Interaction of steroids with D-amino acid oxidase.

1. Progesterone inhibited D-amino acid oxidase (D-amino acid : O2 oxidoreductase (deaminating), EC 1.4.3.3) in competition with its substrate, D-alanine. Binding of progesterone brought about the increase in both fluorescence intensity and fluorescence polarization of FAD, which indicates that the environment surrounding FAD chromophore is modified due to a conformational change in the apoenzyme. 2. Ethinyl estradiol, testosterone, testosterone propionate, corticosterone and aldosterone also inhibited the enzyme slightly in the same manner. Their binding also produced a slight increase in FAD fluorescence without decreasing the fluorescence polarization. 3. Cholesterol did not inhibit the enzyme, though it increased the fluorescence polarization of FAD. This indicates the binding of cholesterol with the enzyme at a site other than the substrate binding site.     

Adenylate regulation of photosynthetic electron transport and the coupling sites of phosphorylation in spinach chloroplasts

The regulation by adenylates of activities of various partial electron transport systems in spinach chloroplasts was studied using systems from H₂O to 2,5-dimethyl-p-benzoquinone, H₂O to 2,6-dichlorophenolindophenol, reduced 2,6-dichlorophenolindophenol to methyl viologen, and H₂O to methyl viologen or ferricyanide. Adenylates regulated all of them. The ratio of the amount of esterified Pi (P) to that of electrons transported (e) in coupling with phosphorylation manifested that there are two phosphorylation sites: one between H₂O and 2,5-dimethyl-p-benzoquinone or 2,6-dichlorophenolindophenol and another between reduced 2,6-dichlorophenolindophenol and methyl viologen, under the proposed stoichiometries,i.e., P/H⁺=0.5 and H⁺/e=1, where H⁺ is the amount of protons pumped by electron transport (= those translocated during phosphorylation), when the basal electron transport (the part not regulated by adenylates) was excluded. The effects of pH, phlorizin, and methylamine on the adenylate regulation of electron transport, and the stimulation profile of electron transport coupled with quasiarsenylation suggested no distinction between the two phosphorylation sites.     

High mutagenicity and toxicity of a diol epoxide derived from benzo[a]pyrene

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP 7,8-diol-9,10-epoxide) is a suspected metabolite of benzo[a]pyrene that is highly mutagenic and toxic in several strains of $\text{Salmonella}\text{typhimurium}$ and in cultured Chinese hamster V79 cells. BP 7,8-diol-9,10-epoxide was approximately 5, 10 and 40 times more mutagenic than benzo[a]pyrene 4,5-oxide (BP 4,5-oxide) in strains TA 98 and TA 100 of $\text{S.}\text{typhimurium}$ and in V79 cells, respectively. Both compounds were equally mutagenic to strain TA 1538 and non-mutagenic to strain TA 1535 of $\text{S.}\text{typhimurium}$. The diol epoxide was toxic to the four bacterial strains at 0.5–2.0 nmole/plate, whereas BP 4,5-oxide was nontoxic at these concentrations. In V79 cells, the diol epoxide was about 60-fold more cytotoxic than BP 4,5-oxide.     

Enhancement factor for oxygen absorption into fermentation broth

Values of the enhancement factor for oxygen absorption into fermentation broth, i.e., the ratio of the liquid phase mass transfer coefficients for oxygen absorption for both cases with and without respiration of microorganisms were predicted theoretically on the assumption of various cell concentration distributions. Calculations indicate that in the usual case the enhancement factor is only slightly or negligibly larger than unity, even when accumulation of microorganisms at or near the gas-liquid interface is assumed. Results of experiments with sparged-stirred fermentors on oxygen absorption into fermentation broths containing resting and growing cells of Candida tropicalis confirmed the theoretical prediction. Except for extreme cases, the effect of respiration of microorganisms on k_La, values can practically be ignored.     

Myosin from molluscan abalone, Haliotis discus. Isolation and enzymatic properties.

Actomyosin was extracted from smooth muscle of molluscan abalone with 0.1 M PPit pH 6.4. Myosin was separated from the actomyosin by centrifugation at 100,000 X g in the presence of 5 mM ATP and 10 mM MgCl2. Myosin in the supernatant was further purified by gel filtration on a Sepharose 4B column. Paramyosin contamination of the actomyosin preparation interfered with the isolation of myosin and complete removal of actin and paramyosin from the myosin has not been accomplished. The myosin appeared to consist of a single f-chain and a single g-chain, as examined by SDS-disc electrophoresis in 8 or 13.7% acrylamide gel. The ATPase [EC 3.6.1.3] activity of this myosin in 0.5 M KCL at neutral pH and at 0 degrees was rather unstable and decreased by 10-20% per day. The effects of rho-chloromercuribenzoate and EDTA on the ATPase activity were similar to those observed with other smooth muscle myosin but the dependence upon pH or KCL concentration was different.     

Structure and function of D-amino acid oxidase. IX. Changes in the fluorescence polarization of FAD upon complex formation.

