A simple method for the release of asparagine-linked oligosaccharides from a glycoprotein purified by SDS-polyacrylamide gel electrophoresis.

A simple method for the release of oligosaccharides from glycoproteins separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) has been developed. Asialo-alpha 1-acid glycoprotein, which was tritiated at the nonreducing terminal D-galactopyranosyl residue by reduction with sodium borotritide after incubation with D-galactose oxidase, was used as a model compound. After electrophoretic separation of the glycoprotein, oligosaccharides were released by the use of a gas-phase hydrazinolysis apparatus. In the first method, the gel was stained with Coomassie Blue and the glycoprotein together with the gel was directly subjected to gas-phase hydrazinolysis after removal of water in a P2O5 desiccator. The recovery of released oligosaccharides was 25.9 +/- 2.4%, based on the amount of the glycoprotein loaded on the gel within the range of 3.5-28.5 micrograms. In the second method, the glycoprotein was electroblotted onto an Immobilon transfer membrane and was visualized by staining with Coomassie Blue. A small piece of the membrane with the corresponding band was cut out, dried in a desiccator and subjected to gas-phase hydrazinolysis. In this case, the recovery of released oligosaccharides was 15.2 +/- 1.0%. These procedures, particularly the first one, should be widely applicable for the isolation of oligosaccharides from glycoproteins separated by SDS-PAGE.     

Covalent alteration of the prosthetic heme of human hemoglobin by BrCCl3. Cross-linking of heme to cysteine residue 93.

Recent studies have shown that a protein-bound heme adduct formed from the reaction of BrCCl3 with myoglobin was due to bonding of the proximal histidine residue through the ring I vinyl of a heme-CCl2 moiety. The present study reveals that BrCCl3 also reacts with the heme of reduced human hemoglobin to form two protein-bound heme adducts. Edman degradation and mass spectrometry provided evidence that these protein-bound heme adducts were addition products in which heme-CCL2 or heme-CCl3 were bound to cysteine residue 93 of the beta-chain of hemoglobin. It appeared that the cysteine residue was bonded regiospecifically to the ring I vinyl group of the altered heme moiety, because the nonprotein-bound products of the reaction included the beta-carboxyvinyl and alpha-hydroxy-beta-trichloromethylethyl derivatives of the ring I vinyl moiety of heme. The absorption spectra of the protein-bound adducts in both the oxidized and reduced states were highly similar to those described for hemichromes, which are thought to be involved in the formation of Heinz bodies and subsequent red cell lysis.     

An Activation of Synaptosomal Na+, K+-ATPase by a Novel Dibenzoxazepine Derivative (BY-1949) in the Rat Brain: Its Functional Role in the Neurotransmitter Uptake Systems

In search of factors mitigating the final outcome of ischemic and epileptic brain damage, we tested a novel dibenzoxazepine derivative (BY-1949), as the compound has been shown to be effective under these two conditions. First, using rat brain, we assessed whether or not BY-1949 affects the Na+,K(+)-ATPase activity. Although in vitro applications of either BY-1949 or its three major metabolites did not cause any apparent effects, both acute and chronic oral administrations of the compound (10 mg/kg) invariably increased the Na+,K(+)-ATPase activity in the synaptosomal plasma membranes by increasing Vmax values. Second, it was shown by this study that the drug treatment caused marked increases in the uptake of both glutamic acid and gamma-aminobutyric acid into the synaptosomes. These results suggest that the activity against ischemic/epileptic brain damage by BY-1949 is explicable, at least partly, in terms of improvement of ionic derangements across the neural membranes via Na+,K(+)-ATPase activation.     

Purification of human placental aromatase cytochrome P-450 with monoclonal antibody and its characterization

A simple and efficient method is described for the purification of microsomal aromatase cytochrome P-450 from human placenta. The enzyme was solubilized with Emulgen 913 and sodium cholate and subjected to chromatography on a column of Sepharose 4B coupled with a specific monoclonal antibody, followed by hydroxyapatite column chromatography. The specific cytochrome P-450 content of purified aromatase was 13.1 (12-14.8) nmol/mg of protein. Aromatase assays were carried out with reconstituted systems of bovine liver P-450 reductase and dilauroyl-L-alpha-phosphatidylcholine with [1 beta-3H,4-14C]-androstenedione as substrate. The specific activity of purified aromatase was 65.0 (50.6-74.3) nmol.min-1.(mg of protein)-1 or a turnover rate of 5.0 (4.3-5.9) min-1. The total recovery of purified aromatase activity was 32.2%, and P-450 recovery was 17.6%. The Km of immunoaffinity-purified aromatase was 12, 210, 41, and 2830 nM for androstenedione, 16 alpha-hydroxyandrostenedione, testosterone, and 16 alpha-hydroxytestosterone, respectively. The very high Km value for 16 alpha-hydroxytestosterone aromatization gives a reasonable indication that estriol is not the directly aromatized product in the fetoplacental unit of human pregnancy. The aromatase P-450 was subjected to SDS-polyacrylamide gel electrophoresis in increasing quantities. Silver stain detection techniques indicated a single band having a molecular mass of 55 kDa with greater than 97% purity. The stability analysis showed a half-life of over 4 years on storage at -80 degrees C.     

