ATP(GTP)-dependent conversion of MVM parvovirus single-stranded DNA to its replicative form by a purified 10 S species of mouse DNA polymerase alpha

A species of DNA polymerase alpha that is active in the ATP(GTP)-dependent conversion of MVM parvovirus single-stranded linear DNA to the duplex replicative form has been purified 4300-fold from Ehrlich ascites mouse tumour cells. The single-stranded----replicative form activity is maintained throughout ammonium sulfate precipitation, DEAE-cellulose, phosphocellulose and hydroxyapatite column chromatography and glycerol gradient sedimentation. Polypeptides with Mr = 230 000, 220 000, 183 000, 157 000, 125 000, 70 000, 65 000, 62 000, 57 000, 53 000 and 48 000 copurify with the single-stranded----replicative form activity, which sediments at approx. 10 S. The Mr = 183 000, 157 000 and 125 000 polypeptides exhibit catalytic activity when assayed in situ following SDS-polyacrylamide gel electrophoresis. The 10 S form of DNA polymerase alpha is functionally distinguishable from an 8.4 S form of the enzyme obtained from the same cells on the basis of single-stranded----replicative form activity. The single-stranded----replicative form activity of the 10 S enzyme is stable at 22 degrees C for up to 3 h, but exhibits a half life of only 5 min at 45 degrees C.     

Iron-induced lipid peroxidation and inhibition of dopamine synthesis in striatum synaptosomes

Crude striatum synaptosomes (P₂ fraction) from Fisher 344 female rats were incubated in the presence of ADP-chelated Fe³⁺ (0.5–50 M) and ascorbate (250 M). Intrasynaptosomal conversion of tyrosine to dopamine (DA) was measured by¹⁴CO₂ evolution froml-[1-¹⁴C]tyrosine in the absence of added cofactors and DOPA decarboxylase. Malondialdehyde (MDA) was measured as an index of lipid peroxidation. A concentration-dependent inhibition of DA synthesis by ADP-Fe³⁺/ascorbate was found with 50% inhibition occurring at 2.5 M Fe³⁺ concentration. This was accompanied by marked accumulation of MDA. Ascorbate or ADP alone did not affect DA synthesis and ADP-Fe³⁺ in the absence of exogenous ascorbate was effective only above 25 M. Exogenously added MDA did not inhibit DA synthesis. Purified synaptosomes were isolated from peroxidized and control P₂ fractions using sucrose gradients. Membrane microviscosity of the purifled synaptosomes was assessed by nitroxyl spin labels of stearic acid using electron paramagetic resonance techniques. There was a significant increase in membrane microviscosity as a result of ADP-Fe³⁺/ascorbate induced peroxidation. Maleimide nitroxide spin-label binding to protein sulhydryls was significantly modified by peroxidation of striatum synaptosomes. The weakly immobilized component of the sulhydryl spin-label (w) was drastically decreased whereas the strongly immobilized component (s) was modified less, thus leading to a marked reduction of w/s ratio. The exposure of striatum synaptosomes to the peroxidizing system resulted in a significant increase in total iron and in a 25% decrease in protein sulhydryl content. It is concluded that ironinduced damage to the DA synthetic system is mediated by alterations of the structural properties of nerve ending membranes.     

The IleL229 → Met mutation impairs the quinone binding to the QB-pocket in reaction centers of Rhodobacter sphaeroides

A spontaneous mutant (R/89) of photosynthetic purple bacterium Rhodobacter sphaeroides R-26 was selected for resistance to 200 M atrazin. It showed increased resistance to interquinone electron transfer inhibitors of o-phenanthroline (resistance factor, RF=20) in UQ_o reconstituted isolated reaction centers and terbutryne in reaction centers (RF=55) and in chromatophores (RF=85). The amino acid sequence of the Q_B binding protein of the photosynthetic reaction center (the L subunit) was determined by sequencing the corresponding pufL gene and a single mutation was found (Ile^L229 Met). The changed amino acid of the mutant strain is in van der Waals contact with the secondary quinone Q_B. The binding and redox properties of Q_B in the mutant were characterized by kinetic (charge recombination) and multiple turnover (cytochrome oxidation and semiquinone oscillation) assays of the reaction center. The free energy for stabilization of Q_AQ_B ^– with respect to Q_A ^–Q_B was G_AB=–60 meV and 0 meV in reaction centers and G_AB=–85 meV and –46 meV in chromatophores of R-26 and R/89 strains at pH 8, respectively. The dissociation constants of the quinone UQ_o and semiquinone UQ_o ^– in reaction centers from R-26 and R/89 showed significant and different pH dependence. The observed changes in binding and redox properties of quinones are interpreted in terms of differential effects (electrostatics and mesomerism) of mutation on the oxidized and reduced states of Q_B.Abbreviations BChl bacteriochlorophyll - Ile isoleucine - Met methionin - P primary donor - Q_A primary quinone acceptor - Q_B secondary quinone acceptor - RC reaction center protein - UQ_o 2,3-dimethoxy-5-methyl benzoquinone - UQ₁₀ ubiquinone 50 This work is dedicated to the memory of Randall Ross Stein (1954–1994) and is, in a small way, a testament to the impact which Randy's ideas have had on the development of the field of competitive herbicide binding.     

A new mutation protocol for obtaining auxotrophic mutants of the yeastPhaffia rhodozyma

Summary A new mutation strategy, which involves -irradiation of cells followed by a selective enzymatic enrichment step, was worked out to obtain auxotrophic mutants from the astaxanthin-producing yeast P. rhodozyma. Under the optimized conditions described, different mutants suitable for strain improvement were isolated.