Identification of porphyrin present in apo-cytochrome c oxidase of copper-deficient yeast cells

Heme a was not detected either in mitochondria isolated from copper-deficient yeast or in the intact cells. Nevertheless, the intracellular concentration of free porphyrins indicated that the pathway of porphyrin and heme synthesis was not impaired in copper-deficient cells. The immunoprecipitated apo-oxidase from copper-deficient cells revealed an absorption spectrum with maxima at 645, 592, 559, 519 and 423 nm, similar to that of purified porphyrin a. When solubilized mitochondria from [³H]leucine and δ-amino[¹⁴C]levulinic acid-labeled copper-deficient yeast cells were incubated with rabbit antiserum against cytochrome c oxidase, a precipitate was obtained. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of this immunoprecipitate showed [³H]leucine associated with six bands and δ-amino[¹⁴C]levulinic acid resolved in a single band. HCl fractionation of copper-deficient mitochondria labeled with δ-amino[¹⁴C]levulinic acid showed a high specific radioactivity in the fraction extracted by 20% HCl, a solvent which extracts porphyrin a. Thinlayer chromatography of the radioactivity found in 20% HCl showed an R_F value identical to that of purified porphyrin a. When δ-amino[³H]levulinic acid-labeled, copper-deficient yeast cells are grown in copper-supplemented medium, the porphyrin a accumulated in copper-deficient cells wa converted into heme a, and this conversion was prevented by cycloheximidine.These observations suggest that porphyrin a is present in the apo-oxidase of copper-deficient cells, but that the conversion to heme a does not occur. This conversion reaction appears to be a point in the biosynthetic pathway of cytochrome c oxidase which is blocked by copper deficieny.     

Energy conservation capacity and morphological integrity of mitochondria in hypotonically treated rabbit epididymal spermatozoa

Hypotonic treatment of rabbit epididymal spermatozoa in 10 mM phosphate buffer disrupts the plasma membrane and removes the cytoplasmic droplet from those cells to which it is still attached. There is, however, no effect on the mitochondria, which retain their helical configuration around the axial filament complex, have intact inner and outer membranes, and show the same cristal morphology as do the mitochondria in untreated cells. Hypotonically treated spermatozoa respire with added malate-pyruvate, succinate, and ascorbate plus N,N′-tetramethyl-p-phenylenediamine, but not with added fructose or NADH. Respiration is inhibited by oligomycin and stimulated by uncoupler, showing that the mitochondria have retained their capacity for energy conservation. The uncoupled respiration rate is not further stimulated by added cytochrome c. Reduced-minus-oxidized difference spectra obtained at −196 °C of the hypotonically treated cells show a full complement of cytochromes, including cytochrome c. This result implies that the cytochrome c lost from mammalian spermatozoa on storage or chilling [Mann, T. (1951) Biochem. J. 48, 386–388] is cytoplasmic cytochrome c not yet incorporated into the mitochondria. The mitochondrial cytochrome c remains firmly held inside the intact outer membrane.     

Sensitivity to detergents and plasmid curing in Enterococcus faecalis

This research reports the sensitivity of a clinical isolate of Enterococcus faecalis to sodium N-lauroylsarcosinate (sarkosyl) and sodium dodecyl sulfate (SDS), as well as the efficiency of these detergents in curing the strain. Compared to Escherichia coli, Enterococcus faecalis was very sensitive to both detergents, with minimum inhibitory concentrations (MIC) for the latter being 100 times lower than for Escherichia coli. The clinical isolate of Enterococcus faecalis used in this study exhibited plasmid-borne resistance to kanamycin (MIC 2 mg/ml) and tetracycline (MIC 50 μg/ml); 3% curing was observed after growth in the presence of sarkosyl but no curing was observed after growth in the presence of either SDS or acridine orange. In contrast, 35% curing of plasmid-bearing Escherichia coli was observed after growth in the presence of either SDS or acridine orange, but none was observed after growth in the presence of sarkosyl.     

Zinc Sulphate Mediates the Stimulation of Cell Proliferation of Rat Adipose Tissue-Derived Mesenchymal Stem Cells Under High Intensity of EMF Exposure

Biological Trace Element Research - Unlike the role of mesenchymal stem cells (MSCs) in regenerative medicine, their application in cell therapy can be complicated by factors such as a reduction in...     

Zinc Oxide Nanoparticles Inhibition of Initial Adhesion and ALS1 and ALS3 Gene Expression in Candida albicans Strains from Urinary Tract Infections

Mycopathologia - The aim of this study was to evaluate effects of zinc oxide nanoparticles (ZnO-np) on initiation adhesion and agglutinin-like sequence (ALS) 1 and ALS3 gene expression, which is...     

Catalytic properties of three L-lactate dehydrogenases from saffron corms (Crocus sativus L)

Three L-lactate dehydrogenase isoenzymes were detected in saffron corms, using potassium ferricyanide as the electron acceptor. Their pH optima were 5.5, 7.5 and 9.5, respectively. All three dehydrogenases were substrate-inhibited by ferricyanide, but at different concentrations; maximum enzymatic activity was observed for 250, 100 and 600 M ferricyanide, at pH 5.5, 7.5 and 9.5, respectively. Catalytic efficiency, calculated per mg corm extract protein, was 1.9, 1.0 and 0.4 min^-1, respectively at pH 5.5, 7.5 and 9.5. Pseudo first order rate constant was also different under the three pH conditions. Malate was an inhibitor for the isoenzyme active at pH 9.5, but had no effect on the others.     

Polyphenol oxidase activity in dormant saffron (<Emphasis Type="Italic">Crocus sativus</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) corm

Crocus sativus L., cultivated since ancient times as the source of saffron, is a triploid plant that can be propagated only via its corms which undergo a period of dormancy. Understanding the processes taking place in the corm is essential to preserve the plant and improve its quality. Color and taste being of prime importance in the quality of the saffron spice, knowledge on polyphenol oxidase (PPO) activity in the plant is of particular interest given the role of the enzyme in fruit and vegetable browning during processing and during the storage of processed food. In this paper, PPO activity was investigated for the first time in extracts obtained from dormant C. sativus L. corms. PPO activity was detectable using l-DOPA, pyrogallol, catechol or p-cresol as substrate, each being oxidized to its corresponding o-quinone; no activity was detectable with l-tyrosine, tyramine or phenol as substrate. Two pH optima, respectively at 4.5 and 6.7, were observed with all substrates and a third one, at 8.5, was found with l-DOPA and p-cresol. Kinetics parameters studied at pH 6.7 indicated the highest catalytic efficiency (in units mg⁻¹ prot mM⁻¹) with pyrogallol: 150, then catechol: 39, l-DOPA: 6.4 and p-cresol: 4.6. The enzymatic activity was inhibited by 50% in the presence of 0.22, 0.35, 0.5 and 0.7 mM kojic acid with, respectively, catechol, pyrogallol, p-cresol and l-DOPA as substrate. When stained for PPO activity, non-denaturing gel electropherograms of extract revealed three distinct bands, indicating the presence of multiple isoenzymes in dormant C. sativus L. corms.