Protective mechanism of sodium molybdate against the acute toxicity of cadmium in rats. II. Prevention of cytoplasmic acidification

In order to clarify the protective mechanism of sodium molybdate against the acute toxicity of cadmium chloride in rat, the effect of in vivo sodium molybdate pretreatment on the cytotoxic action of cadmium in isolated hepatocytes was studied. The cytosolic pH of hepatocytes isolated from untreated rats immediately decreased with incubation in either neutral Hank's balanced salt solution (HBS), pH 7.4, containing 5 µM cadmium chloride minimum or acidic HBS (pH 7.1, 6.8, 6.5, and 6.2). The presence of 5 µM cadmium in HBS adjusted to pH 7.1 aggravated cytosalic acidification induced by the acidic medium alone. Cell viability of hepatocytes incubated in HBS at pH 6.2 was significantly reduced as compared to that of control cells in HBS at pH 7.4, but the presence of cadmium in the acidic HBS had no aggravating action against such a toxic action of the acidic medium although cellular uptake of the metal in the medium increased, as compared to that in HBS at pH 7.4. Molybdenum pretreatment alleviated cytoplasmic acidification induced by the treatment with HBS at pH 7.4 or 7.1 containing cadmium or by extracellular acid load wothout cadmium. This pretreatment also prevented the loss of cell viability induced by the treatment with HBS at pH 6.2 but could not attenuate that when cadmium was present in the medium.These facts suggest that molybdenum pretreatment alleviated the acute toxicity of cadmium in rat by preventing cytoplasmic acidification caused by the harmful metal.     

Enhanced growth of the red algaPorphyra yezoensis Ueda in high CO2 concentrations

Leafy thalli of the red algaPorphyra yezoensis Ueda, initiated from conchospores released from free-living conchocelis, were cultured using aeration with high CO₂. It was found that the higher the CO₂ concentration, the faster the growth of the thalli. Aeration with elevated CO₂ lowered pH in dark, but raised pH remarkably in light with the thalli, because the photosynthetic conversion of HCO ₃ ^? to OH^? and CO₂ proceeded much faster than the dissociation of hydrated CO₂ releasing H⁺. Photosynthesis of the alga was found to be enhanced in the seawater of elevated dissolved inorganic carbon (DIC, CO₂ + HCO ₃ ^? + CO ₃ ^? ). It is concluded that the increased pH in the light resulted in the increase of DIC in the culture media, thus enhancing photosynthesis and growth. The relevance of the results to removal of atmospheric CO₂ by marine algae is discussed.     

Selective Medium for Enumeration of Tannin-Protein Complex-Degrading Streptococcus spp. in Feces of Koalas

A selective agar plate medium (tannin-treated brain heart infusion agar supplemented with colistin-oxolinic acid) was developed to enumerate tannin-protein complex-degrading Streptococcus bovis in the feces of koalas. This medium was successfully used to enumerate strains from fecal samples but failed to enumerate those from pure cultures.     

Distribution of ribonucleic acid coliphages in raw sewage from treatment plants in Japan.

To determine the transmission cycle of ribonucleic acid (RNA) coliphages in their natural habitats, we investigated the distribution patterns of RNA phages in raw sewage collected from treatment plants in various localities in Japan. Most of the sewage samples contained group II and III phages. Samples from treatment plants in Sapporo, Tokyo, and Toyama contained appreciable amounts of group I phages in addition to the group II and III phages. As a whole, raw sewage from treatment plants in Japan contained RNA phages of the three groups in the ratio 1:2:5, group I/II/III. Based on the distribution patterns of RNA phages in sewage from domestic drainage in Japan proper (group II/III, 3:1), in animal feces and sewage from slaughter houses (mostly group I), and in human feces (group II/III, 1:1), it can be reasonably said that group I phages tend to be introduced from animal sources and group II and III phages tend to be introduced from human sources. Raw sewage from treatment plants in Japan consists mainly of human feces, sewage from domestic drainage, and industrial wastewater, and, in part, from slaughter houses. In fact, sewage from slaughter houses together with that from human sources flowed into the treatment plants of Tokyo as far as we could confirm.     

A possible new mechanism of temperature dependence of electron transfer in photosynthetic systems

A new theory for the electron transfer by the non-adiabatic process is formulated taking into account the origin shift and the frequency change of the vibration. The resultant formulas are quite similar to those of Jortner (Jortner, J. (1976) J. Chem. Phys. 64, 4860–4867) except that the free energy gap ΔG is used instead of the energy gap ΔE. By applying this theory to the photosynthetic electron transfer, the role of the remarkable temperature dependence of the electron transfer from cytochrome to P⁺ in Chromatium vinosum and the experimental data were reproduced very well using a small value of the coupling strength in contrast with the previous theory. This implies that proteins play a role to exclude many of the solvent molecules from the region of the electron transfer reaction between the donor and acceptor molecules. The negative activation process in the back electron transfer from Q^?_A to P⁺, the very slow back electron transfer from I^? to P⁺ and the solvent isotope effect on the cytochrome oxidation are also successfully explained by this new theory. It is shown that even a qualitative conclusion as to the molecular parameters obtained from the temperature dependence of the electron transfer is different between the present theory and that of Jortner.