol residue at the reducing terminal. Laminaritol was found to be about equally distributed between the soluble and insoluble fractions. The average d.p. of the laminaran components is 20–25 residues, as determined from the relative amounts of enzymic hydrolysis products and from periodate-oxidation data.     

Separation and characterization of the hemoglobin components ofPterygoplichthys pardalis,the acaribodo

• 1. The four main hemoglobin components of the hemolysate ofPterygoplichthys pardalis have been isolated and characterized.
• 2. The functional properties investigated for the isolated components comprise the effect of pH and ATP on (i) the O₂ equilibrium, (ii) the O₂ dissociation kinetics, (iii) the CO combination kinetics.
• 3. Component I, corresponding to approx 50% of the total hemoglobin, is characterized by functional properties which are distinctly different from those of Components II, III and IV, which are alike
• 4. Thus it is shown, once more, that multiple components in an hemolysate fall into categories of hemoglobins characterized by distinct and complementary functional properties

     

Crystal structure and site-directed mutagenesis of enzymatic components from Clostridium perfringens iota-toxin

Iota-toxin from Clostridium perfringens type E is an ADP-ribosylating toxin (ADPRT) that ADP-ribosylates actin, which is lethal and dermonecrotic in mammals. It is a binary toxin composed of an enzymatic component (Ia) and a binding component (Ib). Ia ADP-ribosylates G-actin at arginine 177, resulting in the depolymerization of the actin cytoskeleton. Here, we report on studies of the structure-function relationship by the crystal structures of Ia complexed with NADH and NADPH (at 1.8 A and 2.1 A resolution, respectively) and mutagenesis that map the active residues. The catalytic C-domain structure was similar to that of Bacillus cereus vegetative insecticidal protein (VIP2), which is an insect-targeted toxin, except for the EXE loop region. However, a significant structural difference could be seen in the N-domain, which interacts with Ib, suggesting an evolutionary difference between mammalian-targeted and insect-targeted ADPRT. The high resolution structure analysis revealed specific NAD conformation (a ring-like conformation of nicotinamide mononucleotide (NMN)) supported by Arg295, Arg296, Asn335, Arg352 and Glu380. Additionally, the mutagenesis study showed that the residues Tyr251, Arg295, Glu301, Ser338, Phe349, Arg352 and Glu380, including a newly identified one, are essential for NAD(+)-glycohydrolase (NADase) activity. At least one residue, Glu378, is an essential residue for ADP-ribosyltransferase (ARTase), but not for NADase. Consequently, the structural feature and these mutagenesis findings suggest that the catalytic mechanism of Ia proceeds via an Sn1-type reaction.