A brown carbon monoxide dehydrogenase from CO-autotrophically grown cells of Acinetobacter sp. strain JC1, which is unstable outside the cells, was purified 80-fold in seven steps to better than 95% homogeneity, with a yield of 44% in the presence of the stabilizing agents iodoacetamide (1 mM) and ammonium sulfate (100 mM). The final specific activity was 474 iimol of acceptor reduced per min per mg of protein as determined by an assay based on the CO-dependent reduction of thionin. Methyl viologen, NAD(P), flavin mononucleotide, flavin adenine dinucleotide, and ferricyanide were not reduced by the enzyme, but methylene blue, thionin, and dichlorophenolindophenol were reduced. The molecular weight of the native enzyme was determined to be 380,000. Sodium dodecyl sulfate-gel electrophoresis revealed at least three nonidentical subunits of molecular weights 16,000 (a), 34,000 (if), and 85,000 (-y). The purified enzyme contained particulate hydrogenase-like activity. Selenium did not stimulate carbon monoxide dehydrogenase activity. The isoelectric point of the native enzyme was found to be 5.8; the Km of CO was 150 LM. The enzyme was rapidly inactivated by methanol. One mole of native enzyme was found to contain 2 mol of each of fiavin adenine dinucleotide and molybdenum and 8 mol each of nonheme iron and labile sulfide, which indicated that the enzyme was a molybdenum-containing iron-sulfur flavoprotein. The ratio of densities of each subunit after electrophoresis (a:p:ry = 1:2:6) and the number of each cofactor in the native enzyme suggest a a2iB2y2 structure of the enzyme. The carbon monoxide dehydrogenase of Acinetobacter sp. strain JC1 was found to have no immunological relationship with the enzymes-of Pseudomonas carboxydohydrogena and Pseudomonas carboxydovorans.Carbon monoxide dehydrogenase (CO-DH) is an enzyme responsible for the oxidation of CO in several gram-positive and -negative carboxydobacteria which are able to grow aerobically with CO as the sole source of carbon and energy (7, 14, 22, 31, 34-36, 39, 43, 58). Studies on the CO-DHs in the purified state, in partially purified preparations, and in extracts revealed that the enzymes from several carboxydobacteria are, with few exceptions, similar in biochemical and immunological properties (2, 6, 14, 22, 23, 25, 34-36, 39, 43).Acinetobacter sp. strain JC1 DSM 3803 is a new carboxydobacterium isolated from soil in Seoul, Korea (7). Preliminary studies with cell extracts showed that the CO-DH of this bacterium is an inducible enzyme which is loosely bound to the inner face of the cytoplasmic membrane (6, 18). The enzyme was found to be unstable outside the cell and to have no immunological relationship with that of Pseudomonas carboxydohydrogena (6), which implies the presence of a novel CO-DH in Acinetobacter sp. strain JC1.In this study, we examined purified CO-DH of Acinetobacter sp. strain JC1 in some detail to determine whether the basis for CO oxidation is diverse in the carboxydobacteria and to learn more about the process. Before the purificat...

Burkholderia sp. TNFYE-5 was isolated from soil for the ability to grow on phenanthrene as sole carbon and energy source. Unlike most other phenanthrene-degrading bacteria, TNFYE-5 was unable to grow on naphthalene. Growth substrate range experiments coupled with the ring-cleavage enzyme assay data suggest that TNFYE-5 initially metabolizes phenanthrene to 1-hydroxy-2-naphthoate with subsequent degradation through the phthalate and protocatechuate and beta-ketoadipate pathway. A metabolite in the degradation of naphthalene by TNFYE-5 was isolated by high-pressure liquid chromatography (HPLC) and was identified as salicylate by UV-visible spectral and gas chromatography-mass spectrometry analyses. Thus, the inability to degrade salicylate is apparently one major reason for the incapability of TNFYE-5 to grow on naphthalene.

Objective The aim of this study was to clarify the characteristic findings in myopic choroidal neovascularization (CNV) and the relationship with lacquer crack (LC). Methods In all, 66 consecutive myopic CNV patients treated with photodynamic therapy and/or intravitreal anti-vascular endothelial growth factor injection in one eye were reviewed. Data from fluorescein angiography (FA) and indocyanine green angiography (ICGA), obtained simultaneously using the Heidelberg retina angiograph 2 (HRA2), were analyzed. Results LCs were associated with a relatively large extent (Z3000 lm) of peripapillary choroidal atrophy and a dark rim, the proliferation of retinal pigment epithelial cells surrounding the neovascular membrane was accompanied by a small extent. Myopic CNV usually developed in the LC area surrounded by tiny crack fragments. In all, 35 patients with LCs received FA and ICGA at least twice during follow-up. LC progression was observed in nine (25.7%) treated eyes and six (23.1%) non-CNV fellow eyes. Crack fragments progressed in three distinct forms such as elongation, branching, or bridging pattern. Newly diagnosed myopic CNV was reported in 18 treated eyes and 3 fellow eyes. Progression of LCs and development of CNV occurred simultaneously in eight eyes. By multivariate Cox's regression, a statistically significant association was observed between recurrence of myopic CNV and the absence of a dark rim on ICGA. Conclusions The HRA2 instrument affords detailed high-resolution images of FA and ICGA. Notably, recurrence of myopic CNV developed in areas surrounded by new small crack fragments and LCs are considered to be important in the development of myopic CNV.

Extracts of heterotrophically grown cells ofPseudomonas carboxydovorans were found to contain an inhibitor of carbon monoxide dehydrogenase (CO-DH). The inhibitor activity was not detected in CO-autotrophically grown cells. The inhibitor was extremely stable to heat treatment based on the extent of inhibition of CO-DH activity. The extent of inhibition was proportional to the amount of cell extract added to the reaction mixture.The inhibition was independent of a prior incubation period of the extracts with CO-DH. The inhibitor was precipitable with ammonium sulfate, phenol, and trichloroacetic acid. It was passed through benzoylated dialysis tubing and Amicon ultrafiltration membrane YM2. Denaturing and nondenturing polyacrylamide gel electrophoresis of CO-DH inactivated by inhibitor revealed that the mobilities of native enzyme and subunits were identical to those of active CO-DH. The inhibitor-treated CO-DH retained its original antigenic sites and exhibited enzyme activity upon activity staining. The CO-DH inhibitor of P. carboxydovorans was also active on CO-DHs from Pseudomonas carboxydohydrogena, Acinetobacter sp. strain JC1, and Pseudomonas carboxydoflava.Carbon monoxide dehydrogenase (CO-DH) is the key enzyme in the utilization of CO as the sole energy and carbon source in several aerobic carboxydobacteria (1,5,6,(15)(16)(17)22). The enzyme has generally been considered as an inducible enzyme, with the exception of the CO-DH in Pseudomonas carboxydoflava (1,2,5,15,17). Biochemical and immunological studies revealed that the enzymes from several carboxydobacteria share similar properties, with few exceptions (3,5,(15)(16)(17).We have recently found that Pseudomonas carboxydovorans growing under heterotrophic condition contains an inhibitor of CO-DH from this bacterium. Since this was the first observation of the presence of CO-DH inhibitor in carboxydobacteria and since nothing is known about the molecule, it was of interest to study the inhibitor in cell extracts. The present work was undertaken to study the physiological and biochemical properties of CO-DH inhibitor in cell extracts of P. carboxydovorans.