Mechanisms of nitroso compound-induced inhibition of superoxide generation in neutrophils: fluorescence quenching of perylene by nitroso-compounds in the membrane fractions of neutrophils.

To investigate the mechanism of nitroso compound-induced inhibition of the respiratory burst in neutrophils, we studied fluorescence quenching of perylene by nitroso-compounds in the membrane fractions of neutrophils at 17, 27, and 37 degrees C and the reagent-induced inhibition of superoxide generation at 28 and 37 degrees C. With increasing temperature, the quenching of perylene fluorescence and inhibition of superoxide generation by nitrosobenzene (NB) were both diminished, while those by 2-nitrosotoluene (NT) were both enhanced. The temperature dependence of the inhibition constants and the quenching constants indicates that the binding of NB is exothermic (deltaH= -27 kJ/mol for inhibition and deltaH= -29 kJ/mol for quenching) and essentially enthalpy-driven.

Effect of aromatic nitroso-compounds on superoxide-generating activity in neutrophils.

Aromatic nitroso-compounds such as nitrosobenzene inhibited the respiratory burst of intact neutrophils induced by various stimulants, including phorbol 12-myristate 13-acetate and a chemotactic peptide. The compounds also inhibited NADPH-dependent oxygen consumption by cell-free preparations of neutrophils. This indicates that nitroso-compounds act directly on the NADPH-oxidase system.

Superoxide production by cytochrome b558 purified from neutrophils in a reconstituted system with an exogenous reductase.

Cytochrome b558, which is considered to be an essential component of the phagocytic superoxide (O2-)-generating system, was highly purified from porcine neutrophils. The isolated cytochrome was resolved into two polypeptides with molecular masses of 60-90 and 19 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For enzymatic reduction of purified cytochrome b558, we utilized hepatic NADPH-cytochrome P450 reductase purified from rat liver microsomes.

Effect of a light-induced pH gradient on purple-to-blue and purple-to-red transitions of bacteriorhodopsin.

Bacteriorhodopsin-containing vesicles that were able to alkalize the extravesicular medium by greater than 1.5 pH units under illumination, i.e.

Effect of partial delipidation of purple membrane on the photodynamics of bacteriorhodopsin.

The effect of lipid-protein interaction on the photodynamics of bacteriorhodopsin (bR) was investigated by using partially delipidated purple membrane (pm). When pm was incubated with a mild detergent, Tween 20, the two major lipid components of pm, phospholipids and glycolipids, were released in different ways: the amount of phospholipids released was proportional to the logarithm of the incubation time; the release of glycolipids became noticeable after the release of approximately 2 phospholipids/bR, but soon leveled off at approximately 50% of the initial content. It was found that the thermal decay of the photocycle intermediate N560 was inhibited by the removal of less than 2 phospholipids per bR.