1. The fluorescence polarization, P, of FAD increased on complex formation with the apoenzyme of D-amino acid oxidase [D-amino acid: O2 ocidoreductase (deaminating), EC 1.4.3.3]. The time course of the increase was monophasic. The values of P were extimated to be 0.04, 0.4, and 0.4 for FAD, the enzyme and the enzyme-benzoate complex, respectively. 2. The value of P of the enzyme is dependent on its concentration, indicating that the degrees of dissociation of FAD in the monomer and dimer are different. The dissociation constant was calculated to be 7 times 10-minus 7 M for the monomeric form of the enzyme. This value is far larger than the value for the dimeric form of the enzyme, 1 times 10-minus 8 M, calculated from equilibrium dialysis data. 3. Changes in fluorescence polarization of the enzyme due to changes in solution pH or temperature can be explained in terms of the monomer-dimer equilibrium.     

Properties of purified hydrogenase from the particulate fraction of Desulfovibrio vulgaris, Miyazaki.

The properties of purified hydrogenase [EC 1.12.2.1] solubilized from particulate fraction of sonicated Desulfovibrio vulgaris cells are described. The enzyme was a brownish iron-sulfur protein of molecular weight 89,000, composed of two different subunits (mol. wt.: 28,000 and 59,000), and it contained 7-9 iron atoms and 7-8 labile sulfide ions. Molybdenum was not detected in the preparation. The absorption spectrum of the enzyme was characteristic of iron-sulfur proteins. The millimolar absorbance coefficients of the enzyme were about 164 at 280nm, and 47 at 400nm. The absorption spectrum of the enzyme in the visible region changed upon incubating the enzyme under H2 in the presence of cytochrome c3, but not in its absence. This spectral change was due to the reduction of the enzyme. The absorbance ratio at 400nm of the reduced and the oxidized forms of the enzyme was 0.66. The activity of the enzyme was hardly affected by metal-complexing agents such as cyanide, azide, 1,10-phenanthroline, etc., except for CO, which was a strong inhibitor of the enzyme. The activity was inhibited by SH-reagents such as p-chloromercuribenzenesulfonate. The enzyme was significantly resistant to urea, but susceptible to sodium dodecyl sulfate. These properties were very similar to those of clostridial hydrogenase [EC 1.12.7.1], in spite of differences in the acceptor specificity and subunit structure.     

Inhibition and uncoupling of the ADP-regulated electron transport in isolated chloroplasts

The effects of energy transfer inhibitors, electron transport inhibitors and uncouplers on the ADP-regulated and ADP-independent activity of ferricyanide reduction in isolated spinach chloroplasts were studied. Phlorizin and sulfate did not affect the ADP-independent ferricyanide reduction. In the ADP-regulated reduction, these reagents did not affect the ADP inhibition process but inhibited the activity restoration process due to phosphorylation. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea and linolenic acid depressed both ADP-regulated and ADP-independent activity of ferricyanide reduction. Gramicidin S and 2-amino-1-butanol depressed ADP-regulated activity and stimulated ADP-independent activity. The decrease in the ADP-regulated ferricyanide-reducing activity (restoration) due to (incomplete) uncoupling paralleled the decrease in phosphorylation activity (P/Δe=1).     

Microorganism Capable of Decomposing N-Acetylglucosaminyl Ribitol Teichoic Acid of Staphylococcus aureus

Enzyme(s) capable of decomposing N-acetylglucosaminyl ribitol teichoic acid prepared from the cell wall of Staphylococcus aureus FDA 209 P was obtained from the culture supernatant of a gram-negative, rod-shaped, spore-forming soil bacterium. Properties of the bacterium were very similar to those of Bacillus circulans.     

DNA methylation profiles provide a viable index for porcine pluripotent stem cells

Porcine induced pluripotent stem cells (iPSCs) provide useful information for translational research. The quality of iPSCs can be assessed by their ability to differentiate into various cell types after chimera formation. However, analysis of chimera formation in pigs is a labor‐intensive and costly process, necessitating a simple evaluation method for porcine iPSCs. Our previous study identified mouse embryonic stem cell (ESC)‐specific hypomethylated loci (EShypo‐T‐DMRs), and, in this study, 36 genes selected from these were used to evaluate porcine iPSC lines. Based on the methylation profiles of the 36 genes, the iPSC line, Porco Rosso‐4, was found closest to mouse pluripotent stem cells among 5 porcine iPSCs. Moreover, Porco Rosso‐4 more efficiently contributed to the inner cell mass (ICM) of blastocysts than the iPSC line showing the lowest reprogramming of the 36 genes (Porco Rosso‐622‐14), indicating that the DNA methylation profile correlates with efficiency of ICM contribution. Furthermore, factors known to enhance iPSC quality (serum‐free medium with PD0325901 and CHIR99021) improved the methylation status at the 36 genes. Thus, the DNA methylation profile of these 36 genes is a viable index for evaluation of porcine iPSCs. genesis 51:763–776. © 2013 Wiley Periodicals, Inc.