Purification and characterization of two forms of microsomal carbonyl reductase in guinea pig liver.

Glycosaminoglycan oligosaccharides generated by treatment of biosynthetically radiolabelled dermatan sulphate and hyaluronic acid with chondroitin AC lyase or testicular hyaluronidase may be resolved into a series of discrete bands by polyacrylamide-gel electrophoresis. Bands were identified by fixation in glacial acetic acid containing 20% (w/v) 2,5-diphenyloxazole followed by fluorography. The bands represented glycans which differed in size by one disaccharide unit. For the larger oligosaccharides (decasaccharides and above) of similar charge: mass ratio, there was a linear relationship between electrophoretic mobility and log Mr. However, the smaller species showed anomalous migration patterns. Consideration of the structures of the fragments produced by the different enzyme treatments suggests that copolymeric and homopolymeric oligosaccharides may be separated by polyacrylamide-gel electrophoresis. There are many potential applications of this technique, foremost amongst them being studies on the molecular size heterogeneity and patterns of enzyme-mediated depolymerization of native glycosaminoglycan chains and investigations into rates of polymer chain elongation and post-polymerization modification reactions so essential to glycosaminoglycan function.     

Purification of bacterial L-methionine gamma-lyase

A chromatographic procedure using sequential ion-exchange columns is described for separating choline, trimethylamine, trimethylamine oxide, and betaine extracted from marine fish tissues; added exogenous carnitine can also be separated by the system. Choline with its positive charge binds to the AG 50W-X8 (Na+, pH 9) column. The column is first eluted with 0.1 N NaOH to collect trimethylamine, trimethylamine oxide, and betaine; choline is then eluted with 0.5 N NaOH. The amines collected with 0.1 N NaOH are subsequently separated using an AG 50W-X8 (H+, pH 4) column eluted with a linear 0-1 M NaC1 gradient.     

A procedure to remove protease activities from Bacillus subtilis sporulating cells and their crude extracts.

A simple procedure was developed to remove both extracellular and intracellular proteases associated with aporulating Bacillus subtilis cells. Cells are washed four times with 1 m KCl before breakage and their crude extracts are treated with 2 mm diisopropylfluorophosphate before passage through a hemoglobin-Sepharose affinity column. RNA polymerase in crude B. subtilis extracts treated by this procedure was stable, functionally and structurally, for more than 1 month at 4°C. This process for removing all proteases should work essentially with any crude extracts containing proteolytic activities.     

Accumulation of O-methyl-L-homoserine by methanol-utilizing bacteria

Summary Several classes of mutants derived from facultative methylotrophs, Microcyclus eburneus and Protaminobacter ruber, accumulate 0.5–3.8 mg/ml of O-methyl-L-homoserine (OMH) in a medium containing methanol as sole carbon source. The wild-type parent strains also accumulate OMH when the medium is supplemented with L-homoserine; the accumulation of OMH by the mutants is ascribed to an increased L-homoserine pool.     

A photochemical fuel cell system using Anabaena N-7363

Summary A blue-green algae, Anabaena N-7363, was immobilized in 2% agar gel. The hydrogen productivity of the immobilized algae was three times higher than that of free algae. The maximum hydrogen production rate by the immobilized blue-green algae was 0.52 moles h^–1 g^–1 (of wet gel) in the medium without nitrogen sources under illumination (10,000 lux). The oxygen evolved was then removed by a reactor containing aerobic bacteria. A photo-current of 15–20 mA was continuously produced for 7 days by the photochemical fuel cell system consisting of the immobilized Anabaena reactor, the oxygen-removing reactor and the hydrogen-oxygen fuel cell. The conversion ratio of hydrogen to current was from 80% to 100%.     

Determination of serum unconjugated estrone, estradiol-17β and estriol during pregnancy by selected ion monitoring

A simple, rapid and highly specific method by selected ion monitoring (SIM), using 9α,11α-[²H₂]estrone, [2,4-²H₂]estradiol-17β and 2,4-[²H₂]estriol as internal standards, was developed for the determination of serum estrogens during pregnancy. Serum samples were submitted to a simple extraction procedure and were analysed after formation of the trifluoroacetic anhydride derivative. The inter-assay coefficients of variation for estrone, estradiol-17β and estriol were 3.73%, 3.42% and 3.49%, respectively. The results obtained by SIM were compared with analysis performed using radioimmunoassay.     

Spore coat protein synthesis in cell-free systems from sporulating cells of Bacillus subtilis.

Cell-free systems for protein synthesis were prepared from Bacillus subtilis 168 cells at several stages of sporulation. Immunological methods were used to determine whether spore coat protein could be synthesized in the cell-free systems prepared from sporulating cells. Spore coat protein synthesis first occurred in extracts from stage t2 cells. The proportion of spore coat protein to total proteins synthesized in the cell-free systems was 2.4 and 3.9% at stages t2 and t4, respectively. The sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis patterns of immunoprecipitates from the cell-free systems showed the complete synthesis of an apparent spore coat protein precursor (molecular weight, 25,000). A polypeptide of this weight was previously identified in studies in vivo (L.E. Munoz, Y. Sadaie, and R.H. Doi, J. Biol. Chem., in press). The synthesis in vitro of polysome-associated nascent spore coat polypeptides with varying molecular weights up to 23,000 was also detected. These results indicate that the spore coat protein may be synthesized as a precursor protein. The removal of proteases in the crude extracts by treatment with hemoglobin-Sepharose affinity techniques may be preventing the conversion of the large 25,000-dalton precursor to the 12,500-dalton mature spore coat protein.     

Subcellular localization of glycolytic key enzymes in germinating castor bean endosperm

Phosphofructokinase and pyruvate kinase activities in castorbean endosperm increased during germination. Subcellular localizationof pyruvate kinase and phosphofructokinase in germinating endospermtissues was studied by differential and sucrose density gradientcentrifugation techniques. Eighty five percent or more of thepyruvate kinase and phosphofructokinase activities were locatedin cytosol. The remaining activities were mainly detected inproplastids. (Received June 30, 1977; )     

Electric field control of lipase membrane activity

Lipase (EC 3.1.1.3., from Pseudomonas sp.) was entrapped in collagen membrane containing liquid crystal (4-methoxybenzilidene-4′-n-butylaniline). The activity of the lipase–liquid crystal membrane at an applied voltage of 4 V was 3.4 compared to a membrane tested without imposition of an external electric field. A linear relationship was observed between the activity of the lipase–liquid crystal membrane and the current. The apparent Michaelis constant (K′_m) of the lipase–liquid crystal membrane under electric field was identical to that of the membrane under ordinary condition. Activation of the lipase–liquid crystal membrane was observed repeatedly, i.e., activation in the presence of an electric field and reversion to a basal level upon removal of the field occurred cyclically. Activity control of immobilized enzymes is desirable for switching devices of a bioreactor. Possible mechanisms of the lipase activation by electric field are discussed.