Three hydroxylations incorporating molecular oxygen in the aerobic biosynthesis of ubiquinone in Escherichia coli.

The biosynthetic origin of the oxygen atoms of ubiquinone 8 from aerobically grown Escherichia coli was studied by 18O labeling. An apparatus was developed which allowed the growth of cells under a defined atmosphere. Mass spectral analysis of ubiquinone 8 from cells grown under highly enriched 18O2 showed that three oxygen atoms of the quinone are derived from molecular oxygen. It was established that the molecular oxygen is incorporated into the two methoxyl groups (at C-5 and C-6) and one of the carbonyl positions of the ubiquinone molecule by demonstrating that only one of the incorporated oxygens will exchange with water under acidic conditions that specifically catalyze the exchange of carbonyl, but not methoxyl, oxygens. That the C-4 carbonyl oxygen is derived from molecular oxygen was shown by the incorporation of three atoms of 18O2 into ubiquinone 8 biosynthesized from added 4-hydroxybenzoic acid. Comparison of ubiquinone 8 and menaquinone 8 from E. coli grown under 18O2 confirmed that the labeled carbonyl oxygen of the [18O2]ubiquinone 8 is incorporated biosynthetically and not by chemical exchange in the cell. It is concluded that the three hydroxylation reactions involved in the pathway for the aerobic biosynthesis of ubiquinone are all catalyzed by monooxygenases. The implications of this study for the anaerobic biosynthesis of ubiquinone 8 in E coli are discussed.     

Assembly of the mitochondrial membrane system XIX

Summary Two F^- mutants deficient in conjugation with F-type donors are isolated and characterized. Phenotypically, these mutants are similar; they have heptose-less lipopolysaccharide and lack some outer membrane protein. Genotypically, they are different. One mutant harbours a point mutation in the 70 to 74 min region, while the other is deleted for the chromosomal region 6.5 to 8.5 min. Comparison of the properties of the conjugation-deficient mutants described in this paper with other such mutants suggests than an outer membrane protein is the receptor for the F-pilus.     

Elevated plasma dopamine beta hydroxylase activity in rats with neurogenic hypertension.

Increased plasma dopamine beta hydroxylase, DBH, activity has been cited as evidence of increased sympathetic function in essential hypertension. Here-to-fore, experimental hypertension in animals has been associated with normal plasma DBH activity. This study shows that rats with neurogenic hypertension, induced by sinoaortic denervation, SAD, have elevated DBH activity; the mean increase in plasma DBH measured 3 days to 11 weeks after operation was 74% higher in the SAD group than in the sham-operated, control group. DBH activity showed a positive correlation with arterial pressure. Mesentery DBH activity was inversely related to plasma enzyme activity in SAD rats, indicating sympathetic nerve terminals in mesentery are a source of plasma DBH. We conclude that plasma DBH activity is an index of increased sympathetic function since it is consistently elevated in rats with neurogenic hypertension resulting from sustained central activation of the sympathetic nervous system.     

The immune response of brown trout (Salmo trutta L.) to injection with soluble antigens.

The immune response of brown trout (Salmo trutta) to horse serum and keyhole limpet haemocyanin was studied. Intraperitoneal and intramuscular injections were used, with and without adjuvant, in 209 fish. Complement-fixing antibodies (CFA) and precipitins were produced to both antigens. CFA were detected after 8 days to haemocyanin and after 13 days to horse serum. Maximum CFA titres to a single intraperitoneal injection of horse serum or haemocyanin were reached at 44 and 43-46 days respectively. Precipitins to a single injection of haemocyanin given intraperitoneally were detected after 19 days using gel diffusion. Similarly using the intramuscular route they were detected after 22 days. However, using counter-current electrophoresis, precipitins were detected after 8 days by the intraperitoneal route and after 9 days by the intramuscular. Precipitins to horse serum given intraperitoneally were demonstrated after 22 days by both gel diffusion and counter-current electrophoresis. Fish given 2 intraperitoneal injections of haemocyanin in adjuvant reached maximum CFA titres after 55 days; a 3rd injection on day 56 did not produce a marked increase in titre. Fish given intramuscular injections of haemocyanin in adjuvant showed maximum CFA titres at day 43. After a 3rd injection on day 56, maximum CFA titres were reached between days 92 and 106. Intramuscular injections gave significantly higher titres than those given by the intraperitoneal route. Some fish which showed no precipitins by gel diffusion were positive by counter-current electrophoresis. Precipitating antibodies to haemocyanin migrating in the beta2-gamma1 region were detected by immuno-electrophoresis.     

Characterization and structural properties of the major biliproteins of Anabaena sp.

Studies are presented of the biliproteins of Anabaena sp. This filamentous cyanobacterium contains three major biliproteins. Whereas two of these, C-phycocyanin and allophycocyanin, are common to all cyanobacteria, the third, phycoerythrocyanin (_max568nm) has hitherto not been described and its distribution among cyanobacteria appears to be limited. Anabaena variabilis and Anabaena sp. 6411 allophycocyanin, C-phycocyanin, and phycoerythrocyanin were purified to homogeneity and characterized with respect to molecular weight, isoelectric point, absorption spectrum and amino acid composition. The and subunits of each of these proteins were also purified to homogeneity and characterized in the same manner. The tetrapyrrole chromophore content was determined for each of the proteins and subunits. The subunit of phycoerythrocyanin carries a novel phycobiliviolin-like chromophore. This chromophore has not previously been detected in cyanobacterial biliproteins, but has been noted as a prosthetic group of a cryptophytan phycocyanin.Sedimentation equilibrium studies show that at pH 7.0, at protein concentrations of 0.2–0.6 mg/ml, allophycocyanin, C-phycocyanin and phycoerythrocyanin, each exists as a trimeric aggregate, ()₃, of molecular weight of approximately 105000. Structural studies of microcrystals of these three biliproteins by electron microscopy and X-ray diffraction reveal a common plan for the construction of higher assembly forms. The major building block appears to be the trimer ()₃. It is proposed that this is a dise-like structure about 3.0×12.0 nm. The individual or subunits are roughly spherical, 3 nm in diameter. Allophycocyanin trimers stack to form bundles of rods which form long needles. Both phycocyanin and phycoerythrocyanin form double dises ()₆ which are visible as ring-shaped structures by electron microscopy. The mode of assembly of the biliproteinstructures in the phycobilisome is, as yet, unknown.Abbreviation Used SDS sodium dodecyl sulfate Dedicated to Prof. Dr. Roger Y. Stanier on the occasion of his 60th birthday.