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A quantitative assay is described for the measurement of cytochrome o in intact cells of E. coli . The procedure involves flash photolysis of the CO-liganded, reduced enzyme in the absence of O2 at temperatures (approx. -100°C) at which the rate of recombination of CO is immeasurably slow. Other CO-binding pigments known to be present, particularly cytochrome d , are excluded from the photodissociation spectrum under these conditions. Measurement of the content of cytochrome o in bacteria separated into size (and thus age) classes by zonal centrifugation shows that the cytochrome accumulates continuously, probably exponentially, throughout the cell cycle and thus constitutes a constant proportion of cell protein during the cycle. The velocity of recombination of CO with cytochrome o at –65 °C is invariant over the cell cycle.
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