Rapid and economical production of microsomal cytochrome P-450 in yeast resuspended in 20% glucose medium: relationship to the biosynthesis of mitochondrial cytochromes [proceedings].

Cytochrome P-450 was originally found in anaerobically grown baker’s yeast and to a lesser extent in yeast grown aerobically to the end of the exponential phase of growth in relatively high-glucose medium (4 %, w/v) (Lindenmeyer & Smith, 1964). Good yields of cytochrome P-450 can be obtained under conditions of glucose repression of mitochondria, for example in 20% glucose medium, and the presence of cytochrome a+a3 (cytochrome oxidase) was noted to be reciprocal with cytochrome P-450 (Wiseman et al., 1975). We now report a rapid and economical production of relatively large amounts of cytochrome P-450 when a heavy inoculum of yeast, previously grown to the end of the exponential phase in growth medium containing as little as 0.5% glucose, is transferred to a relatively small volume of 20% glucose medium (4-fold growth of the yeast occurs). This synthesis, over at least 8h, is accompanied by a failure to resynthesize cytochrome a+a3 (unlike the resynthesis of cytochromes b and c that occurs after a 4h lag phase) despite growth. Theconcentration of cytochrome a+a3 in the yeast was measured by the method of Williams (1964) from the reduced-versus-oxidized difference spectrum. in this method, four simultaneous equations in four unknowns were derived from the knowledge of the contributions in absorbance of each cytochrome to the major maxima and minima of each of these cytochromes. Measurement of the difference in absorbance at four wavelength pairs between 500 and 640nm enables the equations to be solved, this process being speeded up considerably by us by the use of a computer program. Spectra similar to those obtained by Williams (1964) for isolated rat liver mitochondria have been reported for whole baker’s-yeast cells (Chance, 1957) and for Saccharomyces cerevisiae and Candida lipolytica (Skipton et al., 1973). Saccharomyces cerevisiae (N.C.Y.C. no. 240) was grown from a slope culture as described previously (Wiseman et al., 1975), in 0.5 % glucose medium. The yeast was harvested by centrifugation after 48 h and washed once in lOOmM-citrate/phosphate buffer, pH5.8. The yeast thus obtained was divided into 500mg amounts and each resuspended In 15ml of 20% glucose growth medium in a 25ml conical flask and shaken at 30°C as before. At various time intervals up to 24h the yeast suspension was centrifuged and the pressed (wet) weight of yeast was determined. The yeast was then resuspended in 5 ml of distilled water, and 2.5 ml put into each of two 3 ml spectrophotometer cuvettes. A few grains of sodium dithionite were added to the sample cell and Sop1 of 20-volume hydrogen peroxide was added to the reference cell. The difference spectrum between 500 and 640nm was then measured in a Unicam SP. 1800 recording spectrophotometer. The concentration of cytochrome P-450 was measured as described by Wiseman et af . (1975) by reducing the contents of the reference cell with sufficient dithionite. Fig. 1 shows rapid production, and maintenance of a high concentration of cytochrome P-450, especially in view of the 4-fold growth of the yeast with no apparent lag phase, in 20% glucose medium in about 10h. There is no synthesis of cytochrome a+a3 and therefore its concentration per g of yeast decreases in line with the dilution of the